In Vitro Cell. Dev. Biol.ÐPlant 37:701±729, November±December 2001 DOI:10.

1079/IVP2001216
q 2001 Society for In Vitro Biology
1054-5476/01 $10.0010.00

INVITED REVIEW:

IN VITRO CHILI PEPPER BIOTECHNOLOGY

NEFTALI OCHOA-ALEJO1* and RAFAEL RAMIREZ-MALAGON2

1
Departamento de IngenierõÂa GeneÂtica de Plantas, Unidad de BiotecnologõÂa e IngenierõÂa GeneÂtica de Plantas, Centro de InvestigacioÂn y de
Estudios Avanzados del Instituto PoliteÂcnico Nacional (CINVESTAV-Unidad Irapuato), Apartado Postal 629, 36500-Irapuato, Gto., MeÂxico
2
Instituto de Ciencias AgrõÂcolas, Universidad de Guanajuato, Apartado Postal 311, 36500-Irapuato Gto., MeÂxico

(Received 17 May 2000; accepted 13 April 2001; editor G. C. Phillips)

Summary
Chili pepper is an important horticultural crop that can surely benefit from plant biotechnology. However, although it is
a Solanaceous member, developments in plant cell, tissue, and organ culture, as well as on plant genetic transformation,
have lagged far behind those achieved for other members of the same family, such as tobacco (Nicotiana tabacum), tomato
(Lycopersicon esculentum), and potato (Solanum tuberosum), species frequently used as model systems because of their
facility to regenerate organs and eventually whole plants in vitro, and also for their ability to be genetically engineered by
the currently available transformation methods. Capsicum members have been shown to be recalcitrant to differentiation
and plant regeneration under in vitro conditions, which in turn makes it very difficult or inefficient to apply recombinant
DNA technologies via genetic transformation aimed at genetic improvement against pests and diseases. Some approaches,
however, have made possible the regeneration of chili pepper plants from in vitro-cultured cells, tissues, and organs
through organogenesis or embryogenesis. Anther culture has been successfully applied to obtain haploid and doubled-
haploid plants. Organogenic systems have been used for in vitro micropropagation as well as for genetic transformation.
Application of both tissue culture and genetic transformation techniques have led to the development of chili pepper
plants more resistant to at least one type of virus. Cell and tissue cultures have been applied successfully to the selection
of variant cells exhibiting increased resistance to abiotic stresses, but no plants exhibiting the selected traits have been
regenerated. Production of capsaicinoids, the hot principle of chili pepper fruits, by cells and callus tissues has been
another area of intense research. The advances, limitations, and applications of chili pepper biotechnology are discussed.
Key words: Capsicum; plant regeneration; transformation; metabolite production; capsaicinoids.

Introduction
All chili peppers belong to the genus Capsicum of the Solanaceae
family. Pepper, chili, chile, chilli, aji, paprika, and Capsicum are
used interchangeably to describe the plants and fruits of the genus
Capsicum (DeWitt and Bosland, 1993). There are 27 species of
Capsicum, but only five have been domesticated and are currently
cultivated: C. annuum LinneÂ, C. baccatum LinneÂ, C. chinense
Jacquin, C. frutescens LinneÂ, and C. pubescens Ruiz & Pavon.
Barbara Pickersgill has proposed that the first varieties of chili
peppers originated in the remote geologic past in an area bordered
by mountains of southern Brazil to the east, by Bolivia to the west,
and by Paraguay and northern Argentina to the south (DeWitt and
Bosland, 1993). This location is called a nuclear area and has the
greatest concentration of wild species of chili peppers in the world.
The five major cultivated species are derived from different
ancestral stocks found in three distinct centers of origin. Mexico is
the primary center for C. annuum, with Guatemala a secondary
*Author to whom correspondence should be addressed: Email nochoa@
ira.cinvestav.mx
center; Amazonia for C. chinense and C. frutescens; and Peru and
Bolivia for C. baccatum and C. pubescens. Capsicum annuum and
C. frutescens are widely distributed from Mexico through Central
America and throughout the Caribbean region (Greenleaf, 1986).
Capsicum annuum is the most extensively cultivated species in the
world. It is the principal species grown in Hungary, Mexico, China,
Korea and the East Indies. In Mexico, the spanish term chile refers
to both hot and sweet types; therefore, chili pepper will be used
interchangeably in this review to describe the plants and pods of the
genus Capsicum.
Because of the diversity of forms, colors, shapes, flavors,
pungency and aromas, chili pepper fruits are important items
worldwide as ingredients of a wide variety of dishes in many
countries and also as salads, pickles, paprika, chili powder, curry
powder, and pepper sauces. Chili peppers are the most used
condiment and spice in the entire world, having dethroned black
pepper (Andrews, 1995). Chili pepper fruits are among the most
important commercially grown vegetables in the tropics, ranking
only after tomato. They are exported to temperate countries in a
dried form for use as a spice in flavoring sauces and processed
foods. The world production of Capsicum fruits in 1998 was

701
702 OCHOA-ALEJO AND RAMIREZ-MALAGON
approximately 16 000 000 metric tons (FAO, 1998). China and
Nigeria were the leaders in chili pepper cultivation with 352 470
and 195 000 ha, respectively, followed by Indonesia (111 687 ha),
Mexico (110 000 ha), Korea (83 000 ha), Turkey (68 000 ha), USA
(26 568 ha), and Yugoslavia (24 491 ha).
The nutritive value of Capsicum fruits is based on their
significant content of essential nutrients and their high levels of
vitamin C (ascorbic acid) and vitamin A. Chili pepper fruits are also
good sources of the B-vitamin complex (Maga, 1975).
Fruits of Capsicum have been found to exhibit antimicrobial
properties, and physiological and pharmacological effects (Govin-
darajan and Sathyanarayana, 1991). Pungency is apparently the
only pharmacologic property required of chili peppers as a
medicine. Only the more pungent, not sweet peppers, act as
therapeutic agents. Chili pepper extracts, known as oleoresins,
contain the sensory qualities of fresh peppers: color, flavor,
pungency, and aroma. Capsaicinoids, the hot principle of fruits,
are widely used for the preparation of industrial hot sauces (Tabasco
sauce, for example) as well as for elaboration of different
pharmaceutical products including pads for relieving muscular
pains, rubefascient creams, products for alleviating the pain of
diseases like arthritis, post-herpetic neuralgia, diabetic neuropathy,
and non-allergic rhinitis among others. Shampoos to avoid or
prevent hair loss, and antioxidant pills made of chili pepper extracts
are currently sold in the market. Capsaicin or hot chili pepper
extracts are also used as repellents in aerosol sprays against dogs
and thieves. Hot-candies containing chili pepper products are very
popular for children in Mexico.
The extractable colors of chili pepper fruits are used extensively
in the food processing industry to color a wide range of products
such as sausages and other meat products, as well as for cheeses,
butters, salad dressings, condiment mixtures, gelatin desserts and
some other processed foods (Govindarajan, 1986). The consumers
from Latin America prefer fresh eggs with a deep yellow color in the
egg yolks, and a yellow skin color in dressed chickens fed with
rations that contain capsanthin, the red coloring factor of chili
pepper fruits.
Chili pepper production, like any other crop of economic
importance, is affected by different biotic and abiotic factors that
diminish the yields (DeWitt and Bosland, 1993). Most chili peppers
are susceptible, to different extents, to phytopathogenic fungi (fruit
rots of several kinds, damping-off caused by Pythium, Rhizoctonia
and Fusarium, root rot or Phytophthora blight by Phytophthora
capsici, southern blight by Sclerotium rolfsii, anthracnose by several
species of Colletotrichum), bacteria (bacterial leaf spot caused by
Xanthomonas campestris pv. vesicatoria, bacterial soft rot by
Erwinia carotovora pv. carotovora, and southern bacterial wilt
by Pseudomonas solanacearum), and viruses (tobacco mosaic virus,
tobacco etch virus, cucumber mosaic virus, potato virus Y, pepper
mottle virus, tobacco ring spot virus, pepper huasteco virus, Texas
pepper virus, beet curly top virus, among others). Insects and other
pests (European corn borers, flea beetles, fruit worms, grasshoppers,
green pea aphids, grubs, hornworms, leafhoppers, leaf miners,
pepper maggots, pepper weevils, white flies, thrips, spider mites
and root-knot nematodes) can also cause extensive losses in the
yield and quality of peppers. Abiotic factors that can disease and
injure chili peppers include extreme levels of temperature,
moisture, light, nutrients, pH, air pollutants and pesticides.
Breeding programs have been established in countries that produce
chili peppers in order to improve resistance or tolerance against all
the above-mentioned factors and for other horticultural characters,
including earliness, pedicel length, male sterility, and shape, size,
quality, flavor, color and pungency of fruits, among others
(Greenleaf, 1986). Traditional breeding techniques have been of
great value for chili pepper genetic improvement, but biotechno-
logical techniques involving plant tissue culture and recombinant
DNA technologies could be powerful auxiliary tools to accelerate
and achieve this goal. Morrison et al. (1986a), FaÂri (1986), and
Ezura (1997) have previously published excellent reviews on chili
pepper tissue culture, but increasing information has been
generated in recent years, including recent advances in genetic
transformation of Capsicum.
Chili Pepper Tissue Culture: from Cells, Tissues, and Organs
to Plants
In vitro plant regeneration from cells, tissues, and organ cultures
is a fundamental process for the application of plant biotechnology
to plant propagation, plant breeding and genetic improvement.
Despite the economic importance of chili peppers, plant regenera-
tion systems for Capsicum species have not progressed as fast as
those for some other solanaceous crops like tobacco, tomato, and
potato, frequently used as model systems because of their great
capability to regenerate plants from cells, tissues, and organs. In the
case of Capsicum members, they have been shown to be difficult
with regard to regeneration of whole plants from explants and other
tissues. Reports on chili pepper plant regeneration from established
callus lines, cell suspensions, and protoplasts are currently very
scarce, suggesting severe recalcitrant morphogenic problems. Chili
pepper plant regeneration has been achieved mainly through
organogenesis, but recent reports have increased the information on
somatic embryogenesis.
Plant regeneration from protoplasts. Plant regeneration from
chili pepper cells is in its infancy. There are only three published
papers dealing with plant regeneration from protoplasts and just one
from embryogenic cell suspensions. Although chili pepper proto-
plasts can be obtained easily (Fig. 1), cell division and plant
regeneration have been found to be rare events. The importance of
chili pepper protoplast cultures resides in the possibility of
recovering either somaclonal variants or somatic hybrids in cases
of sexual interspecific incompatibility. Somatic hybridization might
provide an alternative method to conventional hybridization for
improving Capsicum.
Saxena et al. (1981) reported the isolation of protoplasts from
axenic shoot cultures of Capsicum annuum L. cv. California
Wonder. Excised shoot tips were cultured on MS medium
(Murashige and Skoog, 1962) supplemented with 13.94 mM kinetin
(Kin), 2.85 mM indoleacetic acid (IAA), and 7% coconut water.
Leaf tissue from shoot cultures was digested with a mixture of 2%
cellulase (Onozuka R-10), 0.4% macerozyme (R-10) in MS liquid
medium supplemented with 0.5 M mannitol as osmoticum. Proto-
plasts were cultured on a medium containing the mineral salts of
DPD (Durand, 1979) or NT medium (Nagata and Takebe, 1971)
with 4.5 mM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.37 mM
naphthaleneacetic acid (NAA) and 4.44 mM benzyladenine (BA),
2% sucrose and 0.5 M mannitol. The density of the protoplast
suspension was adjusted to 5  104 cells per ml, and small drops
were cultured between two layers of 0.7% agar in Petri dishes.
 IN VITRO CHILI PEPPER BIOTECHNOLOGY
Fig. 1. Chili pepper (C. annuum L. cv. TampiquenÄo 74) protoplasts. Cotyledon tissues from 15-d-old seedlings were treated with 2%
cellulase and 0.5% macerozyme in the presence of 0.5 M mannitol, pH 5.6, at 298C for 14 h.
Protoplasts underwent sustained mitotic activity and proliferated to
form callus masses on both culture media, and under dark
conditions. The callus masses differentiated into shoots on the
differentiation medium [22.83 mM IAA, 11.90 mM Kin, and 3%
sucrose] when they were exposed to light. The shoots were induced
to form roots on a medium with 5.71 mM IAA and 0.186 mM Kin.
Flowering plants were recovered 5 mo. after protoplast isolation.
Leaf protoplasts were isolated from axenic shoot cultures
established on MS medium without growth regulators from seedlings
of four cultivars of Capsicum annuum (Americano, Dulce Italiano,
Florida Giant, and Nigrum) and a genotype of C. chinense (DõÂaz
et al., 1988). Shoots were cut into small pieces and plasmolyzed for
1 h in CPW13M medium (Power and Chapman, 1985) and then
treated with an enzyme mixture of 1% cellulase (Onozuka R-10)
and 0.25% macerozyme (R-10) in CPW13M medium. After
incubation and washing, protoplasts were transferred to a mixture
of 2:1 KM8P/KM8 media (Kao and Michayluk, 1975) at a final
plating density range of 5  104 to 1  105 protoplasts per ml. The
protoplast suspension was dispensed as agarose-medium drops into
Petri dishes and bathed by liquid medium. Incubation was carried
out under continuous illumination. Protoplasts of both species
entered division except for Nigrum cultivar. Colonies transferred to
MS medium supplemented with 4.56 mM zeatin (Zea) formed
callus. The calluses were finally cultured on MS medium with
8.88 mM BA. Regenerated shoots were rooted on MS medium
without growth regulators.
A protocol for protoplast isolation from leaf tissue of three
cultivars of Capsicum annuum and two genotypes of C. chinense was
developed (Murphy and Kyle, 1994). The optimized procedure was
as follows: true leaves were sliced, plasmolyzed by submersion in a
mannitol solution of the appropriate concentration, according to
703
the Capsicum material, and shaken at 170 rpm for 10 min at
258C. C. annuum NuMex RNaky and Early Calwonder required
0.6 M mannitol solutions, whereas for Avelar cultivar 0.42 M
mannitol was better for protoplast isolation. Tissues from
C. chinense genotypes were incubated in the presence of 0.43±
0.45 M mannitol for better protoplast yield. The mannitol solution
was decanted and the plasmolyzed leaves were submerged in an
enzyme solution consisting of 1% macerase, 0.25% pectolyase, and
1% cellulysin dissolved in mannitol. The leaves in enzyme solution
were placed on a shaker at 70 rpm and at 258C for 4 h. Yields of
650 000 protoplasts per 0.5 g of leaf tissue were consistently
obtained. Protoplasts from these preparations were found to be
excellent systems for viral RNA infection (mottle potyvirus, tobacco
etch potyvirus, or cucumber mosaic cucumovirus) by electroporation.
Plant regeneration from mesophyl protoplasts of chili pepper,
Capsicum annuum L. cv. California Wonder, was reported by
Prakash et al. (1997). Protoplasts isolated from fully expanded
leaves of 3-wk-old axenic shoots were cultured in TM medium
(Shahin, 1985) supplemented with 5.37 mM NAA ‡ 4:52 mM
2; 4-D ‡ 2:22 mM BA. Division of protoplasts was observed at a
frequency of 20±25%. The antioxidants ascorbic acid and
polyvinylpyrrolidone in the medium and incubation in the dark
helped to overcome browning of protoplasts. Microcalluses and
macrocalluses were formed in TM and MS medium, respectively,
containing 10.74 mM NAA ‡ 2:22 mM BA. Microshoots, two to five
per callus, appeared on MS gelled medium enriched with 2.85 mM
IAA ‡ 5:78 mM GA ‡ 44:4 mM BA. Rooting of shoots was achieved
on half-strength gelled medium containing 5.37 mM NAA ‡
2:22 mM BA. Protoplasts isolated from cotyledons failed to divide.
Plant regeneration from tissues. Through organogenesis. Gunay
and Rao (1978) were the first to report the successful regeneration
704 OCHOA-ALEJO AND RAMIREZ-MALAGON
of shoots and plants from cotyledon and hypocotyl explants of two
C. annuum cultivars (Pimento and California Wonder) and a hybrid
of C. frutescens (Bharath) cultured on MS medium containing the
auxins IAA, NAA, 2,4-D, and the cytokinins BA, Kin, Zea, 6-
benzyl-9-tetrahydropyrane adenine (SD8339), adenine (Ade), and
coconut milk. Presence of 4.52 mM 2,4-D in the culture medium
induced friable callus formation, but had an inhibitory effect on
root and shoot bud formation. Rooting occurred on media
containing 2.85±5.71 mM IAA (thin and long roots) or 5.37 mM
NAA (thick and short roots). Kin, Ade, and SD8339 were found to
be ineffective for inducing bud or shoot differentiation. Zea
promoted bud formation from cotyledons of the C. frutescens hybrid
only. BA was shown to induce shoots from cotyledon explants of all
three genotypes and from hypocotyls of the hybrid. Higher shoot
regeneration was achieved on basal medium supplemented with
2.85±5.71 mM IAA combined with 8.88 mM BA after 6 wk of
incubation. Shoot buds regenerated from the explants also
developed roots on the shoot-regenerating culture medium, and
grew into whole plants that were eventually transferred to soil.
The relationship between position and morphogenetic responses
of hypocotyl explants of C. annuum cultivar T. Havani was studied
by FaÂri and Czako (1981). Hypocotyl sections were cultured on MS
medium supplemented with 8.8 mM BA and 5.7 mM IAA, and
shoot regeneration was observed to occur only in the apical
segments, whereas the middle and basal sections produced only
roots and callus, respectively. Some of the regenerated shoots
developed into whole plants after rooting on culture medium lacking
growth regulators.
Differentiation of multiple shoot buds and plantlets in cultured
embryos of C. annuum var. Mathania was reported by Agrawal and
Chandra (1983). Excised embryos were cultured on MS medium
supplemented with Kin and BA alone or in combination with IAA
and 2,4-D. In the presence of 0.46 mM Kin and 0.57 mM IAA, the
size of cotyledons increased considerably, while 2,4-D alone (2.76±
4.52 mM) or in combination with 2.32 mM Kin promoted friable
callus formation all over the surface of the embryos. Numerous
shoot buds were produced on the margins of the expanded
cotyledons of embryos grown on medium with 22.2 mM BA.
These buds proliferated further into numerous shoot buds when they
were subcultured on fresh culture medium of the same composition.
By subculturing individual buds on a medium supplemented with
0.54±26.86 mM NAA, rooting was induced within 7±10 d, and the
best growth of root and shoot for plant regeneration was observed in
0.54 mM NAA.
Phillips and Hubstenberger (1985) reported organogenesis in
tissue cultures of C. annuum cvs. California Wonder, Yolo Wonder
(sweet bell types), New Mexico no. 6-4 (NM 6-4) and NuMex R.
Naky (long green chili types). The importance of light regime,
growth regulators, and carbon source on shoot organogenesis was
studied. Shoot and root organogenesis in seedling explants was
restricted to primary cultures or those less than 3 mo. old under 12-
and 16-h photoperiod at 258C. Shoot organogenesis was extended to
5 mo. under continuous light at 258C, and to 8 mo. under
continuous light at 28.58C. Shoot elongation and rooting was
promoted in some explants on MS medium containing 0.28 mM IAA
and 0.22 mM BA, while 0.22±17.76 mM and 22.2±44.4 mM BA
stimulated adventitious shoot bud formation. Glucose was found to
be a superior carbon source in comparison to sucrose. Shoot
elongation was shown to be the limiting factor in plant regeneration.
In a series of reports, Sripichit et al. (1987, 1988a, b)
investigated the in vitro shoot-forming capacity of cotyledon
explants in red pepper (C. annuum L. cv. Yatsufusa), the effect of
exposure dose and dose rate of gamma radiation on shoot formation,
and the employment of this system to induce and recover mutant
plants from in vitro cultures. BA was found to be more effective than
Kin in inducing shoot formation in cotyledon explants cultured on
MS medium (Sripichit et al., 1987). BA levels in the range of
13.32±31.1 mM induced the largest number of shoots per explant.
Cotyledon explants excised from 12-d-old seedlings gave the largest
number of shoots per explant and the frequency of shoot formation
was significantly decreased as the age of the seedling increased.
Intact cotyledons with petiole and trimmed cotyledon with petiole
showed a significantly higher frequency of shoot formation than the
blade or petiole of cotyledon. The highest shoot-forming explants
and the largest number of shoots recovered ranged from 90 to 100%
and 8.6 to 11.9, respectively.
In another study (Agrawal et al., 1989), segments of roots,
hypocotyls, cotyledons, stems internodes, stem nodes, leaves, and
shoot tips of C. annuum cv. Mathania were used as explants and
were cultured on MS medium supplemented with BA or Kin alone
or in combination with IAA, indolebutyric acid (IBA), NAA, or
2,4-D. Medium with 2.22±4.44 mM BA induced green compact
and ephemeral callus from explants after 2 wk of incubation. With
increasing concentrations of BA (13.3 mM), shoot buds were
formed directly from both hypocotyl and cotyledon explants, while
stem internode and leaf segments showed scanty callus formation.
With further increased concentration of BA to 22.2 mM, shoot buds
were induced directly in all explants after 2±3 wk. Cotyledon, leaf
and stem explants exhibited higher bud formation than hypocotyls.
Combinations of IAA and BA were the best for shoot bud induction
in comparison with the other auxin/cytokinin combinations tested.
Shoot buds did not elongate on the induction medium. The
maximum size of buds was approximately 1 cm. Adventitious buds
regenerated from different explants on various media were
subcultured on a medium with 22.2 mM BA for further multi-
plication. Isolated shoot buds (ca. 1 cm long) were transferred to
root induction medium consisting of MS medium with NAA
(0.54 mM) or IBA (0.49 mM); under these conditions, rooting was
induced with elongation of shoot buds. Complete plants developed
in 2±3 wk. The chromosome number of all regenerated plants was
found to be diploid …2n ˆ 24†: Histological studies in cotyledon
explants revealed the direct development (without formation of
callus) of meristemoids and shoot buds from epidermal and
subepidermal layers of the explant. Vascular connections between
developing shoot buds and the vascular system of the cotyledon was
shown to occur.
Cultured hypocotyl tissues of chili pepper (C. annuum L. cv.
Esmeralda, ancho type) derived from aseptically germinated
seedlings were evaluated on the basis of their morphogenic
responses to combinations of IAA with BA, Kin or 2-isopentenyl-
adenine (2iP), and NAA or 2,4-D with BA (Ochoa-Alejo and
GarcõÂa-Bautista, 1990). Shoot and root differentiation and callus
formation were observed on the cut ends of the explants after 8 wk
of incubation under continuous light at 25 ^ 28C: Regeneration of
adventitious shoots was more prolific in explants cultured on MS
medium containing 5±50 mM IAA plus either 25±50 mM BA or
2iP, whereas rhizogenesis predominated in hypocotyl segments
incubated on media supplemented with IAA or NAA alone
 IN VITRO CHILI PEPPER BIOTECHNOLOGY
(1±50 mM). NAA produced compact or friable callus depending on the
concentration used, whereas 2,4-D elicited friable callus development.
The influence of chili pepper cultivar on the capacity of
hypocotyl tissues to form adventitious shoots was tested (Ochoa-
Alejo and Ireta-Moreno, 1990). Explants from 16 cultivars,
including pimiento, ancho, serrano, jalapenÄo, pasilla, caloro, and
Anaheim types, were grown on six culture media prepared with the
basal MS medium, supplemented with four combinations of IAA/BA
or two of IAA/2iP. Cultivar differences were observed with regard to
their capacity for in vitro differentiation of shoots and approximately
one-third of the cultivars tested showed relatively high differentia-
tion capabilities (determined by the number of shoots per explant
and the frequency of shoot formation). Optimal shoot regeneration
medium varied with cultivar. Cultivar Anaheim TMR-23 displayed
the highest shoot-regeneration response.
A very extensive study on the effect of explant source
(cotyledons, hypocotyls and zygotic embryos, and different
cultivars), diverse components of the nutrient medium (culture
medium formula, vitamins, and gelling agents), growth regulators,
incubation conditions (continuous light and photoperiod versus
dark) and culture container on Capsicum organogenesis was
reported by Alibert (1990). Histological observations of explants
under organogenic conditions revealed the formation of leafy
structures, but no shoots, on the cut edges of the explants.
Combinations of 5.71 mM IAA with 4.44 and 22.2 mM BA, 4.64
and 23.24 mM Kin, 4.92 and 24.6 mM Zea, or 4.92 and 24.6 mM
2iP were tested for bud and shoot induction. Only bud and foliar
structures were formed on cotyledon and hypocotyl explants and the
highest percentages of organogenesis were achieved in the presence
of 22.2 mM BA or 24.6 mM either Zea or 2iP. Thidiazuron (N-
phenyl-N 0 -1,2,3-thiadiazol-5-yl-urea; TDZ) alone (0, 0.1, 0.5, 1.0,
2.5, 4.54, 9.1, and 100 mM) or in combination with IAA (0, 0.57,
and 5.71 mM) or with either BA (4.44 and 22.2 mM) or Zea (4.56
and 22.8 mM) did not significantly improve the formation of buds,
leafy structures or shoots. Abscisic acid (ABA) decreased the
formation frequency of foliar structures as the concentration
increased from 0 to 10 mM, and did not improve the production
of shoots. No differences in percentages of foliar structure formation
were found to occur when nitrogen source, vitamins or gelling
agents were tested. Similar leafy structures were produced when
cotyledon, hypocotyl, and zygotic embryos were used as explants.
Cultivar differences in the capability of foliar structure formation
were also observed among 15 cultivars of C. annuum and one of
C. baccatum. When five different nutrient formulae were investi-
gated on the effect of organogenesis, MS medium was shown to be
the best. Vitamins from MS medium were also more effective for
organogenesis than those of the anther culture medium reported by
Sibi et al. (1979). Significant differences in organogenesis were
demonstrated using different gelling agents (three types of agar,
Gel-rite, and satiagel), Difco agar being the most effective. A
favorable effect of continuous light as compared with photoperiod
(12 h light/12 h dark) and dark conditions on organogenesis was
also demonstrated as previously reported by Phillips and
Hubstenberger (1985). No influence of culture receptacle (Petri
dishes, test tubes, and bottles) on organogenesis or shoot formation
was observed. This author stressed the difficulties faced when
regenerating plants since just buds and leafy structures were
produced and only one plant was regenerated from the cultured
tissues.
705
In vitro plant regeneration from cotyledons and hypocotyl
explants in two bell pepper cultivars (Capsicum annuum L. cvs.
Pico and Piquillo) was reported by Arroyo and Revilla (1991). Shoot
buds were induced from the explants after 15±20 d of culture on
MS basal medium supplemented with IAA and BA or Zea. Shoot
buds grew into rosettes that rooted in MS plus 0.54 mM NAA and
0.49 mM IBA after 15 d. Plantlets were successfully transferred to
pots and elongation took place when the plantlets were grown in the
greenhouse.
Because of the formation of ill-defined buds, leafy or shoot-like
structures in cotyledon, leaf or hypocotyl explants (Fig. 2), some
approaches have been tested to try to overcome the difficulties for
shoot bud elongation. In one strategy, rooted hypocotyls from three
cultivars of C. annuum were cultured upside down on shoot-
inducing MS medium with three combinations of BA and IAA
supplemented with 10 mM AgNO3 to prevent ethylene action
(Valera-Montero and Ochoa-Alejo, 1992). Shoot buds (4±20) were
formed on the exposed cortex of the hypocotyls in percentages
ranging from 47 to 100 depending on the cultivar and the BA/IAA
combination utilized. Hypocotyls bearing buds, after culture in the
induction medium, were placed in the normal polarity on fresh MS
medium lacking growth regulators to promote shoot elongation and
development. The observed number of elongated shoots per explant
ranged from 1 to 3, depending on the cultivar. Elongated shoots
were excised and rooted on coarse sand, wetted with half-strength
MS medium to regenerate whole plants. A variation of this approach
has been applied for in vitro regeneration of shoots from decapitated
plants of tomato (Lycopersicon esculentum Mill.), eggplant (Solanum
melongena L.), and chili pepper (Capsicum pendulum L.) (FaÂri et al.,
1992).
Ebida and Hu (1993) reported the morphogenetic responses of
seedling explants from C. annuum L. cv. Early California Wonder.
Explants (root, hypocotyl, cotyledon, and shoot tip) from 13-d-old
seedlings were grown on MS medium supplemented with different
levels of NAA and BA. The best responses for multiple shoot bud
regeneration were observed on the cut surfaces of cotyledon, shoot
tip, and hypocotyl explants (1 mo. of incubation) cultured on
medium with NAA/BA (mM) combinations of 0.54/22.2, 0/22.2, and
0.54/44.4 for the above explants, respectively. Root explants did not
form buds. When shoot buds were excised from the explant and
cultured on MS medium with 2.85 mM IAA or 2.15 mM NAA, 70%
rooted in 1 mo. Plantlets were established successfully ex vitro.
Mature seeds have also been used as explants for in vitro chili
pepper plant regeneration through a very simple organogenic
system (Ezura et al., 1993). Shoot formation was achieved in 14 bell
pepper cultivars (C. annuum) from excised explants consisting of
the proximal part of hypocotyl and radicle of mature seeds cultured
on MS medium without growth regulators. Adventitious shoot buds
occurred around the cut surfaces of elongated hypocotyls in a range
of 9±87% and one to four buds per explant, depending on the
cultivar, after 4 wk of culture. When explants bearing shoot buds
were transferred to fresh MS medium, elongated shoots were
developed (0±68%) after an additional 3 wk of culture. Shoots were
rooted on MS medium after a further 2 wk of culture. The
chromosome number of regenerated plants was found to be normal
…2n ˆ 24†: According to the authors, the main advantages of this
system are: (1) simplicity in tissue culture manipulation since only
MS medium without growth regulators is used; (2) the method is
rapid and whole plants can be regenerated in only 9 wk of culture;
706 OCHOA-ALEJO AND RAMIREZ-MALAGON
Fig. 2. Chili pepper shoot organogenesis. a, Hypocotyl, and b, cotyledon explants bearing buds and leafy structures; c, an elongated
shoot regenerated from a cotyledon explant.
(3) the method is applicable to many cultivars; and (4) detectable
variations among the regenerated plants are minimal because the
process does not require exogenous growth regulators. A modifica-
tion of this method was used by Binzel et al. (1996b) for in vitro
regeneration of a mild (cv. New Mexico-6) and a pungent (cv.
Rajpur Hirapur) chili pepper (Capsicum annuum L.). Half-seed
explants were cultured on MS medium with or without cytokinins
(Kin, BA, Zea, or 2iP). Cytokinins in the culture medium
dramatically increased both the percentage of explants forming
buds as well as the number of buds per explant, and also hastened
the rate of bud production. The best response was observed with Zea
in both cultivars. The elongation of leafy buds was severely
inhibited in the continuous presence of high concentrations of
cytokinins (30 mM). Although Kin and BA (10, 20 mM) were less
effective in inducing a large number of buds, the buds developing in
their presence elongated earlier and produced normal shoots.
Within 3±5 wk of transfer to Magenta boxes containing vermiculite
and soil, 70±85% of the rooted hypocotyls developed one or two
elongated shoots. These plantlets grew into normal plants.
Investigations on plant regeneration using the most beneficial
methods and culture media described earlier (Gunay and Rao,
1978; FaÂri and CzakoÂ, 1981; Valera-Montero and Ochoa-Alejo,
1992), and also culture media supplemented with TDZ, were
carried out by SzaÂsz et al. (1995) for important bell pepper cultivars
and hybrids grown in the Carpian basin (10 cultivars) and in Italy
(seven cultivars). Most of the Italian genotypes and two of the
Hungarian genotypes responded well, producing shoots, employing
the system of rooted hypocotyls reported previously (Valera-
Montero and Ochoa-Alejo, 1992). Only two genotypes (one Italian
and one Hungarian) gave weak responses using cotyledon explants
and shoot-inducing culture media described by other authors
(Gunay and Rao, 1978; FaÂri and CzakoÂ, 1981; Valera-Montero and
Ochoa-Alejo, 1992). However, by employing culture media with
TDZ (4±15 mM), direct shoot induction was achieved in cotyledon
explants from two Hungarian and two Italian genotypes that were
found not to be responsive with any other tested combination of
phytohormones. Two different culture media were used for
elongation of shoot buds induced on cotyledonary explants: one
containing 7.32 mM BA ‡ 2:89 mM GA3, and a second one with
2.89 mM GA3. Regenerated shoots were rooted on a medium
supplemented with 2.85 mM IAA.
Organogenic regeneration of chili pepper plants from cotyledon
explants of C. annuum cvs. NM 6-4 and Joe E. Parker has been
documented by Hyde and Phillips (1996). Plants were regenerated
in four stages: bud induction, bud enlargement, shoot elongation,
and shoot rooting. The effect of AgNO3 on bud proliferation and on
 IN VITRO CHILI PEPPER BIOTECHNOLOGY
shoot elongation was also investigated. Cotyledons were excised at
the petioles from 14±16-d-old seedlings, and each cotyledon was
divided transversely into two halves. Primary medium for bud
induction contained MS salts, L2 vitamins (Phillips and Collins,
1979), 3% glucose (Phillips and Hubstenberger, 1985), 2.85 mM
IAA, 8.88 mM BA, and 0.8% Phytagar. Cultures at all stages were
incubated under continuous light and at 28.58C since these
conditions had been found to induce buds (Phillips and
Hubstenberger, 1985). Bud enlargement occurred on a second
medium supplemented with 8.88 mM BA ‡ 5:78 mM GA3, and
5.89 mM AgNO3. Treatment with AgNO3 was necessary for multiple
shoot production and elongation to occur in the third stage of
culture. Most shoots elongated on a third medium without growth
regulators, but also on fresh bud enlargement medium. Elongated
shoots were obtained after incubations for 2, 2, and 4 wk on each
medium. Efficient rooting of shoots was achieved on a medium with
0.54±5.37 mM NAA. Other cytokinins tested were Zea and TDZ;
however, Zea induced few shoots and subsequently few well-
developed plants, whereas TDZ induced multiple shoots after 4 mo.
of culture, but they were small and did not elongate further.
Phytagar proved to be superior to other agars for inducing bud
formation and to avoid explant hyperhydricity.
A study of the relative importance of genotype, explant, medium,
and their interactions of two wild species (C. praetermissum and
C. baccatum) and five cultivars of C. annuum (G4, Bhiwapuri, Sweet
pepper, Cayenne pepper and Hybrid pepper) on three regeneration
media was reported by Christopher and Rajam (1996). Shoots were
induced from hypocotyl, cotyledon, and leaf explants on MS
medium supplemented with 5.7 mM IAA ‡ 13:3 mM BA; 22.2 mM
BA; and 44.4 mM BA. The most significant effect on shoot
regeneration was due to the explant. Leaf explants consistently
generated more shoots than hypocotyls or cotyledons in all
genotypes. Culture medium with 22.2 mM BA was the best growth
regulator for optimal shoot regeneration from leaf explants. Rooting
of regenerated shoots was achieved in 5.7 mM IAA plus 13.3 mM
BA. Complete plants exhibited a diploid number …2n ˆ 24†:
Determination and competence of chili pepper (C. annuum L. cv.
Sweet Banana) hypocotyl cultures during shoot formation seemed to
be dependent on BA and sucrose (Ramage and Leung, 1996). The
simultaneous presence of 22.1 mM BA and 3% sucrose in the MS
liquid medium during the initial stages of shoot initiation have been
found to be obligatory for high frequency shoot formation. The
explants (upper hypocotyl part) are determined for shoot formation
following a minimum of 8 d of culture in the induction medium.
Deprivation of sucrose from day 6 to day 20 of culture had no effect
on shoot-forming response. BA and sucrose seem to act indepen-
dently on different aspects (probably hormonal and energetic,
respectively) of the competence of explants to respond to the
induction medium during shoot initiation. The effect of sucrose was
not due to an osmotic effect since mannitol at the same osmotic
potential was not capable of replacing sucrose.
An improved and reliable plant regeneration system for C.
annuum based on bud formation and shoot bud elongation from
wounded hypocotyls was reported (RamõÂrez-MalagoÂn and Ochoa-
Alejo, 1996) (Fig. 3). Five factors that influence shoot regeneration
were investigated: age of seedlings (9±28 d), hypocotyl wounding
site (apical, middle, and basal), time elapsed between wounding the
hypocotyls and decapitation of seedlings (0±14 d), culture medium
and cultivars (21 cvs.). In general, seedlings at the stage of curved
707
hypocotyls (9 d old) wounded with a syringe needle in the apical
region were the best explants for shoot bud formation and elongation
of shoot buds when they were grown on MS medium without growth
regulators (Fig. 3). Decapitation after wounding also influenced the
shoot regeneration efficiency, with 10±14 d being the best period.
Up to 90% shoot regeneration in cv. Mulato Bajio (ancho type) was
obtained. Statistically significant differences were observed for
shoot formation among the cultivars tested. Regeneration of whole
plants was achieved by rooting the shoots with IBA pulses of
293.3 mM for 3 h and then subculturing on MS medium without
growth regulators. The advantages of this method are: no growth
regulators are needed to induce bud and shoot elongation, elongated
shoots are obtained at 6 wk of culture, efficiency of elongated shoot
regeneration is higher than in previously reported systems (Valera-
Montero and Ochoa-Alejo, 1992; Ezura et al., 1993).
Because of the problems faced with shoot bud elongation, some
other growth regulators have been tested. A plant steroid lactone
(24-epi-brassinolide; EBR) was employed for elongation of shoot
buds of C. annuum L. cvs. Jupiter and Pimiento Perfection (sweet
peppers) (Franck-Duchenne et al., 1998). Shoot buds regenerated
from cotyledon explants of Jupiter cultivar cultured on MS
medium with 34 mM IAA and 35 mM BA, and from Pimiento
Perfection (5.9 mM IAA and 13 mM BA) were placed on media
containing 0.1 mM of the brassinolide in the presence or absence
of 9.1 mM Zea plus 5.2 mM GA3 for further elongation. No stem
elongation was induced on shoot buds with the combination of
Zea and GA3 alone, but normal leaf development was observed.
In this case, more than 50% of the shoot buds developed true
leaves. When EBR at 0.1 mM was present alone in the culture
medium, 36% leaf elongation and 22% stem elongation was
achieved for Jupiter cultivar, whereas 21% leaf elongation and no
stem elongation was recorded for Pimiento Perfection cultivar.
The best treatment for leaf and stem elongation for both cultivars
was double subculture of shoot buds for 2 wk in the presence of
0.1 mM EBR and then a subculture for 2 wk on a medium
supplemented with 0.1 mM EBR ‡ 9:1 mM Zea plus 5.2 mM GA3
where 100% leaf elongation and 38% stem elongation occurred,
respectively, for Jupiter, and 29% leaf elongation and 38% stem
elongation for Pimiento Perfection. It seems that EBR acts
indirectly on stem elongation as an elicitor or enhancer of
elongation in combination or synergism with endogenous or
exogenously added growth regulators. Elongated shoots were
rooted with 0.5 mM NAA and whole plants were regenerated and
transferred to soil to obtain mature plants.
Phenylacetic acid (PAA) has been found also to improve chili
pepper shoot bud elongation (Husain et al., 1999). A three-stage
protocol for the regeneration of chili pepper (C. annuum L.) from
cotyledon explants was established. PAA was included in both the
shoot bud induction and the elongation medium. Induction medium
was supplemented with BA (22.2 or 31.1 mM) and PAA (14.7 mM),
and elongation medium contained BA (8.88 or 22.2 mM) and PAA
(14.7 mM). Shoot bud elongation was achieved in 100% of the
cultures and the elongated shoots were rooted on a medium with
NAA (5.37 mM). Comparatively, cotyledon explants cultured on
medium with BA alone (22.2 or 31.1 mM) or combined with IAA
(1.14±11.4 mM) showed distorted shoots that did not develop into
normal shoots.
Hyperhydricity in chili pepper plants regenerated in vitro was
studied by Fontes et al. (1999). Plants regenerated by the system
708 OCHOA-ALEJO AND RAMIREZ-MALAGON

Fig. 3. Chili pepper plant regeneration from decapitated seedlings. a, Etiolated 9-d-old seedling wounded in the apical part of the
hypocotyl; b, decapitated seedlings showing callus tissue formation at the wounding site; c, adventitious shoot formation at the wounding
site; d, elongated shoots growing on the original hypocotyl explants.

described by Valera-Montero and Ochoa-Alejo (1992) were
investigated by ultrastructural analysis, SDS-PAGE protein electro-
phoresis and immunoblot analysis. In non-hyperhydric leaves the
chloroplasts of the palisade cells had normally developed
thylakoids and grana and low accumulation or absence of starch
grains and plastoglobules, whereas in the hyperhydric plants the
chloroplasts exhibited thylakoid disorganization, low grana number,
an accumulation of large starch grains and low accumulation or
absence of plastoglobules. The structure of mitochondria and
peroxisomes did not change, but the number of peroxisomes did
increase. The SDS-PAGE protein analyses of hyperhydric leaves
revealed the reduced accumulation of a 55±60 kDa protein
(probably the large subunit of 1,5-ribulose bisphosphate carbox-
ylase) and the induction of an 80 kDa protein [binding protein, BiP,
an endoplasmic reticulum (ER)-luminal resident molecular chaper-
one]. Hyperhydricity was also monitored by the induction of the
ER-luminal resident protein. Immunoblotting of total protein using
an anti-soybean BiP serum indicated that the induction of the
80 kDa protein was related to BiP.
Direct and indirect in vitro plant regeneration from chili pepper
(Capsicum annuum L. cv. Soroksari) was reported by Berljak
(1999). Shoot tip, cotyledon, and hypocotyl explants excised from
2-wk-old seedlings were cultured on MS medium supplemented
with B5 (Gamborg et al., 1968) or L2 (Phillips and Collins, 1979)
vitamins, and different content of growth regulators: 2,4-D, BA,
IAA, and Zea. Abundant callus was developed on explants cultured
on media with 2,4-D. Direct shoot regeneration was achieved only
from the basal part of shoot-tip explants cultured on media with
4.4 mM BA or 9.1 mM Zea alone, and with 8.9 mM BA and 2.9 mM
IAA. Regenerated shoots, rooted on MS-B5 medium with IAA, were
successfully transferred to greenhouse conditions. Shoot bud
regeneration and leafy structures were regenerated only from
callus transferred onto medium with BA (17.8 mM) and GA3
(5.8 mM). It was found that a very important step for shoot
development from chili pepper callus was transfer after 2 wk onto
the regenerative medium with lower concentrations of BA (8.9 mM)
and GA3 (2.9 mM). Plants regenerated from callus cultures grown
ex vitro showed differences in their morphological and physiological
traits. This is the first preliminary report of a regenerable callus
system for a Capsicum species.
 IN VITRO CHILI PEPPER BIOTECHNOLOGY 709

TABLE 1

MAIN FACTORS TESTED FOR IN VITRO ORGANOGENESIS AND PLANT REGENERATION FROM CHILI PEPPER TISSUES

Factors most
Topics Tested factors commonly used References
Species C. annuum, C. frutescens, C. baccatum, C. annuum Gunay and Rao (1978); FaÂri et al. (1992);
C. pendulum, C. praetermissum Christopher and Rajam (1996);
Phillips et al. (2000)
Chili pepper types Bell peppers, long green peppers, Bell peppers Ochoa-Alejo and Ireta-Moreno (1990);
and hot peppers Arroyo and Revilla (1991); SzaÂsz et al. (1995)
Explant Cotyledons, hypocotyls, mature seeds, Cotyledons, hypocotyls, FaÂri and Czako (1981); Agrawal et al. (1989);
leaves, shoot tips, stem segments, and roots and mature seeds Ezura et al. (1993); RamõÂrez-MalagoÂn and
Ochoa-Alejo (1996); Phillips et al. (2000)
Culture medium MS, MS/B5 vitamins, and MS/L2 vitamins MS Hyde and Phillips (1996); Berljak (1999)
Growth regulators for bud/shoot BA, 2iP, Zea, TDZ, BA/IAA, 2iP/IAA, BA/IAA, BA Phillips and Hubstenberger (1985);
induction and BA/PAA Agrawal et al. (1989); Ochoa-Alejo and
GarcõÂa-Bautista (1990); SzaÂsz et al. (1995)
Growth regulators for shoot NAA, BA, Kin, GA3, BA/PAA, BA/GA3, BA/PAA SzaÂsz et al. (1995); Hyde and Phillips (1996);
elongation BA/GA3/brassinolide and brassinolide/Zea/GA3 Franck-Duchenne et al. (1998); Husain
et al. (1999); Binzel et al. (1996b)
Growth regulators for rooting NAA, IAA, and IBA NAA Gunay and Rao (1978); Agrawal et al. (1989)
Carbon source Sucrose and glucose Sucrose Phillips and Hubstenberger (1985); Ramage
and Leung (1996)
Ethylene inhibitors AgNO3 AgNO3 Valera-Montero and Ochoa-Alejo (1992);
Hyde and Phillips (1996)
Gelling agent Agar and Phytagel Agar (Phytagel better) Hyde and Phillips (1996)
Light regime Continuous and photoperiod Continuous light Phillips and Hubstenberger (1985)
Temperature 25±28.58C 258C (28.58C better) Phillips and Hubstenberger (1985)

Very recently, Phillips et al. (2000) investigated all reported influence organogenesis and plant regeneration. Despite the variety
regeneration systems for chili pepper and attempted improvements of regeneration systems described, recovery of whole plants remains
for many of them. Key results of this study include: problematic.
Through somatic embryogenesis. Somatic embryogenesis, the
(1) Shoot organogenesis from cotyledon explants. A procedure was
production of bipolar structures resembling zygotic embryos, is an
previously optimized for regeneration of NM 6-4 and Joe E.
alternative morphogenic route for obtaining in vitro plants. Somatic
Parker cultivars from cotyledon explants (Hyde and Phillips,
embryogenesis is theoretically a more efficient morphogenic
1996). A comparison of the protocol developed for Golden
pathway than organogenesis for regenerating and propagating
Tower cultivar (Lee et al., 1993) and that of these authors was
plants with relatively high genetic uniformity. Harini and Lakshmi
carried out, and the new protocol was found to be superior for
Sita (1993) reported for the first time the regeneration of chili
NM 6-4, Joe E. Parker, and Golden Tower, but the response was
pepper plants through direct somatic embryogenesis from immature
better for NM 6-4 and Joe E. Parker than for Golden Tower
zygotic embryos of C. annuum cv. California Wonder. Immature
(Jayashankar, 1998). The main limitation to this procedure was
embryos (globular to cotyledonary stage ranging from 3 to 10 mm)
shown to be the low frequency of shoot elongation. Tests with
were cultured on MS medium supplemented with 2±10% sucrose,
PAA as an alternative auxin (Husain et al., 1999) to potentially
10% coconut water, and 2,4-D (4.53±22.6 mM) or NAA (5.37±
improve the shoot elongation rate gave variable results. It was
26.86 mM). Globular and heart-shaped zygotic embryos failed to
found that some C. baccatum genotypes have superior
survive, whereas cotyledonary stage embryos responded well, but
regeneration capacity than the tested cultivars of C. annuum
the early cotyledon stage (5±6 mm) produced more somatic
(Phillips et al., 2000).
embryos than did larger or smaller embryos. Somatic embryos
(2) Shoot organogenesis from hypocotyl explants. The regeneration
developed from the embryonal axis and cotyledonary margins after
system for chili pepper (Jayashankar, 1998) was reproduced
15 d of culture. Histological studies revealed all the different stages
using hypocotyl explants as described by Valera-Montero and
of embryo development. Initiation and maturation were achieved on
Ochoa-Alejo (1992). This regeneration system was not as
a medium with 8±10% sucrose and 4.53±22.6 mM 2,4-D.
prolific at the induction stage as the cotyledon system, but the
Maturation process was complete within 45 d. After maturation,
shoots were better formed and easier to elongate and root.
the embryos were separated from the explant and were transferred
(3) Shoot organogenesis from half-seed explants. The half-seed
to the germination medium containing 2.89 mM gibberellic acid.
explant regeneration system described by Binzel et al. (1996b)
Plantlets were formed within 15 d and they were transferred to
was modified by including gibberellic acid in the culture
liquid medium without growth regulators for further development.
medium. This modified system produced proliferating callus
Plants were potted in a soil mixture and they reached maturity.
cultures of C. baccatum which retain shoot regeneration
Cytological studies demonstrated that more than 95% of the
capability for several months. This regeneration system is now
dividing cells of the regenerated plants had normal chromosome
being adapted to C. annuum cultivars.
sets …2n ˆ 24†:
Table 1 summarizes the factors most commonly investigated that A system for the induction of direct somatic embryogenesis and
710 OCHOA-ALEJO AND RAMIREZ-MALAGON
TABLE 2
IN VITRO CHILI PEPPER PLANT REGENERATION STUDIES THROUGH SOMATIC EMBRYOGENESIS
Topic Tested factors
Species C. annuum
Chili pepper types Bell peppers and long green peppers
Explant Immature and mature zygotic embryos
Culture medium MS
Growth regulators for embryo initiation 2,4-D, NAA, coconut water, and ABA
and maturation
Growth regulators for embryo germination GA3 and TDZ
Carbon source Sucrose (6±10%)
plant regeneration in C. annuum cvs. New Mexico-6 and Rajur
Hirapur was established using immature zygotic embryos (Binzel
et al., 1996a). Somatic embryos were formed on the zygotic embryo
apex, embryo axis, and cotyledons on MS medium supplemented
with 4±18 mM 2,4-D, 10 mM TDZ, and 6±10% sucrose. The best
response was observed in the presence of 9 mM 2,4-D, 10%
coconut water, and 8% sucrose. The whole process of induction and
maturation was achieved on the same medium. Some embryos were
swollen and distorted, and fasciation of embryos resulting in
multiple embryos occurred often. Secondary somatic embryogenesis
also occurred directly from the primary somatic embryos. More than
70% of the mature normal somatic embryos germinated readily on
MS medium with GA3 or TDZ, alone or in combination, and
generated complete plants.
Recently, a protocol for somatic embryogenesis and plant
regeneration of chili pepper from embryogenic cell suspensions
was developed (Buyukalaca and Mavituna, 1996). Embryogenic
callus masses were induced directly on mature zygotic embryo
explants and were transferred to liquid MS medium containing
4.52 mM 2,4-D and 3% sucrose to establish repetitive embryogenic
cell suspensions. The suspensions were transferred to KNO3-free
MS basal medium supplemented with 9.05 mM 2,4-D and either
K-citrate or K-malate at the concentration of either 6 or 10 g l21
and cultured for 3 wk. The basal MS medium was modified by
reducing NH4NO3 concentration from 20 to 10 mM and adding
6 g l21 proline for embryo initiation from cell cultures. Somatic
embryos at late torpedo stage were matured on paper bridges in
half-strength liquid MS medium containing 1.89 mM ABA and
were converted into plants at up to 97% efficiency.
Phillips et al. (2000) have also reported a comparative
investigation on the published somatic embryogenesis systems
initiated from immature zygotic embryo explants (Harini and
Lakshmi Sita, 1993; Buyukalaca and Mavituna, 1996; Binzel et al.,
1996a). Even though Binzel et al. (1996a) used New Mexico-6 with
up to 80% induction frequencies, no reproducible results were
obtained since only a 3±6% induction frequency was achieved
using NM 6-4 by these authors (Phillips et al., 2000). More
convenient explant sources, such as mature zygotic embryos and
hypocotyls, were evaluated and rare cases (1%) of somatic
embryogenesis were observed. If this regeneration system can be
reproduced routinely for chili pepper at the published levels of
success, and/or if a repetitive embryogenic system can be achieved
for proliferating somatic embryos on a large scale, then this
regeneration approach promises to be more prolific than shoot
organogenesis.
References
Harini and Lakshmi Sita (1993); Binzel et al. (1996a);
Buyukalaca and Mavituna (1996); Phillips et al. (2000)
In conclusion, only a few reports on chili pepper regeneration
through embryogenesis have been published, and it is evident that
more investigation on the factors involved in this process is needed
in order to establish efficient regeneration systems (Table 2).
Plant regeneration from organs. Shoot tip culture. Chili peppers
do not have a natural ability for vegetative or asexual propagation.
Furthermore, in the case of chili pepper, cross-pollination is a very
common event because of the open flower structure of all Capsicum
species. This phenomenon leads to a very high level of
heterozygosity in seed populations, which is an undesirable
characteristic for commercial seed production because of a high
proportion of off-type plants and because it affects the process of
development of new cultivars by breeders. Therefore, in vitro
propagation methods may be used for its clonal multiplication.
Apical shoots, axillary or apical buds and meristems are explants
extensively employed to obtain genetically identical plants in large
numbers through micropropagation. Since chili peppers do not have
a natural ability for vegetative or asexual propagation, in vitro
propagation methods are useful alternative techniques for clonal
multiplication.
Plant regeneration from shoot tips of the domesticated species
C. annuum, C. chinense, C. frutescens, C. baccatum, and C. pubescens
was examined by FaÂri (1986). Shoot tip cultures from accessions of
C. annuum, C. chinense, C. frutescens, C. baccatum, and C. pubescens
were established; however, the C. annuum shoot tips died after the
second or third passage in the media (no data on the composition
was provided), while shoot tip cultures from some accessions of
C. chinense and C. frutescens grew continuously at the same
intensity, even after the eighth or tenth subculture.
A protocol was developed to obtain whole plants from apical
shoot meristems of red pepper (Capsicum annuum L. cv. Bhivapuri)
susceptible to viral infections (Madhuri and Rajam, 1993). The
meristems (ca. 0.8 mm long) from aseptically grown seedlings
(1-mo.-old) were cultured on filter paper bridges in MS liquid
medium supplemented with 8.88 mM BA. Multiple shoots (five to
seven per explant) were produced and the shoots developed further
upon transfer to agar-solidified medium. Complete plants were
obtained after rooting of shoots on MS medium containing 5.37 mM
NAA.
Shoot tips (1 cm long) from C. annuum L. and C. praetermissum
Heiser & Smith were isolated from 3-wk-old axenic seedlings and
were cultured on MS medium in the presence of 5.7 mM IAA
(Christopher and Rajam, 1994). Shoot cultures were subcultured
every 2±3 wk by removing the upper 1 cm part from each plantlet
and transferring them to fresh medium. Shoot tip explants (5±8 mm
 IN VITRO CHILI PEPPER BIOTECHNOLOGY
long) excised from these cultures were inoculated on culture media
containing BA (4.4±177.5 mM) or Kin (4.7±185.9 mM) to promote
shoot proliferation. The highest number of shoots were produced on
medium with 66.6 mM BA or 92.9 mM Kin in the case of
C. praetermissum, and 88.8 mM BA or 116.2 mM in C. annuum
after 4 wk of culture. Addition of 1 mM 2,3,5-triiodobenzoic acid
(TIBA), an inhibitor of polar transport of auxins, and low levels of
BA or Kin, significantly increased shoot number. Regenerated
shoots were rooted on MS medium supplemented with 5.7 mM IAA.
Best rooting (80±100%) was observed in shoots from TIBA plus BA
or Kin treatments, whereas only 40±50% of shoots from the BA or
Kin alone showed root formation. Interestingly, plantlets obtained
from TIBA plus BA or Kin were normal diploids, whereas those
from BA or Kin alone exhibited chromosomal aberrations in root
preparations. Regenerants from TIBA plus BA or Kin treatments
were established in the soil at 86% survival rate and they flowered
and showed normal meiotic behavior with 100% pollen viability.
Shoot tips also have been utilized by Tisserat and Galletta (1995)
to study the in vitro floral and fruit development of California
Wonder, Super Cayenne, and Zippy cultivars grown in MS liquid
medium without growth regulators. Thirty-day-old shoot tips from
seedlings germinated under sterile conditions were cultured in an
automated plant culture system based on a 6-liter polycarbonate
container. Shoot tips were allowed to grow and they started
flowering after 100±120 d and flowered continuously for the rest of
the culture period (280 d). Five to 10% of the flowers set fruits.
Maximum fruit size was approximately 25±75% of the size of fruits
produced by plants grown in vivo. No flowering was found to occur
from shoot tips subcultured every 8 wk in culture tubes containing
25 ml semisolid medium. To follow in vitro fruit development,
immature fruits excised from plantlets grown in the automated
culture system were cultured separately on semisolid medium
containing 0, 0.44, 1.33, or 4.44 mM BA with and without 0.54 mM
NAA. Fruits grew best on MS medium with BA alone and poorest on
medium without growth regulators.
Zygotic embryo culture. Culture of zygotic embryos has been
the approach of choice for the regeneration of plants from abortive
hybrid systems due mainly to incompatibility between embryos and
endosperm. These problems have been found to occur in
interspecies or intergeneric plant sexual crosses. In the case of
chili pepper, there are sexual incompatibilities between different
domesticated and wild species that limit interchange of genetic
traits (pest and disease resistance or important agronomical or
industrial characteristics); these limitations can be overcome by in
vitro hybrid embryo culture. Unfortunately, the information on
zygotic embryo culture and its application to chili pepper genetic
improvement is very limited. FaÂri (1986) reported the culture of
embryos from interspecific crosses between C. annuum and
C. baccatum var. baccatum on a medium containing 0.46 mM Kin ‡
0:45 mM 2,4-D. MS medium with 0.44 mM BA, 2.85 mM IAA,
and 1.44 mM GA3 was the most suitable for keeping the
underdeveloped embryos alive. No further information on the
behavior of the regenerated hybrids or their utilization in breeding
programs was reported.
Anther culture. The importance of haploids stems largely from
their considerable potential for plant breeding. Haploids may be
utilized to facilitate the detection of mutations and the recovery of
unique recombinants. Since most mutations are recessive, they are
difficult to detect in the presence of unmutated dominant genes.
711
Since haploids possess only one set of alleles at each locus, it is
possible for recessive mutants to be detected. Doubling of the
chromosome number of haploids offers a method for the rapid
production of homozygous plants, that in turn may be used for
producing inbred lines for hybrid production (Pickering and
Devaux, 1992). Furthermore, by using F1 hybrids for anther
culture, superior genotypes exhibiting different trait combinations
of both progenitors can be generated in a single step, which may
accelerate the `fixation' of hybrid characters after chromosome
doubling.
Haploids may occur spontaneously in nature or they may be
induced experimentally. The origin of a plant having a single set of
chromosomes is ordinarily atributed to the parthenogenetic
development of an egg or a haploid accessory cell of the female
gametophyte (parthenogenesis or apomixis). In such cases, the
unfertilized egg, the sperm or the synergids start to grow to form a
haploid plant independent of any experimentally applied stimulus.
Induction of haploids can generally be achieved by different
methods, including ionizing irradiation, radioisotopes, thermal
shocks, distant hybridization, delayed pollination, fertilization
with abortive pollen, spraying floral organs with chemicals (growth
regulators, nitrous oxide, etc.), chromosome elimination by culture
of young embryos, in vitro parthenogenesis, culture of microspores
or pollen, and anther culture.
Spontaneous haploids have been found to occur in Capsicum
species (Christensen and Bamford, 1943; Campos and Morgan,
1958; Pochard and Dumas de Vaulx, 1979). The haploids appear
mainly in the form of twin seedlings, at rates ranging from 1 to 10 in
10 000 germinated seeds. Breeding for high rates of parthenogen-
esis through selection of genotypes showing high levels of haploidy,
and applications of nitrous oxide on the floral organs for the
induction of haploids have been reported (Pochard and Dumas de
Vaulx, 1979). Chromosome doubling of the haploid plants by
treatment of cuttings with 0.3% colchicine solution rendered
autodiploid lines (isogenic, homozygous, or pure lines).
Anther culture and regeneration of haploid plants of Capsicum
species has been reported by different authors (George and
Narayanaswamy, 1973; Wang et al., 1973; NovaÂk, 1974; Saccardo
and Devreux, 1974; Sibi et al., 1979; Dumas de Vaulx et al., 1981;
Vagera and HavraÂnek, 1985; Morrison et al., 1986b). A chapter
dealing with in vitro induction of haploids in Capsicum was
published by Vagera (1990). Wang et al. (1973) were the first to
report on chili pepper anther culture and haploid plant regenera-
tion. Anthers of the Yeo Hsien Small Redpepper cultivar with
microspores at the uninucleate stage with the nucleus close to one
side were used in this work. Anthers were cultured on MS medium
modified in some micronutrients and vitamins, and supplemented
with either 4.65 mM Kin, 5.37 mM NAA, or 4.52 mM 2,4-D. Green
seedlings began to appear from the anther sacs after 33 d in culture.
Callus tissue was also produced. The frequency of the callus and
seedling production of anthers inoculated on the medium
supplemented with NAA was 28.6 and 4.8%, respectively, while
that supplemented with 2,4-D was 23.5 and 2.6%, respectively.
Regenerated plantlets were found to be haploid as revealed by the
chromosome number of root tips …n ˆ 12†: In a subsequent work,
Kuo et al. (1973) described the investigation of conditions for
anther culture of red pepper. In this case, anthers were cultured on
NT (Nagata and Takebe, 1971) and MS media, supplemented with
different growth substances, at the microspore stage when the
712 OCHOA-ALEJO AND RAMIREZ-MALAGON
nucleus was situated on one side of the pollen grain. The formation
of proembryoids from uninucleate pollen grains and development
into haploid plantlets was observed and this phenomenon was quite
similar to that of zygotic embryos.
George and Narayanaswamy (1973) reported haploid embryos
and plantlet formation in Capsicum annuum var. grossum from
immature anthers containing uninucleate microspores released just
after the tetrad stage. Young floral buds were surface-sterilized and
inoculated on LS (Linsmaier and Skoog, 1965) basal medium.
Growth supplements such as auxins, cytokinins, coconut milk, yeast
extract, and casein hydrolyzate (CH) were incorporated in the
medium at different levels. Anther cultures were incubated under
constant cool-white, fluorescent illumination at 25 ^ 28C: Sus-
tained division in microspores was observed on media containing
either 1.39 mM Kin ‡ 17:12 mM IAA, 13.9 mM Kin ‡ 5:71 mM
IAA, or 2.85 mM IAA ‡ 400 mg l21 CH ‡ 100 mg l21 inositol.
Exogenous IAA was essential for the initiation of cell division in
microspores oriented towards androgenesis. De-differentiation of
microspores to form multicellular structures resembling pro-
embryoids was observed to occur in only 1% of the microspores
in an anther sac. Frequent transfer of the embryoid-bearing anthers
to medium containing 2.85 mM IAA ‡ 400 mg l21 CH ‡
100 mg l21 myo-inositol was necessary to promote further differ-
entiation into the heart and torpedo stages. Complete plantlets were
obtained in only 0.1% of the several hundred cultured anthers.
Acetocarmine squashes of root tips of two plantlets confirmed the
haploid number …n ˆ 12†:
Callus formation from anther cultures of five sweet pepper
cultivars of C. annuum (Severka, PCR, Sivria 600, Yolo Wonder,
and Sweet Mammouth Red), one cultivar of C. frutescens (Tabasco)
and an interspecific hybrid of C. annuumC. frutescens (Sweet
Mammouth RedTabasco) was investigated by NovaÂk (1974), using
the MS basal medium supplemented with different levels of auxins
(IAA, NAA, and 2,4-D) alone or combined with different levels of
Kin, or two auxins combined with Kin to give 43 different
treatments. Alternatively, 24 treatments were tested using the N
basal medium (Nitsch, 1969) with IAA, NAA, or 2,4-D combined
with Kin or BA and supplemented or not with yeast extract
(100 mg l21) or l-tyrosine (100 mg l21). The influence of floral bud
developmental stage on callus production from anthers was also
investigated in the cultivar Severka. The anthers prepared from
buds at the stage of 1.5±2.5 mm (sepals closed, petals not visible
and various stages of meiosis) became necrotic after 30 d of culture
in all 43 treatments using MS medium. Floral buds of 2.6±5 mm
(growing petals of light green color, uninucleate microspores) or
those over 5 mm (petals of white color with a green tinge, and
maturing binucleate pollen) were the most suitable for callus
formation. Additions of single auxins (IAA and NAA) to both basal
media MS and N induced no growth, whereas in some cultivars
proliferation was weak on the media with 2,4-D (2.26 and
4.52 mM). In these combinations the rate of growth was higher on
the N basal medium. The addition of Kin and BA improved
callus growth; the callus growth was induced on N medium in
combinations with IAA±Kin, NAA±Kin and especially 2,4-D±
Kin, while on the MS basal medium the anthers proliferated only
with 2,4-D±Kin. The most vigorous growth was achieved on MS
medium supplemented with 11.42 mM IAA ‡ 4:52 mM 2,4-D ‡
9:3 mM Kin. Adenine sulfate and l-tyrosine did not affect callus
growth. An inhibitory effect on callus growth with yeast extract
was observed. Interestingly, no shoot or embryo formation was
achieved in any of the treatments using either MS or N basal
medium.
The influence of anther size, cold pretreatment, and photoperiod
on callus and plant regeneration from Yolo Wonder and Quadrato
d'Asti cultivars of C. annuum was investigated (Saccardo and
Devreux, 1974). Cold pretreatments were applied on floral buds
before in vitro anther culture, to try to improve the yield of
regenerated plants. Cytological analysis of microspores from anthers
at different develomental stages (1.5±2.5 mm, yellow; 2.5±3,
yellow; 2.5±3, yellow/blue; and 3±3.5, purple) showed a high
correlation between the length and color of the anther and the
stages of the nucleus of the microspore. Anthers of 1.5±3 mm in
length, characterized by yellow color, exhibited almost only
mononucleate microspores, while the anthers of 2.5±3.5 mm in
length (tips becoming a blue color), contained a large amount of
microspores at the haploid mitotic stage. The anthers from 4 mm to
maturity with completely purple walls were found to present all the
developmental stages of the binucleate gametophyte. Anthers of
2.5±3.5 mm in length containing microspores at the mononucleate
and at the mitotic stages were used as experimental material to test
the influence of cold pretreatment and photoperiod on callus and
plant regeneration. Cold pretreatment did not have any positive
effect on plant regeneration as revealed by the very similar results
observed with treated and untreated anthers from floral buds of
plants grown under greenhouse conditions with a photoperiod of
10 h of natural light + 6 h of additional artificial light and at 258C,
which showed ca. 50% callus formation and no plant regeneration.
On the contrary, anthers from plants grown in a greenhouse with
14 h of natural light and without cold treatment exhibited ca. 96%
callus formation and 1.7% plant regeneration after 40±45 d of
culture (no information was provided on the culture medium and
culture conditions).
Since the techniques previously reported by George and
Narayanaswamy (1973) and by Saccardo and Devreux (1974) had
been shown not to be reproducible, Sibi et al. (1979) investigated
the influence of floral bud stage (eight stages), cold pretreatment
(24 and 48 h at 48C), two culture media, and culture conditions on
the responses of two parental lines and their reciprocal F1. The
best floral stage for successful plant regeneration from anthers was
when the microspores were at the haploid mitotic stage. This floral
stage was achieved when the petal and sepal sizes were similar in
length (5.8 mm) and the tips of anthers were colored by
anthocyanins. For better responses, the floral buds were pretreated
at 48C for 48 h. Two culture media (C and R) differing in
micronutrient levels and in composition of growth regulators were
used for the first and second steps, respectively, of anther culture.
Anther cultures were initially established in culture medium C
(9 mM 2,4-D ‡ 9:3 mM Kin ‡ 0:03 mg l21 vitamin B12) and then
transferred to culture medium R (0.46 mM Kin) after 12 d at 258C
and with a photoperiod of 12 h (fluorescent lamps). In this way the
embryoids developed in a similar fashion as zygotic embyos. The
efficiency of haploid plant regeneration was one to three plants per
100 cultured anthers.
Responses of anthers from 19 cultivars of sweet peppers
(C. annuum L. var. grossum) were studied by Wang et al. (1981).
The basic medium was supplemented with 1.34 mM NAA, 4.65 mM
Kin, 50 mg l21 nucleotide, and 3% sucrose. The androgenic
embryos were further developed into haploid plants after transfer to
 IN VITRO CHILI PEPPER BIOTECHNOLOGY
a medium with 0.57 mM IAA or 0.005 mM NAA, 9.3±18.6 mM Kin
and 17.76 mM BA or 10% fresh coconut milk. Embryoid formation
was observed in 16 cultivars, but normal androgenic embryoids
were obtained only from two cultivars (Square Head and Lu-tai).
The frequency of embryoid induction varied with the cultivar. Low
concentrations of NAA were beneficial to embryoid formation, but
the optimum concentration varied with cultivar. Haploid plants
were doubled with 0.2% colchicine treatment for 24 h.
Stimulation of in vitro androgenesis of chili pepper (C. annuum
L.) by elevated temperature treatments was described by Dumas de
Vaulx et al. (1981). The responses of anthers treated at 358C for 2 or
8 d and the interactions with different 2,4-D and Kin concentra-
tions in the culture medium were investigated. In general,
pretreatment of anthers at 358C for 2 d gave low plant regeneration
efficiency as compared to pretreatment for 8 d at 358C. When 2,4-D
(0.45, 4.52, and 9 mM) was combined with Kin (9.3 mM), the best
response was observed in anthers pretreated at 358C for 8 d and
then cultured at 258C in the presence of 0.45 mM 2,4-D ‡ 9:3 mM
Kin (5% plant regeneration). However, the efficiency of plant
regeneration increased (12%) in anthers pretreated at 358C for 8 d
prior to transfer to 258C and cultured on a medium with 0.04 mM
2,4-D.
Anthers from breeding lines of C. annuum cv. Severka and
Morava were cultured using five different media formulations (MS;
MSH; N; B5 of Gamborg et al., 1968; and SI of Sibi et al., 1979
without FeEDTA) with different additives [200 ml l21 carrot
extract, 1% activated charcoal, FeEDTA or Fe2(SO4)3] (Vagera
and HavraÂnek, 1985). MSH medium was MS medium with one-third
of the normal level of nitrogen, CaCl2 replaced by Ca-acetate, and
increased levels of nicotinic acid and thiamine. A total of 24
treatments were tested. In some cases, isolated floral buds were
kept in the cold (48C) and dark for 3±11 d before extirpation of
anthers. The anthers were cultured in the light (16-h day, 40 W
fluorescent lamps) or in the dark and then in light according to
Dumas de Vaulx et al. (1981). The embryoids which arose were
either left inside the anther or they were isolated and cultured
separately. In general, a low frequency of androgenesis (0.7±7% of
productive anthers, 0.01±0.22 mean number of embryoids per
anther, and 0.014 complete plants per anther) was found to occur in
all tested standard media (complete or minimal). Cold pretreatment
(48C) for 5±11 d did not improve efficiency of androgenesis in
standard media. The frequency of androgenesis was markedly
increased in complete media containing activated charcoal and
even higher in culture media with activated charcoal and carrot
extract. Conversion of embryoids into plants was inhibited by
activated charcoal. The frequency of embryoid formation was higher
using the method described by Dumas de Vaulx et al. (1981) than
the standard method tested in this study. The development of
embryoids into complete plants was better after they had been
isolated and transferred to fresh medium, but most of the embryoids
did not form complete plants even after this transfer. Medium N
without activated charcoal was shown to be most suitable for
polarization of embryoids (greening of the apical part and formation
of long roots). FeEDTA was found to have a beneficial effect on the
formation of globular embryoids and plants.
A double-layer culture medium was reported by Morrison et al.
(1986b) as an approach to regenerate haploid plants from anthers of
a hybrid between Emerald Giant (C. annuum) and CA4 (C. chinense)
that had been found not to grow using the conditions described by
713
Dumas de Vaulx et al. (1982). The basal medium was that of Sibi
et al. (1979) containing FeEDTA and sucrose levels according to
Murashige and Skoog (1962). The C medium was used for the first
12 d of culture and was supplemented with 6% sucrose and
0.01 mM 2,4-D (Dumas de Vaulx et al., 1982). The R medium used
during the remainder of the culture period contained 3% sucrose
and no added growth regulators. The C and R media were gelled
with 1% agarose instead of agar. The double layer charcoal cultures
were based on R medium with 2% activated charcoal and 1%
agarose. Volumes of 25 ml of this culture medium were poured into
Petri dishes and, after gelling, R medium (8±10 ml) without both
charcoal and agarose was poured to cover the charcoal medium
completely. Flower buds were collected from plants when the
corolla was slightly longer than the calyx (microspores at the first
mitotic stage). Flower buds were pretreated at 48C for 100 h.
Anthers were dissected and cultured on medium C at 358C in the
dark for 8 d. The cultures were transferred to 258C in the dark for
an additional 4-d period, and then were cultured on the double-
layer charcoal medium. After transfer to medium C, embryos
appeared in the pollen sacs of cultured anthers in 2±4 wk. Embryos
developed into whole plants after 2±3 wk of culture on R medium.
All plants produced from cultured anthers of the hybrid were
diploid and were found to be homozygous for several loci, as
determined by electrophoretic analysis, indicating their gametic
origin. The double-layer culture method was found to be very
efficient for Calwonder, Yolo Wonder, and Emerald Giant cultivars.
Anther culture from 12 materials of C. annuum L. and F1 crosses
between C. annuum NM 6-4C. chinense NMCA 4 and C. frutescens
(Mexico INIA BG3324)C. chinense (NMCA 4), and BC1
C. annuum(C. annuumC. chinense) were reported by Munyon
et al. (1989). Androgenesis occurred from anthers incubated in a
continuous warm environment (298C) with continuous light. The
hormone compositions of the media reported by Dumas de Vaulx
et al. (1981) were used as controls in all anther culture experiments,
using the MS basal medium with vitamins according to the L2
recipe (Phillips and Collins, 1979). Induction media included
0.045 mM 2,4-D, 0.041 mM picloram (4-amino-3,5,6-trichloro-
picolinic acid), or 0.057 mM IAA in combination with 0.046 mM
Kin or 0.044 mM BA. Embryoids were grown out by transfer to MS
medium including 0.46 or 9.3 mM Kin, or 0.028 mM IAA and
0.022 mM BA. Forty plants and embryoids were retrieved from
anther cultures and analyzed for isozyme markers. Of these, 35
exhibited a single allele for markers suggesting microspore origin,
while five were heterozygous indicating somatic tissue origin.
Chromosome numbers were confirmed for 21 plants, of which 16
were haploid and five were diploid. However, two plants exhibited a
single allele for an isozyme marker, but possessed the diploid
chromosome number, suggesting spontaneous doubling. Anther
cultures also produced slow-growing calluses and fast-growing
calluses. Nearly 92% of the sampled slow-growing calluses were
heterozygous for the isozyme markers indicating somatic tissue origin,
whereas more than 46% of the fast-growing calluses exhibited only
one allele for the marker, indicating microspore origin.
Effects of temperature and photoperiod for donor plant growth on
embryo formation in F1 hybrids (Jetta, Parma, and Trophy) of
Capsicum annuum anther culture were investigated by Kristiansen
and Andersen (1993). Donor plants were grown in greenhouses at
minimum temperatures between 16 and 308C and at photoperiods
between 11 and 19 h. Anthers were collected from individual plants
714 OCHOA-ALEJO AND RAMIREZ-MALAGON
over 5±9-wk periods and cultured as described by Dumas de Vaulx
et al. (1981) to test the significance of donor plant age. Embryos
were obtained at all temperature regimes with a calculated optimum
temperature of 26.48C. Embryo formation was unaffected by
photoperiods tested. Embryo formation varied among successive
samplings. However, a significant decline in anther culture
response with increasing donor plant age was observed in three
different experiments.
An improved in vitro anther culture method based on the use of
young mother plants and frequent subcultures was reported by
Mityko et al. (1995). Four chili pepper breeding lines (C. annuum
L.), seven cultivars, and four F1 hybrids were used. The donor
plants were grown under greenhouse conditions. Flower buds
(corolla of the same size as calyx) were harvested only during the
4 wk after the first flower buds had appeared. The in vitro anther
culture method developed by Dumas de Vaulx et al. (1981) was
applied. Surviving anthers on R medium were transferred each
month onto fresh R medium. Well-developed embryos were
separated from the anthers and transferred onto V3 medium
(Dumas de Vaulx et al., 1981) without hormones for further
development. The first haploid embryos appeared 30±40 d after the
isolation of anthers, in contrast to the 20±30 d for the original
protocol. Anther response for embryoid formation ranged from 0 to
48% depending on the cultivar, and plant regeneration efficiency
per 100 anthers varied from 0 to 102.
An integrated molecular linkage map of chili pepper (Capsicum
annuum L.), including mainly restriction fragment length poly-
morphism (RFLP) and randomly amplified polymorphic DNA
(RAPD) markers, was constructed by alignment of three intra-
specific linkage maps generated by segregating doubled-haploid
progenies (Lefebvre et al., 1995). No details on the generation of
doubled-haploids were mentioned.
The induction of pollen embryogenesis in Capsicum annuum L.
has been studied at the cellular level using various in situ
approaches with several molecular probes for DNA, RNA, and
proteins (GonzaÂlez-Melendi et al., 1996). The late vacuolated
microspore stage was found to be the most responsive to
embryogenesis induction. The proliferating cell nuclear antigen
(PCNA) was immunolocalized at the electron microscopy level in
order to map replication sites in relation to the fine structure of
chromatin. These studies revealed that the late vacuolated
microspore was in a period of replication. Other in situ studies
were performed to characterize the state of nuclear activity at the
specific developmental stages in which the embryogenic induction
occurred. The modern in situ terminal-deoxy-nucleotidyl transfer-
ase (TdT) reaction for DNA, the immunolocalization of various
nuclear antigens [small nuclear ribonucloproteins (snRNPs),
fibrillarin, RNA], and the structural in situ hybridization using
18S and 25S ribosomal probes provided valuable data about the
specific features displayed by the functional nuclear compartments
of the microspore, and the young vegetative and generative cells.
They were related not only to the state of gene activity but also with,
probably, the ability to switch to the sporophytic pathway at specific
developmental times of their gametophytic program.
Anther-derived spontaneous doubled-haploid (DH-R1) plants
were developed from an intercultivar F1 hybrid (Holland) of cv.
Mazurka (Capsicum annuum L. var. annuum, 2n ˆ 24) cultured on
the nutritive medium described by Dumas de Vaulx et al. (1981)
containing 0.045 mM 2,4-D and 0.046 mM Kin (Gyulai et al.,
2000). Sugar sources and concentrations were changed according to
the modifications of GeÂmesne et al. (1998). Cultures were incubated
for 8 d at 358C in the dark, then transferred to 258C with a 12 h
photoperiod. Responsive anthers with embryoids on the surface
were transplanted onto 2,4-D-free plant regeneration medium
supplemented with 0.46 mM Kin (Dumas de Vaulx et al., 1981).
Regenerants from anther culture were grown in greenhouses for
seed production to R2-generation (DH-R2) prior to flow cytometric
analysis which revealed DH-R1 plants with haploid …n ˆ 12†;
doubled-haploid, DH …2n ˆ 24†; tetraploid …2n ˆ 48†; and aneu-
ploid genomes. Pollen grains of anther cultures showed a high
decrease in viability during the first 2 wk of incubation time. The
DNA samples of the 47 DH-R2 individuals were bulked in nine
groups and subjected to PCR analysis for estimating the level of
homogeneity versus heterogeneity in comparison with the control of
anther donor bulk of five plants of F1 hybrid chili pepper cv.
Mazurka (DH-R0). In total 26 primers were applied, 22 of them
revealed scorable PCR profiles with 54 scorable PCR bands. Of
these, 19 (35.2%) bands generated by seven primers were
polymorphic. All of the nine bulks of DH-R1 plants were
discriminated at least by one primer.
We have applied the technique described by Morrison et al.
(1986b) to the anther culture of Mexican varieties of chili pepper
and the best results were obtained with C. annuum L. cv. Mulato
Bajio (ancho type) in which 1.4% embryo formation (14 embryos
regenerated from 1000 anthers) was achieved, but from these
embryos only four germinated and gave whole plants (0.4% plant
regeneration) (Fig. 4). Anther-regenerated plants produced notably
smaller fruits than those from the mother plants under the same
conditions of growth; scarce seed formation was observed in the
fruits from anther-regenerated plants as has been reported by
Vagera (1990) (Fig. 4).
A summary of aspects taken into account for in vitro anther
culture of chili pepper is shown in Table 3.
Applications of Plant Regeneration Systems
Generation of cytoplasmic mutants exhibiting resistance to
antibiotics, resistance to herbicides or alterations in chlorophyll
characteristics are useful for designing selection schemes for
somatic hybrid recovery and organelle transfer. Streptomycin
resistance is the most widely used marker in higher plants. A
shoot regeneration system combined with chemical mutagenesis was
used to generate chili pepper mutants resistant to streptomycin
(Subhash et al., 1996). Resistant shoots and plants were regenerated
from cotyledon explants of C. annuum cv. G4 treated with
ethylmethane sulfonate (EMS). Seeds of chili pepper were
incubated for 18 h in MS medium containing 0.1% EMS. These
treated seeds were washed, surface-sterilized, and germinated
aseptically on MS basal medium. Cotyledons from 3-wk-old
seedlings were inoculated on shoot regeneration medium with
2.85 mM IAA and 13.3 mM BA, and 500 mg l21 streptomycin as
the selection agent. Cotyledon explants from mutagenized seedlings
regenerated albino (chlorophyll deficient; 9.5%) and green
streptomycin-resistant shoots (5.4%). The mean number of
regenerated shoots per explant was 3.4. Green shoots regenerated
from mutagenized cotyledons cultured on medium with 500 mg l21
streptomycin were rooted on MS medium with 5.71 mM IAA.
Interestingly, leaf sections from the regenerated green plants when
 IN VITRO CHILI PEPPER BIOTECHNOLOGY 715

Fig. 4. Androgenetic embryogenesis from chili pepper (C. annuum var. Mulato Bajio) anther culture. a, Haploid embryos at different
developmental stages; b, root development in haploid embryos; c, an anther-regenerated plant (left) and a mother plant (right) bearing
fruits; d, fruits from an anther-regenerated plant (top) and from a mother plant (bottom).

cultured on medium with a higher streptomycin concentration
(1000 mg l21) were able to regenerate shoots from green callus.
These results confirmed the stability of streptomycin resistance. All
regenerated plants (45) were shown to be diploid …2n ˆ 24†: The
inheritance of streptomycin resistance, studied by reciprocal
crosses, was found to be under the control of cytoplasmic DNA
(chloroplasts).
A high frequency of plastid-encoded antibiotic-resistant variants
of Capsicum annuum have been isolated from seeds and explants
mutagenized with EMS (0.1%) or with nitrosomethylurea (5 mM
NMU) (Rao et al., 1997). Non-mutagenized cotyledons (controls),
mutagenized cotyledons from EMS-treated 3-wk-old seedlings, and
NMU-treated cotyledons were cultured on shoot regeneration
medium based on MS medium supplemented with 5.71 mM IAA
and 13.3 mM BA, and streptomycin (500 mg l21) or lincomycin
(100 mg l21) as the selective agents. The antibiotics caused
716 OCHOA-ALEJO AND RAMIREZ-MALAGON
MAIN FACTORS TESTED FOR IN VITRO PLANT REGENERATION FROM CHILI PEPPER ANTHER CULTURES
Topics Tested factors
Species C. annuum, C. annuum var. annuum,
C. annuum var. grossum, C. chinense,
C. frutescens
Materials Bell cultivars, several lines, and hybrids
Floral bud stage Floral buds with yellow or blue color tips
Anther stage Uninucleate and early binucleate
microspores
Floral pretreatment Cold (48C), 35/258C (358C first and then 258C),
and 28.58C continuous
Culture medium MS, MSH, MS/L2 (MS salt and L2 vitamins),
LS, N, B5, SI and C-R (Medium C first and
then Medium R)
Growth regulators 2,4-D, IAA, NAA, Picloram, IAA/Kin,
NAA/Kin, 2,4-D/Kin, Kin, BA, IAA/BA
Organic additives Coconut water, carrot extract, yeast extract,
casein hydrolyzate
bleaching of the explants and suppression of adventitious shoot
initiation in control explants. After mutagenesis, occasional green
resistant shoots appeared at the cut edges of the explants. NMU-
treated cotyledons gave a higher frequency of variants than
cotyledons from EMS-treated seedlings. The mutagenic effect of
EMS was more pronounced using whole seeds rather than
cotyledons; in contrast, NMU was more effective in inducing
variations in cotyledons than in seeds. The stability of streptomycin
resistance and lincomycin resistance in the variants was confirmed
by the leaf assay. Pieces of leaves from the regenerants were
subcultured three times on regeneration medium containing
2.85 mM IAA and 13.3 mM BA and the same levels of antibiotics
used for the selection of variants. Production of green shoots from
the leaf segments was evidence of a resistant phenotype. The
resistant plants had normal morphology, ploidy, and fertility. No
inheritance data of the recovered variants were provided.
Unfortunately, there is limited information at this time on the
application of in vitro regeneration systems for genetic improvement
of Capsicum through chemical or physical mutagenesis. This
approach might lead to the recovery of interesting chili pepper
mutants with important agronomical or industrial characteristics in
the near future. Only two papers have been published dealing with
this aspect.
A shoot regeneration system was employed to study the effect of
gamma radiation on organogenesis (Sripichit et al., 1988a). Twelve-
day-old seedlings were irradiated (gamma radiation) with varying
exposure doses (0±2.5 krad) and dose rates (1±10 krad h21), and
cotyledons were excised and cultured on MS medium supplemented
with 13.3 mM BA for shoot regeneration. Percentages of shoot-
forming explants and the number of shoots per explant decreased as
the exposure dose increased. The exposure dose that caused 50%
reduction in the number of shoots per explant (RD50) was around
TABLE 3
Factors most
commonly used References
C. annuum Wang et al. (1973); George and Narayanaswamy
(1973); Sibi et al. (1979); Morrison et al. (1986b);
Munyon et al. (1989)
Bell peppers Saccardo and Devreux (1974); Sibi et al. (1979);
Wang et al. (1981); Dumas de Vaulx et al. (1981);
Morrison et al. (1986b); Munyon et al. (1989);
Mityko et al. (1995)
Floral buds with blue Saccardo and Devreux (1974); Sibi et al. (1979);
color tips Mityko et al. (1995)
Uninucleate microspores George and Narayanaswamy (1973); Dumas de Vaulx
et al. (1981); Vagera and HavraÂnek (1985)
35/258C Saccardo and Devreux (1974); Sibi et al. (1979);
Gyulai et al. (2000); Dumas de Vaulx et al. (1981);
Munyon et al. (1989); Vagera and HavraÂnek (1985)
MS and C-R George and Narayanaswamy (1973); Sibi et al.
(1979); Munyon et al. (1989); Vagera and HavraÂnek
(1985)
2,4-D/Kin (Medium C) and Wang et al. (1973); George and Narayanaswamy
Kin (Medium R) (1973); Sibi et al. (1979); Dumas de Vaulx et al.
(1981); Morrison et al. (1986b); Mityko et al. (1995);
Gyulai et al. (2000)
Rarely used George and Narayanaswamy (1973); Vagera and
HavraÂnek (1985)
1.0 krad at the dose rates of 1 and 5 krad h21, whereas RD50 was
0.75 krad at the rate of 10 krad h21. Seedlings (12-d-old) were
irradiated by gamma radiation at exposure doses ranging from 0 to
1.25 krad delivered at the dose rate of 5 krad h21, and cotyledon
explants were cultured for shoot induction (Sripichit et al., 1988b).
The shoots were rooted and the regenerated plants were grown
under greenhouse conditions to maturity. Visible variants were
found among the primary regenerants or M1 plants at the frequency
of 4.4%. Variations in plant type (dwarf and indeterminate growth
habit), leaf (viridis, variegated mottled, bushy variegated, and
filiform), flower (yellow anther, partial male sterility, and narrow
petal) and fruit morphology (blunt apex fruit and oblate fruit) were
observed. The rate of chimerism based on visual characters was
considerably high, indicating the multicellular origin of adventi-
tious shoots. Most of the variant characters were transmitted to the
M2 progeny and were considered as mutants.
Genetic transformation. Plant genetic transformation is cur-
rently the approach of choice for transfer of specific genes encoding
some agronomically important traits. Application of genetic
transformation requires the development of efficient techniques
for both the transfer of foreign genes into plant cells, and for
regeneration of whole plants from the transformed cells. In general,
Agrobacterium tumefaciens has been used as the vector for genetic
transformation of diverse dicotyledonous species, but biolistic
bombardment has been a very useful technique to introduce foreign
DNA into plant cells of monocotyledonous and dicotyledonous
plants. In the case of chili pepper, genetic transformation is
certainly an important goal to facilitate genetic improvement
against several diseases caused by phytopathogenic fungi, bacteria,
and viruses, as well as for improvement against different pests.
However, advances in this area have been limited because of low
efficiency for in vitro plant regeneration of the reported systems.
 IN VITRO CHILI PEPPER BIOTECHNOLOGY
A decade has passed since Liu et al. (1990) published the first
results on chili pepper genetic transformation. They used six bell
pepper cultivars (C. annuum L.) and one Guatemalan wild
accession of C. annuum var. glabriusculum. Explants (hypocotyls,
cotyledons, and leaves) from in vitro seedlings (14±21-d-old) were
cocultured with individual suspensions of the wild tumorigenic
strains A281 and C58 of A. tumefaciens, and with a disarmed strain
bearing the plasmid pGV3850 containing the neomycin phospho-
transferase gene (nptII) and the b-glucuronidase gene (gus) under
the control of the 35S promoter from cauliflower mosaic virus
(CaMV). Tissues cocultured for 24±48 h with the tumorigenic
strains were subsequently cultured on MS medium without growth
regulators, while explants cocultured with the disarmed strain were
cultured on a selective medium containing 44.4 mM BA ‡ 5:71 mM
IAA ‡ 150±200 mg l21 kanamycin. Formation of tumors (galls) on
explants infected with the wild species, or the regeneration of shoots
on selective medium with expression of GUS activity in tissues
infected with the disarmed strain, were the criteria for genetic
transformation. Gall formation on explants was mainly dependent on
the A. tumefaciens strain (higher with C58 than with A281).
Production of kanamycin-resistant cell lines was more effective for
cotyledons and leaves than for hypocotyls. Only cotyledon and leaf
tissues formed callus, leaf-like structures, and occasional shoot
buds in the presence of 200 mg l21 kanamycin. Although a number
of kanamycin-resistant shoot buds were obtained, no further
elongation and plant formation occurred. Sections from leaf-like
structures and shoot buds showed positive in the GUS assay. No
GUS actvity was detected in shoot buds from control explants.
A patent describing a complex and time-consuming method for
genetic transformation and plant regeneration of chili pepper was
registered by Engler et al. (1993). Cotyledons from surface-
sterilized mature seeds were removed and cut into three or four
sections which were submersed in liquid medium OMSG (MS salts,
B5 vitamins, 1.6% glucose, and 600 mg l21 MES). The liquid
medium was removed and explants were cocultured with a
suspension of the LBA4404/p5T35AD strain of A. tumefaciens.
This strain contained the binary vector p5T35AD in which the
CaMV 35S promoter drives a double mutant form of the acetolactate
synthase (ALS) gene which confers resistance to the herbicide
chlorsulfuron. Explants were transferred onto the 10BIAS culture
medium (OMGS medium supplemented with 44.4 mM BA, 5.71 mM
IAA, 200 mM acetosyringone, and 2.5 g l21 Gel-rite) and incu-
bated at 288C during the first 16 h and then at 248C for a total of
3 d in the dark. Following the cocultivation period, explants were
washed in OMSG medium supplemented with 500 mg l21 carbe-
nicillin, 500 mg l21 cefotaxime, and 500 mg l21 vancomycin. The
washed explants were grown on selective 10BI/cscf medium
(10BI ‡ 50 mg l21 chlorsulfuron 1 500 mg l21 cefotaxime). Cul-
tures were incubated in the dark at 288C for 24 d. The explants
were then cultured for 14 d on fresh medium of the same
composition, but containing 15 mM silver thiosulfate. Explants
bearing shoot buds were subcultured on elongation medium 5BSTS/
csc (OMSG/cs medium supplemented with 50 mg l21 chlorsulfuron,
22.2 mM BA, 500 mg l21 carbenicillin, and 15 mM silver
thiosulfate). Cultures were moved to incubation conditions of
288C, 500 lux, and 16 h photoperiod. Nineteen days later, explants
were transferred to fresh medium of the same composition. After
1 wk of incubation, cultures were moved to the same conditions as
before, but with a light intensity of 2500 lux. Twenty days later, the
717
shoot buds thus produced were removed from the original explant
with a scalpel, and transferred to OMSG/cs medium supplemented
with 15 mM silver thiosulfate and 500 mg l21 carbenicillin. Two
months later, shoot buds were transferred to MSOT medium (MS
salts, MS vitamins, 3% sucrose, 1 g l21 inositol, 1 mg l21
thiamine.HCl, and 2.5 g l21 Gel-rite) supplemented with
50 mg l21 chlorsulfuron. Six weeks later …total ˆ 6:5 mo:†; shoots
were rooted on OMSSN medium (MS salts, B5 vitamins, 3%
sucrose, 600 mg l21 MES, 0.54 mM NAA, and 2.5 g l21 Gel-rite).
Two months later …total ˆ 8:5 mo:†; shoots were cultured on fresh
OMSSN medium lacking NAA. One month later …total ˆ 9:5 mo:†;
plants were removed and transferred to the greenhouse. The plant
regeneration efficiency of this system was as follows: from 720
explants, just five shoots were still alive and in the process of
elongating and normalizing on the selective medium MSOT/cs, and
from these, only two elongated shoots produced roots and eventually
plants after 9.5 mo. The five regenerated shoots, which were able to
grow on the selection culture medium, were confirmed to be
transgenic as revealed by PCR analysis; therefore, the transforma-
tion efficiency was 0.7%. Leaf tissues from the two regenerated
transgenic plants were cultured on a selective callus-inducing
medium (OMSG/cs supplemented with 5.37 mM NAA, 0.91 mM
2,4-D, 1.82 mM Zea, and 250 mg l21 cefotaxime); the leaf tissues
produced callus confirming the transgenic phenotype.
Further modifications to this technique were introduced to
increase the transformation efficiency (Engler et al., 1993).
Cotyledons from 9-d-old seedlings of the cultivar Vegisweet were
cocultured with p5T35AD for 5 d at 248C and the vancomycin
concentration of the OMSG medium was reduced to 100 mg l21.
The culture medium used for selection/induction was 10BI/cscf
(10BI ‡ 50 mg l21 chlorsulfuron 1 500 mg l21 cefotaxime). Fol-
lowing 24 d of culture, 43 out of 200 explants exhibited shoot bud
formation. Explants were transferred to 10BI/cscf medium for an
additional 16 d. At this time, 54 out of 200 explants showed shoot
bud regeneration (putative transformation efficiency ˆ 27%). The
explants were cultured on B4/cscf2 medium (MS salts, B5 vitamins,
1.6% glucose, 600 mg l21 MES, 1 mg l21 inositol, 108.6 mM
adenine sulfate, 200 mg l21 CH, 17.76 mM BA, 28.9 mM GA3,
15 mM silver thiosulfate, 50 mg l21 chlorsulfuron, and 250 mg l21
cefotaxime). Three weeks later, shoots were removed from the
explants and transferred to fresh B4/cscf2. Two weeks later, 17 of
the shoots had elongated and were cultured on MSOT/cs medium
(MS salts, MS vitamins, 3% sucrose, 1 g l21 inositol, 50 mg l21
chlorsulfuron, and 2.5 g l21 Gel-rite). These shoots were rooted on
OMSSN medium and the plants were finally transferred to the
greenhouse for growth. Confirmation that the shoots exhibited the
transformed phenotype was done by the callusing assay. Forty-one
and 47 d after cocultivation was begun, leaf tissues from six and
eight additional plants, respectively, were tested for callus
formation and they all produced prolific callus indicating their
transgenic phenotype. However, no inheritance data were pre-
sented, nor were molecular evidences of transgene integration
provided.
At least two other binary vectors carrying genes encoding
kanamycin (pSLJ1911) and hygromycin (pWTT2039) resistance,
respectively, and b-glucuronidase as the reporter gene were tested
for genetic transformation (Engler et al., 1993). One shoot that was
regenerated in the presence of 250 mg l21 kanamycin showed
positive GUS activity while another one selected at 500 mg l21
718 OCHOA-ALEJO AND RAMIREZ-MALAGON
kanamycin appeared faintly blue, indicating that transformation
occurred in the first case but it was not surely confirmed in the
second case. It was also shown that kanamycin at the selection
concentrations used (50 mg l21) for other Solanaceous species like
Nicotiana tabacum, was not effective, since 13 shoots regenerated
using this concentration of antibiotic did not show positive GUS
activity. Similarly, eight shoots formed on 100 mg l21 and one
shoot regenerated on 200 mg l21 kanamycin did not exhibit GUS
activity, indicating that they were escapes. One out of two shoots
regenerated on 300 mg l21 gave positive GUS activity. In the case
of explants of California Wonder cocultured with pWTT2039, one
out of 150 and two out of 180 explants exhibited shoot bud
formation on 500 mg l21 spectinomycin as selector after 2 mo. of
culture. One of the regenerated shoots was assayed for GUS activity
and this gave a positive reaction.
In vitro plant regeneration and Agrobacterium-mediated transfor-
mation from cotyledon explants of hot pepper (Capsicum annuum
cv. Golden Tower) were described by Lee et al. (1993). For
transformation, cotyledon explants were cocultured with Agrobac-
terium tumefaciens strain LBA4404 carrying a binary vector pRok1/
105 harboring the cDNA of cucumber mosaic virus I17N-satellite
RNA. CMV satellite RNAs are small RNAs that are not infectious
by themselves, are unable to replicate and encapsidate in host cells
without the presence of a helper virus, are not necessary for the
replication of the helper virus, and have no appreciable sequence
homology with the helper virus genome. After coculture (3 d), the
explants were cultured on MS medium supplemented with 0.27 mM
NAA plus 9.1 mM Zea for shoot induction (4 wk) and then on
medium containing 0.054 mM NAA plus 9.1 mM Zea for shoot
elongation; in both cases kanamycin sulfate (100 mg l21) and
carbenicillin (250 mg l21) were added to the medium. Shoots were
recovered 2 mo. after coculture and were rooted on a medium
supplemented with kanamycin sulfate (50 mg l21). Regenerated
plants were transplanted to potting soil. Transformation frequency
was about 4% of total regenerants. PCR and Northern analyses
showed that the introduced gene was integrated and stably
expressed in the regenerated plants.
According to Zhu et al. (1996), fertile transgenic sweet pepper
plants (Capsicum annuum var. grossum) were regenerated at a
relatively high rate from different explants of Zhong Hua no. 2
cultivar cocultured with A. tumefaciens strain GV3111-SE harbor-
ing a plasmid containing the cucumber mosaic virus coat protein
gene (CMV-CP). Explants from young leaves were dissected from
3-wk-old seedlings and were cocultured for 3 d. The explants were
inoculated on MS medium supplemented with 35.5 mM BA,
11.4 mM IAA, 50 mg l21 kanamycin, and 500 mg l21 carbenicil-
lin. Explants were subcultured every 2 wk on the same medium.
When regenerated buds were 0.2±0.3 cm long (3±4 wk of culture)
they were cut from the explants and incubated on elongation
medium (MS ‡ 8:88 mM BA ‡ 5:78 mM GA3) until the stem
reached 0.5 cm in length (approximately 10 d). Individual buds
were cut and cultured for 10 d on MS medium containing
8:88 mM ‡ 5:78 mM GA3 ‡ 1:89 mM ABA. Shoots larger than
2 cm in length were transferred to root-inducing medium (MS
basal medium 1 50 mg l21 kanamycin 1 500 mg l21 carbenicil-
lin 1 0.54 mM NAA). Regenerated plants were transferred to pots
and maintained in a growth chamber. Southern analysis showed
that at least three out of five R1 progeny plants reacted positively
with the CMV-CP gene probe. Western blot analysis of CMV-CP
containing R1 progeny plants showed that two of them accumulated
significant levels of coat protein while another two expressed it only
at low levels. No data were provided on the resistance of these
transgenic plants against infection by CMV.
Satellite RNA-mediated resistance to CMV in transgenic plants
of hot chili pepper was reported by Kim et al. (1997). Transgenic
plants were obtained by infection with a strain of A. tumefaciens
harboring the plasmid pRok1/105 containing the cDNA of CMV
satellite RNA driven by the CaMV 35S promoter and the nptII gene
as the selection marker (Lee et al., 1993). Two independent T0
primary transgenic lines which produced CMV satellite RNA were
self-fertilized to produce T1 seeds (Kim et al., 1997). Segregation
analysis in the progeny was determined by germinating seeds on MS
medium with 200 mg l21 kanamycin and recording the resistant
and sensitive seedlings after 6 wk of incubation. PCR analysis for
detection of nptII sequence was carried out in 43 T1 lines derived
from the two T0 lines to confirm the transgenic character. Twenty-
six out of 43 seedlings (ca. 50%) were positive in this test. To
estimate virus resistance, 17 T1 transgenic plants were mechani-
cally infected with two variants of CMV (CMV-Y or CMV-Kor).
Symptom development was monitored for 2 mo. after inoculation.
Typical systemic symptoms were observed in non-transgenic plants,
whereas symptoms in transgenic plants were noticeably attenuated
and delayed.
A protocol for regeneration and genetic transformation has been
established for chili pepper (C. annuum L. var. Pusa jwala) by
Manoharan et al. (1998). Shoot bud regeneration was achieved
using cotyledonary leaf explants cultured on MS medium wth
2.27 mM TDZ. Elongation of shoot buds and subsequent rooting was
achieved on half-strength MS medium with 2.85 mM IAA. A.
tumefaciens strain EHA105 carrying a binary vector plasmid
(pBI121) was used for genetic transformation. Cotyledonary
explants were cocultured for 2 d. Shoot buds were produced after
30±40 d on regeneration medium supplemented with 50 mg l21
kanamycin and 400 mg l21 cefotaxime. Shoot bud elongation and
rooting processes were achieved after 90 d in the presence of
25 mg l21 kanamycin. Four transgenic plants were regenerated
from 10 explants bearing shoot buds from a total of 200
cotyledonary explants. The transgenic nature of the regenerated
plants was confirmed by histochemical staining of GUS, PCR, and
Southern analysis of nptII gene.
Phillips et al. (2000) have attempted to reproduce the reported
chili pepper transformation protocols, and they have evaluated
alternative methods for transformation. Key results from this group
include:
(1) Agrobacterium tumefaciens-mediated transformation. Attempts
to reproduce the published pepper transformation protocols
reported by Lee et al. (1993), Engler et al. (1993), Zhu et al.
(1996), and Manoharan et al. (1998) with chili pepper, failed to
recover transformed plants despite the use of sufficient numbers
of explants to have achieved less than 0.1% transformation
efficiency (Jayashankar, 1998). However, these authors did
recover stably transformed callus lines (from cotyledons) and
roots (from hypocotyls) at about 0.1% frequency, which
expressed the transferred GUS reporter gene and kanamycin
resistance selectable marker gene, but protocols have not been
available to date to regenerate pepper plants from callus or root
tissues. These authors concluded that chili cell types capable of
 IN VITRO CHILI PEPPER BIOTECHNOLOGY 719

shoot regeneration only rarely overlap with cell types capable of
being transformed by A. tumefaciens (Phillips et al., 2000).
(2) Agrobacterium rhizogenes-mediated transformation. Transforma-
tion of chili pepper by A. rhizogenes has been achieved
(Jayashankar et al., 1997; Jayashankar, 1998). A binary vector
system in which the wild-type Ri plasmid conferred the hairy
root phenotype, and the binary vector carried the reporter and
selectable marker genes, was used for transformation. Stably
transformed hairy roots were recovered that expressed both the
reporter and selectable marker genes at a 16% cotransformation
rate, which is significantly better than the A. tumefaciens or
biolistics methods (see below). However, no transgenic shoots
have been recovered from the hairy roots. Modifications were
made to the regeneration procedure to avoid hairy root
formation and to encourage direct shoot regeneration, and
consequently an 8% transformation rate in shoot buds was
obtained but none of these buds elongated. This approach for
chili transformation appears to be promising. The availability of
a regenerable callus system and/or a method to improve shoot
elongation from buds induced on cotyledon explants, combined
with this A. rhizogenes delivery method, could potentially lead
to a more reliable and efficient chili transformation protocol
(Phillips et al., 2000).
(3) Biolistics-mediated transformation. The biolistics approach to
direct gene transfer in chili pepper has been tested
(Jayashankar, 1998). Transient expression (within 72 h follow-
ing biolistic bombardment) of the GUS reporter gene in chili
cell suspensions was marginally efficient, but no long-term
reporter gene expression was obtained. Stable transformation
was observed at low frequency (5%) in limited regions of
cotyledon and hypocotyl explants, and in a few roots
regenerated from bombarded hypocotyls (Phillips et al., 2000).
transformation. Hypocotyls from etiolated 9-d-old seedlings of C.
annuum cv. Mulato Bajio (ancho type) were infected by wounding
the apical part with a syringe needle containing a suspension of
either strain A208 (pTiT37::pMON9749) or LBA4404 (pBI121) of
A. tumefaciens. These plasmids contained the nptII and gus genes.
The infected seedlings were cultured on MS medium without growth
regulators and kanamycin in order to allow bud formation and
further shoot elongation. No kanamycin was included in the culture
medium because we had found that this antibiotic did not inhibit
shoot formation unless it was added at very high concentrations
(1.6 g l21). Each elongated shoot was tested for GUS activity. None
of the regenerated shoots from seedlings infected with LBA4404
(pBI121) showed positive GUS activity, while only one shoot per
1000 regenerated shoots from 1500 seedlings was positive in
the GUS assay in those seedlings infected with A208
(pTiT37::pMON9749) (data not shown). Progeny (T1) derived from
five GUS-positive regenerants (T0) exhibited an approximately 3:1
GUS(+)/GUS(2) ratio, indicating a monogenic Mendelian inheri-
tance of the gus gene. Interestingly, an increase in transformation
efficiency was observed when seedlings from the T1 progeny that
segregated as negative for GUS activity were infected by the same
procedure and shoots were regenerated. In this case, three out of 87
regenerated shoots (3.4%) from 200 seedlings showed GUS activity.
Five plants that were found to exhibit GUS activity were further
analyzed by PCR and Southern blot analyses to detect the presence
of nptII gene sequences and all of them were positive for these tests
(Fig. 5).
Table 4 summarizes the current status of chili pepper
transformation. Although some advances have been made, the
efficiency for recovering transformed plants using A. tumefaciens
remains low. No transformed plants using A. rhizogenes or physical
systems (biolistic, electroporation, etc.) have been obtained.

Our own experience on chili pepper transformation using A.

tumefaciens has not been very optimistic (RamõÂrez-MalagoÂn, 1997; Applications of Cell and Tissue Cultures
Abraham-JuaÂrez, 1999). We have used the chili pepper plant
regeneraton method previously described (RamõÂrez-MalagoÂn and Cell and tissue cultures of chili pepper have been used as models
Ochoa-Alejo, 1996) as the system for Agrobacterium-mediated to study different physiological, biochemical, or molecular

TABLE 4

PRESENT STATUS OF CHILI PEPPER GENETIC TRANSFORMATION

Species/cv. Transformation system Results and remarks References

C. annuum/Six bell cultivars;
C. glabriusculum/wild-type accession
C. annuum/Vegisweet and
California Wonder
C. annuum/Golden Tower
C. annuum var. grossum/Zhong
Hua no. 2
C. annuum/Mulato Bajio
C. annuum/ Pusa jwala
A. tumefaciens/wild-type
strains A281 and C58; pGV33858
plasmid (nptII, gus, 35S promoter)
A. tumefaciens/strain LB4404,
p5T35AD (acetolactate synthase
gene, gus, 35S promoter); pSLJ1911
(nptII, gus, 35S promoter); pWTT2039
(hpt, gus, 35S promoter)
A. tumefaciens/strain LB4404, pRok1/105
(cucumber mosaic virus I17N-Satellite RNA,
nptII, 35S promoter)
A. tumefaciens/strain GV3111-SE
(CMV-CP, nptII, 35S promoter)
A. tumefaciens/strain A208, pTiT37::pMON9749
(nptII, gus, 35S promoter); and strain LBA4404,
pBI121 (nptII, gus, 35S promoter)
A. tumefaciens/strain EHA105, pBI121
(nptII, gus, 35S promoter)
Tumor formation in the absence Liu et al. (1990)
of growth regulators. Callus and
leaf-like structures GUS+.
Shoots and plants GUS+. Engler et al. (1993)
0.5±0.7% plant transformation
efficiency
Regenerated plants with attenuated Lee et al. (1993);
symptoms against CMV. 4% plant Kim et al. (1997)
transformation efficiency
Regenerated plants expressing Zhu et al. (1996)
CMV-CP
Regenerated plants GUS+. 0.1% RamõÂrez-MalagoÂn (1997)
plant transformation efficiency
Regenerated plants GUS+. 2% Manoharan et al. (1998)
plant transformation efficiency
720 OCHOA-ALEJO AND RAMIREZ-MALAGON

Fig. 5. Agrobacterium tumefaciens-mediated chili pepper (C. annuum var. Mulato Bajio) genetic transformation. a, A transgenic plant
grown under greenhouse conditions. b, PCR analysis of a nontransformed plant (lane 2) and transformed plants (lanes 3±7) using
oligonucleotides for amplification of a central fragment of 517 bp of nptII gene; a molecular marker (1 kb DNA ladder) was included in
lane 1. c, Southern blot analysis of a nontransformed plant (lane 2) and transformed plants (lanes 3±8); plant DNA was digested with
HindIII and hybridized with an nptII probe. A molecular marker (1 kb DNA ladder) is shown in lane 1.

processes as well as systems for the isolation of variant cells
exhibiting specific biochemical or agronomical characteristics.
Fine suspension cultures are usually necessary for several
purposes (immobilization, clonal cell selection, etc.). A method for
rapidly obtaining fine chili pepper cell suspensions has been
described (Williams et al., 1988). The method uses a Waring
blender for aseptic homogenization of cultures and has been shown
to be adequate for the establishment of fine suspensions from cell
and callus cultures.
Effect of Abiotic Stresses on Cell Cultures and Isolation of Resistant
Variant Cells
Withers and Street (1977) reported on the capacity of C. annuum
cell cultures to survive to freezing temperatures after an osmotic
pretreatment with mannitol (1.7 or 5.2%) and a previous incubation
with cryoprotectants (2±10% DMSO 1 5±20% glycerol). Capsicum
cells were shown to be highly sensitive to chilling injury (0 viability
after 4.5 h at 28C). Freezing damage was reduced by pretreatment of
cells with mannitol and also by cryoprotectant treatments, resulting
in cells capable of maintaining 20±30% viability after treatment at
21008C and short-term storage at 2968C.
Capsicum annuum cell lines capable of growing in liquid media
containing 1 or 2% sodium chloride were isolated by Dix and Street
(1975). The cell lines were selected as colonies by plating cell
suspension on Petri dishes with media including 1% NaCl. Twenty
colonies were subcultured on medium without salt until they
produced callus. The resistance of these callus cultures was tested
by inoculating small pieces of callus on agar plates containing 1, 2,
or 3% NaCl. All the callus recovered from 1% NaCl grew in the
presence of this level of salt. Two lines of callus resistant to 1%
NaCl and one recovered from medium with 0% NaCl were
established as cell suspensions. These cell lines were tested for
growth in 0, 1, or 2% NaCl. Differences were found in capacity to
grow in salt since the cells selected in the presence of 1% NaCl
were shown to be more resistant than the control cells. Cultures in
the third and fourth passage in medium with 1% NaCl yielded cells
capable of growing in 2% NaCl. The selected cells retained the
 IN VITRO CHILI PEPPER BIOTECHNOLOGY
resistance trait when they were subcultured in the absence of NaCl
and then challenged in the presence of salt, indicating stability of
the resistance. Changes in the morphology of the selected resistant
cells were observed (small size, and in cell aggregates).
By using the same approach, Dix and Street (1976) also selected
chili pepper cell lines with enhanced chilling resistance. Cell
cultures were exposed or not to the mutagen EMS and then
subjected to chilling for 21 d at 238C and 58C, and the cell lines
derived from the surviving cells were tested for resistance to
chilling. Some of the cell lines when again submitted to the chilling
stress retained their resistance after an extended period of growth at
248C. Treatment with EMS promoted the isolation of stable resistant
cell lines. Differences in the respiratory activity of isolated
mitochondria from a resistant and a sensitive cell line were
detected.
Cell clones of chili pepper with enhanced resistance to osmotic
stress were isolated by us (Santos-DõÂaz and Ochoa-Alejo, 1994a)
using 15% polyethylene glycol (PEG) as the selection agent. Cell
clones were isolated by plating the cell suspension on filter paper
discs supported by polyurethane foam that were bathed with culture
medium containing 15% PEG. Two cell clones (T6 and T7), chosen
based on their characteristics of growth and friability, were
established as suspension cultures in the presence of 15% PEG
and were subsequently subcultured in increasing concentrations of
osmoticum. By this approach the cell clones were capable of
growing in the presence of 20 and 25% PEG, respectively. The cell
clone T7 was found to grow better in the presence of 5±10% PEG
after a period of subculturing in the absence of osmoticum,
indicating that the resistance trait was stable. The tolerant cell
clones exhibited a 3±3.5-fold decrease in osmotic potentials in
comparison with the nonselected cells, suggesting that osmotic
adjustment occurred. K+ was the major solute contributing to the
osmotic potential, and was found to be 1.3±3 times higher in the
PEG-resistant cell clones than in the control cells. Proline and
glycine betaine levels showed a positive correlation with the degree
of resistance to osmotic stress. Differences in the electrophoretic
pattern of cell wall proteins between clone T7 and the nonselected
cells have been reported (Quintero-Higuera et al., 1997). After
treatment with 15% PEG, T7 cells accumulated three major
proteins of 9, 11, and 14 kDa bound to structural cell wall polymers
by noncovalent links or disulfide bonds. Regarding covalent-bound
proteins, the main difference was the presence of a new protein of
10 kDa in the cell walls of the T7 clone. We are currently using this
T7 clone as a system to investigate the molecular mechanisms
involved in osmotic stress resistance at the plant cell level
(VeraÂstegui-PenÄa, 1999) and we have observed selective expression
of some genes in resistant cells. We are currently isolating these
genes in order to investigate their regulation by drought or osmotic
stressors and also to demonstrate their role in the resistance
mechanisms.
Chili pepper cell cultures also have been used as a comparative
drought-sensitive model to study drought resistance of the
xerophytic species Larrea tridentata (creosote bush), an evergreen
plant that grows in the Sonoran desert (Santos-DõÂaz and Ochoa-
Alejo, 1994b). Cell cultures of both species were exposed to osmotic
stress imposed by the presence of PEG in the culture medium. Cell
cultures of L. tridentata showed higher resistance to PEG (6.6 times
higher) than chili pepper cells. More negative osmotic potentials
were found to occur in cell cultures of L. tridentata in response to
721
PEG as compared to chili pepper cells. A positive correlation was
observed between proline accumulation and the capacity of cell
cultures to grow in conditions of osmotic stress.
Use of cell suspensions to study pathogenic defense responses. The
responses of plants to pathogen infections are generally character-
ized by metabolic and structural changes associated with the
occurrence of defense reactions. The biosynthesis of phytoalexins is
believed to be one of the major defensive systems in higher plants.
Capsidiol is the major phytoalexin produced by inoculation of chili
pepper and tobacco plants with pathogenic fungi. The biosynthetic
pathway of capsidiol has been partially elucidated using tobacco
cultured cells. It has been demonstrated that all organs and callus
tissue of chili pepper are able to produce capsidiol (Brooks et al.,
1986). Cells suspension cultures of chili pepper offer the
opportunity to study the processess involved in capsidiol produc-
tion under controlled conditions. Hoshino et al. (1994) have studied
phytoalexin induction in green chili pepper cell cultures cultured
in MS medium and treated with arachidonic acid (1 mM), an active
elicitor which is a component of Phytophthora infestans. Twelve
hours after addition of arachidonic acid, green chili pepper cell
cultures appeared brown. Addition of arachidonic acid to green
chili pepper (Capsicum annuum) cell suspension cultures induced
the extracellular accumulation of the phytoalexins capsidiol (major
component) and rishitin. Capsidiol and rishitin accumulation in the
culture medium reached significant levels some 12 h after addition
of elicitor and attained a maximum by 24±36 h. The intracellular
accumulation of phytoalexins (less than 10% of total phytoalexins)
followed a similar time course as that observed in the culture
medium. The cytochrome P450 inhibitors ancymidol and ketoco-
nazole suppressed the synthesis of both phytoalexins in elicited cell
suspension cultures, suggesting that a part of the capsidiol and
rishitin biosynthetic pathways of green chili pepper is regulated by
a cytochrome P450-dependent process.
GarcõÂa-PeÂrez et al. (1998) reported a study on the defense
response of chili pepper (Capsicum annuum) suspension cells to the
fungus Phytophthora capsici. Cell cultures of three cultivars of chili
pepper with different degrees of sensitivity to Phytophthora capsici
responded to elicitation by both lyophilized mycelium and fungus
filtrate. Cell suspensions exhibited conductivity changes, browning,
production of capsidiol and synthesis, or accumulation of
pathogenesis-related (PR) proteins with glucanase and chitinase
activities. The greatest rate of capsidiol accumulation occurred after
18 h in the mycelium-treated cells and after 12 h in those elicited
with the filtrate. The inhibition threshold of fungal growth [300 mg
(g dry weight)21] was reached only in the resistant cultivar Smith-5,
but not in the sensitive cultivars Americano and Yolo Wonder. An
intracellular glucanase and an extracellular chitinase were induced
only in the resistant cultivar 24 h after elicitation, suggesting that
these enzymes are involved in the resistance to Phytophthora
capsici. The results also indicate that elicitation of chili pepper cell
cultures by signal molecules from P. capsici exhibited properties of
a multicomponent dynamic system. The differential responses to
P. capsici of cell cultures derived from resistant and sensitive chili
pepper cultivars correlated well with those observed at the plant
level.
Capsaicinoid production by cells and tissues cultured in
vitro. One of the main characteristics of chili pepper fruits
is their hot taste due to the presence of a group of compounds
known as capsaicinoids. These compounds are synthesized from
722 OCHOA-ALEJO AND RAMIREZ-MALAGON

Fig. 6. Capsaicinoid biosynthetic pathway. Enzymes involved in the pathway are: phenylalanine ammonia lyase (PAL), cinnamic acid
4-hydroxylase (Ca4H), coumaric acid 3-hydroxylase (Ca3H), caffeic acid O-methyltransferase (COMT), and capsaicinoid synthetase (CS).

l-phenylalanine through the phenylpropanoid biosynthetic pathway
to render vanillylamine, which is ultimately linked to the branched
fatty acid residues that are synthesized from l-valine or l-leucine
(Fig. 6) to give the five capsaicinoid analogs: capsaicin,
dihydrocapsaicin, nordihydrocapsaicin, homocapsaicin, and homo-
dihydrocapsaicin. In nature, capsaicin and dihydrocapsaicin
account for 90% of the total capsaicinoid content in chili pepper
fruits (Suzuki et al., 1981).
The biosynthetic capacity of in vitro-cultured cells and tissues
to produce capsaicinoids has been investigated by different groups.
Incorporation of l-[14C]valine and l-[14C]leucine into the
intermediates of the branched chain fatty acids in protoplasts
isolated from placental tissues of C. annuum var. annuum cv.
Karayatsubusa was demonstrated, indicating the role of these amino
acids as precursors of the branched fatty acids pathway (Suzuki
et al., 1981). Fujiwake et al. (1982) further studied capsaicinoid
formation in a protoplast suspension (ca. 105 cells per 2 ml)
incubated with either l-[U-14C]phenylalanine or l-[3,4-3H]valine at
388C for 120 min. After incubation, the protoplast suspension was
analyzed for incorporation of the precursors into capsaicinoid
biosynthetic intermediates by HPLC and also by scintillation
spectrometry. When protoplasts were fed with radiolabeled
 IN VITRO CHILI PEPPER BIOTECHNOLOGY
phenylalanine, all the phenylpropanoid intermediates and capsaicin
were found to be radioactive, indicating that phenylalanine was the
precursor for the biosynthesis of the vanillylamine moiety.
Protoplasts incubated with [3H]valine exhibited incorporation only
into capsaicin, demonstrating that the branched fatty acid moiety of
capsaicin is derived from this amino acid.
Accumulation of capsaicinoids in cultured cells and tissues has
been manipulated by altering the physical organization of liquid-
suspended cells as occurs when they are immobilized, and also by
reducing the growth rate (omitting growth regulators or nutrients),
by addition of precursors, by selection of cell lines, and by cell
elicitation (Holden et al., 1987).
Immobilization of C. frutescens cells by entrapment in poly-
urethane foam has been reported by Lindsey et al. (1983) and by
Mavituna and Park (1985), and the characteristics of immobilized
chili pepper cells in bioreactors has been described by Mavituna
et al. (1987). Cell suspensions were found to invade, and were
strongly retained in, the polyurethane foam particles over a 21-d
culture period (Lindsey et al., 1983). The viability of the
immobilized cells was 70±80%. The immobilized cells produced
significantly more capsaicin than suspended cells (50, 100, and
1000 times, depending on the replicates). Almost all the capsaicin
was released into the culture medium. Increases of five times more
capsaicin were found by the addition of 5 mM isocapric acid
(8-methylnonanoic acid) to the immobilized cultures. Further
studies by this group (Lindsey and Yeoman, 1984a) revealed that
immobilized cells retained a high level of viability, as determined
by respiratory activity, esterase activity (by staining with
fluorescein diacetate), nutrient uptake, and secondary metabolic
activity. Accumulation of capsaicin was retained during prolonged
culture periods (up to 12 wk). In the absence of specific precursors
to capsaicin, immobilized cells were found to produce two to three
orders of magnitude higher yields of capsaicin than did suspended
cells (Lindsey and Yeoman, 1984b). These results were positively
correlated by an increased rate and extent of incorporation of
l-[U-14C]phenylalanine into capsaicin in immobilized cells as
compared with cell suspensions, and an inverse relationship was
found to occur between incorporation of l-[U-14C]phenylalanine
into protein and capsaicin. The accumulation of capsaicin was
shown to be increased by supplementing the medium with
precursors of capsaicin such as phenylalanine (6 and 8.6 times
higher for cell suspension and immobilized cells, respectively) and
isocapric acid (44.5 times higher for immobilized cells), and by
reducing the growth rate of immobilized cells through the omission
of growth regulators (2.76 mM 2,4-D, 10.72 mM p-chlorophenoxy-
acetic acid, and 0.46 mM Kin) from the culture medium (Lindsey
and Yeoman, 1984b).
Nutrient limitation also has been found to increase capsaicin
accumulation in immobilized cells (Lindsey, 1985). Cells were
cultured in media containing reduced concentrations of essential
nutrients, in an attempt to manipulate the rates of protein synthesis.
Cells cultured in the absence of phosphate for 15 d showed
significant reduction in the incorporation of [U-14C]phenylalanine
into proteins. Immobilized cells deprived of nitrate and phosphate
exhibited a higher reduction of [U-14C]phenylalanine incorporation
into proteins, but a higher incorporation into capsaicin than control
cells growing in the complete medium. Cell cultures of C. annuum
immobilized in calcium alginate also have been shown to
accumulate higher levels of capsaicin when they were cultured in
723
medium without either phosphates (4-fold), nitrates (13-fold), or
sucrose (1.5-fold) (Ravishankar et al., 1988).
Incorporation of l-[14C]phenylalanine and l-[14C]coumaric acid
into capsaicin produced by suspended and immobilized cells of
C. frutescens was investigated taking into consideration that
phenylalanine is used for protein and capsaicin biosynthesis,
while coumaric acid is involved in capsaicin biosynthesis as well as
in cell-wall metabolism (Lindsey, 1986). Under growth-promoting or
growth-inhibiting conditions, immobilized cells generally incorpo-
rated higher levels of radioactivity into capsaicin from both
precursors, but under growth-promoting conditions the relative
amount of radioactive capsaicin accumulated was related to the
phase of the growth cycle. The accumulation of radioactive and total
capsaicin was reduced in culture conditions promoting cell division.
It was suggested that both protein and cell-wall metabolisms are
potential sinks for capsaicin precursors, and that these sinks can be
manipulated experimentally.
A comparative study of accumulation of phenylpropanoids and
capsaicinoid compounds in cell cultures (freely suspended and
immobilized cells) and fruits of chili pepper revealed that the
phenolic precursors of capsaicin are present in chili pepper cells at
extremely low levels (Hall et al., 1987). In fruits, capsaicin is
notably absent in the early stages of development and is
accumulated at a maximum rate as the fruit approached the end
of the phase of growth (40 d). Radioactive tracer studies in
immobilized cell cultures indicated that the majority of the phenolic
derivatives of phenylalanine are ultimately bound to the insoluble
fraction of the cells.
An approach to increase the pools of phenylalanine and the
phenylpropanoids in chili pepper cells has been the isolation of
cells with resistance to the phenylalanine analog p-fluorophenyl-
alanine (PFP) (Salgado-Garciglia and Ochoa-Alejo, 1990). Cell
suspensions of chili pepper (C. annuum cv. TampiquenÄo 74; serrano
type) were plated onto Petri dishes on MS medium containing
increasing levels of the analog (0±500 mM). Friable and actively
growing callus colonies were selected on medium with 10 mM PFP.
Four cell lines with different levels of resistance to PFP were
serially selected in increasing concentrations of the analog and
suspension cultures were established from each of them. Resistance
to PFP was retained even after 75 d of culture in the absence of
analog, indicating the stability of the selected characteristic. PFP-
resistant cell lines accumulated higher levels of capsaicin than
sensitive lines even after prolonged culture in PFP-free medium.
Capsaicin production in non-selected cells was only 26.8% of that
found in one cell line resistant to 500 mM PFP. The capsaicin
contents in the non-selected cell suspension and in one of the
resistant cell lines were 6.7 and 24.9%, respectively, that of
maximum value in fruits. These cell lines were further characterized
regarding the activity of phenylalanine ammonia-lyase (PAL) which
is the first enzyme of the phenylpropanoid pathway, and the levels
of free phenylalanine, phenolics, and phenylpropanoid acids
involved in capsaicin biosynthesis (Ochoa-Alejo and Salgado-
Garciglia, 1992). In this case, a non-selected cell line, a PFP-
sensitive line (CA-02), a moderately resistant cell line (CA-29) and
two resistant cell lines (CA-04 and CA-16) were studied. Higher
PAL activities and higher levels of phenolics and phenylalanine
were found in the PFP-resistant cell lines even after a minimum of
nine subcultures (15 d each) in the absence of PFP, indicating that
the selected trait was stable. PFP-resistant cell lines accumulated
724 OCHOA-ALEJO AND RAMIREZ-MALAGON
higher amounts of capsaicin precursors (cinnamic, caffeic, and
ferulic acids) than either the nonselected cells or the sensitive cell
line. Overall, accumulation of free phenylalanine correlated well
with PAL activity, phenolics, phenylpropanoids, and capsaicin
levels, suggesting an active flow through the phenylpropanoid
pathway in the PFP-resistant cells of chili pepper.
In an attempt to identify possible key control points in the
capsaicin biosynthetic pathway, experiments were carried out in
which immobilized cells of chili pepper were incubated in the
presence of capsaicin for 24 h after which l-[U-14C]phenylalanine
was added (Hall and Yeoman, 1991) and the fate of radioactivity
was followed. A marked reduction (2.4-fold less) in the incorpora-
tion of l-[14C]phenylalanine into capsaicin was observed in the
treated cells. On the other hand, addition of sinapic acid, an
immediate derivative of ferulic acid and a component of the lignin
biosynthetic pathway, led to an increase (2-fold) in the accumula-
tion of labeled capsaicin. This result indicated that sinapic acid
inhibited the diversion of l-[14C]phenylalanine and their inter-
mediates into lignin.
Variations in the biosynthetic activity of cloned cell cultures of
C. frutescens and their response to an exogenously supplied elicitor
was reported by Holden and Yeoman (1994). Twenty clones
established from single cells of a suspension culture of C. frutescens
were maintained as callus and suspension cultures. The clones
showed marked differences in growth, chlorophyll, and phenolic
content (chloroform-soluble fraction). Elicitation of cell cultures
with an elicitor prepared from spores of the fungus Gliocladium
deliquescens increased PAL activity, reduced the incorporation of
l-[U-14C]phenylalanine into the chloroform-soluble fraction of the
culture medium, and increased incorporation into the methanol-
soluble fraction (capsaicin?) of the cells in 10 suspension clones.
Addition of ferulic acid or vanillylamine, the two intermediates at
the end of the phenylpropanoid pathway, to immobilized cell
cultures of C. frutescens revealed that these compounds were
converted into vanillin and capsaicin, respectively (Sudhakar
Johnson et al., 1996). This trend was expected since ferulic acid
and vanillylamine are the nearest precursors to vanillin and
capsaicin, respectively. It was shown that immobilized cells were
capable of carrying out oxidative deamination, a reversible reaction
which was evidenced by conversion of vanillylamine to vanillin.
Callus tissues have been used as systems to study capsaicin
biosynthesis in a few cases. For example, Weathers et al. (1992)
investigated the influence of light on the formation of capsaicin in
callus tissue of C. annuum (jalapenÄo type). They found that
increasing light intensities from 0 to 270 and 570 mmol m22 s21
inhibits capsaicin accumulation in callus tissues cultured on SH
medium (Schenk and Hildebrandt, 1972) supplemented with
2.26 mM 2,4-D ‡ 0:46 mM Kin ‡ 10:72 mM p-chlorophenoxyacetic
acid.
In order to determine the functionality of the capsaicinoid
biosynthetic pathway in callus cultures of chili pepper (C. annuum
cv. TampiquenÄo 74; serrano type), the enzyme activities of PAL,
cinnamic acid-4-hydroxylase (CA4H), p-coumaric acid-3-hydro-
xylase (CA3H), caffeic acid-O-methyltransferase (CAOMT), and
capsaicinoid synthetase (CS) were determined in both callus tissue
and in developing and mature fruits (Ochoa-Alejo and GoÂmez-
Peralta, 1993). The maximum activities of PAL, CA4H, and CA3H
in callus cultures were similar to those of chili pepper fruits, but the
values of CAOMT and CS were six times lower than those of fruits,
suggesting that the low activities of these enzymes are probably the
limiting steps for capsaicin biosynthesis in nondifferentiated
tissues.
Differentiated tissues also have been utilized to study capsaicin
production. Suspension cells and placental tissues from fruits of
C. annuum were immobilized in calcium alginate (Sudhakar
Johnson et al., 1990). Immobilized placental tissues exhibited
greater potential for capsaicin synthesis than immobilized cells.
Production of capsaicin reached 1345 mg g21 fresh weight
immobilized cells by day 14 and 2400 by day 30. Addition of
2.4 mM ferulic acid to immobilized placental cells increased
capsaicin production two-fold, whereas addition of phenylalanine,
l-valine, or caffeic acid did not cause any significant increase in
capsaicin accumulation over the control. Immobilized cell cultures
were treated with different elicitors to increase capsaicin
accumulation. Extracts of Aspergillus niger and Rhizopus oligosporus
stimulated capsaicin production in immobilized placental tissues.
A comparative study between capsaicin accumulation in free and
immobilized placenta was reported (Sudhakar Johnson et al., 1995).
In immobilized placenta, maximum accumulation of capsaicin was
on the 14th day of culture with a production of 2045 mg per culture,
whereas in free placenta, the accumulation reached maximum on
day 7 (2050 mg per culture), indicating that immobilization is not
necessary for capsaicin production. Pungency threshold of
capsaicin produced by in vitro-grown placenta was carried out by
Scoville heat units (SHU), and it was found that in vitro-produced
capsaicin was slightly lower in pungency compared to standard
natural capsaicin. In vitro-produced capsaicin was chemically
identical to standard natural capsaicin.
Biotransformation using plant cell cultures has received
increasing attention as a method for the synthesis of products
which are not normally extracted from the plant or for formation of
novel products. Cell suspension cultures and immobilized cells can
be adopted for the production of food additives or pharmaceuticals
by biotransformation processes. In the case of chili pepper,
Ramachandra Rao and Ravishankar (1999) have recently tested
biotransformation of isoeugenol to vanilla flavor metabolites and
capsaicin in suspended and immobilized cell cultures of Capsicum
frutescens. Suspended and immobilized cell cultures accumulated
vanilla flavor metabolites (vanillin, vanillic acid, and ferulic acid)
when fed with isoeugenol, the starting material in the manufacture
of vanillin. The addition of isoeugenol also stimulated its
biotransformation to capsaicin. Maximum levels of vanillin
accumulated at 566 mg per 40 ml culture on the 6th day in
isoeugenol- (2.5 mM) treated cultures. This value was twice the
increase achieved by suspended cultures of the same day. The
formation of ferulic acid (58 mg) was observed on the fifth day in
immobilized cultures treated with isoeugenol. The addition of b-
cyclodextrin, a compound capable of forming stable inclusion
complexes with organic molecules, together with isoeugenol, each at
2.5 mM, resulted in increase in the accumulation of vanillin
(919 mg per 40 ml culture), which corresponds to 1.62 times more
than in cultures treated with isoeugenol alone. Isoeugenol-treated
immobilized cells, when elicited with aqueous mycelial extract of
Aspergillus niger, yielded maximum vanillin concentrations (735 mg
per 40 ml culture), whereas addition of medium filtrate of A. niger
led to marginal increase in the vanillin (610 mg per 40 ml).
Accumulation of capsaicin reached a maximum, 121 and 92 mg, on
the 10th day in isoeugenol and elicitor-added cultures, respectively.
 IN VITRO CHILI PEPPER BIOTECHNOLOGY
Biotransformation of isoeugenol was more effective in immobilized
cells, and this was enhanced by the addition of b-cyclodextrin and
fungal elicitor.
Ramachandra Rao and Ravishankar (2000) have also investi-
gated the conversion of protocatechuic aldehyde and caffeic acid
into vanillin and capsaicin in freely suspended and immobilized
cell cultures of Capsicum frutescens. It was found that the exogenous
phenylpropanoid intermediates protocatechuic aldehyde and caffeic
acid were biotransformed to vanillin and capsaicin. It was noted
that this culture biotransformed externally fed protocatechuic
aldehyde to vanillin more than its conversion to capsaicin, whereas
caffeic acid-treated cultures accumulated more capsaicin than
vanillin. The maximum accumulation of vanillin (5.63 mg l21) and
capsaicin (3.83 mg l21) was recorded, respectively, in immobilized
cell cultures treated with protocatechuic aldehyde, which was 1.8
and 1.4 times higher than in protocatechuic aldehyde-treated cell
suspensions. Caffeic acid-treated immobilized cells accumulated
maximum vanillin and capsaicin at 2.68 and 3.03 mg l21 culture,
respectively, which was 1.65 and 1.33 times over freely suspended
cells treated with caffeic acid. The addition of S-adenosyl-l-
methionine, a methyl donor, to protocatechuic aldehyde-treated
immobilized cell cultures, resulted in accumulation of vanillin
(14.08 mg l21), which was 2.5-fold higher than that in cultures
treated with protocatechuic aldehyde alone, suggesting the
influence of S-adenosyl-l-methionine on O-methylation of proto-
catechuic aldehyde, resulting in more vanillin accumulation. The
increase in vanillin accumulation was well correlated with an
increase in specific activity of caffeic acid O-methyltransferase in
protocatechuic aldehyde and S-adenosyl-l-methionine-treated
immobilized cell cultures.
Conclusions
Cell, tissue, and organ culture techniques of chili pepper have
been established by several groups; however, Capsicum species
have remained a recalcitrant group with respect to in vitro plant
regeneration, since very few cases of plant regeneration from cells
(protoplasts and cell suspensions) have been reported. In general,
all of these systems are dependent upon either an adventitious
proliferation system (somatic embryos in suspension) or on short-
term memory mimicking adventitious shoot regeneration from
explants (protoplasts). Although variant cells exhibiting different
levels of resistance against abiotic stresses have been isolated from
cell suspensions, no plants showing these traits have been
regenerated. It is possible that longer-term cultures of recovered
colonies (callus) did not regenerate because of the loss of
totipotency. Furthermore, no plant regeneration from callus tissues
has been achieved (except for a work with C. annuum and a
preliminary report in C. baccatum); this problem has also limited
the utilization of somaclonal variation as an alternative strategy for
genetic improvement.
Few examples of chili pepper plant regeneration from somatic
embryogenesis currently exist in the literature. In vitro embryo
formation and plant regeneration have been more often observed in
gametic cells (microspores) from anther cultures.
Direct organogenesis has been the most frequently used
morphogenic route for in vitro chili pepper plant regeneration;
however, the major problem faced to achieve this goal has been the
failure of elongation of the induced shoot buds. Shoot buds and
725
rosettes are not well formed during the induction step, perhaps
because of a lack of true apical meristems. The partial success
achieved by others using alternative auxins (PAA, brassinolide)
suggests that there may be a problem in auxin perception and/or
signal transduction leading to malformed meristems. Strategies to
overcome this problem during induction may ultimately improve
both elongation and plant recovery. Some approaches have been
tested to overcome this problem, and in some cases successful
elongation of shoot buds and further plant regeneration has been
described, but these approaches seem difficult to replicate in
different laboratories. Micropropagation of chili pepper through
shoot tip culture has been successfully established.
Chili pepper transformation via Agrobacterium tumefaciens
infection has been reported, but the efficiency is far below the
efficiencies achieved with other members of the Solanaceae family.
Both the limitations of the reported regeneration systems, as well as
apparent difficulties in targeting transformation-competent cell
types in chile pepper, contribute to the difficulties in achieving
transformation of pepper. This inefficiency has limited the wide
application of genetic transformation for genetic improvement or
genetic manipulation of this important crop. Utilization of
A. rhizogenes as a vector for genetic transformation of chili pepper
is in its infancy. Information in the literature on genetic
transformation by bioballistic means is limited, or by alternative
methods (electroporation, silicon fibers) is null.
Cell suspension cultures of chili pepper have been utilized in
only a few cases to investigate plant cell±pathogen interactions.
Cell cultures have been shown to respond against fungal infection or
to elicitation in a similar way as tissues, organs or plants. Therefore,
chili pepper cell suspension cultures can be excellent models to
study the biochemistry and molecular biology of these responses.
Chili pepper cells and tissues have been used as model systems
to study capsaicinoid production capacity. In general, low levels of
these pungency compounds have been found to accumulate in
freely-suspended cells as compared to chili pepper fruits.
Immobilization of cells and tissues, nutrient limitation, addition of
precursors and intermediates, treatment with elicitors, and cell
selection have been applied to increase in vitro capsaicinoid
accumulation. However, the levels of capsaicinoids in cell and
tissue cultures have never reached the levels in fruits. Diversion of
precursors and intermediates of capsaicinoid biosynthesis to other
pathways (protein, lignin, etc.), and low activities of some of the
enzymes involved might be the limiting factors for adequate
capsaicinoid accumulation in nondifferentiated in vitro cultures.
Chili pepper cell cultures have been also found to be capable of
bioconverting precursors into interesting industrial products other
than capsaicin such as vanillin flavor components.
Perspectives
During the past decade, significant efforts have been focused on
the establishment of conditions for in vitro chili pepper plant
regeneration and genetic transformation. Several groups have
described important advances in the development of organogenic
and embryogenic systems for plant regeneration. It is a matter of
time before optimization of these systems will allow chili pepper to
reach the comparative efficiency values achieved for other
important crops. This also will be true for the development and
optimization of genetic transformation methods.
726 OCHOA-ALEJO AND RAMIREZ-MALAGON
Perhaps we have not been able to identify the adequate
environmental and chemical factors that mimic the conditions
necessary for inducing morphogenic events in vitro that occur
naturally in chili pepper. What are the precise levels and
combinations of growth regulators necessary for in vitro induction
of embryos or shoot buds, and their further elongation in chili
pepper cells and tissues? It is an open question, but some
qualitative and quantitative analysis of the endogenous growth
regulators produced during in situ organogenesis or zygotic embryo
formation might reveal interesting data; for example, isopentenyl
adenosine, zeatin, zeatin riboside, and the N9-glucosides of zeatin
and isopentenyl adenine have been shown to be the dominant
endogenous cytokinins in rapidly expanding leaves of chili pepper
(Nielsen and Ulvskov, 1992; Ulvskov et al., 1992). These growth
regulators have not been extensively used for in vitro chili pepper
plant regeneration. In vitro shoot bud formation and elongation have
been found to be independent of exogenous growth regulators in
some regenerative systems (Ezura et al., 1993; RamõÂrez-MalagoÂn
and Ochoa-Alejo, 1996); these systems could be used to investigate
the type of endogenous growth regulators involved in the
organogenic processes that lead to plant formation. The utilization
of growth inhibitors or growth regulators like ABA, silver nitrate,
2,3,5-triiodobenzoic acid, phenylacetic acid, or brassinosteroids to
modulate in vitro developmental processes, has been shown to
improve chili pepper plant regeneration in certain cases. The
genetic background of the chili pepper plants used as explant
sources is obviously another important factor to be considered for
the optimization of regeneration systems.
Chili pepper transformation methods will be of enormous value
not only as a tool for genetic improvement, but also as an approach
for genetic manipulation of some biosynthetic pathways that operate
in Capsicum species fruits (capsaicinoid and carotenoid syntheses).
It is necessary to test the best cultivar±Agrobacterium tumefaciens
interactions in order to increase the efficiency of the reported
systems. It is also advisable to explore some other transformation
techniques (A. rhizogenes infection, silicon fibers, etc.) that might
help to overcome the low efficiencies reported to date.
Protection of chili peppers against diseases caused by viruses,
bacteria, and fungi is of major importance. The first transgenic chili
pepper plants exhibiting cucumber mosaic virus resistance have
been obtained and it is expected that more examples of this kind of
protection will be produced in the near future. No transgenic chili
pepper plants resistant to bacteria, fungi, and pests have been
described as yet. Some genes involved in protection against
microorganisms have been isolated from Capsicum; among these,
defensin genes, a family of small proteins (5 kDa) related to insect
defensins with activity against fungi, have been reported (Meyer
et al., 1997; Houlne et al., 1998; Aluru et al., 1999). These genes
and some others (glucanase and chitinase) might be useful to confer
resistance against fungal diseases.
Regarding manipulation of the capsaicinoid pathway, recently a
number of cDNAs that are differentially expressed in placenta
tissues of hot fruits (C. chinense) have been isolated (Curry et al.,
1999). These cDNAs encode for PAL, CA4H, and OMT (Curry et
al., 1998), and also a clone for an aminotransferase (Aluru et al.,
1998b), predicted to synthesize vanillylamine, and another one for a
3-keto-acyl acyl carrier protein synthase (Aluru et al., 1998a),
predicted to elongate branched-chain fatty acids, were identified.
These genes could be engineered to transform cells or tissues in
order to increase (by overexpression of the genes) or decrease
(antisense constructs) the levels of capsaicinoids in cells and
transformed plants. This approach also might be useful for the
manipulation of carotenoid biosynthesis, since significant progress
has been made in recent years to characterize genes involved in
carotenoid accumulation (DerueÁre et al., 1994a, b; Kuntz et al.,
1998).
Acknowledgments
We would like to thank Conacyt, Mexico (project 3017-N9306), Concyteg
(projects 98-03-01-048 and 99-03-201-037), and SIHGO-Conacyt (projects
ALIM/16 and 19990201008) for financial support.
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