Direct Somatic Embryogenesis

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10
Direct Somatic Embryogenesis in Coffea canephora
Francisco Quiroz-Figueroa, Miriam Monforte-Gonzlez,
Rosa M. Galaz-valos, and Victor M. Loyola-Vargas
Summary
Somatic embryogenesis (SE) provides a useful model to study embryo development in
plants. In contrast to zygotic embryogenesis, SE can easily be observed, the culture conditions
can be controlled, and large quantities of embryos can be easily obtained. In Coffea spp several
model systems have been reported for in vitro SE induction. SE for coffee was first reported in
Coffea canephora. Several systems have been developed since then, including SE from callus
cultures derived from leaf explants; a two-phase experimental protocol for SE from leaves of
Coffea arabica; and from leaf explants of Arabusta or C. arabica using a medium with cytokinins. Here we report a protocol using young leaves from in vitro seedling pre-conditioned with
growth regulators. This is a simplified method to obtain a faster and more efficient protocol to
produce direct somatic embryos in C. canephora.
Key Words: Coffee; Coffea spp; somatic embryogenesis; direct; leaves.

1. Introduction
The capacity to produce morphologically well-formed and normally developed embryos from somatic cells resided uniquely within the plant kingdom
(1) up to the present time. In addition, the development of somatic and zygotic
embryos is highly similar (1,2). Therefore, somatic embryogenesis (SE) provides a useful model to study embryo development in plants. In contrast to
zygotic embryogenesis, SE can easily be observed, the culture conditions can
be controlled, and large quantities of embryos can be easily obtained (3).
Two types of SE are recognized. The term direct is applied to explants
that undergo a minimum of proliferation before forming somatic embryos,
whereas indirect refers to explants that undergo an extensive proliferation
before the development of somatic embryos (4). It has been suggested that in
From: Methods in Molecular Biology, vol. 318: Plant Cell Culture Protocols, Second Edition
Edited by: V. M. Loyola-Vargas and F. Vzquez-Flota Humana Press Inc., Totowa, NJ

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Quiroz-Figueroa et al.

direct embryogenesis, embryogenic cells are present and simply require favorable conditions for embryo development, whereas indirect embryogenesis
requires the re-determination of differentiated cells (5). However, the terms
direct and indirect are still useful in describing cases in which either very
little or a great deal of explant proliferation precedes embryogenesis although
not necessarily indicating fundamental differences in the cells involved (6).
In Coffea spp several model systems have been reported for in vitro SE
induction. SE In Coffea canephora was first reported by Staritsky (7), who
described the induction of callus tissue from orthotropic internodes. Subsequently, Herman and Haas (8) obtained SE for Coffea arabica from callus cultures derived from leaf explants. Sndahl and Sharp (9,10) developed a
two-phase experimental protocol for SE from leaves of C. arabica. Dublin
(11) reported SE from leaf explants of Arabusta using a medium with cytokinins (6-Benzylaminopurine [BA] and kinetin). Whereas, that of Yasuda et al.
(12) induced embryogenic calli and somatic embryos from C. arabica leaf
explants using only BA.
The physiological and morphological maturity of the tissue, as well the components of the culture medium, determine the time of occurrence and types of
possible responses in coffee SE (13). Using young leaves from in vitro seedling pre-conditioned with growth regulators, we (14) have shorted the embryogenic response time in direct SE of Coffea spp and preconditioning with growth
regulators was critical for embryogenic response, because, no embryos were
observed when explants came from plants cultured in the absence of growth
regulators. The protocol presented here is a simplified method to obtain a faster
and more efficient protocol to produce direct somatic embryos in C. canephora.
2. Materials
1. Flow cabinet, dry-hot sterilizer, scissors, Petri plates, scalpel, forceps, sterile cotton, 70% (v/v) ethanol.
2. Seeds of C. canephora.
3. Sterile distilled water by autoclaving (121C during 20 min), commercial bleach.
4. MS basal medium is based on formula of Murashige and Skoog (15). The vitamins, amino acids, and plant growth regulators are prepared in independence
stocks solutions. The medium is semi-solidified by addition of 0.25% (w/v)
gelrite as gelling agent.

3. Methods
3.1. In Vitro Plantlets
1. Seeds are washed and imbibed for 2448 h in sterile distilled water (see Note 1).
2. Seeds are disinfected with commercial bleach (1.25% v/v free sodium hypochlorite) for 20 min with agitation. Remove the solution and rinse the seed three times
with sterile water.

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3. Isolate the zygotic embryos (see Note 2) using a scalpel and forceps, the embryos
are puts onto zygotic germination medium (see Table 1). Ten embryos are placed
in each Magenta plastic box (see Note 3) containing 40 mL of medium and cultivated at 25 2C under a 16/8-h (light/dark) photoperiod. Subculture with fresh
medium is doing every 60 d. The boxes are sealing with strips of cling film.
4. The zygotic plantlets are growing in Magenta boxes containing 40 mL of zygotic
plantlet medium (see Table 1) and cultured at 25 2C under a 16/8-h (light/
darkness) photoperiod and transferred to fresh medium every 60 d. One plantlet
is put for each container.
5. When the plantlet has six pairs of leaves (size) it can be used as an explant.

3.2. Direct Somatic Embryogenesis

1. Plantlet leaves are cut avoiding mid-vein and edges (see Fig. 1A).
2. The foliar explants (ca. 0.25 cm2) are placed on direct embryogenesis induction
medium (Table 1) in glass bottle, aluminum sheet or plastic are used as tap (see
Note 4) and sealed with cling film strip (see Fig. 1B). Those bottles are incubated
under photoperiod (16 h/8 h light/darkness) at 25 2C.
3. Somatic embryo can be observed after 3 wk of induction, however, on wk 6 the
edge explant is cover with somatic embryos (see Fig. 1C,D).

3.3. Germination of Somatic Embryo

1. Cotiledonary somatic embryos are separate from explants using forceps and put
onto developmental medium (Table 1) for their germination, ensure that embryos are in good contact with the medium (see Fig. 1E and Note 5).
2. The subculture of embryos in germination fresh medium is done every 8 wk.
3. The plantlets obtained (see Fig. 1F) can be maintenanced in the same developmental medium.
4. Direct embryogenesis induction (see Notes 6 and 7) can be done following the
same protocols as in Subheading 3.1., step 5 to Subheadings 3.3., step 3 for
somatic plantlets.

4. Notes
1. The beans are imbibed in water with aim of to take off the parchment. The coat is
removed by manual manipulation.
2. The developmental stage of zygotic embryo affects response of germination.
Optima embryo stage is cotyledonary.
3. Each magenta box has a thread in border (see Fig. 1F, arrow) that service as filter
to gasses interchange.
4. When glass bottle is cover with aluminum the embryogenic yield is higher than
when use plastic tap.
5. After 5 wk somatic embryos have develop their radicular system (see Fig. 1E,
arrows) and 5 wk after can be seen the first pair leaves (see Fig. 1E, headarrows).
6. The position of leaves on the plant is very important, because the first two pairs
of leaves do show poor embryogenic response as compared with the mature

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0.1 (0.54)
0.5 (2.32)

0.5 (2.32)

1.12 (5)
0.5 (2.32)

0.1 (0.54)

30 (87.64)

30 (87.64)

30 (87.64)
30 (166.48)
0.1 (0.54)

25 (158)

25 (158)

2 (9.72)
2 (16.24)
4 (11.85)
100 (550)

25 (158)

1 (4.86)
1 (8.12)
10 (29.6)
100 (550)

0.125 (0.5)
3.100 (50)
6.83 (40)
0.05 (0.2)
4.3 (15)
21 (75.53)
27.9 (74.95)

0.830 (5)
0.025 (0.1)
0.250 (1)
6.200 (100.27)
22.300 (80.5)
0.025 (0.1)
8.100 (28)
27.950 (100)
37.230 (100)

1650 (20.61)
1900 (18.79)
440 (2.99)
170 (1.249)
370 (1.500)

4 (11.86)
100 (550)

0.830 (5)
0.025 (0.1)
0.250 (1)
6.200 (100.27)
22.300 (80.5)
0.025 (0.1)
8.100 (28)
27.950 (100)
37.230 (100)

0.830 (5)
0.025 (0.1)
0.250 (1)
6.200
(100.27)
22.300 (80.5)
0.025 (0.1)
8.100 (28)
27.950 (100)
37.230(100)

412.0 (5.15)
475.0 (4.7)
110.0 (0.748)
85.0 (0.624)
92.5 (0.375)

DEI

10 (29.6)
100 (550)
0.01 (0.041)

1650 (20.61)
1900 (18.79)
440 (2.99)
170 (1.249)
370 (1.500)

1650 (20.61)
1900 (18.79)
440 (2.99)
170 (1.249)
370 (1.500)

ZP

been adjusted.
Abbr:ZG, zygotic germination medium; ZP, zygotic plantlet medium; DEI, direct embryogenesis medium, D, developmental medium.

aThe pH of all medium are adjusted to 5.8 before autoclaving (20 min, 110C). Gelrite is used as gelling agent at 0.25% (w/v) and is added after the pH has

Vitamins -mg/L (M)Piridoxine HCl

Nicotinic acid
Thiamine HCl
Myo-inositol
Biotin
Amino acids -mg/L (M)Cysteine
Sugars g/L (M)Sucrose
Glucose
Growth regulator -mg/L (M)Naphthaleneacetic acid
6-Benzyl-aminopurine
Kinetin

Macro salts -mg/L (mM)NH4NO3

KNO3
CaCl22H2O
KH2PO4
MgSO4.7H2O
Micro Salts -mg/L (M)Kl
COCl26H2O
Na2MO42H2O
H3BO3
MnSO47H2O
CuSO45H2O
ZnSO47H2O
FeSO47H2O
Na2EDTA

ZG

Table 1
Composition of Mediums-MS Medium: Inorganic and Organic Salt (15),
Vitamins and Others Supplements

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Quiroz-Figueroa et al.

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Fig. 1. Direct embryogenesis system in Coffea spp. (A) Cut usual of explant leaf
avoiding midvein and edges. (B) Glass bottle with plastic tap where direct embryogenesis is induced. (C) Direct somatic embryos obtained after 6 wk embryogenesis
induction. (D) Close up of C, showing a cotyledonary somatic embryo. (E) Different
stages of embryo germination, arrows radicular system, head arrows first pair of leaves.
(F) Plantlet from somatic embryo after 25 wk. Arrow, thread of cotton.

leaves. Also, explants coming from the distal part of the leaf are less responsive
than those coming from the basal part of the leaf.

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Quiroz-Figueroa et al.

7. The preconditioning of plantlet for 2 wk in medium with 0.54 M

naphthaleneacetic acid and 2.33 M kinetin is critical for the induction of direct
somatic embryogenesis. Reduced embryogenic response is observed when the
plantlet is not preconditioning.

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