Second Report

Chemical Hybridizing Agents

Identification of Chemical Hybridization Agents (CHA) suitable for commercial
hybrid seed production in specific vegetable crops (enlisted as priority crops in
the PC-I) along with mode of application.

Research By

Muhammad Boota Sarwar

For

Ministry of Food and Agriculture

Facilitation Unit for Participatory Vegetable Seed & Nursery
Production Program
 Contents

Sr. Page
No. Title No.

1 Introduction 3

2 Chemicals for Restoration of Male Fertility in Male Sterile 6

Plants

3 Some Important Features of Vegetable Hybrid 9

Development Program

4 Role of Genetically Engineered System of Male Sterility 13

in Hybrid Production of Vegetables

5 Male Gametocide for Coriander 33

6 CUCUMBER: Sex Expression and Chemical Modifiers 44

2
 INTRODUCTION

Exploitation of heterosis or hybrid vigor to increase crop yields started

early in the twentieth century with maize. From inbreeding a number
of crop plants including maize, George H. Shull developed a
perspective on heterosis that he outlined in a 1908 publication
entitled “Composition of a Field of Maize.” He argued persuasively
that by inbreeding isolated homozygous lines, these lines could be
crossed to capitalize on heterosis. East (1908) discussed the danger
of inbreeding. Commercially feasible F1 maize hybrids were
developed following Jones’s 1918 proposition of the doubled cross.
Average yields increased twofold after double crosses replaced the
open-pollinate cultivars (OPCs) during 1935–1965 and increased
another twofold after single crosses replaced the double crosses
during 1966–1996 (Hallauer 1999).

The T cytoplasmic male sterility (CMS) had been used to produce

hybrid seeds during 1950–1970. Wide use of this single cytoplasm
resource resulted in the epidemic of Southern leaf blight in 1970
caused by a new virulent Race T of Helminthosporium maydis, and
an estimated 15% of the maize crop in the United States was
destroyed. Hybrid maize is now based on hand detasseling, which
avoids the use of CMS female parents. The success of hybrid maize
is due to the ability of major seed companies such as Pioneer Hi-Bred
International, Inc., to select for yield stability based on extensive
testing and the efficiency of seed production that insures profitability.
Now over 90% of maize in the United States is hybrid (Duvick 1999).

Hybrid rice was first commercialized in the early 1970s in China.

Unlike maize, rice is self-pollinated and hermaphroditic. Hybrid rice
breeding has been based on using CMS or environment-induced
genic male sterility (EGMS). A breeding system using three-lines
(CMS line, CMS maintainer, and CMS restorer) was established by
using a male sterile plant discovered from a wild rice species (Oryza
rufipogon Griff. Or O. f. spontanea) in 1970 by Prof. Longping Yuan
and his assistant (Li and Yuan 2000). A two-line hybrid rice system
using EGMS was established by using a photoperiod-sensitive genic
male sterility (PGMS) mutant discovered in 1973 by Mingsong Shi

3
from a japonica cultivar ‘Nongken 58’ (Shi 1981). Now over 50% of
rice in China is hybrid (Li and Yuan 2000).

Afterwards hybrid vegetables are also commercialized in US and

other countries and hybrid breeding programs have been established
in several Asian, South American and North American countries.

Hybrids provide many advantages in a crop production system. The

principal benefit is increased yield. In open-pollinating species, one of
the most often overlooked benefits is uniformity, an element which
has allowed for the rapid expansion of production in many crop plants
such as the vegetables. Additional benefits may include stress
tolerance and pest resistance and other performance characteristics.
Breeders of hybrid crops can react faster and with more options to
meet changing markets, customer needs, and production demands.
Other advantages of hybrids include the ability to combine useful
dominant genes available in different inbred lines, to optimize the
expression of genes in the heterozygous state, and to produce
unique traits. Yield increases accounted for about 92% of increased
cereal production in the developing world between 1961 and 1990.
The yield advantage of hybrids ranged from 15% (maize) to 50%
(sunflower), compared to non-hybrids (Duvick 1999). However, yield
growth rates are stagnating in some areas and, in a few cases,
falling. A slowdown in the rate of yield increase of major cereals
raises concern because increased yields are expected to be the
source of increased food production in the future (Reeves et al.
1999). Utilization of heterosis and the improvement of the efficiency
of hybrid breeding is one of the ways by which we can attempt to lift
the yield ceiling.

Producing F1 hybrids depends on the development of an efficient

system for hybridization. There are several approaches to production
of hybrids: CMS, EGMS including PGMS and thermo-sensitive genic
male sterility (TGMS), protogyny, self incompatibility, chemical
hybridization agent (CHA), and hand-emasculation systems. No
matter which system is used, the central task is to develop and
maintain two parents that have desirable traits. One serves as a
pollen receptor and the other as a pollen donor for hybrid seed
production. All desirable traits for hybrid seed production and

4
heterosis itself, which are not required in pure line breeding, are
difficult to measure, compared to other agronomic traits.

Genomics in vegetable crops has created a substantial information

base for their improvement. Marker-assisted selection (MAS), the first
benefit that breeders can obtain from genomics, has been receiving a
great deal of attention and will play an important role in hybrid
breeding. The objective of this paper is to discuss the development of
molecular marker strategies in breeding hybrid rice by drawing upon
information obtained from maize and other crops. Information on the
types of marker systems available and their relative merits is
included, as is the use of markers in (1) evaluation and
characterization of germplasm resources, (2) selection for different
types of traits, (3) gene introgression, (4) prediction of hybrid
performance, and (5) monitoring of seed quality in the seed
production process.

Induction of male sterility and restoration of fertility through use of

chemical hybridizing agents have paved the way for bypassing
cumbersome process of inserting cytoplasmic male sterility trait in
female lines and restorer gene in male lines through traditional
breeding techniques.

This second report covers some other aspects of chemical

hybridizing agents that were not taken up in first report hence will be
useful for the stakeholders opting for use of such chemicals in their
hybrid seed production programs.

5
Chemicals for Restoration of Male Fertility in Male Sterile Plants

The evaluation of histochemical localization of histones, DNA and

proteins in the anthers of male-fertile as well as cytoplasmic, genic
and chemically induced male-sterile plants of Allium cepa, Beta
vulgaris, Capsicum annum, Cucumis melo, Cucurbita maxima, Datura
alba, Ranunculus muricatus, Sesamum indicum, Solanum melongena
and Triticum aestivum revealed that the anthers of male-sterile plants
were markedly deficient in these substances. It is concluded from
these observations that this deficiency, acting as limiting factor for
pollen development in all probability, causes the inhibition of vascular
supply in sterile anthers.

The evaluation of distribution patterns of histones, DNA and total

proteins in the anthers of various stages of development in male
fertile (MF), cytoplasmic male sterile (CMS), genic male sterile (GMS)
and induced male sterile (IMS) plants can be carried out using
standard protocols of Alkaline Fast Green Test for histones, Feulgen
Reaction for DNA and Ninhydrin-Schiff’s Reaction for total proteins.

Anther development has been divided into six stages. The

sporogenous cells are initially evident and the last stage is near
anthesis when pollen grains become engorged with reserves.

A: Sporogenous tissue stage: In most parts of an anther of MF plants,

the concentration of histones, DNA and total proteins at this stage is
low. However in tapetal and sporogenous cells it is high and
moderate respectively. On the other hand, the concentration of these
substances in various parts of an anther including tapetal and
sporogenous cells is low in MS plants.

B. Meiosis I&II stage: During meiotic division, there is a considerable

increase in the concentration of these substances in the anther of MF
plants and the maximum being in the tapetal cells. On the contrary,
the anthers of MS plants fail to exhibit any appreciable increase as
compared to their fertile counterparts.

C. Tetrad stage: At this stage, in the anthers of MF plants tapetal

cells show a slight decline in the protein contents. However the

6
concentration of histones and DNA in these cells reach the maximum.
There is a reduction in the amount of these substances in the young
microspores enclosed in the callose wall. At this stage, the
concentrations remain low in all the parts of an anther including
tapetum and microspores in sterile plants.

D. Vacuolate microspore stage: During this period the microspores

are released from the common callose wall with rapid growth and
vacuolation and the concentration of histones, DNA and total proteins
in the microspores of MF plants decline spectacularly for a while, but
with age, these substances increase gradually. Intense localization of
histones, DNA and total proteins is exhibited by mature microspores.
Tapetal cells and other parts of an anther of MF plants show high and
moderate or low intensity of various reactions respectively. There is
no marked change in the concentration of these substances in the
anthers of various MS plants as it remains either low or moderate in
different parts including tapetum and microspores.

E. Vacuolate pollen stage: By this stage the tapetal cells in the

anthers of MF plants almost break down and their remaining
fragments show only low concentration of histones, DNA and total
proteins. However the spore histones, DNA and proteins increase
considerably. The other parts of the anther show low concentration
except endothecium which shows intense reactions. On the other
hand, the intensity of various reactions fails to accelerate in the
anthers of MS plants. The malformed tapeta and non-viable pollen
grains exhibit lower amounts of histones, DNA and total proteins.
Similarly, in other parts the concentrations are far below.

F. Engorged pollen stage: When pollen grains become engorged with

reserves in the anthers of MF plants, the concentration of histones,
DNA and total proteins reach the maximum. The non-viable pollen
grains in MS anthers, on the contrary, exhibit the same reaction as
shown during the preceding stage. The hypertrophied tapetal cells or
pseudo-periplasmodial also show poor reactions.

Various studies have indicated that application of specific histones

deficient in male sterile plants may lead to restoration of male fertility
in such plants; and so there is no need of developing maintainer for
the female parent line of the hybrid.

7
References:

1. Chauhan, S. V. S; 1976: Morphological and histochemical

studies in natural and chemically induced male-sterile plant.
Current Science 45(7): page 274-275

2. Chauhan, S. V. S. and Toshiro Kinoshita; 1979: Histochemical

localization of Histones, DNA and Proteins in the Anthers of
Male-fertile and Male-sterile Plants. Japan Journal of Breeding
29(4): pages 287-293.

3. Chauhan, S. V. S; R. K. Dhingra and Toshiro Kinoshita; 1982:

Effect of Phytogametocidal Compouds on the Development of
Endothecium in Some Plants. Japan Journal of Breeding 32(2):
pages 139-145.

8
 Some Important Features of Vegetable Hybrid Development
Program

A. Hybrid Prediction

The process of plant breeding has developed through several key

phases, including unconscious selection in Neolithic times, empirical
art during the development and expansion of agriculture, and a
predictive science-based approach practiced today. In general,
prediction in plant breeding started from the evaluation of progeny
performance. Pedigree methods and use of statistical tools in
assessing progeny performance brought about a way for plant
breeders to quantitatively separate the heritable portion of variation
from the non-heritable and thus make parental choices based on
heritability (Goldman 2000). As a result, exploitation of additive
genetic variance during the inbreeding process, and dominance
variance during the test-crossing phase when assessing performance
across multiple testers, has become a standard for cross-pollinated
crops. There are several breeding practices that also make plant
breeding more predictive. The breeding procedure known as
backcrossing makes the progeny highly predictive. Identification of
general and specific combining ability in vegetables helps identify
superior inbred lines that can be used more efficiently in breeding
new inbreds and hybrids. Wide area testing of hybrids makes it
possible to develop widely adapted hybrids and improves the
efficiency of vegetable breeding operations.

Hybrid vegetable breeding has a very different story. Breeders of self-

pollinating crops have been highly successful in breeding inbred
cultivars. Although inbred breeding procedures were established and
used for many years, hybrid vegetable breeding attempted a
completely different approach and the hybrid breeding procedures
established for open-pollinated crops were suitable for vegetables.
Vegetable breeders have been searching for more reliable prediction
methods in their breeding programs.

High performance of hybrid plants results from the complementary

action of both parents. Thus, parents with excellent performance per
se may not produce desirable hybrids; superior hybrids may come
from low-yielding parental lines. The evidence from maize indicates

9
that inbred grain yield is not highly correlated with hybrid grain yield
(Hallauer and Miranda 1988). Correlations between midparent values
and hybrid means for the 91 crosses studied by Dudley et al. (1992)
ranged from 0.46 for ear height to 0.71 for plant height. For grain
yield, the correlation was 0.56. These correlations are too small to be
of practical use in a breeding program. Despite their low values, the
inbred-hybrid yield correlations were positive. They indicated a
tendency for high-yielding inbreds to produce high-yielding hybrids.
For a specific breeding program, however, the tendency does not
make any kind of prediction meaningful.

The second reason for the unpredictability is the lack of a full

understanding of the genetic basis of heterosis, which affects all
aspects of hybrid performance. As noted in the published literature,
hybrid performance is positively related to the genetic distance
between the parental lines when comparison is based on crosses
derived from the intra-heterotic group. This “prediction” does not
work, however, when comparison is based on the crosses derived
from parental lines belonging to different heterotic groups, ecotypes,
or subspecies. Although many hypotheses, such as physiological
complementary, over-dominance, dominance, and partial dominance,
have been proposed as the genetic explanation of heterosis,
“isolated” research conclusions cannot be extended to other
populations from the same varietal group, let alone the hybrids
derived from very different crosses or species. As a result, limited and
less-reliable information often provides contradictory results. As will
be discussed later, prediction of hybrid performance depends on a
thorough understanding of heterosis from different aspects, including
genetics, physiology, and developmental biology.

B. Selection for Hybrid Performance

Hybrid performance depends on genes, and their interactions and

combinations. Selection for hybrid performance in breeding programs
is based on test-crossing and progeny testing. That is, we breed
hybrids through selection of parental lines with desirable agronomic
traits. To associate the parental phenotype with hybrid performance,
breeders have to cross their candidate breeding lines with several
testers, and from the hybrid progeny, determine if the candidates
contain the genes required for hybrids and whether the parental

10
combinations produce useful hybrids. This indirect selection, based
on test-crossing and progeny testing, is time-consuming and very
expensive. Furthermore, the association between the parental line
and hybrid from one cross cannot be used to make a prediction about
other associations.

A cross of two extremely low-yielding inbreds can give a hybrid with

good mid-parent or high-parent heterosis but poor performance,
whereas a cross of two high-yielding inbreds might exhibit less mid-
parent or high-parent heterosis but nevertheless produce a hybrid
with good performance. High-yielding hybrids owe their yield not only
to heterosis but also to other heritable factors that are not necessarily
influenced by heterosis. For effective selection, one needs to know
the relative importance of each genetic contribution—of heterosis and
non-heterosis—in individual hybrids (Duvick 1999). Furthermore,
when examining yield trends in a time series of successively released
hybrids, breeders need to know what portion of the genetic yield
gains (if gains are made) is due to increase in heterosis, and what
portion to increases in non-heterosis (Duvick 1999).

C. Seed Production and Commercialization

In cultivar breeding, once an outstanding line is identified to be

superior to the commercial control, it can be registered as a new
commercial cultivar for production. In hybrid breeding, however, a
superior hybrid requires not only a high yield potential and desirable
agronomic traits such as disease resistance and good quality, but
also an economically viable seed production and maternal production
system for each specific hybrid. Simply to exhibit good hybrid
performance is insufficient. To be commercially viable, potential lines
must produce excellent hybrids and have many traits desirable for
seed production, such as high out-crossing rate and good flowering
habits. This allows the cost of seed production to be low enough so
that the seed producer benefits from the production and sale of hybrid
seed.

Requirements for activities such as roguing, emasculation, pollination

assistance, and other special management systems for seed
production and parent multiplication can affect the cost and quality of
the final product. This is very important to crops such as vegetables

11
where reducing seed cost becomes critical for successful
commercialization. Thus, a good seed production system will
determine the cost of seed production and therefore the final
commercialization of the hybrid.

D. Seed Production

In hybrids, commercial produce is produced from hybrid plants, but

hybrid seed is produced from inbred parents. The agronomic practice
used for the two production systems could be very different. For
example, hybrid cauliflower may have a very different nitrogen
response than the parental lines and thus different kinds of nitrogen
management are required, depending on the environments in which
the hybrid is growing. Low potassium can be a problem for hybrid
carrot in many areas, although this element may be sufficient enough
for inbred lines (Yuan and Chen 1988).

Modern vegetable hybrids differ from open-pollinated and earlier

hybrids primarily in their response to stress. New hybrids have
improved water stress performance, are much less prone to flowering
delay, have significantly lower respiration rates during flowering, have
longer periods of fruiting, and are higher-yielding under both low- and
high-input environments (Tomes 1998). Severe droughts in the
American Cornbelt during 1934 and 1936 resulted in poor maize
crops; however, hybrids often out-performed their open-pollinated
counterparts under these conditions (Goldman 1999). This is due to
the strong root system and lodging resistance of hybrids. These
characteristics helped hybrid maize quickly replace OPCs.

In conclusion, hybrid breeding consists of all the components of

inbred line breeding plus the additional complexity of recombining
desired traits and efficient seed production capacity.

12
Role of Genetically Engineered System of Male Sterility in Hybrid
Production of Vegetables

1. Introduction

Many of our modern-day vegetables are so-called "hybrids". These

originate from parent plants that exhibit significant genetic
differences. The aim of such hybrid-cultivation measures is to avoid
the familiar negative effects of "in-breeding" (the cross-breeding of
closely related organisms). Hybrids produce higher yields and are
less susceptible to disease and environmental stress than non-hybrid
varieties. A technical implication is encountered in the cultivation of
hybrids is the fact that many plants bear both male and female
reproductive organs which exhibit a tendency to auto gamy. In the
production of hybrid seeds, therefore, efforts are made to use purely
female – or more precisely "male-sterile" – plants for cultivation
purposes. The pollen is transferred to the female organs manually.

Male-sterile plants have been developed both by conventional

cultivation procedures as well as by genetic modification. The manual
removal of flowers or portions thereof and manual fertilization of
female flowers by breeders are therefore no longer necessary. The
genetic modification technique involves the introduction of genes that
are active only in the male reproductive organs and that prevent the
emergence of male flowers (or parts thereof). This results in purely
female ("male-sterile") plants that can then be used for cultivation
purposes and for the production of hybrid seeds. The production of
hybrid seeds, the male-sterile plants of one parent is planted
alongside unmodified plants of the other. The seeds developing in the
male-sterile plants therefore can have developed only by crossing of
two intended parents. An essential feature here is that hybrid plants
those grow from the hybrid seeds go on to form pollen themselves to
the extent that the seed of hybrid plants indeed constitutes the crop to
be harvested (leaves, stems, roots, etc.), the restoration of the pollen
fertility is not absolutely necessary (e.g. sugar beet, animal-feed
grasses).

Male sterile plants don't produce pollen. That makes it easier to breed
improved hybrids that yield and perform better, and to produce hybrid
seed more economically. Sterility also helps ease concerns that

13
genetically modified crops will spread their enhanced genetic
characteristics, such as herbicide resistance, to wild plants. Scientists
have long tried to develop male sterile plants through a variety of
techniques, from tapping natural mutations to inducing sterility
through radiation and chemical methods. And this characteristic can
be unstable - some types of sterile plants can revert to fertility, which
causes problems for growers.

Sally Mackenzie (2003) a plant geneticist at University of Nebraska-

Lincoln thinks she's found a genetic key to sterility. It promises to
work for a wide range of crops and horticultural products. Scientists
have long known that, in nature, changes in the cells' mitochondrial
DNA cause the sterility mutation. Mackenzie and her team followed
that genetic trail to recreate the mutation in the lab. They found a
gene in the cell's nucleus that controls genetic changes in the
mitochondria, which are the cell's energy producers and also contain
DNA. By inserting foreign DNA into this gene, they turned it off,
observed changes in the mitochondria and pinpointed which change
actually triggers male sterility. They tracked down the gene in
Arabidopsis, which they use as a model plant because its genetic
code is known, but their findings have broad potential. Because all
plants carry this gene that affects the mitochondria, these NU Institute
of Agriculture and Natural Resources scientists can use their
technique to trigger male sterility in others.

Mackenzie now is growing transgenic tomatoes to search for

additional male sterile. "The really cool thing about this is that once I
induce a male sterile, it's stable," Mackenzie said. After removing the
foreign DNA that caused the original genetic change, the plant
remains sterile. But by eliminating the foreign DNA, the plant is no
longer considered transgenic. "That's the beauty of it," she says.
"Nobody has to have any qualms about using GMO technology."
Agriculture would benefit if this method of inducing male sterility
proves successful. Mackenzie wants consumer to benefit, too. She's
applying her findings to develop a sterile, seedless green bean that
vegetable buyers should appreciate. Without seeds, the pod is tender
and more easily digestible. Sterility also tricks the plant into producing
three times the number of pods, increasing yields. While genetically
modified crops have helped to reduce the need for agricultural
pesticides, consumers have yet to benefit directly, she said. "If we hit

14
the market with our male sterile and, at the same time, come up with
our new seedless bean," said Mackenzie, "I think the consumer is
going to say, 'this is nice engineering'.” Researchers hope to work
with an agribusiness to make sterile males commercially available in
a variety of vegetable crops.

2. Manifestations of Male Sterility

a. Absence or malformation of male organs (stamens) in bisexual

plants or no male flowers in dioecious plants. b. Failure to develop
normal microsporogenous tissue- anther. c. Abnormal
microsporogenesis--deformed or in viable pollen. d. Abnormal pollen
maturation; inability to germinate on compatible stigmata. e. Non
dehiscent anthers but viable pollen,- sporophytic control. f. Barriers
other than incompatibility preventing pollen from reaching ovule.

3. Kinds of Male Sterility

Genic male sterility:

It is also known as nuclear male sterility as controlled by nuclear
genes whose action and expression are not influenced by the
plasmon or cytoplasmic genes. Therefore, inheritance pattern and
expression exhibit no reciprocal differences, low environmental
impact, or nominal genomic influence. Hence, sterility is stable,
reliable, and replicable. Predominantly, single recessive Mendelian
genes control this type of sterility, through two to three recessive
genes or control by one to two dominant genes (Kaul, 1986). Most of
the naturally occurring or induced male sterile mutants are recessive
in nature with few exceptions in cole vegetables (e.g. cabbage and
broccoli) and genetically transformed male sterile lines.

Certain mutants, which although produce functional pollen, pollen fail

to self fertilize, either due to non-dehiscence of pollen or their special
flower morphology e.g. positional sterility in tomato (Atanassova,
1999) and functional male sterility in eggplant (Phatak and Jaworski,
l989). The occurrence of predominantly recessive male sterility
clearly indicates that gms is the result of mutation in any gene(s)
controlling microsporogenesis (pollen development), stamen
development or microgatnetogenesis (male gamete development
process).

15
4. Environmental sensitive genic male sterile (EGMS) Line

Certain GMS lines are conditional mutants, meaning thereby in a

particular environment male sterile mutant plants turn into male
fertile. Alter determination of critical environment (usually temperature
or photoperiod) for sterility and fertility expression, such GMS
mutants are classified under environmental sensitive genic male
sterile (EGMS) lines. In vegetable crops, mostly temperature
sensitive genic male sterile lines have been reported in cabbage,
brussel sprout, broccoli and tomato (Kumar et al. 2000). From
practical application view point, it is necessary to identify critical
temperature or photoperiod for the fertility/ sterility expression in
temperature and photoperiod sensitive genetic male sterility,
respectively (Table-1).

Table 1: Environmental sensitive male sterility mutants

TGMS-Thermo sensitive genic male sterility, TCMS- Thermo sensitive

cytoplasmic male sterility, PGMS- Photo sensitive genic male sterility

Like the spontaneously arisen genic male sterility mutations, majority

of the induced male sterile described by Kumar et al., 2000 (Table 2).

16
Although it appears possible to induce male sterility in any vegetable
crop, being easier in diploids than in polyploids because of the
genome multiplication and high gene interactions, induction following
mutagenesis has been very limited and difficult, the main reason
being the low frequency of male sterile, absence of marker genes,
gradual diminution, and ultimate elimination of male sterile due to
self-sterility.

Table 2: Cytoplasmic- nuclear male sterility in selected vegetable

species

* exploited at commercial scale

A perusal of Table -3 reveals that both physical and chemical

mutagens, singly or in combination, induce male sterility. Sterility,
irrespective of the ploidy level, in almost all these is conditioned by
single recessive ms genes. Although y-rays and EMS treatments
have induced male sterility in maximum species, this does not reflect
the effectiveness, efficacy, or efficiency of these two mutagens, but
indicates their wide usage. In fact, the use of other mutagens for
inducing male sterility has been limited and many mutagens have
been tested neither on the same species nor on any single species. It

17
is desirable to determine the male sterility induction potential of
different doses of various potentials/chemical mutagens.

Utilization of GMS

Since GMS is maintained through backcrossing in hybrid seed

production field. 50% male fertile segregants (MSMS) need to be
identified and removed before they shed pollen.

Table-3 Nuclear male sterility in selected vegetable species Source

Kumar (2002) * exploited at commercial scale

18
In some gms lines, ms genes are tightly linked with the recessive
phenotypic marker genes. Such marker genes, especially which
expresses at seedling stage are good proposition for the identification
of sterile/fertile plants at seedling stage. Hybrid seed production using
EGMS line is more attractive because of the ease in seed
multiplication of male sterile line. Seeds of EGMS line can be
multiplied in an environment where it expresses male fertility trait
while hybrid seeds can be produced in other environment, where it
expresses male sterility. Because of more tedious maintenance
process and non-availability of suitable marker gene among the
vegetable crops utilization of GMS is restricted only in few vegetables
(Table-3). The identification of fertilizing cytoplasm for specific
nuclear male sterile gene, is an interesting research area, which upon
success, may provide opportunity for most efficient utilization of gms
lines, like cms line.

Cytoplasmic male sterility

Cytoplasmic male sterility is controlled solely by the specific

cytoplasm genes (S-genes) whose action is not influenced by nuclear
or other genes. Therefore, as long as the cytoplasm of an individual
has these specific genes, it is male sterile cytoplasm (S). In the
absence of these genes, the cytoplasm is normal (N) or fertile (F).
Therefore, irrespective of the nuclear genes, plants having the S-
cytoplasm are male sterile. True cytoplasmic male sterile ideally
should remain uninfluenced by nuclear genes; but such stable and
true cytoplasmic male sterile are unknown as fertility restorer genes,
and maintainer nuclear genes have been detected in all plants
labeled CMS (Kaul, 1986).

Cytoplasmic Sterility

Compared to the large number of plants in which genic male sterility

has been induced by mutagenic treatments, the number of plants in
which genic cytoplasmic male sterility has been induced is
exceedingly low. This is because, whereas predominantly the
mutation of a single specific nuclear gene leads to genic male
sterility, for gene-cytoplasmic male sterility the induction of two
specific and either simultaneous or successive gene mutations, one
at the nuclear and the other at the cytoplasmic level, are required.

19
The probability of the occurrence of two such simultaneous mutations
in specific genes is exceedingly low. In the species where gene-
cytoplasmic male sterility has been induced , the nuclear fr genes
were already present in the N-cytoplasm having the C gene.
Therefore, in all cases, a cytoplasmic gene mutation has led to male
sterility. Among all the chemical mutagens used, ethidium bromide
has been successful in inducing the cytoplasmic mutation.

Utilization of CMS

The CMS system is the most commonly utilized male sterility to

produce commercial hybrid seeds of several vegetables (Table-3) .
The cms based hybrid development is often term as three lines
method of hybrid breeding involving A line (male sterile S-rfrf), B line
(maintainer: N-rfrf) and C or R line (restorer: S. or N-RfRf). As
mentioned cms line without restorer male parent can not be utilized in
fruit producing vegetables (e.g. chilli), but it can be utilized in
vegetables where vegetative part is of economic value (e.g. onion,
Cole crops, radish, leafy vegetables etc). Besides several factor
vulnerability of sterile cytoplasm to specific diseases is a major risk).

Mechanisms of male sterility (GMS and CMS)

In both GMS and CMS systems, male sterility is the consequence of

breakdown of tightly regulated pollen development and fertilization
processes at any of the pre- or pro-meiotic stages i.e. during meiotic
process during the formation of tetrad, during the release of tetrad, at
the vacuolate microspore stage, at pollen dehiscence stage.
Expression of male sterility trait is associated with a large number of
morphological, physiological histological, biochemical and molecular
changes of microsporogenesis and microgametogenesis.

Role of Tapetum

Tapetum cells are innermost layer of the anther wall that surrounds
anther locule possessing sporogenous cells (developing pollen).
These cells are involved in the transmission of nutrients/energy to
sporogenous cells and associated with synthesis of callase enzyme.
The most striking histological feature associated with the majority of
male sterile plants (GMS and CMS) is persistence or premature

20
breakdown of tapetum. Aberration in tapetum development leads to
the failure of tapetum function, consequently failure of pollen
development and, therefore expression of male sterility. Ever since
the first report on abnormal developments of tapetum in male sterile
plant, there is growing experimental evidence in favors of strong
association between abnormal tapetum behavior and nuclear or
Cytoplasmic male sterile plants. It has been analyzed constituents of
C14 material, in male sterile microspores (pollen) and observed very
less C14 material, although there was normal supply of this material
in the tapetum. Therefore, they speculated disruption in the transfer
of C l4 material from tapetum to the developing microspores.
Behavior of some genetically transformed male sterile plants supports
failure of tapetum development and its function leading to male
sterility. In fact, knowledge on importance of tapetum has actually
been utilized to develop gene construct that selectively destroys the
tapetum and thereby induces male sterility in transformed plants.
Several other experimental evidences see are also discussed under
specific vegetables at appropriate places. Albeit alterations its
tapetum form and function are considered as the primary causes of
male sterility, there are male sterile lines in which both tapetum form
and function are normal (Kaul, 1989).

Role of Callase

Callase is an enzyme required for breakdown of the callose that

surrounds the PMCs. Thus helps in release of microspores (pollen)
from tetrad after meiosis. Early or delayed callase activities have
been found to be associated with male sterility. Mistiming of callase
activity led to the pre mature or delay release of meiocytes and
microspores, resulting in male sterility.

Role of Esterase

Esterase enzymes are believed to play role in the hydrolyses of

sporopollenin, the polymer required for pollen formation. Decreased
activity of esterase in male sterile plants has been observed in
petunia. Hence it has been proposed that decreased activity of
esterase has adverse effect on pollen development (Sawhney, 1997)

21
Role of Plant Growth Regulators (PGRs)

Endogenous PGRs play very important role in stamen and pollen

development. Male sterility has been reported to be associated with
change in a number of PGRs rather than any specific substance and
perhaps it is the altered balance of PGRs that effects the pollen
development process. Reduced level of Abscissic acid associate with
seeds of GMS and CMS plants indicates that both kind of male
sterility system probably involve some common pathways. On one
hand exogenous supply of reduced substances in several male sterile
has been found to restore fertility(tomato), on the other , in several
cases expected fertility restoration could not be obtained (Sawhney,
1997)

Approaches for Development of Male Sterility

The specific mechanisms causing male sterility in plants vary from

species to species and are subject to influence by environment, and
nuclear and cytoplasmic genes. Male sterility may be permanent
(heritable) or transient (CHAs). It can be induced by anther culture
(Kaul, 1986), somatic cell culture (Mariana et al. 1990), and somatic
hybridization. However, the most significant development is the
possibility of engineering male sterility by inserting cloned gene
sequences which can disrupt any or more than one step during
microsporogenesis. Commercial exploitation of hybrid vigour in
hermaphrodite crops is facilitated by the availability of CMS lines to
affect pollination control. As the CMS genotype results from
alloplasmic substitutions or by harnessing the mitochondrial
mutations, they are generally associated with yield penalty and
imperfect fertility restoration in the hybrids. These imperfections in
CMS-fertility restorer systems have stimulated considerable research
effort to engineer new and perhaps more efficient male sterility
systems (Kaul 1998; Perez-Prat and van Lookeren Campagne,
2002). Most of these efforts take advantage of the fact that
microsporogenesis is a complex developmental process sensitive to
mutations. Disruption of any of the steps of microsporogenesis or its
premature termination results in male sterility. Male sterile plants
have been produced by aberrant spatial or temporal expression of
degrading enzymes or through inhibition of specific enzymes via
antisense strategy. In the present write up an attempt has been made

22
to review current realities and emerging possibilities in the arena of
genetically engineered male sterility in crop plants.

A. Cell Cytotoxicity

1. Dominant Nuclear Male Sterility (Barnase-Barstar System)

Intensive studies revealed that the TA29 gene 5 region programmed

the expression of the RNase] and barnase gene specifically to anther
tapetal cells, which caused selective ablation of the tapetal cell layer
that surrounds the pollen sac, presumably by hydrolyzing the tapetal
cells. This disrupted the normal pollen formation and caused male
sterility. Of these genes, barnase seemed more effective than
RNase-T1 as only one copy of TA29-barnase was required to
produce male sterile plants, where, in contrast, at least four copies of
the TA29-RNaseTl gene were required for the same level of male
sterility expression. Male sterile flowers showed noticeable reduction
in length of stamen filament, petal size, reduction in bud diameter, ad
tapetal cell content. Sterile anthers contained empty exiles.

To restore fertility, the barnase-specific RNase inhibitor, barstar, was

used (Mariana et al. 1992). Upon crossing the genetically engineered
male sterile plants with transgenic fertile plants carrying TA29-barstar
gene, the F1 progeny showed co expression of both genes in the
anthers of the male fertile plant. It was found that barstar gene is
dominant to the barnase gene, and fertility restoration was due to the
formation of tapetal cell-specific barnase and barstar complexes.
Female fertility was not affected, and transformed plants had normal
morphology. By coupling the TA29-RNase gene to a dominant
herbicide resistance gene, uniform populations of male sterile plants
could be produced. For this, the two genes coding for ribonucleases
RNase and barnase were linked independently to the “bar” gene,
which confers resistance to the herbicide bialophos
(phosphinothricine or PPT), to obtain a marker for male sterility
(Denis et al. 1993).

The barnase-barstar male sterility-fertility restoration system is

available in cauliflower and tomato (Banga and Raman 1998).
Reversion to fertility from male sterile plants has been observed in
some cases. It was found that RNase-T1-mediated male sterility is

23
unstable at temperatures higher than 25°C. Jagannath et al. (2001)
could overcome these problems by use of spacer DNA between
barnase gene (driven by a tapetum specific promoter) and the CaMV
35S promoter-driven bar gene. This helped in insulating the tissue
specific expression of the barnase gene over all developmental
stages of transgenic plant. These newly developed male sterile lines,
however, could not be restored by transgenic carrying wild type
barstar. For that plants carrying modified barstar constructs were
developed (Jagannath et al., 2002).

2. Male Sterility through Hormone Engineering

Drastic changes in endogenous levels of auxins have been

demonstrated to cause male sterility in tomato (Sawhney 1997) and
several other crops. Induction of male sterility by manipulating
endogenous hormone levels (Spena et al. 1987). Such male sterile
can be maintained by linking the gene for herbicide resistance to the
male sterilizing “rol c”. Male fertile segregants can be chemically
rouged out by selective elimination in maintainer population.

3. Pollen Self-Destructive Engineered Male Sterility

Theoretically, it is feasible to genetically engineer plants having

altered endogenous auxins indole acetic acid (IAA) levels with pollen
exhibiting self-destructive mechanisms. To achieve this, McCormick
et al. (1989) and Wood (1990) transformed plants with a chimeric
gene consisting of pollen-specific promoter (LAT59) and a gene
(fins2) that converts indole acetamide (IAM) into IAA. Although the
detailed characterization of such a transgenic was not reported, it
was argued that, if this system works, plants carrying the LAT59-fins2
gene when sprayed with IAM will selectively convert IAM into IAA at
very high concentrations to kill the pollen and render the plants male
sterile. Although this technology will avoid the problems of elimination
of maintenance of male sterile and the restoration of fertility in F1
hybrids, the complications such as are associated with the use of
chemical hybridizing agents would be there, except the necessity of
spraying gametocides/CHAs repeatedly at a specific growth stage
and interval.

24
Another possibility of inducing such male sterility is the transformation
of plants with chimeric gene involving TA-29 promoter and coding
region of β-glucuronidase (GUS). Such transformants, when sprayed
with protoxins like sulfonyl urea or maleic hydrazide, cause male
sterility through their breakdown in the tapetum by the β-
glucuronidase enzyme. The transgenic plants not sprayed with
protoxins remain fertile, so a fertility restoration system like TA29-
barstar is not required. Shihshieh et al. (2003) used tissue-specific
promoters expressing CKX1 and gai, genes involved in oxidative
cytokinin degradation and gibberellins (GA) signal transduction,
respectively, to study the roles of cytokinin and GA in male organ
development.

4. Male Sterility Using Pathogenesis-Related Protein Genes

Pathogenesis-related (PR) protein β-1,3 glucanase (callase) is known

to dissolve specific cell wall made of callase, a β-1,3-linked glucan
between cellulose cell wall and plasma membrane and tetrads
synthesized by microsporocyte. The β-1, 3 glucanase (callase)
secreted by the tapetum helps to release free microspores into
locular space by breaking down the callase wall. The genetic
alteration of this mechanism in plants caused male sterility. Callase
appearance and distribution was normal in male sterile transgenic
plants up to prophase I, whereupon callase was prematurely
degraded. Electron microscopy revealed a thick callase wall
surrounding each microspore of the tetrad in fertile anthers, whereas
it was clearly absent in sterile microspores. The premature dissolution
of callase indicated that the modified glucanase is secreted from the
tapetum and is active within the anther locule. Transgenic tobacco
plants expressing the β-1, 3 glucanase under the control of CaMV
35S promoter showed normal fertility despite the expression of
modified glucanase. This was attributed to either lower expression of
the modified gene in the tapetum or inappropriate timing of the
modified callase synthesis during the process of microsporogenesis,
stronger (Rf-WA-1) being located on chromosome 10. It has also
been possible to identify 6 RAPD markers linked to another gene for
fertility restoration (Rf-WA-3), three of these mapped on
chromosome1. These marker-aided selections are for fertility-
restoring ability.

25
B. Male Sterility through Modification of Biochemical Pathways

1. Flavonoids

Among the three major flower pigments, (flavonoids, carotenoids, and

betalains), the flavonoids are the most common and most important.
Apart from their role in color development, flavonoids are important in
plant reproduction and defence-related mechanisms. In legumes,
they may act as signal molecules in the interaction with nitrogen-
fixing bacteria. The biochemical pathways of flavonoids synthesis
have shown that these are produced as phenyl-propanoid based
secondary metabolites via chalcone synthase (Chs). A large number
of genes involved in the biochemical pathway of flavonoids synthesis
have been cloned and characterized (Forkmann 1991). However, it
was not concluded whether the anther box on its own directs the
expression in the tapetum cells. It was argued that modules that
confer organ specificity to the Chs or CaMV 35S promoter may act in
concert with anther box. About 14% of transgenic plants showed a
clear reduction of anther and pollen pigmentation, which confirmed
the involvement of anther box in tapetum-specific expression. The
copy number and orientation of the inserted anther box did not affect
the antisense phenotype in a quantitative or a qualitative way.

2. Jasmonic acid

Jasmonic acid (JA), synthesized from linolenic acid (LA) through

octadecanoid pathway (Weiler 1997), plays an important role in
anther dehiscence and pollen maturation (Sanders et al. 2000; Stintzi
and Browse 2000; Ishiguru et al. 2001). The triple fad
(fad3,fad7,fad8) mutants lacking LA had indehiscent anthers and
hence showed functional male sterility (McConn and Browse 1996)
Recently, Ishiguru et al. (2001) linked impaired dehiscence in
Defective Anther Dehiscence I (DAD I) mutant (Sanders et al. 2000;
Stintzi and Browse 1996) to mutation in DAD I gene that encoded a
chloroplastic phospholipase AI to supply free LA at the initial step in
JA biosynthesis, they further suggested that JA also regulated flower
opening, anther dehiscence as well as pollen maturation.

Exogenous application of JA reversed the impaired dehiscence. Of

the 25 transgenic plants obtained, 3 showed delayed anther

26
dehiscence and inviable pollen. Segregation of the transgenes in the
selfed progeny was consistent with the Mendelian inheritance of two
copies of the transgenes at different loci. The male sterile phenotype
could be reversed by application of JA (0.5 μm) as well as LA:
Genetic male sterility based on Br DAD I gene has many advantages;
most important being the ability to induce normal pollen production
and dehiscence. This is critical for developing lines homozygous for
the male sterilizing allele. Moreover, this system involves suppression
of a single gene and floral morphology is not adversely affected. The
stability of expression over a range of environmental conditions,
however, remains to be investigated.

3. Carbohydrates

Carbohydrates play a critical role in the anther and pollen

development by sustaining growth as well as signal pathways. Their
transportation from photo synthetically active source tissues to
developing sinks is regulated by extra cellular invertase. This class of
invertase is encoded by small gene familiar with differential regulation
and expression patterns. The extra cellular invertase Nin 88 of
tobacco shows specific temporal and spatial expression patterns in
developing anthers. At early stages of flower development, the
invertase is present exclusively in the tapetum cell layers of the
anthers followed by a distinct expression pattern during pollen
development.

The tissue specific antisense repression of Nin 88 under the control

of corresponding promoter in plant caused male sterility by blocking
pollen development during early stages of microsporogenesis (Goetz
et al. 2001). Exogenous supply of carbohydrates in an in vitro
maturation assay was able to partially overcome this block, thus
opening up the possibility of maintaining this male sterility system.
Fertility restoration is essential for its use in hybrid seed production,
can theoretically be achieved by crossing this GMS system with
transgenic plants expressing distantly related invertase (may be from
bacteria), which is not under the control of the antisense repression
through a plant invertase. Alternatively introduction of a sucrose
transporter could bypass the requirement for extra cellular sucrose
cleavage. Male sterility caused by antisense expression of Nin 88
was not associated with any morphophysiological abnormality.

27
C. Transgenic induction of mitochondrial rearrangements for
Cytoplasmic male sterility in crop plants:

Recently Ajay et al. (2007) found that stability of the mitochondrial

genome is controlled by nuclear loci. In plants, nuclear genes
suppress mitochondrial DNA rearrangements during development.
One nuclear gene involved in this process is Msh1. Msh1 appears to
be involved in the suppression of illegitimate recombination in plant
mitochondria. To test the hypothesis that Msh1 disruption leads to the
type of mitochondrial DNA rearrangements associated with naturally
occurring cytoplasmic male sterility in plants, a transgenic approach
for RNA was used to modulate expression of Msh1 in tobacco and
tomato. In both species, these experiments resulted in reproducible
mitochondrial DNA rearrangements and a condition of male (pollen)
sterility. The male sterility was, in each case, heritable, associated
with normal female fertility, and apparently maternal in its inheritance.
Segregation of the transgene did not reverse the male sterile
phenotype, producing stable, non transgenic male sterility. The
reproducible transgenic induction of mitochondrial rearrangements in
plants is unprecedented, providing a means to develop novel
cytoplasmic male sterile lines for release as non-GMO or transgenic
materials.

D. Engineering Cytoplasmic Male Sterility via the Chloroplast

Genome

Naturally-occurring Cytoplasmic male sterility (CMS) has been known

for over 100 years. CMS systems are used to produce commercial F1
hybrid lines. Ruiz and Daniel (2005) recently reported the first
engineered cytoplasmic male sterility system in plants. They studied
the effect of light regulation of the phaA gene coding for β-
Ketothiolase engineered via the chloroplast genome. The phaA gene
was efficiently expressed in all tissue types examined, including
leaves, flowers, and anthers. The transgenic lines were normal
except for the male sterile phenotype, lacking pollen. Scanning
electron microscopy revealed a collapsed morphology of the pollen
grains. Transgenic lines showed an accelerated pattern of anther
development, affecting their maturation, and resulted in aberrant
tissue patterns. Abnormal thickening of the outer wall, enlarged

28
endothecium, and vacuolation decreased the inner space of the
locule, affected pollen grain, and resulted in the irregular shape or
collapsed phenotype. Reversibility of the male sterile phenotype was
observed under continuous illumination, resulting in viable Pollen and
a copious amount of seeds. This study offers a new tool for transgene
containment for both nuclear and organelle genomes and provides an
expedient mechanism for F1 hybrid seed production.

Advantages of the chloroplast transformation system

A chloroplast genetic engineering approach offers a number of

attractive advantages, including high-level transgene expression,
multi-gene engineering in a single transformation event, transgene
containment via maternal inheritance, lack of gene silencing, position
effect due to site-specific transgene integration, and lack of
pleiotropic effects due to sub-cellular compartmentalization of
transgene products. Genetically engineered cytoplasmic male sterility
via the chloroplast genome may be used for the safe integration of
foreign genes via the nuclear genome and in those rare cases in
which plastid genomes are paternally or biparentally transmitted.
Recently, plastid transformation was demonstrated in carrot with their
ability to grow in very high saline conditions (up to 400 mμ sodium
chloride). Additionally, plastid transformation of recalcitrant crops
such as cotton and soybean allows the application of the cytoplasmic
male sterile system to commercially important crops.

Havey (2004) has documented the worldwide use of CMS to produce

competitive hybrid cultivars. Major investments of time and resources
are required to backcross a male-sterility-inducing cytoplasm into elite
lines. These generations of backcrossing could be avoided by
transformation of an organelle genome of the elite male-fertile inbred
line to produce female inbred lines for hybrid seed production.
Because the male-fertile parental and male-sterile transformed lines
would be developed from the same inbred line, they should be highly
uniform and possess the same nuclear genotype, excluding
mutations and residual heterozygosis.

Therefore, the male-fertile parental line becomes the maintainer line

to seed-propagate the newly transformed male-sterile line. A few
generations of seed increases would produce a CMS-maintainer pair

29
for hybrid seed production. An additional advantage of organelle
transformation would be the diversification of CMS sources used in
commercial hybrid-seed production. Transformation of the chloroplast
genome would allow breeders to introduce different male-sterility-
inducing factors into superior inbred lines. Introduction of a male-
sterility inducing transgenes into one of the organelle genomes of a
higher plant would be a major breakthrough in the production of male-
sterile inbred lines. This technique would be of great potential
importance in the production of hybrid crops by avoiding generations
of backcrossing, an approach especially advantageous for crop
plants with longer generation times. Moreover, transgenes that are
engineered into annual crops could be introgressed into wild crops,
persist in the environment, and thereby have negative ecological
consequences. Therefore, it may be necessary to engineer a male
sterility system that is 100% effective. For vegetable, fruit, or forage
crops, restoration of male fertility in the hybrid is not necessary. This
simplifies the production of hybrids because effort can be
concentrated on maintainer line development, without concern over
whether the pollinator restores male fertility in the hybrid.

For crops with seeds as the economically important product, such as

canola, sunflower, or maize, one or both of the hybrid’s parents must
bring in male-fertility restoration factors or the male-sterile hybrid
seed must be blended with male-fertile hybrid seed. In currently
available cytoplasmic male sterile lines, the nuclear genome controls
various restoration factors and such factors are often located at
multiple loci and are poorly understood. However, the authors report
restoration of male fertility by changing conditions of illumination.
Therefore, this is a novel approach for creating male sterile
transgenic plants, which may help advance the field of plant
biotechnology through effective transgenes containment.

Conclusions

Genetically engineered male sterility provides tremendous

opportunities to the breeders for enforcing pollination control in hybrid
seed production systems. On the other hand these systems have
some disadvantages like availability of efficient gene construct,
possible dispersion of transgene to other related species, availability
of efficient transformation technique and very high initial investment.

30
Much will, however, depend upon large scale availability of the
desired constructs/genes as at present most of the genes are
patented and not available to large number of breeders. Apart from
barnase-barstar system, no other system has reached the
commercial stage. Further researches and their subsequent
commercialization are expected.

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32
 Male Gametocide for Coriander

INTRODUCTION

Coriander is a facultative cross pollinated crop. The inflorescence is a

compound umbel. Peripheral florets of the umbellets are
hermaphrodite and central florets are staminiferous or sometimes
sterile (Diederichsen, 1996). Romanenko et al. (1991) showed that
plants that were not emasculated but were pollinated with pollen of
other plants still had a degree of selfing of 25%. Crossing in coriander
is acknowledged as cumbersome due to difficult process of
emasculation (Romanenko et al., 1992). Hence, new varieties of the
crop are traditionally bred using either mass selection or pure line
selection or recurrent selection or through mutation breeding.
Attempts to use hybridization to combine desirable traits were
scarcely successful due to inherent disadvantages in the crop for
successful emasculation and crossing. Hence, using male
gametocides as an alternative to hand emasculation offers immense
scope for development of new varieties.

Several gametocides have been reported effective in inducing pollen

sterility in various crops. Sodium methyl arsenate, 2,3-
dichloroisobutyrate, sodium 2,2-dichloropropionate, Gibberellic acid,
Maleic Hydrazide (1,2-dihydropyridazine, 3-6-dione), 2,4-dichloro
phenoxy acetic acid, ethyl 4-fluorooxanilate, Trihalogenated
methylsulfonamides, ethyl and methyl arsenates, and many other
chemicals were reported to have male gametocide effects in several
crops. Salgare (2004) reported that foliar application of all the
concentrations of maleic hydrazide above 50, 200, 800 μg/ml
suppressed cent per cent pollen germinability of Phaseolus mungo,
P. aureus, Cyamopsis tetragonoloba, respectively showing prospect
for use as male gametocide. Garcia Torres et al. (1979) have shown
that GA3 150 ppm induced of maximum pollen sterility in sunflower.
Lakshmi Praba and Thangaraj (2005) reported that ethrel (800 ppm),
salicylic acid (600 ppm) and maleic hydrazide (0.2%) induced a
significantly higher percentage of male sterility in the TGMS lines of
rice. Singh (1999) in rice, Chauhan and Vandana Singh (2002) in
mustard and Gangaprasad et al. (2004) in Niger (Guizotia abyssinica)
reported induction of very high pollen sterility at concentrations of one
to six per cent of detergent.

33
Due to its herbaceous nature of coriander with small and delicate
flowers, the crop is sparsely amenable for crossing. In view of this,
the present study is taken up to assess five male gametocides viz.
Gibberellic acid, 2, 4-D, Maleic Hydrazide, Ethrel and Surf Excel at
different concentrations using Dilpazir as test variety.

Table 1: The composition of the Pollen Germination medium for each

1000 ml of water

Sr. Ingredient Quantity

1 Sucrose 100 g
2 Boric acid (H3BO3) 100 mg
3 Calcium nitrate (Ca (NO3)2. 4H2O) 300 mg
4 Magnesium Sulfate Heptahydrate (MgSO4. 7H2O) 200 mg
5 Potassium nitrate (KNO3) 100 mg

MATERIALS AND METHODS

The experiments were conducted during late Rabi seasons of 2009 -

2010. In general the methodology provided by Giridhar Kalidasu et al
was fully observed.
Chemicals evaluated and test variety

Five gametocides viz. Gibberellic acid, 2, 4-D, Maleic Hydrazide,

Ethrel and Surf Excel Quick Wash (active ingredient: sulphonated
methyl ester) were evaluated. GA at 50 and 300 ppm; 2, 4-D at 50,
100 and 500 ppm; Maleic Hydrazide at 125 and 250 ppm; Ethrel at
2000 and 5000 ppm; Surf Excel at 5% and distilled water spray as
control were used. The test variety chosen was Dilpazir which is a
medium duration variety. The variety comes to 50% flowering
between 50 - 60 days. The variety shows flower primordia initiation
between 35 - 45 days. All the gametocides were sprayed once at the
time of flower primordia initiation using uniform quantity of spray fluid
of 22 ml per one meter length of plant row. Three meter lengths of
rows containing 30 plants were used for applying the gametocides.
Care was taken so that all the plants in the row are evenly sprayed.

34
Immediately after spraying, twenty plants were randomly selected
and labeled for subsequent observations.

Pollen collection, assessment of pollen sterility and pollen

germination

In all the treatments fresh pollen was collected in the field from
recently opened anthers showing fresh pollen, from the florets of the
treated plants. While collecting the pollen, care was taken so that the
pollen was from at least five different plants. The pollen was mixed to
make a single lot. A tiny droplet of the 1% acetocarmine stain was
placed on the slide. Using a teasing needle, a small amount of pollen
of this lot was placed on stain and thoroughly mixed to ensure
uniform penetration of the stain into the pollen. Cover slips were
gently placed on to different slides for each treatment. The slides
were then observed under a microscope.

The stained pollen was examined under microscope and at least five
hundred pollen grains were counted in each sample by selecting
random fields. Tests were repeated whenever there was a full
staining of all the pollen grains or partial dying was observed. Tests
were initiated on the tenth day of the spraying and repeated for three
times with an interval of four days. Pollen sterility estimated thus was
converted to percentage from the number of non-dyed and total
pollen.

In vitro pollen germination was assessed using Pollen Germination

Medium (PGM). The PGM was prepared using Brewbaker and
Kwack’s (1963) preparation method. The composition of the medium
for each 1000 ml of water is presented in Table 1. The medium was
prepared from dissolving the above ingredients in one liter of distilled
water.

Freshly collected pollen was spread on a cover slip and was placed
on a slide having a tiny droplet of PGM solution. The slides were
placed in an incubator at 25°C. Each treatment was assessed for
germination after three and six hours of incubation. A minimum of 100
pollen grains were examined for each observation. Germination
frequencies were recorded by counting germinated and non-
germinated pollen grains. Pollen grains with development of pollen

35
tube (germinated) and without are counted, and germination was
expressed in percentage.

Observations on anther dehiscence were also recorded using a

stereomicroscope. The specific findings on anther dehiscence were
further verified using another test variety Irani by spraying Maleic
Hydrazide at 125 ppm on fifty pre-labeled plants.

Phytotoxicity and related plant growth assessment

Phytotoxicity was assessed periodically starting from the second day

of the spraying with seven days interval. Toxicity symptoms on
leaves, stem, and inflorescence were recorded.

Evaluation of MH use in chemical emasculation

The effect of maleic hydrazide in preventing the pollen dispersal

through pollen agglutination which was observed during first year of
testing was further evaluated for its use as chemical emasculation
agent in test variety Sudha. Three spray schedules, that is maleic
hydrazide 100 ppm at weekly interval from 25 DAS to cessation of
flowering, maleic hydrazide 125 ppm at weekly interval from 25 DAS
to cessation of flowering and maleic hydrazide 100 ppm at 25 DAS
followed by maleic hydrazide at 125 ppm subsequently at weekly
interval to cessation of flowering were tested to compare the efficacy
of maleic hydrazide in sustaining the pollen agglutination. Pollen
agglutination from the treated samples was monitored periodically
through the use of stereomicroscope during post-spray period until
the cessation of flowering. Bagging of umbels of treated plants was
carried out to observe any seed set for confirming the visual
observations.

Assessment of Maleic Hydrazide use in chemical emasculation

The maleic hydrazide use in breeding programs was assessed using

Dilpazir as male parent and Irani as female parent. The female parent
was sprayed with maleic hydrazide 100 ppm at weekly interval from
25 DAS onwards until the end of flowering cessation. The pollen
agglutination was monitored through the use of stereomicroscope
periodically.

36
Table 2: Phytotoxicity symptoms observed on treated plants.

Sr. Chemical Phytotoxicity Symptoms

1 GA No phytotoxicity.
50 ppm
2 GA 300 Elongation of plant and inflorescence. Plants are tender
ppm and weak.
3 2,4-D Similar symptoms as at Sr.2 to a lesser extent.
50 ppm
4 2,4-D Severely convoluted plants. Stem thickens showing
100 ppm some pink pigmentation. Leaves elongated and
drooping. Thickened leaves. Distortion of leaf shape from
normal, showing less pinnation and uneven pinnation.
Abnormal thickening and shape of umbel and umbellets.
Abnormal floret shape. Shortening of flower stalk. Higher
number of male flowers in umbels.
5 2,4-D Mortality followed by the symptoms described for 2, 4-D
500 ppm 100 ppm below.
6 MH Reduction in plant growth and foliage. Reduction in
125 ppm number of umbellets and hermaphrodite florets.
7 MH Scorching of stem and leaves. Shortening of plant and
250 ppm reduction in plant growth. Reduction in foliage size and
number. Increased number of umbels but reduced
number of umbellets. Reduction in number of umbellets
and hermaphrodite florets.
8 Ethrel Chlorosis of leaves with occasional drying of older
2000 ppm leaves. Stem pigmentation and slenderness.
Early and higher pinnation of leaflets. General growth
suppression.
9 Ethrel Severe chlorosis of leaves. Drying of older leaves. Stem
5000 ppm pigmentation and slenderness. Early and higher
pinnation of leaflets. General growth suppression. Early
senescence.
10 Surf Excel Scorching of leaves on the edges.
5.0 %

Further, crossing was taken up by dusting of pollen of selected male

parents on umbels of female parents. Twenty umbels were used for
each cross. Number of hermaphrodite flowers was recorded before
the pollination. Repeat pollination was taken up for four consecutive
days to ensure inner florets also received pollen from selected male

37
parent. Such crossed umbels were bagged, tagged and assessed for
seed set. Selfing was recorded in both maleic hydrazide treated and
untreated female parents.

RESULTS AND DISCUSSION

Male gametocides were found quite effective in various crops.

However, there is a paucity of information on the use of male
gametocides in coriander. The present investigation threw several
insights on the use of male gametocides in coriander.

Phytotoxicity on treated plants

Among the gametocides evaluated, GA at 50 ppm did not show any

symptoms of phytotoxicity. All other concentrations of the chemicals
evaluated caused phytotoxicity (Table 2). Most pronounced was the
effect of 2, 4-D at 500 ppm that caused severe phytotoxicity and plant
mortality within three weeks of spraying. Salgare (1999) who worked
on P. mungo for induction of male sterility using 2, 4-D, reported that
2,4-D concentration ranging from 200 to 5000g per ml caused
mortality of all plants, which is similar to the present study. Plants in
all the treatments except for 2, 4-D 500 ppm, completed life cycle with
a distinct flowering phase.

Pollen sterility and pollen germination

Among the gametocides studied none have shown any significant

gametocide effect on pollen except for 2, 4-D. Pollen sterility among
the treatments ranged from 0-25%. The chemicals Gibberellic acid at
lower concentrations showed less than 3% sterility. However, 2, 4-D
at 100 and 50 ppm and maleic hydrazide at 250 ppm showed 25%,
7.0 and 9.5% of pollen sterility respectively (Table 3).

Only 2, 4-D at 100 ppm did show considerable degree of pollen

sterility (25%). However, the sterility recorded with 2, 4-D at 100 ppm
was not continuous but with a peak at fifteen days after spraying.
Rustagi and Mohan Ram (1971) reported the occurrence of rhythms
of pollen non-viability interspersed with periods of restoration of
viability with the use of Dalapon and Mendok in Linseed. Salgare
(2004) reported that maleic hydrazide at 800 μg/ml failed to suppress

38
cent per cent pollen fertility in P. mungo, P. aureus, Cyamopsis
tetragonoloba and Vigna mungo.

Observations on pollen germination showed cent percent germination

in all the treatments tried except for of maleic hydrazide above 3000
μg/ml. This supports that concentration of maleic hydrazide used in
the experiment may be well below the toxic levels for pollen
germination and repeated spraying of maleic hydrazide should also
be assessed for pollen germination. But, pollen germination alone is
not sufficient for normal formation of zygote because further growth of
tube in the style leading to fertilization should take place. Whether
these chemicals have any role on growth and fertilization of coriander
needs to be further evaluated.

Though none of the chemicals had induced pollen sterility or

suppressed pollen germination, Maleic Hydrazide at 125 and 250
ppm caused severe suppression of anther dehiscence. The chemical
also suppressed anther protrusion and prevented the petal opening.
A series of effects were observed in the florets under the influence of
the maleic hydrazide over time. One of the primary effects was pollen
agglutination. Pollen agglutination was first observed on 10th day
after spraying. Poor opening of florets and prevention of protrusion of
anthers were observed during 10th to 22nd day. During this phase,
severe agglutination of the pollen was observed which caused
prevention of pollen dispersal (suppression of anther dehiscence).
The effect relapsed on 23rd day after spraying. This phenomenon
was confirmed using another test variety Irani. The effect of maleic
hydrazide on Irani followed the similar trend as with the case of
Dilpazir (Table 4).

As early as 1969, Tadahiko Hirose reported that sodium 2, 2-

dichloropropionate caused suppression of anther dehiscence in bell
pepper which started about two weeks after treatment. However,
there is poor information on effect of gametocides on anther
dehiscence of crop plants. Rustagi and Mohan Ram (1971) reported
functional male sterility with the use of Mendok (sodium 2, 3-
dichloroisobutyrate) and Dalapon (sodium 2, 2-dichloropropionate) at
250, 500 and 1000 ppm in linseed where functional male sterility
resulted from lack of anthesis, fusion and non-dehiscence of fertile

39
anthers and agglutination of pollen. The results of the present
investigation are similar to the report of Giridhar Kalidasu et al.

Table 3: Pollen sterility (%) and pollen germination (%) in treated

plants

Sr. Chemical % sterility * % pollen germination**

1 GA 300 ppm 0 100%

2 GA 50 ppm 1.0 100%
3 2,4-D 500 ppm Mortality Mortality
4 2,4-D 100 ppm 25 Severe distortion of florets
On assessment day
5 2,4-D 50 ppm 7.0 97%
6 MH 250 ppm 9.5 100%
7 MH 125 ppm 3.0 100%
8 Ethrel 5000 3.5 100%
ppm
9 Ethrel 2000 0 100%
ppm
10 Surf Excel 5.0 0.5 100%
%
11 Control 1.0 100%
* Average of five tests starting from tenth day of spraying with an interval of 4 days.
** Average of two tests starting from sixteenth day of spraying with an interval of 2 days.

Table 4: Maleic Hydrazide influence on anthesis and anther

dehiscence

Sr. Days after spraying Observations on varieties Sadhana

1 6th day to 9th day
2 10th day-22nd day in
Dilpazir
3 10th day-21st day in
Irani
4 23rd day in Dilpazir
5 22nd day in Irani
and Swathi
Normal anthesis, normal pollen
dehiscence
Poor opening of petals, anthers do not
protrude.
Pollen agglutination and suppression of
dehiscence.
Normal mode restored
Normal mode restored

40
Table 5: Percentage of fruit set in maleic hydrazide assisted crossing

Cross combination %age fruit set

Dilpazir x Irani 41.0
Irani x Dilpazir 52.0

Use of Maleic Hydrazide in chemical emasculation of coriander

In the treatments with repeated spraying of maleic hydrazide, the

observations recorded through stereo microscope revealed that the
persistence of severe pollen agglutination continued from the first day
of anthesis to cessation of flowering in both the treatments. This is
significant as the entire flowering phase could be brought under the
influence of maleic hydrazide thus forcing pollen agglutination which
prevented selfing. Bagging of the umbels on treated plants did not
result any fruit set further confirming the microscopic observations. In
contrast, selfing in untreated female parents was 9.0% in Dilpazir and
to 15% in Irani indicating that, without assisted pollination, no fruit set
takes place in the umbels of treated plants due to prevention of
anther dehiscence.

Successful crosses with the use of Maleic Hydrazide

Two cross combinations were attempted involving two varieties

(Dilpazir and Irani) using the maleic hydrazide as chemical
emasculation agent which resulted in 41% fruit set involving with
female parent Dilpazir and 52% fruit set involving female parent
Irani demonstrated the utility of the technology (Table 5). The
success in obtaining seed of desired parentage through maleic
hydrazide assisted crossing revealed that the huge amount of hybrid
seed can be produced in coriander through the use of maleic
hydrazide as chemical emasculation agent with relative ease.

Conclusion

The present investigation identifies maleic hydrazide as potential

male gametocide and an effective alternative for cumbersome hand
emasculation in coriander. Spraying of maleic hydrazide 100 ppm
from 25 DAS on wards until the cessation of flowering is an effective

41
alternative to cumbersome emasculation in coriander and for quick
generation of large quantity of breeding material.

REFERENCES

Bots M, Mariani C (2005). Pollen viability in the field. COGEM. Radboud

Universiteit Nijmegen, Netherlands. pp: 14-28.

Brewbaker JL , Kwack BH (1963). The essential role of calcium ion in pollen

germination and pollen tube growth. Am. J. Bot., 50: 747-858.

Chauhan SVS. , Vandana Singh (2002). Detergent induced male sterility and bud
pollination in Brassica juncea (L.) Czern and Coss. Current Sci., 82(8): 918-920.

Diederichsen A (1996) Promoting the conservation and use of underutilized and

neglected crops. 3. Coriander (Coriandrum sativum L.) pp: 11.

Gangaprasad S, Sreedhar RV, Salimath PM, Ravikumar RL (2004). Induction of

Male Sterility in Niger (Guizotia abyssinica Cass.). New directions for a diverse
planet: Proceed. of the 4th Int. Crop Sci. Congr. Brisbane, Aust., 26 September -
1 Oct 2004.

Garcia Torres L, Dominguez Gimenez J, Fernandez Martinez J (1979). Male

sterility and female sterility induced in sunflower with GA3. Anales del Institute
Nacional de Investigeiones Agrarias, Production Vegetal 9: 147-169.

Kalidasu Giridhar, C. Sarada, P. Venkata Reddy and T. Yellamanda Reddy

(2009); Use of male gametocide: An alternative to cumbersome emasculation in
coriander (Coriandrum sativum L.); Journal of Horticulture and Forestry Vol. 1(7)
pp.126-132 September, 2009

Lakshmi Praba M, Thangaraj M (2005). Effect of Growth Regulators and

Chemicals on Pollen Sterility in TGMS Lines of Rice. Plant Growth Regulation
46(2): 117-124.

Romanenko LG, Nevkrytaja NV, Kuznecova EJU (1991). Features of pollination

in coriander [in Russ.]. Sel. Semenovod. (Moskva) pp. 16-17.

Romanenko LG, Nevkrytaja NV , Kuznecova EJU (1992). Self fertility in

coriander [in Russ.]. Sel. Semenovod. (Moskva) pp: 25-28.

Rustagi PN, Mohan Ram HY (1971). Evaluation of Mendok and Dalapon as Male
Gametocides and their Effects on Growth and Yield of Linseed New Phytologist
70(1): 119-133.

42
Salgare SA (2004). Evaluation of MH as male gametocide and a new method of
plant breeding - a critical review. Recent trends in biotechnol. 9(2): 263-267.

Singh AK (1999). Male gametocidal effect of synthetic detergent in rice. The Ind.
J. Genet. Plant Breed 59(3): 371-373.

Stanley RG, Linskens HF (1974). Pollen Springer-Verlag, Berlin Heidelberg New

York.

Tadahiko HIROSE 1969 Studies of chemical emasculation in pepper. I.

Suppression of anther dehiscence and other morphological changes caused by
sodium 2, 2-dichloropropionate. Engei Gakkai zasshi. 38(1): 29-35.

43
 CUCUMBER: Sex Expression and Chemical Modifiers

Cucumber is an annual deep-rooted (ca. 3 ft) crop with tendrils and

hairy leaves. The plants may have an indeterminate, determinate, or
a compact plant habit. The compact growth habit consists of plants
with shorter internode length than plants with indeterminate or
determinate growth habit. Optimum growth occurs between 70-75F
(20-25C), with growth reduction occurring below 60F (16C) and
above 90F (30C).

Several flowering habits exist in cucumbers. Most cultivars are

monoecious, with separate male and female flowers in the same
plant. Gynoecious or "all-female" cultivars produce only female
flowers resulting in up to 13 times more female flowers than those
obtained in monoecious cultivars. The so-called "PF" hybrids produce
predominantly female flowers but also produce a small number of
male flowers. Often "pollination" plants are supplied to insure
fertilization on "PF" types. Many cultivars grown in greenhouses such
as European cucumbers are parthenocarpic. Parthenocarpic varieties
require no pollination for fruit production. In fact, pollination of these
cultivars causes an off-shaped appearance of the fruit.

The first flowers of monoecious plants are staminate or 'male'

followed by pistillate or 'female' flowers from which fruits are born.
Sex expression in cucumber may be affected by several factors such
as plant density, plant stress, temperature, and light intensity.
Reduced rates of female flowers in gynoecious cultivars may result
from exposure to stress caused by high plant population densities,
insect attack, wind damage, and combinations of low light intensity
and high ambient temperatures. The commercially available
hormone ethephon, at 125-250 ppm, increases the production of
pistillate or female flowers in gynoecious cultivars. Cucumbers
will interbreed with other cucumber cultivars but neither with melons
nor squash. Some markets, such as the Japanese market, prefer
'bloomless' fruits, or fruit free of the natural film or powdery tissue that
covers the skin of cucumbers and several other vegetables.

44
Flowering and Sex Expression

Normally, cucumber plants are monoecious, producing both male and

female flowers separately on the same plant. The male (staminate)
flowers have very short stems and are borne in clusters of three to
five. These are located mostly on the main stem, while female
(pistillate) flowers are located on the laterals, as well as on the main
stem, and can be recognized by the ovary at the base of the flower
which develops into the fruit. The male flowers are the first to appear
and in considerably larger numbers than the female flower.
Monoecious cucumbers generally go through three phases of sex
expression:

1) an initial period when only male flowers are produced,

2) a long period when equal numbers of male and female flowers are
borne, and
3) a final relatively short phase when female flowers largely
predominate.

New cucumber types incorporate a gynoecious flowering habit,

whereby only female flowers are produced. These hybrids, often
referred to as "all-female," tend to be early in maturity, out-yield
standard cultivars, and produce a concentrated fruit set, making them
well-suited for mechanical harvest.

Somewhat similar to the gynoecious lines are the predominantly

female types commonly referred to as "PF" cultivars. These hybrids
are not completely gynoecious but produce some male blooms. The
"PF" expression is more typical of most present-day gynoecious
hybrids. The number of male flowers is generally far less than those
of monoecious plants, but can vary widely depending on cultivar and
environmental conditions. The highly female expression of the hybrid
"PF" plants concentrates fruit set so plants are well suited for once-
over harvest.

Sex expression in cucumbers can be modified by the use of

several chemicals. Silver nitrate and gibberellic acid will
promote male blooms on gynoecious plants. The growth
regulator ethephon induces female flowering and causes
monoecious plants to exhibit gynoecious expression.

45
Environmental factors can also have significant influences on sex
expression in cucumbers. High temperatures and long days favor
male blooms, while short days and low temperatures promote female
flower development. As a general rule, environmental factors that
result in stress in the plant, such as increased plant populations and
low moisture, will tend to increase male flowering in "PF" types and
will sometimes cause some male flowers to develop in gynoecious
lines.

Pollination

In most cucurbits, the pollen is rather sticky and heavy, and

pollination by insects is required. Honeybees are the principal means
of pollination since most other insects are not reliable for adequate
pollen transfer. Monoecious cultivars and the newer hybrid "PF" types
require one hive of bees per 50,000 plants per acre. When fully
gynoecious hybrids become more available, two or three times as
many bees may be required. Since no male flowers are produced,
these types must be inter-planted with monoecious types that
produce male and female blooms to ensure an adequate supply of
pollen. Approximately 10-15% of the crop should consist of the
monoecious pollinator in order to ensure adequate pollination. Seed
dealers usually add the male parent of the gynoecious cultivar to the
seed lot to serve as the pollinator seed.

Cucumber flowers normally open only one day. Bee activity is

generally greatest in the morning up until early afternoon. Wet, cool
weather significantly reduces bee activity and is a major reason for
poor fruit set in certain years. Overhead irrigation is detrimental to
bee activity, and during flowering watering should be withheld until
late in the day or at night if possible.

Bee colonies should not be placed in the field before the first female
flowers appear. If placed too early, the bees will visit plants in bloom
outside the field and be less effective on the cucumber crop to be
pollinated. In experiments with cucumbers for machine harvest,
delaying pollination for as much as 12 days resulted in a significant
increase in the number of fruits per plant. Generally, colonies should
not be set out until 3-6 days after blooming starts. This ensures that
flowering will be sufficiently well along to attract bees and will result in

46
a more concentrated and uniform fruit set. Also, for maximum
pollination, bee colonies should have a nearby source of water,
preferably within 0.25 mile of the site.

Highly toxic insecticides applied to other crops will kill bees visiting
the crop. Bees are attracted to sweet corn when it sheds pollen and
are easily killed by carbaryl applied for earworm control. The carbaryl
remains highly toxic to bees for several days, since it is mixed with
pollen, which bees store in the hive to feed their young. Insecticides
should be applied late in the day or at night when there is little or no
bee activity.

Contrary to popular belief, cucumbers, melons, and squash will not

cross-pollinate with one another; however, cultivars within each
species will inter-breed.

Seedless or parthenocarpic cucumbers are another distinctive

type. The plants are gynoecious with a fruit borne at each axil. They
are grown on a trellis in protected, screened culture to prevent
bees from introducing foreign pollen, which would cause seeds
to develop. Fruits are long, straight, smooth, thin-skinned, and
medium to dark-green in color. A slightly restricted "neck" at the stem
end of the fruit serves to readily identify this unique type.

Parthenocarpic varieties require no pollination for fruit production. In

fact, pollination of these cultivars causes an off-shaped appearance
of the fruit.

Parthenocarpy can be induced in cucumber Cucumis sativus L. by

foliar application of morphactin. Morphactin (chlorfluorenol) is most
effective in inducing parthenocarpy when applied.

47