PROCEDURE OF HYBRIDIZATION

Oncethe breeder has decided the

There are seven steps inVolved inobjectives of programme, he is
ready to begin
hybridization: choice of parents. (2)
(1) hybridizatiot
evaluation
 197
parents,(3) emasculation, (4)bagging, (5) tagging, (6) pollination, and (7) harvesting and
seed.
storage of F
10.6.1. Choice of Parents
The choice of parents mainly depends upon the
addition to other objectives, increased yields are
objectives of breeding programme. In
Refore, at least one of the parents involved in a crossalways an objective of the breeder.
should be a well adapted and proven
rietyin thearea for which the new variety is being developed. The other variety
baving the characters that are absent in this variety. In combination breeding, theshould be
genetic
iversity of parents is not important, but in transgressive breeding genetic diversity is of great
importance. For transgressive breeding, it should be made sure that the parents differ for
many genes affecting yield or some other character of importance. However, it would be
desirable that the performance of parents is good and that they are well adapted in the areas
where they are commonly grown. Further, some parents produce superior F,'sandF;'s, while
others do not. This property of the parents is known as combining ability and can be
estimated by one of the many biometrical techniques. Combining ability of the parents serves
as a useful guide in the selection of parents for a hybridization programme.
It is essential that all the characters sought to be improved are present in one or the other
parent. If necessary, three or more parents may be included in a complex cross. One should
remember that a character absent in the original parents will generally not appear in the
segregating generations, except for the possibility of transgressive segregations and some
choice of parents is the basic
gene interactions, e.g., complementary gene action. Thus the else, determines the success
anything
step in a hybridization programme and often, more than
or failure of the programme.
10.6.2. Evaluation of Parents
performance of parents in the area where breeding is to be done is known,
If the
in the area is not known, it should be
evaluation is not necessary. But if their performance contribute and for disease
expected to
determined, particularly for the characters they are parent may be susceptible to
reaction is important because an introduced
resistance. Disease area, or even to new diseases present in the
pathogen occurring in the
the new races of the known. New strains should also be checked
for
reaction may not be
area for which its the crop species showsabout 5 per cent more
cross
mechanical mixture and for heterozygosity if
necessary to self-pollinate a parent for one or
pollination. In the latter case, it may be
heterozygous.
generations if it is suspected to be
10.6.3. Emasculation
or anthers or the killing of pollen grains of a flower without
The removalof stamens reproductiveorgans is known as [Link] purpose
4jecting inany way the female in the flowers of the line/variety
self-fertilization
to be used as
Ofemasculation is to prevent male plants are removed, while in monoecious species
the female parent. In dioecious plants,
male inflorescence, e.g., in maize, are removed to
castor, or the done in
e male flowers. e.g.. in essential in bisexual flowers. It may be
emasculation is
Pevent self-pollination. But method suitable for a species is largely determined by the size
any one of several ways. The
 The technique for 199
that is about to emerge emasculation
from the
in barley(H.
flag leaf wouldvulgare) is described in some detail. Aspike
The flag leaf is carefully be generally appropriate
from the lower and the peeled away, but not for emasculation.
the two side spikelets are
upper 2-3 nodes are clipped removed, to expose the spike. The spikelets
off. From the
otigmas of central and sideremoved remaining
leaving the central spikelets only. nodes, generally
lemma and palea of each ofspikelets become receptive at different [Link]
is because the
top one-third of
nresent in each spikelet are now the remaining spikelets is
then clipped off. The three
Care must be taken not to easily accessible and are removed with anthers
the help of forceps.
intact from allthe spikelets. damage the stigmas and the ovary, and to remove all the
anthers
[Link]. Suction Method. This method is
Emasculation is done in the morning just before or useful in species with small flowers.
petals are generally removed with forceps immediately after the flowers open. The
or exposing
glass tube attached to a suction hose is used tothe anthers and the stigma. Athin rubber
is also passed over the stigmas to suck any suck the anthers from the flowers. The tube
pollen
may be produced by an aspirator attached to a grains present on their surface. The suction
water tap or a small suction pump. The
amount of suction used is very important. The suction should by be
and pollen grains, but not the flowers or the gynoecium. With enough to suck the stamens
suction method, considerable
amount of self-pollination (up to 15 per cent) is likely to occur. Washing the stigma
of water may help in reducing self-pollination. However, self-pollination cannot be with a jet
in this method.
eliminated
[Link]. Hot Water Emasculation. Pollen grains are more sensitive than the female
reproductive organs to both genetic and environmental factors. This property is uilized to kill
the pollen grains with hot water or other agents like alcohol or cold water treatment without
damaging the female reproductive organs. In the case of hot water emasculation, the
temperature of water and the duration of treatment vary from crop to crop, and must be
determined for every species. For jowar (S. bicolor), treatment with water at 42-48°C for ten
minutes is found tobe suitable. In case of rice (0. sativa), a10minute treatment with-water at
40-44°C is adequate. Hot water treatment is given before anthers dehisce and prior to the
opening of flowers. The hot water is generally carried inthermos flasks and the whole spike is
immersed in the water. Emasculation with hot water is generally effective in killing all the
duration are used.
pollen grains provided the correct temperature and treatment
emasculation. The
[Link]. Alcohol Treatment. It is not a commonly used method of
inflorescence in alcohol of a suitable
method consists of immersing the flower or the
sweet clover, immersion
concentration for a brief period, followed by rinsing it with water. In
of the inflorescence in 57 per cent alcohol for 10seconds was highly effective; the percentage
selfing was only 0.89. It is a better method of emasculation than the suction method.
of Even a slightly prolonged period
duration of treatment is of utmost importance.
However, the recommended, would greatly reduce seed set.
few seconds more than the
of treatment. say a
organs would also be killed by alonger treatment.
This Is because the female reproductive like hot water treatment, kills pollen grains
[Link]. Cold Treatment. Cold treatment,
In case of rice, treatment with cold water at 0-6°C kills pollen
without damaging gynoecium.
gynoecium. Keeping wheat plants at 0-2°C for
15-24 hours kills the
grains without affecting
 Plant BT
198
thods
set per fruit and
the purpose
flowers, the amount of seed needed, the number of seeds
of its
larger
for which the hybrid seeds are required. In species with relatively
emasculation may be adequate in most hybridization programmes. On the other hand, flowers,
handin
Species with small flowers hand emasculation is generally difficult, iring and time
Onsumng. Similarly, when large quantities of hybrid seed are needed, hand emasculation i
impractical in most self-pollinated crop species, except in those that set many seeds in one
Iruit, e.g., tobacco (about 2,000 seeds per fruit)., tomato, brinjal, etc. Similarly, for accurate
genetic studies, hand emasculation is desirable because with other mnethods there may be
some self-pollination.
Anefficient emasculation technique should prevent self-pollination and result in a high
percentage of seed set on pollination of the emasculated flowers. The etficiency of an
emasculation technique may be tested by bagging the emasculated flowers without
pollination. The amount of seed thus set would indicate the frequency of chance self.
pollination during emasculation, (It is assumed here that the crop species does not show
apomixis. Why ?) If the seeds are to be used in genetic studies, there should be no self.
pollination during emasculation. But in most of the crop improvement programmes and in the
production of hybrid seed, a small amount of self pollination may be permissible. However, it
1sdesirable that self-pollination should be kept to the minimum, preferably to the zero level.
The various techniques of emasculation are briefly outlined below.
[Link]. Hand Emasculation. In species with relatively large flowers, stamens or
anthers are removed with the help of forceps. The exact detailsof the procedure vary from
one crop species to the other. Emasculation is done before the anthers are mature andthe
stigma has become receptive; this minimises accidental self-pollination. Üsually, stigma
receptivity is at its peak during the morning hours when the flowers open, but different crop
species show considerable variation in the duration for which their stigmas remain highly
receptive. Therefore, emasculation is generally done in the evening, e.g., between 4 and 6
P.M., one day before the anthers are expected to dehisce or mature and the stigma is likely to
become fully receptive. Therefore, the flowers selected for emasculation are those that are
likely to open the next morning. With some experience, the breeder should be able to select
such flowers without much difficulty. Generally, it is desirable to remove the
older and the
younger flowers located close to the flower to be emasculated in order to avoid
the identification of crossed fruits/pods, etc. confusion in
A generalised procedure for hand emasculation is as
follows. The corolla of the selected
flowers is opened and the anthers are carefully removed with the help of
many crop species, the androecium is epipetalous, e.g., in fine-tip forceps. In
tomato, potato, bhindi, etc. In such cases, sometimes the cotton, jute, brinjal, sweet potato,
corolla
alongwith the epipetalous stamens. In cereals, one-third of the emptymay be totally removed
off with scissors to expose the anthers. In glumes may be clipped
largeflorets per spikelet are left; the other wheat (T. aestivum) and oats (A. sativa), only twO
all the anthers from the jlowers florets are removed, Care must be taken to remove
without breaking them and, the most
gynoecium must not be injured. The first precaution is necessary to important, ne
while latter is necessary for seed set after the prevent
desired pollination. Further. self-pollination,
the
Bouge the minimum damage to the it is desirable 0
flowers during emasculation.
 201
anther dehiscence falls within the duration of
with the opening of flowers. Anthers stigma receptivity and both generally coincide
withthe species. The pollination generally dehisce during morning; the exact time varies
anthers ofthe male parent and procedure consists of collecting pollen from. freshly dehisced
This may be done in one of the
dusting this pollen onto the stigmas of emasculated flowers.
1 Pollen grains are
following several ways.
collected in a bag, and are used for dusting the
inflorescence, e.g., in maize, bajra, etc., or of emasculated flowers. stigmas of female
9 Mature anthers are collected from the flowers of male
and applied to the stigmas with the help of a parent. The pollen is liberated
camel hair brush, piece of paper, tooth
pick or forceps.
3 Anthers are collected and allowed to burst directly over the
stigmas. In rice, oats,
wheat and barley, one anther is generally inserted into each floret where it dehisces
and covers the stigmas with pollen grains.
4. The spike of male inflorescence is shaken over the emasculated inflorescence just
when the anthers are about to dehisce. AS a result, the exposed stigmas are covered
with [Link] is commonly done in wheat and barley where the lemma and palea
are clipped off to expose the stigmas of emasculated flowers. [Link] palea of
the spike of male parent are also clipped off to expose the anthers, which are used as
the source of pollen.
5. In species like maize, the male inflorescence may be detached and enclosed in the bag
covering the female inflorescence. In case of bajra and jowar, panicles from the male
parent may be enclosed in the same bags that enclose the panicles of female parent.

10.6.7. Harvesting and Storingthe F, Seeds

heads or pods should be harvested and threshed. The seeds should be dried
The crossed
stored to protect them from storage pests. Proper care should be taken to avoid
and properly seeds. The seeds from each cross should be kept
contamination of the hybrid seed with other
kept along with the original tags.
Separately and, preferably, the seeds should be

10.7. RAISING THE F GENERATION

differ for genes affecting seedling characteristics, the parent
If the parentsinvolved in a crosscharacter should be used as the male parent. This would
allow
with the dominant form of the generation. The breeder should take advantage of
seeds in the F
the identification of selfed available inthe cross he has planned.
such marker genes if they are ; . Allard (1960)feels that even 12 E,
regarding the si1ze of
There is some controversy programmes, except the backcross. But Elliot (1958) is of
breeding the only
seeds are enough for most should be as large as possible because F, is
the opinion that the F population for which the twO parents
differ would be in the heterozygous
rare
generation where all the genesthe size of F1, the greater will be the opportunity for is
condition. Thus the larger larger F size is not used because (1) hand crossing
Generally, a unmanageable, (3)
recombinations to occur. (2) with a large F population,the F2 becomes
tedious and time taking,
 treatment. 1 he amount of sale
F 8lans. Cold treatment is less effectivethan hot water
POnation is generallygreater in coldtreatment than that in the case Of not water treatment
[Link]. Genetic Emasculation, Genetic or cytoplas1mic male sterility may be used to
Cminate necessity of emasculation
theemasculation (Chapter 6). Many species are selr-lncompatible, In
such cases, is not necessary because self-fertilization will not take place. In
certain genotypes and under certain environments, the male sterility and
self-incompat
Systems may breakdown partially. However, for commercial hybrid seed production, male
ibilit
sterility is the most feasible method of emasculation. Protogyny facilitates crossing without
y
emasculation. Since the stigmas become receptive before the anthers mature, hand pollination
ensures Seed set from cross-Dollination and prevents self-fertilization, i.e., in bajra (p
glaucum).
[Link]
Immediately after emasculation, the flowers or the inflorescences are enclosed in suitable
bags of appropriate size to prevent random cross-pollination. In cross-pollinated crops, like
maize, the male flowers are also bagged to maintain the purity of pollen used for pollination.
Ihe bags may be made of paper, butter paper, glassine or fine cloth. Butter paper or vegetable
parchment bags are the most commonly used. Cloth bags are generally not preferred since
they permit some degree of chance cross-polination. The bags are tied to the base of
inflorescence or to the stalk of flower with the help of thread, wire or pins designed for the
purpose. The moisture and temperature are generally higher inside the bags as compared to
the outside. Therefore, bagging may promote fungus development on the fruits or the spikes.
This may be prevented by removing the bags usually 2-3 days after pollination after the
danger of cross-pollination is over.

[Link]
The emasculated flowers are tagged just after bagging. Tags are
sizes. In most of the crops, circular tags of about 3 cm available in different
cm are used. In crops like maize (Z. mays), bajra and diameter, or rectangular tags of 3 x 2
cm) are used. The tags are attached to the flower or the jowar (S. bicolor), bigger tags (6 x3
The following information is recorded on the inflorescence with the helo of thread.
tags with a carbon pencil: (1) date of
emasculation, (2) date of pollination, and (3)
name of the female parent is written first, and names
that of
of the female and the male
parents. The
Bdenotes that A is the female the male parent written later, e.g., Ax
is
parent and B is the male parent.
10.6.6. Pollination
The two most important operations that
are determine the amount of seed set in hybridization
emasculation and pollination. During emasculation,
organs must be avoided. And during damage to the female reproductive
male parent should be placed on pollination, mature, fertile and viable pollen from the
fertilization. The duration of pollenreceptive stigma of emasculated flowers to bring about
snecies to another, e.g., a few minutesviability after anther
in wheat and oats to dehiscence varies greatly from one
dvisable that fresh pollen from mature a few hours in maize.
Therefore, 1t
anthers should be used for
pollination.