In this article we will discuss about the purposes and techniques of hybridization in plants.
Purpose of Hybridization:
The objectives of hybridization can be categorized under 3 major criteria:
1. Development of productive varieties:
One of the objectives is to create new variability which is further used for development of improved varieties with respect to existing varieties.
This is achieved through the following methods of breeding or selection of the segregating progeny of F1 hybrid:
(a) Pedigree method
(b) Mass-pedigree method
(c) Bulk progeny
(d) Bulk population breeding.
2. Removal of Bottle-Neck Genes:
The appearance of new problems (pathogen, insect) to yield can be protected through the creation of new recombinants which will help to come out from this kind of situation.
3. Development of Heterotic F1 Hybrids:
In order to exploit hybrid vigour, hybridization between two chosen inbred parents can be resorted to produce F1 hybrids. In case of self-pollinated crops two methods of breeding are used for attaining this objective.
(a) Combination Breeding:
The main objective of this method is to transfer one or more characters into a single variety from other varieties. In this breeding the genetic divergence between the two parents is not the major consideration, here among the two parents one must have the character to be transferred and another parent is the popular variety.
(b) Transgressive Breeding:
This method aims at improving yield or contributing characters through transgressive segregation. It is the production of plants in the F2 generation which will be superior to both the parents for one or more characters.
Assembly of all productive genes in transgressive progenies requires the following genetic situations:
(i) The character must be polygenic;
(ii) Parents should be completely homozygous;
(iii) Parents should be complementary to each other;
(iv) There should not be any linkage between the chosen characters.
General Technique of Hybridization:
Before the beginning of any hybridization programme the breeder should decide the objective of the programme.
The whole hybridization programme involves the following procedures (Fig. 5.1):
(a) Choice of parents
(b) Crossing schedule
(c) Emasculation
(d) Bagging
(e) Tagging
(f) Pollination
(g) Harvesting and storage of F1 seed.
Choice of Parents:
Parents which are chosen for hybridization provide the requisite variability for isolating desirable segregants in subsequent generations.
The following criteria are essential for choosing parents for hybridization:
(i) Agronomic Base:
Any well-established local cultivar used as seed parent or female parent should have agronomic base for that area.
(ii) Complementary Pollen Parent:
The male parent should be complementary to the female parent, i.e., it must possess the complementary attributes in their intense form.
(iii) Homozygosity of Parent:
The parent chosen for hybridization must be homozygous for the character, i.e., pure-line. Though there may be cryptic variability.
(iv) Multiple Parentages:
When the desired attributes are not present in the above two parents, more than two parents can be chosen for crossing.
(v) Combining Ability:
Experimental evidences suggest that cross-combinations involving high and low combiners (H x L) or both high combiners (H x H) often provide better opportunity for superior recombinants.
(vi) Genetic Divergence:
Choice of parents based on genetic divergence will be helpful to get the divergent parents in segregating generations. In conclusion, the breeder should have a well-defined, clear cut objective, based on the present and expected future needs, in developing a new variety. Breeder should select the parents accordingly and use them in a suitable hybridisation programme.
Crossing Schedule:
Crossing schedule entails development of hybrids by emasculation and pollination which helps in increasing the spectrum of variation among the hybrids, this heterogeneity is narrowed down by developing homozygous pure line.
Type of Crosses:
The parents involved in hybridization should belong to the same species; they may be of two strains, varieties or races of the same species. When only two parents are crossed to produce the F1 then it is called simple cross; the F1 hybrid may be selfed or back crossed with homozygous parent.
More than two parents are also crossed to produce the F1 hybrid, which is then used to produce F2 or is used in a back cross. Such a cross is also known as convergent cross because this crossing programme aims at converging, i.e., bringing together genes from several parents to a single hybrid. This is the complex cross where multiple convergences are attempted.
Number of Crosses:
How many (single, double or multiple) crosses should be developed would depend upon the objectives defined for the breeding programme, floral biology of the crop and the quality of parents chosen. Based on the recent concept of genetics, the correct choice of parents can cut short the number of crosses to develop the wanted hybrid.
Emasculation:
In case of hybridization programme for self-pollinated crops, it is very much essential to emasculate the plant to avoid the self-pollination. Removal of male sex organs or killing that part of the flower without any damage or disturbances to female reproductive organ is known as emasculation.
Before going to hybridization programme the efficiency of emasculation technique may be tested by bagging the emasculated flowers without pollination. The amount of seed thus set would indicate the frequency of self fertilisation occurring during emasculation.
There are various techniques of emasculation:
(i) Hand Emasculation:
A General Procedure for Hand Emasculation is as follows:
(a) Emasculation is done before the anthers mature and the stigma has become receptive to minimise self-pollination.
(b) The corolla of selected flowers is opened with the help of fine tip forceps and the anthers are removed.
(c) In case of epipetalous stamen the corolla lobes are removed carefully keeping the gynoecium not injured at all.
(ii) Suction Method:
This method is useful in case of small flowers where hand emasculation is not possible. The petals are generally removed with forceps exposing the anthers and stigma. A thin rubber tube or glass tube attached to a suction hose is used to suck the pollen grains from its surface.
The suction may be produced by an aspirator or by a small suction pump. Care must be taken that suction should be enough to suck the stamen and pollen grains but not the gynoecium. But this method is not very efficient, as 15% of self-pollination takes place. Washing the stigma with water may also reduce self-pollination.
(iii) Hot Water Emasculation:
Pollen grains are more sensitive to hot water than the female reproductive organs. So treatment with hot water at particular temperature and for fixed time period is helpful for killing the pollen grains without damaging the female organ.
Treatment with water at 42-48°C for 10 minutes is effective in jowar, treatment at 40-44°C for 10 minutes is effective in rice; in both the cases the whole spike is immersed in thermo-flask containing hot water.
(iv) Alcohol Treatment:
It is not very popular method; a particular concentration of alcohol is used for a fixed time period to kill the pollen grains. But a little bit more exposure, i.e., few seconds more than the recommended time period will reduce the female receptivity, i.e., seed set, as female organs would also be killed by this treatment.
(v) Cold Treatment:
Like hot water treatment, cold treatment can also kill pollen grains without damaging the gynoecium. Keeping rice plant at 0-6°C kills the pollen grains, and also wheat plants at 0-2°C for 15-24 hrs. kills the pollen grains. But cold treatment is less effective than hot treatment.
(vi) Genetic Emasculation:
Genetic or cytoplasmic male sterility may be used to eliminate the necessity of emasculation, i.e., male sterile plants are naturally emasculated. For self-incompatible species emasculation is not necessary, but in certain genotypes male sterility can be induced by gene manipulation in cytoplasmic or nuclear genome.
Bagging:
Immediately after emasculation, the flower or the inflorescence is enclosed in suitable bag to prevent random cross pollination. The bags may be made up of paper, butter paper or parchment paper, and tied at the base of the inflorescence with thread, pin or wire. As the moisture and temperature become higher within the bag so fungus may develop, which may be prevented by removing the bag after 2-3 days after pollination?
Tagging:
The emasculated flower or inflorescence is tagged after bagging. The tags are made up of light weight tin-plate and are written in carbon pencil.
The tag should bear the information:
(a) Date of emasculation;
(b) Date of pollination;
(c) Names of female and male parents, consecutively in the cross.
Pollination:
Mature, fertile and viable pollen from donor plant (Male parent) should be placed on the receptive stigma to bring about fertilisation. During pollination pollen viability is a major factor and also is the time duration for receptivity of stigma. Both timing should match during pollination. The detail procedure for pollination varies in case of different crops.
Harvesting and Storing F1 Seeds:
The seeds are harvested from crossed heads or pods. The seeds should be dried and properly stored to protect them from storage pests. Proper care should be taken to avoid contamination of the hybrid seed with other seeds. The seeds from each cross should be kept separately and preferably, the seeds should be kept along with the original tags.
Raising F1 Generation:
In a hybridization programme it is expected that the F1 generation will be in heterozygous condition. The size of the F1 generation is a crucial matter, large F1 population is difficult to handle, but the large F1 population has the greater opportunity of rare recombination to occur. Usually F1 is allowed to self-pollinate to get the F2 but in back cross programme it is crossed to one of the parents.
Selfing:
The technique of selfing varies from one crop to the other depending upon their mode of reproduction. In self-pollinated crops selfing is not very troublesome as it is the natural mode of reproduction. But in cross pollinated crops the flowers are bagged to prevent cross pollination. If the inflorescence bears bisexual flowers then bagging is very much helpful for self-pollination also.
Consequences of Hybridization:
Segregation and recombination of genes would produce several new genotypes in F2, in addition to parental types. The number of recombinant genotypes increases rapidly with the increase in number of segregating genes. The Fig. 5.2 indicates the reduction in heterozygocity by 50% in each successive generation on selfing.
