Mechanisms for Hybrid Seed Production | Vegetable Breeding

After reading this article you will learn about the mechanism for hybrid seed production.

This is the most simple mechanism of producing F₁ hybrid seed provided:

(i) Emasculation and pollination are easy to perform.

(ii) More seeds per pollination are produced.

(iii) Price of hybrid seed is economical in relation to the cost of manual hybrid seed production.

(iv) Seed rate is low.

Accordingly, this method is in use in hot pepper tomato, eggplant, capsicum, okra, cucurbits, maize, etc.

Genetic Male Sterility:

Genetic male sterility has been discovered in several crops like cotton, carrots, tomato, chilli, muskmelon, winter squash, pumpkin, watermelon, pigeonpea, wheat, corn, sorghum, etc. The male sterility gene usually is recessive and the male sterility may be due to pollen abortion, failure of anther dehiscenee, anther abortion, pistillody of the anthers or several other possible causes.

Using genetic male sterility to produce hybrids on wider scale does not eliminate all hand labour. Through a combination of crossing, selfing and collecting seed set on sterile plants, a population can be created which segregates in 1:1 ratio for Msms.

Msms individuals as given below:

Now, onwards the seed set on msms will always segregate into 1 fertile: 1 sterile. This stock seed will be used as the female parent in hybrid seed production. However, 50% male fertile plants will have to be rogued from the seed production rows prior to pollen shedding.

With crops in which male sterile plants cannot be detected until pollen is shed and stigma receptivity and pollen shedding are simultaneous, commercial production of hybrid seed using genetic male sterility is nearly impossible.

In such cases it helps to have a marker gene closely linked to the male sterility gene so that the male fertile plants could be detected prior to anthesis and rogued out. This technique, so far has not got wider acceptance and commercialisation, except in chilli.

Cytoplasmic Genetic Male Sterility:

Cytoplasmic genetic male sterility results from the interaction of cytoplasm with nuclear genes. The potential of CMS for F₁ hybrid seed production was first realised following the discovery of a male sterile plant in a Californian crop of the onion cv Italian Red by H.A. Jones and S.L.Emsweller in 1936.

Subsequently its inheritance was determined by H.A. Jones and A.E. Clarke in 1943. The cytoplasmic factor S interacts with recessive nuclear gene ms and produces a male sterile line of the genotype Smsms which is male sterile and is known as ‘A’ line in the hybrid seed production programme.

The genotype with normal cytoplasm is known as Nmsms and is male fertile. This is referred to as ‘B’ line and is used as a maintainer for the male sterile line, Smsms (A line). After repeated backcrossing, the male sterile ‘A’ line and the maintainer ‘B’ lines become almost isogenic. To produce hybrid seed the ‘A’ line is inter-planted with the pollinator or ‘C’ line of the genotype NMsMs.

These steps are demonstrated as follows:

The CMS system can easily be used to produce seed of single, double or 3-way hybrids as given in Fig. 6.4 following Riggs (1988)

Although discovery of cytoplasmic genetic male sterility in onion by Jones and Clarke has been instrumental in application of this system in hybrid seed production on large scale in several crops, particularly the 4 crops, namely, onions, pear millet, sunflower and grain sorghum, and now rice the contributions of F.V. Owen, M.M. Rhoades, L.M. Josephson, M.T. Jenkins, H.L. Everette, P.C. Mangelsdorf, J.S. Rogers, J.R. Edwardson, J.C. Stephens and R.F. Holland have been of considerable significance to provide several vital necessary details for overall application of CMS towards hybrid seed production.

Self-Incompatibility:

The term ‘self-incompatibility’ is defined as inability of a plant to set seed when self-pollinated, even though it can form normal zygotes when cross-pollinated and its pollen can fertilize other plants. According to Frankel (1973) self-incompatibility is of wide occurrence in many species of flowering plants. The genetic control of SI may be gametophytic or sporophytic.

The gametophytic incompatibility is controlled by pollen grain, whereas in the sporophytic system the reaction is between the exine of the pollen grain, which is sporophytic in origin and the papillae of the stigma. This incompatibility is determined by the diploid nucleus of the sporophyte.

In other words, the behaviour of each pollen grain is determined by the diploid genotype. As a result, patterns of inter-allelic interaction ranging from independence to complete dominance may occur in both pollen and style. All kinds of brassicas have a sporophytic SI system being strongest in kale and weakest in summer cauliflower. The S-allele system is complex with about 50 alleles at a single locus.

It is known that glycoproteins have an important role in causing self-incompatibility. Increased rate of synthesis of these S- locus specific glycoproteins coincides with the onset of the incompatibility reaction.

Therefore, while developing parental inbred lines this reaction is overcome by pollinating the flowers in the bud stage during selfing. The commercial F₁ hybrids are produced by inter-planting 2 self-incompatible but cross-compatible inbred lines. The inbred lines are maintained through bud-pollination.

A simple system is shown below:

Thompson (1964) recommended production of ‘triple-cross’ hybrid kale, a hybrid type which can be produced only with the sporophytic SI. The triple-cross is a hybrid between 2 three- way crosses, that is (ABxC)x(DExF).

With this type of cross there is a minimum of bud- pollination (needed to maintain inbred lines), and the simple cross, three-way cross and triple-cross can also be made without emasculation if proper self-incompatibility alleles are present in the 6 inbred lines. If kale has gametophytic system of incompatibility, the triple-cross could have been impossible because some plants in each three-way cross could be cross compatible.

In cole crops (Brassica oleracea L), hybrid seed production makes use of the sporophytic incompatibility mechanism. Due to the great number of alleles, cabbage populations, generally, consist of plants that are heterozygous at the incompatibility locus. With bud pollinations, inbred lines may be developed from such a population.

If inbred lines with the constitution S₁ S₁ and S₂ S₂ which have been tested for specific combining ability, are produced by bud-selfing and grown next to one another, seeds only with the allelic combination S₁ S₂ will be formed.

This is due to the fact that the S₁ pollen cannot fertilize any egg cells of the S₁ S₁ plants, nor can S₂ pollen fertilize any egg cells of the S₂ S₂ plants. To prevent occurrence of pseudo-fertility and pollination of parent lines by selfing during hybrid seed production, it is necessary to develop inbred lines with strong dominant alleles.

If the seed production after a single cross is too low and therefore, uneconomical, double crosses can also be produced according to the following scheme:

As a result of the pseudo-fertility, e.g. due to high temperatures or late flowers, pollination within parental lines leading to the development of selfed or sibbed seeds, must always be taken into account. Depending upon the cole crop species, the plant material, environmental conditions, and modifying genes, their proportion may amount to as high as 40%.

In addition to this, mutations leading to self-fertility alleles S_F occasionally, occur, rendering the self-incompatibility mechanism ineffective. “Wrong” alleles, which enter the population uncontrolled, may considerably disturb the developed system. Therefore, a strict supervision of the self-sterility in the developed material is indispensable.

Seed companies are forced to ascertain the sib proportion in each hybrid seed lot which must not be more than 5% in EC countries and in the USA. In general, this is done by growing a representative seed sample under field conditions until the sib portion can be positively recognized.

Experimental results of isozyme analysis for manifestation of the proportion of sibs in cole crop hybrids have been reported in several publications. Application of recessively inherited wax-lessness, which has been found in kohlrabi, has also been suggested for determination of sibs in hybrid seed lots as a marker.

Manipulation of Sex Expression:

Production of hybrids in certain crops like cucurbits is possible through manipulation of sex expression. Cucumber plants are typically monoecious producing male and female flowers on the same plant. Now gynoecious lines are available. These lines produce only female flowers and can be easily used as female parent to produce F₁ seed of cucumber on large scale.

Multiplication of such lines is made possible by induction of male flowers through spraying of gibberellic acid i.e., GA 4/7 (2000 ppm) or Ag NO₃ on seeding leaves. This has been possible through the efforts of L.M. Pike, G.E. Tolla and C.E. Peterson.

On the other hand, monoecious lines of squash can be rendered female by applying ethephon to suppress male flower production as demonstrated by S. Shannon and R.W. Robinson in 1979. Two applications of ethephon @ 600 ppm at the 2-and 4-leaf stage result in complete male flower suppression during the fruiting stage. Ethephon is being used on commercial scale for production of hybrid seeds in squash.

Chemical Hybridizing Agents (CHAs):

The CHAs are defined as the chemicals which cause pollen abortion and render the treated plants male sterile without affecting ovule fertility. These chemicals are also called as gametocides. To be really useful in F₁ hybrid seed production, CHAs should have no mutagenic effect, be easy and economical to apply, have wider applications and have no harmful side effects.

Although CHAs have relatively larger application in major cereal crops, their use in vegetables is yet to be commercialized. Positive responses have been achieved with GA3 and GA 4/7 in lettuce and onion, with maleic hydrazide in tomato and onion, and with sodium 2, 3-dichloroisobutyrate in tomato as reported by J. Sneep and his colleagues in 1979.

Biotechnological Approaches in Hybrid Seed Production:

The conventional F₁ hybrid seed production systems have depended on hand emasculation, genetic, cytoplasmic genetic male sterility and self-incompatibility, etc. In most cases these systems have some disadvantages, involving cost, efficacy, ease of use, reliability and effect of environmental factors leading to breakdown of the systems.

Plant breeders have therefore, looked for improvement to these systems and for completely new systems which may ideally be suitable for any species. The first transgenic male sterility system was developed by Mariani and coworkers (1990, 1992) in tobacco and rapeseed (Brassica napus). They had expression of a bacterial gene, barnase (ribonuclease coding gene from Bacillus amyloliquefaciens) under the control of TA-29 promoter (derived from tobacco).

This system led to the production of cell-toxic ribonuclease enzyme in the tapetal cells (which nourish cells that develop into male gametes), thereby causing male sterility. For technical reasons, mother gene, bar conferring resistance to herbicide glufosinate was hooked to the barnase cytotoxic gene.

This gene cassette helps in retaining only male sterile plants from mixed progenies in commercial hybrid seed production plots. For commercial crop production, the F₁ hybrids have to bear normal bisexual flowers. To restore male fertility in the F₁ hybrids, they used barstar gene from the same bacterium, B. amyloliquefaciens. The product of barstar forms a complex with barnase enzyme and nullifies the toxic effect of Rnase.

Incorporating barstar in the male parent which can be used to pollinate barnase carrying male sterile, but female fertile plants provided a perfect system for commercial hybrid seed production. This system has been patented by M/s Plant Genetic Systems (PGS) Ltd., Belgium.

In 1994, Proagro Seed Company Ltd. (PSCL) made a joint venture company with Plant Genetic Systems, Belgium to develop transgenic plants in mustard (Brassica juncea) and in vegetables. The technology of Plant Genetic Systems known as Seed-link technology was already tested in B. napus in Canada.

Proagro Seed Company has converted B. juncea lines with the help of transgenes, barnase, barstar and bar, and stabilized the hybridisation system in mustard to produce hybrid seed on commercial scale. The limited field study trials for environmental and food safety as per the requirements of Department of Biotechnology (DBT) were conducted in India but commercial release did not materialize.