Hybrids Seed Production in Cole Crops
Author: Arvind Nagar
Cole crops are an important group of cool season vegetables. Initially, these crops were confined to Europe and other temperate regions. But, after the Second World War, their cultivation has spread to tropics and sub tropics as well. All the members of this group have a common ancestry and that is the reason they are fully cross-compatible with each other. In general, these are highly cross-pollinated crops and show preponderance of non-additive gene action for most of the economic traits. Hence, heterosis breeding has turned out to be of more relevance. The genetic phenomena of sporophytic self incompatibility and male sterility (particularly cytoplasmic male sterility) have proved instrumental in commercialization of hybrid seed production.
The term self-incompatibility refers to the partial or complete incapacity of a plant, producing functional gametes, to set viable seed on self pollination. Alternatively, it is the failure of the genetically identical gametes to unite in fertilization. In cole crops, the sporophytic self-incompatibility (SSI) exists wherein the incompatibility reaction is governed by the genotype of the pollen producing plant and it is controlled by a series of alleles at a single locus 'S'. SSI system was confirmed in kale (Thompson, 1957), sprouting broccoli (Sampson, 1957), cabbage (Adamson, 1965) and cauliflower (Hoser-Krauze, 1979). However, its use in cabbage hybrid seed production had been demonstrated much earlier (Odland and Noll, 1950).
Basic steps in the use of SSI
a) Identification of self-incompatible plants in diverse population/ genotypes.
b) Development of homozygous self-incompatible lines.
c) Identification of S-alleles in the homozygous self-incompatible lines.
d) Establishment of inter-allelic relationships among the S-alleles.
e) Identifying the best combining lines.
f) Maintenance of parental self-incompatible lines.
g) Commercial hybrid seed production.
Identification of self-incompatible plants is carried out by making different types of pollinationviz., self pollination in freshly opened flowers (OP), compatible pollination with unrelated S-allele pollen (CP) and bud pollination to ascertain whether the plant in question is male and female fertile and also to get selfed seeds to raise its progeny. The plants are categorized as self-incompatible/compatible based on seed set in OP in relation to CP (watts, 1963). For quicker (within 24-48 hours of pollination) identification of self-incompatible plants, fluorescence microscopy can be used (du Crehu, 1968; Vidyasagar and Chatterjee, 1984). Wallace (1979) suggested the use of seed set as well as pollen tube penetration (fluorescence microscopy) data.
Homozygous self-incompatible lines are developed by making intra-progeny crossings/pollinations in freshly opened flowers in the progeny of a self-incompatible plant in full diallel by utilizing at least 7 progeny plants (Mackay, 1977). The intra-progeny crossings/pollinations reveal heterozygous nature of the original self-incompatible plant. After grouping the progeny plants on the basis of compatible/incompatible reaction, we get at least two (one homozygous and one heterozygous) or all the three (both homozygous and one heterozygous) phenotypic groups. By selecting plants from the homozygous groups, homozygous lines could be developed in the next 2-3 generations.
Information about the inter-allelic relationships among the S-alleles is important to ascertain their level of dominance and S-allele interactions. The S-alleles high in the dominance series are likely to give minimum selfs and sibs in hybrid seed. S-allele interactions (in pollen and stigma) are worked out by using the data (seed set as well as pollen tube penetration) obtained from the pollinations carried out in freshly opened flowers reciprocally between a heterozygote (SxSy) and its two corresponding homozygotes (SxSx and SySy). Four types of S-allele interactions viz., type I (same S-allele dominant over the other in both pollen and stigma), type-II (one S-allele dominant over the other in pollen but codominant in stigma), type-III (one S-allele dominant over the other in stigma but codominant in pollen) and type-IV (both S-alleles codominant in pollen as well as stigma) have been reported.
In order to have heterotic hybrids, it is essential to identify the best specific combining S-allele lines. This can be ascertained from the SCA studies and per se performance. Self-incompatible lines can also be combined with self-compatible lines but the hybrid seed quantities shall be lesser since the hybrid seed will be harvestable on the plants of self-incompatible lines only.
Maintenance of self-incompatible lines is a costly affair. The various methods being followed are large scale seed production through manual sib mating in bud stage, carbon dioxide gas treatment (3-5% conc.) for 8-24 hours at 100% relative humidity in air-tight growth chambers and tissue culture by using meristem as explant. Of late, sprays of sodium chloride (3-5%) have been reported to be effective in temporary breakdown of self-incompatibility (Kucera, 1990; Yang et al., 1995 and Kucera et al., 2006). This method is being followed at IARI to produce the seeds of the parental self-incompatible line ScSc which is one of the parents of Pusa Hybrid -2 of cauliflower (personal communication).
Commercial hybrid seed production is done by way of developing single cross, three-way cross or double cross. In single cross, two self-incompatible but cross-compatible best combiners are planted in alternate rows in isolated plots. The hybrid seed is harvested on both the lines. In three way cross, one single cross and a self-incompatible line are planted in alternate rows. Similarly in double cross, two single crosses are used. In USA, for hybrid seed production of cabbage, top cross is being used. For every 2 or 3 rows of a self incompatible line, one row of a good open pollinated (OP) cultivar as a pollen parent is provided. However, the hybrid seed is harvested from the self incompatible plants only.
The main problems being faced in hybrid seed production are depression in S-allele lines by continuous inbreeding, pseudo-compatibility, the effect of environmental factors on the level of self-incompatibility and higher proportion of selfs/sibs in hybrid seed due to lack in proper synchronization of flowering. These could be managed by resorting to vegetative propagation, using the S-allele lines which behave stable under diverse environments and selecting the parental lines which have perfect synchronization in flowering.
Male sterility is defined as the deviant condition in normally bisexual plants (monoecious as well as hermaphrodite) when no viable pollen is formed. Genic male sterility which is governed by recessive nuclear genes has been reported in cabbage (Rundfeldt, 1960), cauliflower (Nieuwhof, 1961) and other members of the cole group as well. In view of the problems associated with the maintenance of genic male sterile lines, its commercial use has been rather limited. In cauliflower, it has been utilized for hybrid production by maintaining the male sterile lines clonally (Grout, 1988).
Pearson (1972) reported male sterility controlled by Brassica nigra nuclear-cytoplasm interaction but this could not be used on commercial scale due to partial opening of petals and less developed nectar glands leading to poor bee-activity. Ogura male sterility (Ogura,1968) was reported in Japanese radish. No restorer nuclear genes could be found in Japanese genotypes but the same could be obtained in European radish. Bannerot et al. (1974) could introduce cabbage nucleus into the cytoplasm of Ogura male sterile radish (R-cytoplasm) by repeated back crossing. This CMS in cabbage had the problem of chlorosis at low temperature (below 120C). This drawback was overcome by replacing radish chloroplasts with Brassica oleracea chloroplasts through protoplast fusion by Robertson et al. (1987). Sigareva and Earle (1997) standardized the protoplast fusion procedure to transfer desirable male sterility cytoplasm from broccoli into cabbage much more rapidly than conventional back cross method. No fertility restorer genes have been found for R-cytoplasm (Ogura) induced male sterility in cole crops (Brassica oleracea). Ogura-CMS is stated to be a mitochondrial DNA encoded male sterility. This implies that all Brassica spp. including cole crops act as maintainers of R-cytoplasm (Ogura) induced male sterility. Fang et al. (1997) have also reported the presence of dominant male sterile gene in cabbage. Its usage may not be preferred over R-cytoplasm (Ogura) induced male sterility because of the possible problems associated in maintaining the male sterile lines in homozygous dominant condition. R-cytoplasm (Ogura) induced male sterility already present/introduced in any genotype of a cole crop can be transferred into the desired genetic background through back cross method.
Basic steps in the use of CMS
1. A-line (female parent) of desired genetic background. (genetically S-msms).
2. B-line (maintainer): This is an isogenic line in the genetic background as that of A line and its function is in the maintenance of A-line (genetically N-msms).
3. C-line (male parent): This is a male parental line and also the best specific combiner with A line. Genetically this could be N/S-Ms/- (fertility restoration) or N-msms since the economic product in cabbage and cauliflower is not the true seed.
4. Maintenance of A, B and C lines.
5. Hybrid seed production.
The first increase of the seeds of the lines A, B and C must be in insect proof cages/structures followed by an increase in the open but not more than twice to obtain a high degree of purity in the stock seeds of the parental lines used for the production of F1 hybrid seeds. The seeds of lines A and B can be produced in the same cage while the line C must be grown separately.
Hybrid seed production on commercial scale is carried out in the open by providing recommended isolation distance of at least 1000m. Usually for every 2 or 3 rows of A-line, one row of C-line is planted. The lines A and C must have perfect synchrony in flowering for good pollination and seed set. The hybrid seed is harvested from the plants of A-line only.
The main problem being faced is the lower quantity of hybrid seed on account of honey-bees preference for the pollen fertile C-line. This could be overcome to a certain extent with appropriate flower morphology and manipulating the ratio of plants of A and C lines, spacing and planting design. Inbreeding depression is another problem which results in low seed production of inbred parental lines and also that of single cross hybrids. Three-and four way (double cross) crosses may prove economic.
Self-incompatibility versus cytoplasmic male sterility
Both the genetic phenomena have their own merits and demerits. The use of self-incompatible lines ensures the harvest of hybrid seed on all the plants in the hybrid seed production block. In CMS system, the hybrid seed will be harvested only from 67-75% of the plants in the hybrid seed production block. The latter phenomenon (CMS) has been considered better in avoiding selfs in the hybrid seed. However, in CMS lines also, some pollen production has been noticed especially at relatively higher temperatures nearing end of flowering stage thereby creating conditions for the possible selfs in the ultimate hybrid seed in this system as well. In my opinion, both the genetic systems (SSI and R/Ogura-CMS) will prove equally good provided the S-allele and CMS lines are tested over space and time in variable weather conditions for their stability and consequent use in the hybrid seed production programme.
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Ph D student, IARI, New Delhi
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