ABSTRACT

Interspecific cross was attempted between Brassica carinata (BBCC, 2n=34) and B. tournefortii (TT, 2n=20) aimed at broadening the genetic basis of these species and combining their valuable traits in a genotype. Hybrid between these species could be obtained successfully when B. carinata was used as a female parent. Morphologically, the hybrid was intermediate between parent species but its leaves resembled more with B. carinata. The F₁ plant was tall but grew very slowly. It was found to be resistant/ tolerant to white rust and Alternaria diseases. Hybrid was almost sterile showing only 2.3 per cent pollen stainability. Meiotic studies of trigenomic haploid hybrid (BCT, 2n = 27) showed various chromosome configurations including quadrivalents (0-1), trivalents (0-2), bivalents (2-9) and univalents (5-23). An average chromosome association observed at diakinesis/ metaphase I was 0.02 quadrivalents, 0.77 trivalents, 6.69 bivalents and 11.20 univalents. Out of the maximum of nine bivalents observed in the BCT hybrid, at least two could be explained by allosyndesis indicating inter-genomic pairing among these genomes. This indicated the possibility of transferring genes across the species through interspecific hybridization. Trivalents and quadrivalents noticed in hybrid revealed secondary polyploid origin of B, C and T genomes.

KEYWORDS Crossability, interspecific cross, cytomorphology, B. carinata, B. tournefortii

INTRODUCTION

Ethiopian mustard (Brassica carinata A. Braun) has favourable attributes like drought resistance (Kumar et al., 1984), resistance to pod shattering and disease (Alonso et al., 1991) and better performance under saline and late sown conditions (Malik, 1990). These features make it a better oilseed crop for higher productivity and sustainability under biotic and abiotic stress (Raut, 1996). However, due to late maturity and poor quality of both oil and meal, this species does not appear suitable for direct introduction in India.

Wild turnip (B. tournefortii Gouan), grown sporadically in few pockets, has been reported as a good source of tolerance/resistance against aphids (Singh et al., 1965); Alternaria blight & White rust (Yadav et al., 1991); and drought (Salisbury, 1991). However, lower test weight (2-3 g/ 1000 seed) and susceptibility to downy mildew (Rajpurohit and Choudhary, 1995) are the major constraints in spread of this species.

Both B. carinata and B. tournefortii species have a limited genetic variation. Therefore, an interspecific cross between B. carinata and B. tournefortii was attempted to examine crossability between these two species, carry out genomic homology through meiotic analysis in F₁ hybrid and to generate genetic variation.

MATERIALS AND METHODS

The interspecific cross was attempted involving two diverse genotypes of B. tournefortii (RBT 58 and RBT 63) and BCN of B. carinata during winter, 1991-92 at Agricultural Research Station, Mandor. Emasculation and pollination were done as per standard procedure. At least 100 flower buds in each species cross combination were pollinated. Seeds obtained from crosses were grown along their parents for evaluation. Based on pollen fertility and morphological attributes, only one hybrid plant from cross BCN x RBT 58 obtained was selected for further studies.

Pollen fertility of the hybrid was estimated by pollen grain stainability in acetocarmine (1%). For meiotic analysis, young flower buds were fixed in freshly prepared solution of propionic acid: absolute ethanol (1:3) containing ferric chloride as a mordent. After 24 hours the rinsed buds were stored in 70% ethanol until slides were made by the acetocarmine (1%) squash technique. Chromosome associations were observed at diakinesis/ metaphase I.

RESULTS

Crossability

The seed setting was almost double in the cross B. carinata x B. tournefortii as compared to its reciprocal combination (Table 1). Out of 112 flowers buds of B. carinata pollinated with pollen of B. tournefortii, 22 seeds were collected giving rise to a single hybrid plant. On contrary, in reciprocal combination, out of 122 buds of B. tournefortii pollinated by B. carinata pollen, only 11 seeds were obtained which did not yield any true hybrid. The crosses attempted involving RBT 58 as a parent resulted in higher seed set in comparison to cross where in RBT 63 was involved.

Table 1. Per cent hybrids obtained in cross B. carinata x B. tournefortii

Cross combination	Number of					Per cent
	Buds pollinated	Seeds harvested	Hybrid plants obtained	Hybrids/ pollination	of hybrids recovery
B. carinata x B. tournefortii
BCN x RBT 63	57	8	-	-	-
BCN x RBT 58	65	14	1	0.015	1.54
Total/ Mean	112	22	1	0.009	0.89
B. tournefortii x B. carinata
RBT 63 x BCN	53	3	-	-	-
RBT 58 x BCN	69	8	-	-	-
Total/ Mean	122	11	-	-	-

Cytomorphology of F₁ hybrids

Morphology: The phenotype of the hybrid was intermediate between parent species (Fig. 1a). It was tall but grew very slowly and took as many as 95 days to flower and 168 days to mature completely. The size and shape of leaves of the hybrid were more closer to B. carinata (Fig. 1b). They were obovate-lanceolate, petiolate, pinnatified to pinnatipartite with entire to slightly dentate margin, obtuse tip, sparsely hairy and dark green in colour. The flowers were medium in size with pale yellow petals but their shape was similar to B. tournefortii. The F₁ plant exhibited enhancement in values of attributes like primary and secondary branches, main raceme length, siliquae on main raceme, siliqua intensity and siliquae per plant. On the other hand, siliqua size, seeds per siliqua, seed weight and seed yield were recorded much lower in the hybrid as compared to either of the parents. Hybrid plant was found resistant/tolerant to White rust, Alternaria blight and Downy mildew.

Fig. 1a-b. Morhpology of hybrid B. carinata x B. tournefortii, a. F₁ plant and b. lower leaves (from left to right) of B. carinata, the hybrid and B. tournefortii.

Cytology: Amongst 59 pollen mother cells (PMCs) of the hybrid analyzed at diakinesis/ metaphase I, not a single PMC was observed with the expected chromosome configuration of 27 I (Table 2). At least two bivalents were encountered in all the PMCs, but the maximum of nine bivalents were observed in 17 per cent of cells (Fig. 2b). Many of them exhibited chiasmatic configurations (Fig. 2b). Multivalents in the form of trivalents (0-2) and quadrivalents (0-1) were noticed in majority of PMCs (59.4%). Only five univalents and the rest be trivalents and bivalents were observed in one cell. The mode of chromosome association observed was 2 III + 7 II + 7 I (16.9%), 9 II + 9 I (11.9%), 1 III + 8 II + 8 I (8.5%) and 7 II + 13 I (8.5%). On an average, each PMC had association of 0.02 IV + 0.77 III + 6.69 II + 11.20 I .

Fig. 2a-c. Meiosis in pollen mother cells of hybrid B. carinata x B. tournefortii a. Metaphase I (M I), 3 II + 21 I ; b. M I, 1 III + 9 II + 6 I , and c. Anaphase I with bridge-fragment

Table 2. Chromosome associations at diakinesis/metaphase I in BCT hybrid (2n=27) of cross B. carinata (BBCC, 2n = 34) x B. tournefortii (TT, 2n = 20)

Chromosome association	PMCs observed
(Number/PMC)	Number	Frequency (%)
1 IV + 1 III + 6 II + 8 I	1	1.7
2 III + 8 II + 5 I	1	1.7
2 III + 7 II + 7 I	10	16.9
1 III + 9 II + 6 I	3	5.1
1 III + 8 II + 8 I	5	8.5
1 III + 7 II + 10 I	8	13.6
1 III + 4 II +16 I	3	5.1
1 III + 3 II + 18 I	4	6.8
9 II + 9 I	7	11.9
8 II + 11 I	4	6.8
7 II + 13 I	5	8.5
6 II + 15 I	4	6.8
3 II + 21 I	2	3.4
2 II + 23 I	2	3.4
Total number of cells analysed	59	100

The stickiness and delayed terminalization of the chiasmata made it difficult to decide exact nature of the chromosome association observed (Fig. 2a). Further it was not always possible to distinguish between the relatively small bivalents of B and T genome(s) and relatively large univalents of C genome. In majority of the cases, well defined metaphase plate could not be recognized as the bivalents did not congress at equatorial plate but distributed randomly through out the cell. Bivalent and trivalent associations were observed even at late anaphase I. Distribution of chromosomes at anaphase (I/II) was random due to unequal disjunction. Majority of cells had laggards at both anaphase I and II. Bridge-fragment configurations were observed in 12.5 per cent of cells (Fig. 2c). The complete process of meiosis was found irregular. Most of the cells did not divide synchronously, monads and dyads were observed in good frequency. These meiotic irregularities might be the reason for mostly sterile pollen. The percentage of stainable pollen recorded in the hybrid was only 2.3 per cent.

DISCUSSION

Crossability

The hybrid from cross B. carinata x B. tournefortii was obtained only when B. carinata was used as a female parent. This was not anticipated as in the majority of interspecific crosses reported in Brassica, the success was more when the species with the higher basic chromosome number was used as a female (Quazi, 1988). The hybrid B. carinata x B. tournefortii obtained in the present study is in contradiction Harberd theory that interspecific crosses with B. tournefortii were only possible when it was used as a female parent (Harberd, 1976).

The interspecific trihaploid BCT hybrid obtained from cross B. carinata x B. tournefortii has not been reported earlier. The hybrid plant was observed to be intermediate in many phenotypic features of both the parents. It is an advantage, since it would allow better selection for specific attributes in segregating progenies.

Meiotic studies of BCT hybrid (2n=27) attempted for first time, revealed various chromosome configurations including quadrivalents (0-1), trivalents (0-2) and bivalents (2-9). High amount of pairing observed in trihaploid hybrid might be due to auto/allosyndesis pairing within and among these genomes. This might be attributed structural similarities and duplications of some of the chromosome (Robbelen, 1960). Heteromorphic appearance of bivalents in the PMCs observed and presence of only five univalents instead of 27 expected, indicated the extent of homology among B, C and T genomes. Bridges at anaphase I observed in present materials might have resulted from pairing between partially chromosomes of the parent genomes. Such inter-genomic pairing in Brassica hybrid was also observed by Howard (1938).

Based on earlier studies on haploid of genomes B, C and T, a total of seven bivalents were theoretically possible due to autosyndesis in these three genomes (Armstrong and Keller, 1982; and Prakash, 1973, 1974). Hence, out of the maximum of nine bivalents observed in the present hybrid, at least two should be due to allosyndesis among B, C and T genomes. Mizushima (1968) noticed maximum of three bivalents in TC hybrid and opined that one of them might be due to allosyndesis between these two genomes, while Ljungberg et al. (1993) observed maximum of 1 III + 3 II + 10 I in the similar hybrid and suggested the possibility of allosyndetic pairing in T and C genomes. Narain and Prakash (1972) reported maximum of three bivalents in BT hybrid but did not say anything regarding the nature of origin of such bivalents. Sarla and Raut (1991) found a maximum of 1 III + 5 II + 4 I in BC hybrid and demonstrated homology between B and C genomes. Pairing in trihaploid hybrid has assumed to occur mostly between B and C genomes. However, less than ten univalents were counted in 45.8 per cent of total PMCs, some T chromosomes of B. tournefortii might have been involved in the pairing with B and C chromosomes. The occurrence of multivalent association in high frequency in the hybrid studied, indicated that parts of the chromosomes of these genomes possess sufficient affinity to allow multivalent association (Newell et al., 1984). This confirmed the hypothesis of secondary polyploid origin of the B, C and T genomes. Inter-genomic chromosomes association noticed in the hybrid indicated the possibility of transferring genes across the species through interspecific hybridization.

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