ABSTRACT

Crosses among three Brassica species viz., B. campestris (AA, 2n = 20), B. juncea (AABB, 2n = 36) and B. napus (AACC, 2n = 38) were attempted with the objective to find out crossability among them, analyses chromosome association in interspecific hybrids, generate genetic variability and combine desirable characters in a targetted genotypes/species. The crossability of B. campestris with B. juncea and B. napus showed higher success when amphidiploid species were used as a female parent. B. napus showed higher cross compatibility then B. juncea with B. campestris. The hybrids, in general, were vigorous and intermediate in morphological attributes. The meiotic studies in F₁ plants of hybrids AAB (B. juncea x B. campestris), AAC (B. napus x B. campestris) and AABC (B. juncea x B. napus) exhibited ten bivalents in majority of PMCs analyzed. It suggested pairing between chromosome of A-A genomes derived from each of parent species. Maximum chromosome association of 11 II in AAB, 12 II in AAC and 14 II in AABC hybrids and the presence of multivalent associations were attributed to the auto- and allosyndetic nature of pairing within and among A, B and C genomes.

INTRODUCTION

Indian mustard (Brassica juncea L. Czern & Coss) is the dominant species covering around 85 per cent of area under rapeseed-mustard in India while rest of the area covered by three ecotypes of B. campestris L. namely Brown sarson, Yellow sarson and Toria (Prakash and Chopra, 1996). Both the species are well adopted to drier conditions and mature earlier than other species (Kimber and McGregor, 1995) but the available varieties of these species do not have a plant type which can be exploited to achieve a substantial increase in yield under intensive cultivation (Prakash and Raut, 1983). Secondly, they are susceptible to aphids, Alternaria blight and White rust. Both of the species have a limited genetic variation for resistance to these factors (Kumar et al., 1997). On the other hand, B. napus L. (rape or rapeseed) known for its higher yield potential in favourable environments (Mendham and Salisbury, 1995) is characterized by higher oil content, better oil quality, resistance against White rust, Alternaria blight and Downy mildew (Landge and Khalatkar, 1996). However, it suffers from high photosensitivity, late maturity, pod shattering and drought susceptibility problems and needs extensive alternation to rectify the undesirable attributes for its wider adaptability in Indian conditions.

All the three species under consideration have both desirable characteristics and deficiencies. Interspecific hybridization is the way which could combine the valuable features of parental species into the hybrid. The present study was attempted to examine crossability and genomic homology between B. campestris, B. juncea and B. napus.

MATERIALS AND METHODS

The interspecific crosses were attempted involving three genotypes each of B. juncea (RSM 98, RSM 151 and RSM 152), and B. campestris (BCBS of Brown sarson, BCT of Toria and BCYS of Yellow sarson) and BN 15 of B. napus during winter, 1991-92 at Agricultural Research Station, Mandor. Seeds obtained from crosses were grown in field along with their parents for evaluation. Based on pollen fertility and morphological attributes, seven hybrids viz., RSM 151 x BCT & RSM 152 x BCYS of combination B. juncea x B. campestris, BN 15 x BCBS, BN 15 x BCT & BN 15 x BCYS of combination B. napus x B. campestris and RSM 98 x BN 15 & RSM 152 x BN 15 of combination B. juncea x B. napus were selected for further studies.

Pollen fertility of the hybrids 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 and slides were made by the acetocarmine (1%) squash technique. Chromosome associations were studied at diakinesis/metaphase I.

RESULTS

Hybridization and crossability

B juncea x B. campestris: Hybrids in this cross were obtained in both the directions, frequency being higher when B. juncea was used as a female parent. As many as 18 F₁ plants were obtained from 301 pollinations in cross B. juncea x B. campestris, while the lone hybrid grown from 316 pollinations in the reciprocal cross did not survive till maturity (Table 1). The frequency of hybrids varied with the cultivars of B. juncea as well as the form of B. campestris. The cross B. juncea cv. RSM 152 x B. campestris var. Yellow sarson cv. BCYS gave maximum hybrid plants (22%) while not a single hybrid was recovered in cross RSM 151 x BCYS. Similarly, in cross B. juncea cv. RSM 151 x B. campestris var. Toria cv. BCT, 14 per cent plants were hybrid while not a single hybrid was obtained when RSM 152 used as one of the parent.

B napus x B. campestris: Hybrids in this cross were more frequent when B. napus was used as a female parent. Out of 55 seeds obtained by crossing 161 buds of B. napus with pollen of Yellow sarson, Toria and Brown sarson forms of B. campestris, 19 hybrid plants were recovered. Whereas, in reciprocal cross, out of 26 seeds produced from 173 pollinations, only two infirm hybrid plants were obtained. Amongst the forms of B. campestris involved in the cross combinations Yellow sarson gave maximum hybrid plants (18%) followed by Brown sarson (11.32%) and the least (6.90%) with Toria (Table 1). Secondly, all the F₁ plants of cross B. napus x B. campestris were healthy and survived till maturity, whereas the hybrid plants raised from reciprocal cross were weak, grew slowly and died before blooming.

B. juncea x B. napus: From 62 seeds obtained through 157 pollinations in cross B. juncea x B. napus, 21 plants were true hybrids (Table 1). It was the most successful cross with mean of 13.38 per cent hybrid recovery. As regards varietal differences, the efficiency percentage was much higher when female parent used was RSM 152 (24%) in comparison to RSM 98 (17.65%). In reciprocal combination, out of 24 seeds produced from 153 pollinations, only two F₁ plants recovered were infirm which died during the vegetative phase.

Table 1. Per cent hybrids recovery in crosses attempted among Brassica species

Cross combination	Number of				Per cent
	Buds pollinated	Seeds harvested	Hybrid plants obtained	Hybrids/ pollination	of hybrids recovery
B. juncea x B. campestris
RSM 151x BCBS	58	9	-	-	-
RSM 151 x BCT	50	19	7	0.140	14.00
RSM 151 x BCYS	46	13	-	-	-
RSM 152 x BCBS	53	21	-	-	-
RSM 152 x BCT	44	0	-	-	-
RSM 152 x BCYS	50	25	11	0.220	22.00
Total / Mean	301	87	18	0.060	5.98
B. campestris x B. juncea
BCBS x RSM 151	52	18	-	-	-
BCBS x RSM 152	43	0	-	-	-
BCT x RSM 151	55	3	-	-	-
BCT x RSM 152	49	0	-	-	-
BCYS x RSM 151	50	3	1*	0.020	2.00
BCYS x RSM 152	47	9	-	-	-
Total / Mean	316	33	1*	0.003	0.32
B. napus x B. campestris
BN 15 x BCBS	53	18	6	0.113	11.32
BN 15 x BCT	58	14	4	0.069	6.90
BN 15 x BCYS	50	23	9	0.180	18.00
Total / Mean	161	55	19	0.118	11.80
B. campestris x B. napus
BCBS x BN 15	67	9	-	-	-
BCT x BN 15	55	10	-	-	-
BCYS x BN 15	51	7	2*	0.039	3.92
Total / Mean	173	26	2*	0.012	1.16
B. juncea x B. napus
RSM 98 x BN 15	51	26	9	0.176	17.65
RSM 151 x BN 15	56	13	-	-	-
RSM 152 x BN 15	50	23	12	0.240	24.00
Total / Mean	157	62	21	0.134	13.38
B. napus x B. juncea
BN 15 x RSM 98	42	8	-	-	-
BN 15 x RSM 151	57	5	1*	0.018	1.75
BN 15 x RSM 152	54	11	1*	0.019	1.85
Total / Mean	153	24	2*	0.013	1.31

* Hybrid plants were infirm and could not servived till maturity

Cytomorphology of F₁ hybrids

Hybrid B. juncea x B. campestris: In B. juncea x B. campestris cross combination, two hybrids viz., RSM 151 x BCT and RSM 152 x BCYS obtained were intermediate as compared to the parents. The lower leaves of the hybrids were elliptic to ovate, sessile, lyrately pinnatifid to pinnatipartite with sinuate-dentate margin and obtuse tip. The upper leaves were auricled and similar to B. campestris. The flowers were intermediate in size with yellow petals. Hybrid vigour was manifested as taller plants, profuse branching, longer main raceme, higher siliqua intensity, more number of siliquae on main raceme and siliquae per plant in comparison to better parent. On the contrary, siliqua size, seed set, seed weight, seed yield and oil content were very much reduced in the hybrids.

The meiotic analysis of AAB hybrid was characterized by chromosome association of 10 II + 8 I in majority of pollen mother cells (PMCs) analyzed (71.8 %) (Fig. 1a). A maximum of 11 bivalents were observed in four out of 142 cells analyzed. Another deviation was nine or more univalents instead of expected 8 I in 24 per cent of PMCs. Multivalent configurations in the form of trivalents, quadrivalents and even a pentavalent association (Fig. 1b) were found in 14.1 per cent of PMCs. The average chromosome association per PMC recorded in hybrid was 0.01 V + 0.06 IV + 0.09 III + 9.59 II + 8.29 I (Table 2).

Fig. 1a-d. Meiosis in pollen mother cells of hybrid B. juncea x B. campestris, a. Metaphase I (M I), 10 II + 8 I; b. M I, 1 V + 9 II + 5 I; c. M I, 7 II + 14 I and d. Anaphase I ( 14 : 14)

Chromosome orientation and disjunction observed at anaphase (I/II) were found abnormal. Sometimes well defined metaphase plates could be recognized, but in majority of cases, the bivalents did not align themselves on the equatorial region. The bivalents disjunction was normal, while univalents either lagged or got incorporated in any of the polar group. Although majority of the cells (61.5 %) observed had laggards at anaphase (I/II) yet 15 .4 per cent of cells found with equal distribution and without laggards (Fig. 1d). Bridge-fragment configuration was observed in three out of 65 cells analyzed at anaphase. Average pollen fertility recorded from 18 F₁ plants was 18.5 per cent.

Hybrid B. napus x B. campestris: All the three hybrids viz., BN 15 x BCBS, BN 15 x BCT and BN 15 x BCYS obtained from B. napus x B. campestris combination were more vigorous than their respective better parent. Degree of clasping of leaves and siliqua attachment on inflorescence was intermediate between parental species. The lower leaves of F₁ plants were ovate, auriculate, lyrately pinnatifid with sinuate margin and obtuse tip. The flowers were larger in size with deep yellow petals. Heterosis was manifested as greater plant height, profuse branching, longer main raceme, more number of siliquae on main raceme as well as siliquae per plant. On the other hand, sharp reduction in siliqua length, seeds per siliqua, seed weight, seed yield and oil content was noticed in the hybrid. The F₁ plants showed resistance/tolerance against White rust.

In meiotic analysis of AAC hybrids (2n=29), 153 out of 183 PMCs analyzed, depicted 10 II + 9 I chromosome association (Fig. 2b). A maximum of 12 bivalents were observed in 4.9 per cent cells (Fig. 2a), while 11 II was found in two PMCs. Multivalent association in the form of trivalent or quadrivalent along with compensating number of bivalents and univalents was noticed in 8.2 and 2.2 per cent of cells analysed. The average chromosome association observed was 0.01 IV + 0.10 III + 10.03 II + 8.55 I (Table 2). The bivalents and even trivalents associations persisted through late anaphase I. In many cells, distinction between metaphase and anaphase was difficult as the chromosomes lay scattered from one pole to another (Fig. 2b).

Fig. 2a-d. Meiosis in pollen mother cells of hybrid B. napus x B. campestris, a. Metaphase I (M I), 12 II + 5 I; b. M I, 10 II + 9 I; c. Anaphase I ( 15 : 14) and d. Anaphase I with bridge-frigments configuration.

At anaphase (I/II), majority of cells showed laggards (58.8%), while in 26.9 per cent of PMCs analyzed, the laggards were not observed (Fig. 2c). Bridge-fragment configuration was noticed in 14.3 per cent of cells (Fig. 2d). The pollen stainability in the hybrids of cross B. napus x B. campestris varied among three different genotypic combinations. The hybrid BN 15 x BCT showed an average of 17.2 per cent pollen stainability while BN 15 x BCBS and BN 15 x BCYS exhibited 21.6 and 27.4 per cent pollen fertility, respectively.

Table 2. Chromosome association (Mean ± SE) at meiosis in pollen mother cells (PMCs) of interspecific hybrids in Brassica

Cross combination	Hybrid genome	No. of PMCs	Chromosome association
		analysed	V	IV	III	II	I
B. juncea x B. campestris	AAB, (2n=28)
RSM 151 x BCT		60	-	0.13±0.05	0.10±0.04	9.62±0.14	7.93±0.18
				(0-2)*	(0-1)	(4-11)	(4-12)
RSM 152 x BCYS		82	0.01±0.01	0.01±0.01	0.07±0.03	9.56±0.10	8.55±0.19
			(0-1)	(0-1)	(0-1)	(7-11)	(5-14)
Total / Mean		142	0.01±0.01	0.06±0.02	0.09±0.02	9.59±0.08	8.29±0.14
			(0-1)	(0-2)	(0-1)	(4-11)	(5-14)
B. napus x B. campestris	AAC, (2n=29)
BN 15 x BCBS		63	-	0.04±0.03	0.06±0.03	10.13±0.08	8.37±0.18
				(0-1)	(0-1)	(8-12)	(5-10)
BN 15 x BCT		52	-	0.02±0.02	0.15±0.06	9.94±0.08	8.58±0.15
				(0-1)	(0-2)	(8-12)	(5-9)
BN 15 x BCYS		68	-	-	0.09±0.04	10.01±0.06	8.71±0.13
					(0-2)	(8-12)	(5-9)
Total / Mean		183	-	0.01±0.01	0.10±0.03	10.03±0.04	8.55±0.09
				(0-1)	(0-2)	(8-12)	(5-10)
B. juncea x B. napus	AABC, (2n=37)
RSM 98 x BN 15		97	-	0.05±0.02	0.08±0.03	10.79±0.15	14.96±0.29
				(0-1)	(0-1)	(9-14)	(9-17)
RSM 152 x BN 15		65	-	0.02±0.02	0.11±0.04	10.46±0.15	15.69±0.30
				(0-1)	(0-1)	(9-14)	(9-17)
Total / Mean		162	-	0.04±0.01	0.09±0.02	10.66±0.11	15.20±0.22
				(0-1)	(0-1)	(9-14)	(9-17)

* Range

Hybrid B. juncea x B. napus: The hybrid plants of both the crosses viz., RSM 98 x BN 15 and RSM 152 x BN 15 of combination B. juncea x B. napus were quite vigorous, bushy and intermediate to their putative parents for inflorescence and other morphological attributes. Basal leaves of the hybrid resembling more with B. napus, were ovate-lanceolate, short petiolate, lyrately pinnatifid with sinuate-dentate margin, obtuse tip and dark green in colour. The flowers with deep yellow petals were intermediate in size. As regards quantitative traits viz., plant height, primary and secondary branches, main raceme length and siliquae per plant, the hybrids were superior, while for siliqua intensity, siliqua length, seeds per siliqua, seed weight, seed yield and oil content, they were inferior than better parent. The hybrid plants were observed to be free from White rust.

In meiotic analysis of AABC hybrids (2n=37), 10 II + 17 I was the most frequent chromosome association noticed in 103 out of 162 PMCs analyzed. The maximum number of 14 bivalents were noticed in 10.5 per cent and 11, 12 and 13 bivalents were observed in 4.9, 2.5 and 5.6 per cent of PMCs, respectively. Multivalents in the form of trivalent and quadrivalent were seen in 9.3 and 3.7 per cent of cells, respectively. On an average, chromosome association per PMC noticed was 0.04 IV + 0.09 III + 10.66 II + 15.20 I (Table 2). The meiotic division observed was abnormal.

Fig. 3a-d. Meiosis in pollen mother cells of hybrid B. juncea x B. napus, a. Metaphase I (M I), 11 II + 15 I; b. Anaphase I with bridge-frigments configuration; c. Anaphase II with laggards, and d. Tetrad with six microspores (hexads)

At anaphase (I/II), besides unequal chromosome disjunction, varying number of chromatin bridges, lagging and precociously dividing chromosome associations were observed (Fig. 3c &d). Frequencies of bridges and laggards found were 9.7 and 66.7 per cent, respectively. Dyads, triads, tetrads and even hexads (Fig. 3d) were observed, leading to large variation in the size of pollen grains. The mean pollen fertility of two hybrids of this cross combination was 21.5 per cent. In the cross RSM 98 x BN 15, nine plants examined had, on an average, 25.7 per cent good pollen, while 12 plants of the cross RSM 152 x BN 15 showed 18.4 per cent mean pollen fertility.

DISCUSSION

Hybridization and crossability

Cross-compatibility of B. campestris with amphidiploid species was much better when amphidiploid species used as a female parent. Whereas, reciprocal cross combinations generally unsuccessful. Similar observations were also made by Ramanujam and Srinivasachar (1943), Olsson (1960a) and Quazi (1988). The hybrid recovery from cross of B. campestris with B. napus was higher than with B. juncea, indicated more affinity of genome A with C than with B genome as also hypothesized by Olsson (1960b). Between amphidiploid species crosses, B. juncea x B. napus was most successful while the hybrid recovered from its reciprocal cross was infirm, very slow in growth and died during the vegetative phase. Rao et al. (1993) also attributed similar findings to very slow growth in the initial stage of hybrid when B. napus was used as a female parent. Other reasons for failure of interspecific crosses to set seeds could be non-synchrony in flowering and environmental conditions (Olsson, 1960b).

The success rate of cross-fertility of three ecospecies of B. campestris with B. juncea and B. napus was in order of Yellow sarson > Toria > Brown sarson. Differences were also observed at varietal level. Amongst crosses attempted involving three genotypes of B. juncea (RSM 98, RSM 151 and RSM 152) with B. napus (BN 15), RSM 152 produced maximum hybrids (24%) followed by RSM 98 (17.65%) while not a single hybrid was obtained when RSM 151 used as a parent. These observations clearly demonstrated that the success of interspecific crosses depended not only on the species but also on the ecospecies and genotypes of the species involved. The present observations were supported opine made by Mizushima (1952), Olsson (1960b) and Akbar (1989).

Cytomorphology of F₁ hybrids

Hybrid B. juncea x B. campestris: Morphological observations in both the hybrids of this cross were in agreement with Olsson (1960a), and Mohapatra and Bajaj (1988). However, Das et al. (1984) reported higher influence of B. campestris than that of B. juncea while, Mamatha (1989) found greater resemblance of F₁ plants to B. juncea in most of the morphological attributes. Such differences could be accounted for by the genotypes of the variety used. Most frequent chromosome association of 10 II + 8 I in AAB hybrids (2n=28) indicated pairing between 10 chromosome of each of the A genome derived from B. juncea (AABB, 2n=36) and B. campestris (AA, 2n=20) leaving eight chromosomes of B genome as univalents. Morinaga (1929) also concluded that 10 chromosomes of the B. juncea were homologous to B. campestris chromosomes. The occurrence of only four to nine instead of expected 10 bivalents in 24 per cent of PMCs, was not reported earlier in any of the such hybrids analyzed (Morinaga, 1929; Sikka, 1940; Ramanujam and Srinivasachar, 1943; Mizushima, 1950; Olsson, 1960a and Mamatha, 1989). Such a low association might be due to cryptic structural changes occurred naturally in some of the chromosomes of the A genome of both the species (Sybenga, 1972).

Excess of bivalents noticed eleven against the expected ten, indicated autosyndesis within B genome (Prakash, 1973a). The multivalents in the form of trivalents, quadrivalents and even pentavalent observed in the present materials were reported very rarely (Sikka, 1940). However, genomic formulae suggested by Robbelen (1960) for A and B genomes, provided theoretical possibility of formation of even octavalent association in AAB hybrid. Occurrence of multivalent associations supported existence of partial homoeology between A and B genomes (Olsson, 1960a; Prakash, 1973b). This affinity suggested that the chromosomes of these genomes were able to pair intergenomically. Such genetic exchanges should make possible selection of desirable segregants.

Hybrid B. napus x B. campestris: The observations recorded on morphological attributes in the hybrid were in close agreements with the reports of Olsson (1960b), Nwankiti (1970) and McNaughton (1973). Occurrence of 10 II + 9 I chromosomes association in majority of PMCs (86.3%) in AAC hybrids (2n=29) could be attributed to homology between both A genomes each derived from B. napus (AACC, 2n=38) and B. campestris (AA, 2n=20), while the nine chromosomes of C genome remained as univalents. More or less identical synaptic behaviour of chromosome of B. campestris vars. Yellow sarson, Brown sarson and Toria with the 10 homologous chromosomes of A genome in B. napus suggested that there was no appreciable differences in the chromosomes of A genome of the above subspecies. The higher chromosome association of 12 II + 5 I instead expected of 10 II + 9 I and multivalent associations found in these hybrids indicated allosyndetic pairing between chromosomes of A and C genomes as also hypothesized by Kamala (1976). However, autosyndesis could not be ruled out within genome C (Armstrong and Kellar 1982). Bridges with/without fragments observed in hybrids indicated delayed terminalization of nonhomologous segments at the terminal/subterminal portion of the bivalents (Nwankiti, 1970). The structural alterations resulted in differences between certain A-C or A-A chromosomes which might have led to the formation of bridge in the present hybrid.

Hybrid B. juncea x B. napus: Closer resemblance of leaves in this hybrid with B. napus was in conformity with the reports of Sharma and Singh (1992) but contrary to findings of Rao et al. (1993) who observed that F₁ plants of these two species resembled more with B. juncea.

In meiotic analysis of AABC hybrids (2n=37), occurrence of 10 II + 17 I in high frequency (63.6%) might be attributed to preferential pairing between the 10 chromosomes of A genome of B. juncea (AABB, 2n=36) with that of B. napus (AACC, 2n=38). The maximum number of bivalents noticed in hybrids was fourteen. The four additional bivalents over the expected of ten, might have originated from pairing between and/or within chromosomes of B and C genomes. This involved not only the bivalents from autosyndesis but also those resulting from allosyndesis between B and C genomes. Similar indications were made by Mizushima (1950), Prakash and Chopra (1988), and Rao et al. (1993). As discussed earlier, in AAB hybrid maximum number of bivalents recorded was 11 while AAC hybrid had 12 bivalents. Assuming that these additional three bivalents observed were because of autosyndesis within B and C genomes, there would be 13 bivalents expected in hybrid AABC. Thus fourteenth bivalent observed should be due to allosyndesis between B and C genomes. Mizushima (1950) opined that three out of four bivalents noticed in B and C genomes could be due to allosyndesis. The present study demonstrated that it was an overestimation and the homology between the two genomes was less.

CONCLUSION

The present cytological survey of the hybrids involving three Brassica species viz., B. campestris, B. juncea, and B. napus indicated inter-relationship among the three basic genomes A, B and C. Among the three hybrids studied, B. napus x B. campestris and B. juncea x B. napus displayed fairly good seed set on open pollination. The low fertility of the F₁ plants, in general, could be due to chromosomal and genetic imbalance and/or cytoplasmic-nuclear interaction. Such sterility promoted out-crossing and selection of chromosomally balanced genotypes by sieving out unbalanced gametes. The occurrence of multivalents in form of trivalents and quadrivalents in AAB, AAC and AABC hybrids, reaffirmed the homoeology among A, B and C genomes, which provided opportunity for interspecific transfer of gene/gene-complexes. These association also confirmed the hypothesis of secondary polyploid origin of the A, B and C genomes. Further, higher frequency of bivalents noticed in hybrids AAC as compared to AAB, revealed close affinity of A with C in comparison to B genome.

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