S. S. Banga

Potential use of random amplified polymorphic DNA (RAPD) technique to study the genetic diversity in Indian mustard (Brassica juncea) and its relationship to heterosis.

RAPD assays were performed, using 34 arbitrary decamer oligonucleotide primers and six combinations of two primers, to detect inherent variations and genetic relationships among 12 Indian and 11 exotic B. juncea genotypes. Of 595 amplification products identified, 500 of them were polymorphic across all genotypes. A low level of genetic variability was detected among the Indian genotypes, while considerable polymorphism was present among the exotic ones. Based on the pair-wise comparisons of amplification products the genetic similarity was calculated using Jaccards similarity coefficients and a dendrogram was constructed using an unweighted pair group method was arithmetical averages (UPGMA). On the basis of this analysis the genotypes were clustered into two groups, A and B. Group A comprised only exotic genotypes, whereas all the Indian genotypes and four of the exotic genotypes were clustered in group B. Almost similar genotypic rankings could also be established by computing as few as 200 amplification products. In general, a high per cent of heterosis was recorded in crosses involving Indian x exotic genotypes. On the other hand, when crosses were made amongst Indian or exotic genotypes, about 80% of them exhibited negative heterosis. Results from this study indicate that, despite the lack of direct correlation between the genetic distance and the degree of heterosis, genetic diversity forms a very useful guide not only for investigating the relationships among Brassica genotypes but also in the selection of parents for heterotic hybrid combinations.

Transfer of Ogu cytoplasmic male sterility to Brassica juncea and improvement of the male sterile line through somatic cell fusion

Male sterility conferred by ogu cytoplasm of Raphanus sativus has been transferred to Brassica juncea cv ‘RLM 198’ from male-sterile B. napus through repeated backcrossing and selection. The male-sterile B. juncea is, however, highly chlorotic and late. It has low female (seed) fertility and small contorted pods. To rectify these defects, protoplasts of the male sterile were fused with normal ‘RLM 198’ (green, self fertile). Four dark green, completely male-sterile plants were obtained and identified as putative cybrids. All the plants were backcrossed three times with ‘RLM 198’. Mitochondrial and chloroplast DNA analysis of backcross progeny confirmed hybridity of the cytoplasm. The restriction pattern of the chloroplast DNA of progeny plants of three cybrids (Og 1, Og 2, Og 3) was similar to that of the green self-fertile ‘RLM 198’ and indicated that the correction of chlorosis resulted from chloroplast substitution. The chloroplast DNA of the lone progeny plant of the fourth cybrid (Og 10) could not be analyzed because the plant was stunted and had only a few leaves. When total cellular DNA was probed with mitochondrial probes coxI and atpA it was found that the cybrids had recombinant mitochondria. The chlorosis-corrected plants were early flowering and had vastly improved seed fertility.

Cotyledon assay as a rapid and reliable method of screening for resistance against Sclerotinia sclerotiorum in Brassica napus genotypes

Sclerotinia sclerotiorum is a major pathogen of many crops, including oilseed rape (Brassica napus), and there is keen interest worldwide to identify Brassica genotypes with resistance to this pathogen. However, field testing to identify resistance in B. napus germplasm is expensive, time-consuming and at times unreliable due to variability in field environmental conditions and plant architecture. To address this, we aimed to examine the feasibility of utilising for B. napus a cotyledon test already developed for Sclerotinia disease on legumes. Initially, cotyledons of 32 B. napus genotypes were drop-inoculated using macerated mycelium (104 mycelial fragments/mL) under controlled environmental conditions. Significant differences were recorded between B. napus genotypes, and the experiment was repeated twice using genotypes selected from the first experiment. Certain genotypes responded with a distinct hypersensitive reaction (lesions <1 mm diameter), either always (cv. Mystic) or frequently (cv. Charlton), which is the first report of this phenomenon in the B. napus-S. sclerotiorum pathosystem. Responses of genotypes between the three screening experiments were significantly and positively correlated. Results obtained in the first experiment were compared with those from our earlier field screening for stem rot that utilised the same strain of S. sclerotiorum and the same B. napus genotypes. In particular, there was a significant positive correlation (r=0.62, P < 0.01) between published field data for stem rot and our cotyledon test results across genotypes in common. This indicates the usefulness of this cotyledon assay to provide a relatively reliable indication of field performance of genotypes. We believe that this is the first report demonstrating that a cotyledon assay can be successfully applied to rapidly differentiate the reactions of B. napus genotypes against S. sclerotiorum.

The importance of the type and time of inoculation and assessment in the determination of resistance in Brassia napus and B. juncea to Sclerotinia sclerotiorum

Sclerotinia stem rot (SSR) is a significant agricultural problem worldwide. Finding sources of resistance is crucial to the ongoing search for better management of this disease. Brassica germplasm from Australia, China and India was screened for resistance to SSR under Western Australian field conditions following stem inoculation, application of a spray of mycelial suspension, or as a consequence of myceliogenic germination originating from sclerotia resident in soil. Significant differences in response were observed among 53 genotypes using each of the three screening methods. There was a variable impact of the time of inoculation on the disease level depending upon time of assessment post-stem inoculation. However, this impact could be reduced to an insignificant level provided the assessment after stem inoculation was delayed until 3 weeks post-inoculation. The results of these studies indicate that the use of appropriate inoculation and assessment methods could significantly reduce variability in the responses commonly observed in screening for resistance in crop plants against Sclerotinia sclerotiorum.

Alloplasmic male-sterile Brassica juncea with Enarthrocarpus lyratus cytoplasm and the introgression of gene(s) for fertility restoration from cytoplasm donor species.

Abstract. A new cytoplasmic male sterility (CMS) source in Brassica juncea (2n = 36; AABB) was developed by substituting its nucleus into the cytoplasm of Enarthrocarpus lyratus (2n = 20; EℓEℓ). Male sterility was complete, stable and manifested in either petaloid- or rudimentary-anthers which were devoid of fertile pollen grains. Male sterile plants resembled the euplasmic B. juncea except for slight leaf yellowing and delayed maturity. Leaf yellowing was due mainly to higher level of carotenoids rather than a reduction in chlorophyll pigments. Female fertility in male-sterile plants varied; it was normal in lines having rudimentary anthers but poor in those with petaloid anthers. Each of the 62 evaluated germplasm lines of B. juncea was a functional maintainer of male sterility. The gene(s) for male-fertility restoration (Rf) were introgressed from the cytoplasm donor species through homoeologous pairing between A and Eℓ chromosomes in monosomic addition plants (2n = 18II+1Eℓ). The percent pollen fertility of restored F1 (lyr CMS × putative restorer) plants ranged from 60 to 80%. This, however, was sufficient to ensure complete seed set upon by bag selfing. The CMS (lyr) B. juncea compared favourably with the existing CMS systems for various productivity related characteristics. However, the reduced transmission frequency of the Rf gene(s) through pollen grains, which was evident from the sporadic occurrence of male-sterile plants in restored F1 hybrids, remains a limitation.

Expression and relationships of resistance to white rust (Albugo candida) at cotyledonary, seedling, and flowering stages in Brassica juncea germplasm from Australia, China, and India

White rust (Albugo candida) is a highly destructive disease of oilseed Brassicas such as Brassica juncea and B. rapa. Most commercial B. juncea or B. rapa varieties are highly susceptible and yield losses from combined infection of leaves and inflorescences can be up to 20% or 60% in Australia and India, respectively. In Australia, canola-quality B. juncea has been developed to extend oilseed Brassica production into lower rainfall areas, with the first commercial B. juncea canola-quality variety planned for release in 2006. It is essential to identify useful sources of host resistance in B. juncea as breeding and/or selection of material for resistance is the most cost-effective method of delivering control for farmers. Three experiments were undertaken under controlled-environmental conditions to identify the best methods of characterising host resistance and to identify sources of resistance in B. juncea germplasm from Australia, China, and India. Forty-four B. juncea genotypes, viz. 22 from India, 12 from Australia, and 10 from China, were tested. Four Chinese genotypes (CBJ-001, CBJ-002, CBJ-003, CBJ-004) and one Australian genotype (JR049) consistently showed high resistance to A. candida across the different plant growth stages against a pathotype prevailing in Australia. Similarly, the most susceptible genotypes (viz. Indian genotypes RH781, RL1359, RH819) were extremely susceptible irrespective of the plant growth stage. Overall, although disease severity on cotyledons and leaves at the different growth stages was significantly and positively correlated, there was, however, no significant correlation between the number of stagheads and any of the other disease parameters measured. Our study demonstrates that controlled-environmental conditions are suitable for rapid identification of resistant genotypes and that genotypes with high levels of resistance can be reliably identified at the cotyledonary, seedling, or flowering stages.

Hybrid cultivar development.

Heterosis: An Introduction / Male Sterility: Classfication and Concept / Male Sterility: Molecular Characterisation / Self-Incompatibility / Monoecious and Dioecious Floral Morphologies / Chemical Hybridizing Agents / Biotechnology and Hybrid Breeding/ Molecular Genetic Markers / Rice / Wheat / Maize / Barley / Pearl Millet / Sorghum / Cotton / Sunflower / Rapeseed and Mustard / Castor/ Pigeonpea / Tomato / Onion / Cole Crops / Capsicum / Muskmelon/ Watermelon.

Male sterility in Indian mustard (Brassica juncea (L.) Coss.) — a biochemical characterization

SummaryBiochemical studies were conducted on some male sterile and their fertile F1 analogues in Indian mustard. The variation in pH activity during microsporogenesis was normal, except in MS-3. Male sterile anthers had deficient sugar metabolism. Cytochemical analysis of sporogenous and tapetal tissue suggested an effect of sterility elements on the anabolic and catabolic fate of DNA and protein during microsporogenesis. Leaves of male sterile lines had a higher chl a/b ratio. Leaf peroxidase activity was low and different isozymes appeared when separated by starch electrophoresis.

Development and characterization of Brassica juncea – fruticulosa introgression lines exhibiting resistance to mustard aphid (Lipaphis erysimi Kalt)

BackgroundMustard aphid is a major pest of Brassica oilseeds. No source for aphid resistance is presently available in Brassica juncea . A wild crucifer, Brassica fruticulosa is known to be resistant to mustard aphid. An artificially synthesized amphiploid, AD-4 (B. fruticulosa × B. rapa var. brown sarson) was developed for use as a bridge species to transfer fruticulosa resistance to B. juncea. Using the selfed backcross we could select a large number of lines with resistance to mustard aphid. This paper reports cytogenetic stability of introgression lines, molecular evidence for alien introgression and their reaction to mustard aphid infestation.ResultsMajority of introgression lines had expected euploid chromosome number(2n= 36), showed normal meiosis and high pollen grain fertility. Well-distributed and transferable simple-sequence repeats (SSR) markers for all the 18 B. juncea chromosomes helped to characterize introgression events. Average proportions of recipient and donor genome in the substitution lines were 49.72 and 35.06%, respectively. Minimum alien parent genome presence (27.29%) was observed in the introgression line, Ad3K-280 . Introgressed genotypes also varied for their resistance responses to mustard aphid infestations under artificial release conditions for two continuous seasons. Some of the test genotypes showed consistent resistant reaction.ConclusionsB.juncea-fruticulosa introgression set may prove to be a very powerful breeding tool for aphid resistance related QTL/gene discovery and fine mapping of the desired genes/QTLs to facilitate marker assisted transfer of identified gene(s) for mustard aphid resistance in the background of commercial mustard genotypes.

Heterosis as Investigated in Terms of Polyploidy and Genetic Diversity Using Designed Brassica juncea Amphiploid and Its Progenitor Diploid Species

Fixed heterosis resulting from favorable interactions between the genes on their homoeologous genomes in an allopolyploid is considered analogous to classical heterosis accruing from interactions between homologous chromosomes in heterozygous plants of a diploid species. It has been hypothesized that fixed heterosis may be one of the causes of low classical heterosis in allopolyploids. We used Indian mustard (Brassica juncea, 2n = 36; AABB) as a model system to analyze this hypothesis due to ease of its resynthesis from its diploid progenitors, B. rapa (2n = 20; AA) and B. nigra (2n = 16; BB). Both forms of heterosis were investigated in terms of ploidy level, gene action and genetic diversity. To facilitate this, eleven B. juncea genotypes were resynthesized by hybridizing ten near inbred lines of B. rapa and nine of B. nigra. Three half diallel combinations involving resynthesized B. juncea (11×11) and the corresponding progenitor genotypes of B. rapa (10×10) and B. nigra (9×9) were evaluated. Genetic diversity was estimated based on DNA polymorphism generated by SSR primers. Heterosis and genetic diversity in parental diploid species appeared not to predict heterosis and genetic diversity at alloploid level. There was also no association between combining ability, genetic diversity and heterosis across ploidy. Though a large proportion (0.47) of combinations showed positive values, the average fixed heterosis was low for seed yield but high for biomass yield. The genetic diversity was a significant contributor to fixed heterosis for biomass yield, due possibly to adaptive advantage it may confer on de novo alloploids during evolution. Good general/specific combiners at diploid level did not necessarily produce good general/specific combiners at amphiploid level. It was also concluded that polyploidy impacts classical heterosis indirectly due to the negative association between fixed heterosis and classical heterosis.