V. Dinesh Kumar

Plant regeneration via somatic embryogenesis in chickpea (Cicer arietinum L.).

Efficient plant regeneration via somatic embryogenesis has been developed in chickpea cultivar C235. Leaf explants, on MS medium supplemented with 1.25 mg/l 2,4-D and 0.25 mg/l kinetin, yielded somatic embryos with high efficiency during dark incubation. MS medium supplemented with B5 vitamins, 0.125 mg/l IBA and 2 mg/l BAP was found suitable for embryo maturation. The well formed embryos germinated into plantlets on basal B5 medium supplemented with 0.25 mg/l BAP. Further development into healthy plantlets was obtained on basal B5 medium. Hardened plantlets produced normal, fertile plants upon transfer to soil.

Brassica coenospecies: a rich reservoir for genetic resistance to leaf spot caused by Alternaria brassicae

Development of leaf spot resistant mustard cultivars is a relevant objective in view of heavy crop losses caused by this pathogen. Thirty-eight species belonging to 9 genera, including cultivated and wild allies, of the genus Brassica were evaluated under epiphytotic conditions for two years. Inoculations were done on whole plants (in vivo) and on detached leaves (in vitro). Data on incubation period, number of lesions per leaf, lesion size and leaf area covered by lesions were recorded. Species which never produced disease symptoms throughout the growing period in pots and until 72 hours after inoculation in detached leaf assays during both years were treated as resistant, while those that produced symptoms were classified as moderately resistant, susceptible or highly susceptible depending upon incubation period, size of lesions and leaf area covered by disease symptoms. Eight species (Brassica desnottesii, Camelina sativa, Coincya pseuderucastrum, Diplotaxis berthautii, D. catholica, D. cretacea, D. erucoides, and Erucastrum gallicum) were found completely resistant, whereas others were classified as moderately resistant (12), susceptible (11) or highly susceptible (9). Since resistance is unavailable within the cultivated species, these 8 resistant wild species could be used as donor parents for introgressing resistance to leaf spot disease in Indian mustard.

EST-SSR marker-based assay for the genetic purity assessment of safflower hybrids

Full potential of any hybrid can be exploited by ensuring the supply of genetically pure seeds. Conventionally, hybrid seed purity assessment is done through grow out test (GOT) which is based on the morphological and floral characters of plants grown to maturity. Being land and labor intensive, time consuming and influenced by the environment, there is an immense need to replace GOT with a simple, rapid, unbiased and cost-effective DNA based assay for hybrid purity assessment. With this objective, the parental lines of three commercial safflower hybrids of India (NH-1, NH-15 and DSH-129) were screened using 74 safflower EST-SSR markers and five markers were found to be polymorphic. A PCR-based assay with these markers showed both alleles of the parental lines in pure hybrids proving the heterozygosity, while the off-types were identified by the presence of either of the parental alleles. This assay could accurately determine the genetic purity in a predetermined sample of hybrids constituted by deliberately mixing seeds of parental lines. This is the first report demonstrating the utility of EST-SSR markers for the assessment of genetic purity of hybrids in crop plants.

Cytoplasmic male sterility in alloplasmic Brassica juncea carrying Diplotaxis catholica cytoplasm: molecular characterization and genetics of fertility restoration

Abstract The present study was aimed at characterizing cytoplasmic male sterility (CMS) and identifying the fertility restorer gene for CMS (Diplotaxis catholica) Brassica juncea derived through sexual hybridization. The fertility restorer gene was identified by crossing the CMS line with progeny plants derived from somatic hybrids of B. juncea and D. cathoilca. The CMS line is comparable to the nuclear donor B. juncea in all respects except for flower and silique characteristics. In CMS plants, the flowers have smaller nectaries, and anthers are converted into petals or tubular structures. Gynoecium exhibits a crooked style and trilocular ovary. Seed fertility was reduced in the CMS line. Genetic segregation data indicated that a single, dominant, nuclear gene governs fertility restoration. Restored plants showed a high female fertility and lacked gynoecium abnormalities. In fertility-restored plants, petal development was found to be variable; some flowers had the normal number of four petals, while others had zero to three petals. Interestingly, the trilocular character of the ovary was found to co-segregate with CMS and became bilocular upon male-fertility restoration. Thus, this trait appears to be affected by the interaction of nuclear and mitochondrial (mt) genomes. Restriction fragment length polymorphism analysis indicated that mt-genome of D. catholica is highly divergent from that of B. juncea. However, in Northern analysis, out of eight mt genes studied, an altered transcript pattern was recorded for only atpA. In fertility-restored plants, the atpA transcript became shorter, thereby showing its association with CMS.

Chloroplast substitution overcomes leaf chlorosis in a Moricandia arvensis-based cytoplasmic male sterile Brassica juncea

Abstract A male sterile Brassica juncea line based on Moricandia arvensis cytoplasm was developed previously by backcrossing the somatic hybrid M. arvensis+B. juncea, and the gene for restoring fertility was introgressed. The CMS line is very severely chlorotic because of the presence of alien chloroplasts and flowering is delayed by 30–40 days, making it unsuitable for the exploitation of heterosis. We have resorted to another cycle of protoplast fusion between green fertile B. juncea and chlorotic male sterile B. juncea, and developed green male-sterile plants. Molecular analysis revealed that in green male-sterile plants chloroplasts of M. arvensis origin were substituted by those from B. juncea, giving rise to intergeneric cytoplasmic hybrids with mitochondria of M. arvensis origin. With the development of dark-green male-sterile plants, the CMS fertility restoration system is suitable for the production of hybrid mustard.

Random chloroplast segregation and mitochondrial genome recombination in somatic hybrid plants of Diplotaxis catholica+Brassica juncea

Abstract Detailed molecular analysis of the somatic hybrid plants of Diplotaxis catholica+B. juncea indicated random chloroplast segregation. One of the five hybrid plants analyzed derived its chloroplasts from D. catholica and two hybrids had chloroplasts of B. juncea origin. Two hybrid plants maintained mixed population of chloroplasts. The mitochondrial (mt) genomes of the fusion partners had undergone recombinations. Occurrence of fragments specific to both the parents in HindIII digestion followed by atp 9 probing, as in hybrid DJ5, provided evidence for intergenomic mitochondrial recombination between D. catholica and B. juncea. Similar mt genome organization in two hybrids (DJ3 and DJ6) suggested that intergenomic recombination may be preferred at specific sites. Hybrid DJ1 had about 70% similarity to D. catholica in mt genome organization. mt genomes of hybrids DJ2, 3, 5, and 6 differed from B. juncea by 14.3–28%. The significance of these novel mt genome organizations in developing novel male sterility systems is discussed.

A Simple Protocol for Regenerating Mesophyll Protoplasts of Vegetable Brassicas

We report here a simple protocol for regenerating plants from leaf protoplasts of vegetable Brassicas, viz., cabbage, cauliflower and broccoli. Protoplasts from in vitro grown leaf material were cultured in Kao’s medium with a supplementation of 2,4-D, NAA, BAP and glucose, initially in dark for 3d and subsequently in light. Dilution of protoplast cultures was effected on the 7th, 10th and 13th day of culture initiation with Kao’s medium supplemented with sucrose, and reduced 2,4-13 content; NAA was omitted. Micro-colonies were plated on a K3 medium having 2,4-D, BAP and sucrose gelled with agarose. Transfer of calli to another K3 medium with zeatin regenerated shoots from cauliflower protoplast derived calli, whereas a medium with kinetin and zeatin supported shoot regeneration in cabbage and broccoli. Shoot regeneration occurred within 6-6 weeks of culture initiation. Shoots were easily rooted on MS medium without growth regulators.