Lalitha Anand

Assessment of genetic relatedness among mango cultivars of India using RAPD markers

Summary DNA-based RAPD (Random Amplification of Polymorphic DNA) markers have been used extensively to study genetic relationships in a number of fruit crops. A wide genetic diversity exists in the mango fruit in India. Present day commercial cultivars originated mainly from this subcontinent. In this study, 18 commercial mango cultivars, traditionally grown in western, southern, northern and eastern parts of India, were selected to assess genetic relatedness. Total genomic DNA was extracted and subjected to RAPD analysis using 30 arbitrary 10-mer primers. Of these, 27 primers amplified mango genomic DNA. None of these primers produced unique band pattern for each cultivar. RAPD data were used to calculate a squared Euclidean distance matrix, and based on this cluster analysis was done using a minimum variance algorithm. Cluster analysis clearly showed two groups—the first consisting of western, northern and eastern mango cultivars and the second group consisting of southern cultivars. From the analysis of results, it appears the majority of mango cultivars originated from a local mango genepool and were domesticated later.

Development of new microsatellite markers from Mango (Mangifera indica) and cross-species amplification

PREMISE OF THE STUDY Microsatellite markers were developed and characterized to assess the genetic diversity among mango (Mangifera indica) cultivars and to test their amplification in closely related species. METHODS AND RESULTS Thirty-six microsatellite (simple sequence repeats; SSR) loci were isolated by a microsatellite-enriched partial genomic library method. Primers designed for these loci were characterized using 30 diverse mango cultivars. The number of alleles ranged from 3 to 19 with an average of 9.2 alleles per locus. Polymorphic information content values ranged from 0.185 to 0.920 with a mean of 0.687. The total value for the probability of identity was 2.42 × 10(-31). CONCLUSIONS The newly identified SSRs would be useful in genetic diversity studies, finger-printing, and mapping. Loci from five related species, M. odorata, M. anadamanica, M. zeylanica, M. camptosperma, and M. griffithii, were successfully amplified using these SSR primers, showing their potential utility across species.

Analysis of genetic diversity among Indian short-day onion (Allium cepa L.) cultivars using RAPD markers.

Summary Randomly amplified polymorphic DNA (RAPD) markers were used to estimate genetic diversity among 24 cultivars of short-day onions. Total genomic DNA was extracted and subjected to RAPD analysis using 90 arbitrary 10-mer primers. Of these, 15 primers were selected which yielded 137 bands, 91.24% of which were polymorphic. None of the primers produced a unique banding pattern for each cultivar. RAPD data were used to calculate a Squared-Euclidian Distance matrix which revealed a minimum genetic distance between cultivars ‘AFLR-722’ and ‘PBR-140’, and a maximum genetic distance between cultivars ‘PBR-139’ and ‘A.Kalyan’, and ‘MS-48’ and ‘A.Kalyan’. Based on the distance matrix, cluster analysis was done using a minimum variance algorithm.The dendrogram thus generated, based on Ward’s method, grouped the 24 onion cultivars into two major clusters. The first cluster consisted of cultivars from the northern region, and the second of cultivars from the southern region of India. The present study shows that there is high diversity among the onion cultivars selected and indicates the potential of RAPD markers for identification and maintenance of onion germplasm for crop improvement purposes.

Movement and distribution pattern of the residues of granular insecticides in tropical soil and okra plants

SummaryThe increased downward mobility of phorate, quinalphos and carbofuran residues was detected in soil with increase in depth of soil column whereas aldicarb was found to remain localised mainly in 0–7.5 cm and 7.5–15.0 cm layers. Persistence of organophosphate insecticides was higher as compared to carbamates in all the soil layers. Residues of all the four insecticides got distributed in all parts of okra plant through uptake but accumulated in higher amounts in fruits only.

Persistence and safety evaluation of synthetic pyrethroids on sweet orange fruits

The residues of four synthetic pyrethroids from two spray concentrations per compound on sweet orange fruits dissipated at the respective half-life values of 2.45 and 3.15 days for 0.015 and 0.02% fenvalerate; 2.28 and 2.58 days for 0.015 and 0.02% permethrin; 1.73 and 2.12 days for 0.0075 and 0.01% cyper-methrin ; and 2.6 and 2.9 days for 0.0015 and 0.002% deltamethrin, respectively. However, the comparative persistence of synthetic pyrethroids on the rind of sweet orange fruits in the tropical belt in India was found to be in the order of fenvalerate = permethrin > cypermethrin > deltamethrin. No residues were detected in the fruit pulp.

Transgenic Chili Possessing Baculovirus Chitinase Gene Exhibits in vitro Fungal Inhibition

Cultivation of chili (Capsicum annuum L.) is limited by several fungal diseases, the serious among them being anthracnose. In the absence of a source of resistance for anthracnose in C. annuum background, the transgenic approach, i.e., introduction of a gene encoding pathogen cell wall-degrading enzyme, viz., chitinase, into the host plant against the test fungus Alternaria alternata as well as anthracnose (Colletotrichum capsici) was explored. Chili being recalcitrant to in vitro regeneration, the regeneration protocol was optimized in cotyledonary explants using different cytokinins, viz., 6-benzyl amino purine (BAP), thidiazuron (TDZ), or zeatin, in combination with gibberellic acid (GA3) and/or auxins, viz., indole-3-acetic acid (IAA) or phenyl acetic acid (PAA). Agrobacterium-mediated transformation of cotyledonary explants with Autographa californica baculovirus chitinase gene under the control of 35S promoter resulted in seven putative transformants under kanamycin selection. The presence of transgene was confirmed in two of the seven primary transformants. One of the transformants exhibited enhanced inhibition of the fungus Alternaria alternata in in vitro bio-assays at T0 (primary transformant) stage. The two transgenic plants were then advanced to T1 generation (progenies of T0) and their fruits were examined for resistance to anthracnose. A few lines among the progenies of each of the primary transformants exhibited higher tolerance to anthracnose than the rest of the lines and untransformed control. Higher endochitinase activity in one of the primary transformants was correlated to better expression of anthracnose tolerance in its progenies, suggesting the possibility of chitinase gene being used for developing transgenic lines tolerant to anthracnose.

In vitro regeneration and transient gene expression in mango cv. ‘Vellaikolumban’

SUMMARY A protocol to regenerate shoots through somatic embryogenesis was developed in the polyembryonic mango cv. ‘Vellaikolumban’, and the feasibility of gene transfer using the βglucuronidase (GUS) reporter gene was demonstrated. Young, 25–30 d-old fruits were found to be ideal for culture initiation. MS and B5 media were equally effective for initiation of nucellar cultures. A higher percentage of somatic embryo induction was obtained from nucellar cultures using B5 or MS medium supplemented with 20% (v/v) coconut water compared to MS medium without coconut water. Maturation of somatic embryos, 1.0 – 1.5 cm in length, was optimised in M3 medium composed of B5 salts, 400 mg l–1 L-glutamine and 1 mg l–1 abscisic acid. Faciation and necrosis of embryogenic cultures could be effectively controlled through the use of 0.1 mg l–1 salicylic acid. For germination of mature somatic embryos, with well-developed roots and shoots, a semi-solid medium was found to be superior to a bilayer medium. Various stages of nucellar culture were transformed with Agrobacterium tumefaciens strain LBA 4404 containing the binary vector, pCambia 2301, harbouring the npt II gene as a selectable marker and GUS as a reporter gene. During kanamycin sensitivity tests, it was found that 200 mg l–1 kanamycin completely inhibited regeneration of fresh new callus in nonco-cultivated embryogenic callus derived from nucellus. Among the various stages of nucellar culture tested, embryogenic callus was found to be amenable to Agrobacterium-mediated gene transfer. Maximum transformation was obtained using 150 µl bacterial culture with 3 d of co-cultivation. The expression of the reporter transgene was confirmed by GUS assay.

Pathogenesis – related proteins of tomato: comparative studies on protein profiles of resistant and susceptible lines of tomato following infection with Alternaria solani

Plants elaborate a number of inducible defence responses following microbial attack. These include synthesis of phytoalexins, reinforcement of cell wall and production of the so-called “PR-proteins” or pathogenesis-related proteins (Lamb et al., 1989). These proteins have been detected in various plant species (Van Loon, 1985). Some of these proteins have been shown to be proteinase inhibitors or lytic enzymes like chitinases and Beta-1, 3-glucanases (Kombrink et al.,1988; Legrand et al., 1987). Chitinases and glucanases have attracted considerable attention as defence weapons in recent years (Boller, 1987), owing to the fact that these enzymes have the ability to directly attack specific structures of pathogens. The enzyme chitinase has been shown to be induced in tomato plants following infection with Verticillium albo-atrum (Pegg and Young, 1982). Preliminary investigations carried out in our laboratory have demonstrated the induction of chitinase in tomato plants following infection with the fungus Alternaria solani,the causal organism of early blight disease of tomato, as well as the ability of partially purified preparations of tomato chitinase to hydrolyse cell walls of A.solani and inhibit its mycelia] growth. In the present investigation, an attempt has been made to examine the role of chitinase in the defence response exhibited by two susceptible and four resistant lines of tomato following A.solani attack. The protein profiles of these lines have also been examined in order to compare the differential response of resistant and susceptible lines of tomato at the level of protein expression.