Identification of unique subset of cashew germplasm using DNA marker analysis

363-374Cashew (Anacardium occidentale L.) is an important edible nut crop of India and other tropical regions. Its utility depends on characterization of germplasm resources by molecular and morphological markers. A study was initiated on 172 cashew germplasms to assess their genetic diversity and population variation based on a combination of both RAPD and ISSR markers. Using selected 9 and 10 primers of RAPD and ISSR, a total of 46 and 56 polymorphic bands with 86.9 and 91.8% polymorphism were generated, respectively. For better genetic differentiation, markers data from both (RAPD+ISSR) were combined and obtained a total of 107 bands, of which 96 bands (89.7%) were polymorphic with an average of 5.05 polymorphic bands per primer. High percentage of polymorphism (86.9-91.8%) observed with different markers indicated high level of genetic variation existing among the accessions. Co-efficient of genetic similarity (Jaccard’s) between different pair of accessions varied from 0.43 to 0.94 in RAPD, 0.38 to 0.89 in ISSR and 0.38 to 0.86 with combined markers suggested a genetic diversity (dissimilarity) ranging 6 to 57%, 11 to 62% and 14 to 62%, respectively. An average diversity around 50% indicated moderate diversity existing among the accessions. The cluster diagram based on combined markers distinguished 172 accessions into 17 clusters. Some correspondence was observed between the molecular groupings and the morphological clusters. Among the accessions, NRC-432 and NRC-119 were highly divergent and NRC-216 and NRC-235 were highly similar. A ‘Subset’ of 63 accessions were identified, which included unique genotypes like CNSL free types, purple genotype, dwarf types, wild species and diverse genotypes, which need to be conserved and used. Population differentiation based on number of alleles, Shannon’s information index and percentage of polymorphic loci indicated high genetic diversity in the collections and it was relatively more in Karnataka group, followed by Kerala and Andhra Pradesh; while genetic diversity was low in Orissa and West Bengal groups. Based on analysis of molecular variation (AMOVA), maximum (96%) genetic variation was observed within the population and least (4%) genetic variation was noticed between the populations (subsets). On the basis of present study, suggestions were made for the type of collections to be used for the conservation of cashew

Micrografting of Protea cynaroides

The inability to induce rooting of in vitro-established Protea cynaroides microshoots has prevented the production of complete plantlets. A successful shoot-tip micrografting technique was developed using in vitro-germinated P. cynaroides seedlings as rootstocks and axenic microshoots established from pot plants as microscions. Thirty-day old seedlings, germinated on growth-regulator-free, half-strength Murashige and Skoog medium, were decapitated and a vertical incision made from the top end. The bottom ends of microshoots established on modified Murashige and Skoog medium were cut into a wedge (‘V’) shape, and placed into the incision. The micrografted explants were cultured in a growth chamber with the temperature adjusted to 25 ± 2°C, with a 12-h photoperiod. Best results were obtained by placing the microscions directly onto the rootstock without any pre-treatments. Dipping the explants in anti-oxidant solution or placing a layer of medium around the graft area led to the blackening of the microscion. Abbreviations EDTA Ethylenediaminetetraacetate - BAP 6-Benzylaminopurine - GA3 Gibberellic acid - PAR Photosynthetic active radiatio

In vitro production of Indian citrus ringspot virus (ICRSV) free kinnow plants employing phytotherapy coupled with shoot tip grafting

This paper reports the elimination of Indian citrus ringspot virus (ICRSV) from “kinnow” (Citrus nobilis Lour × Citrus deliciosa Tenora) employing phytotherapy coupled with shoot tip grafting under in vitro conditions. Nodal segments from infected mother plant (indexed by indirect enzyme-linked immunosorbent assay [ELISA] and reverse transcriptase PCR [RT-PCR]) were cultured on Murashige and Skoog medium containing 2iP (1 mg/l or 4.9 μM) and malt extract (800 mg/l) along with different concentrations of aqueous extracts from leaves of Azadirachta indica (Neem), Sorghum vulgare (Jowar), and roots of Boerhaavia diffusa (Punarnava). Shoot tips were excised from the nodal sprouts and grafted on to rough lemon (Citrus jambhiri) under aseptic conditions. Maximum effect (50% virus elimination) was seen for aqueous leaf extracts of A. indica followed by B. diffusa root extract (42.86%) and S. vulgare leaf extract (31.58%). Plants/plantlets were considered virus-free only when showing negative reactions by both indirect ELISA and RT-PCR