Ajay Kumar Thakur

Genetic engineering strategies for biotic and abiotic stress tolerance and quality enhancement in horticultural crops: a comprehensive review

Genetic engineering technique offers myriads of applications in improvement of horticultural crops for biotic and abiotic stress tolerance, and produce quality enhancement. During last two decades, a large number of transgenic horticultural crops has been developed and more are underway. A number of genes including natural and synthetic Cry genes, protease inhibitors, trypsin inhibitors and cystatin genes have been used to incorporate insect and nematode resistance. For providing protection against fungal and bacterial diseases, various genes like chitinase, glucanase, osmotin, defensin and pathogenesis-related genes are being transferred to many horticultural crops world over. RNAi technique has been found quite successful in inducing virus resistance in horticultural crops in addition to coat protein genes. Abiotic stresses such as drought, heat and salinity adversely affect production and productivity of horticultural crops and a number of genes encoding for biosynthesis of stress protecting compounds including mannitol, glycine betaine and heat shock proteins have been employed for abiotic stress tolerance besides various transcription factors like DREB1, MAPK, WRKY, etc. Antisense gene and RNAi technologies have revolutionized the pace of improvement of horticultural crops, particularly ornamentals for color modification, increasing shelf-life and reducing post-harvest losses. Precise genome editing tools, particularly CRISPR/Cas9, have been efficiently applied in tomato, petunia, citrus, grape, potato and apple for gene mutation, repression, activation and epigenome editing. This review provides comprehensive overview to draw the attention of researchers for better understanding of genetic engineering advancements in imparting biotic and abiotic stress tolerance as well as on improving various traits related to quality, texture, plant architecture modification, increasing shelf-life, etc. in different horticultural crops.

In vitro plant regeneration studies and their potential applications in Populus spp.: a review

Genus Populus comprises about 25–35 species of deciduous flowering plants in the family Salicaceae which are widely distributed in temperate climates of the Northern Hemisphere. Populus species are important resources in certain branches of industry and have a special role for the scientific study of biological and agricultural systems. The poplar is known for its remarkable significance among the commercially propagated tree species such as teak, eucalyptus, wild cherry, red wood, and radiata pine. In vitro regeneration refers to growing and multiplications of cells, tissues and organs on defined liquid/solid media under aseptic and controlled environments. In vitro clonal propagation of forest trees, due to the high multiplication rate, is an attractive alternative for rapid propagation of elite genotypes of those species that could not easily be propagated through conventional methods. Owing to their widespread uses at the industrial level and for meeting the ever-increasing global demand for biomass p...

SSR marker variations in Brassica species provide insight into the origin and evolution of Brassica amphidiploids

BackgroundOilseed Brassica represents an important group of oilseed crops with a long history of evolution and cultivation. To understand the origin and evolution of Brassica amphidiploids, simple sequence repeat (SSR) markers were used to unravel genetic variations in three diploids and three amphidiploid Brassica species of U’s triangle along with Eruca sativa as an outlier.ResultsOf 124 Brassica-derived SSR loci assayed, 100% cross-transferability was obtained for B. juncea and three subspecies of B. rapa, while lowest cross-transferability (91.93%) was obtained for Eruca sativa. The average % age of cross-transferability across all the seven species was 98.15%. The number of alleles detected at each locus ranged from one to six with an average of 3.41 alleles per primer pair. Neighbor-Joining-based dendrogram divided all the 40 accessions into two main groups composed of B. juncea/B. nigra/B. rapa and B. carinata/B. napus/B. oleracea. C-genome of oilseed Brassica species remained relatively more conserved than A- and B-genome. A- genome present in B. juncea and B. napus seems distinct from each other and hence provides great opportunity for generating diversity through synthesizing amphidiploids from different sources of A- genome. B. juncea had least intra-specific distance indicating narrow genetic base. B. rapa appears to be more primitive species from which other two diploid species might have evolved.ConclusionThe SSR marker set developed in this study will assist in DNA fingerprinting of various Brassica species cultivars, evaluating the genetic diversity in Brassica germplasm, genome mapping and construction of linkage maps, gene tagging and various other genomics-related studies in Brassica species. Further, the evolutionary relationship established among various Brassica species would assist in formulating suitable breeding strategies for widening the genetic base of Brassica amphidiploids by exploiting the genetic diversity present in diploid progenitor gene pools.

High Efficiency Plant Regeneration from Cotyledon Explants of Pomegranate (Punica granatum L.) cv. Kandhari Kabuli

A high frequency plant regeneration pro-tocol via direct organogenesis has been develo-ped for Punica granatum L. cv. Kandhari Kabuli, a medicinally important horticultural tree. Cotyle-don explants procured from 15-20-days-old in vitro grown seedlings were used as explants. MS medium supplemented with 2.0 mg/l 6-benzylaminopurine (BAP) and 1.5 mg/l naphtha-lene acetic acid (NAA) was optimal for shoot re-generation, which resulted into highest frequen-cy of shoot regeneration (67.89%) along with maximum average number of shoots formed per explant (5.38) and average shoot length (3.62 cm). Shoot multiplication and elongation took place on the same medium. Half strength MS medium containing 0.1 mg/l NAA and 0.02% ac-tivated charcoal was found most effective for rooting of shoots resulting into 80.12% root re-generation frequency. Regenerated plantlets were transferred to plastic cups containing auto-claved peat moss and successfully acclimatized with 85% survival rate. This is the first report on in vitro plant regeneration through direct orga-nogenesis in P. granatum L. cv. Kandhari Kabuli using cotyledon explants.

Understanding genetic relationship and population structure of Indian soybean varieties using microsatellite markers

Thirty-nine simple sequence repeat markers have been used to estimate the genetic diversity and population structure of 78 soybean varieties released in India. A total of 140 alleles were detected and an average of 3.59 alleles per locus was recorded. Polymorphism information content values for all the polymorphic markers across 78 soybean varieties ranged from 0.073 (Satt415) to 0.931 (Satt084), with an average value of 0.59. Mean similarity coefficient was recorded as 0.41 for all the genotypes. Marker index values ranged from 0.051 (Satt498) to 5.556 (SOYSHP176) with an average value of 2.54. The resolving power value ranged from 0.102 (satt498) to 2.086 (GMABABE) with an average value of 1.64. Both the phylogenetic and population structure analysis classified soybean varieties into five main clusters and five sub-populations, respectively. Principal coordinate analysis results are in agreement with both of these methods, which grouped the varieties on the basis of their origin, pedigree and releasing centre.

Transgenic Development for Biotic and Abiotic Stress Management in Horticultural Crops

Abstract Genetic engineering offers one of the most potential techniques for modification of many horticulturally important traits, which have been found impossible so far to incorporate in elite backgrounds via conventional breeding methods without affecting their phenotypic traits. Thus this technique assists in circumventing the problems of a long generation period, which hampers the scope of genetic improvement particularly in perennial horticultural tree species. Advancements in molecular biology and genetic transformation have made it possible to identify, isolate, and transfer desirable genes from any plants or microbes. The introduction or enhancement of desirable traits is traditionally done by breeding. This is time consuming and also not very precise. On the other hand, genetic engineering creates plants with specific changes in the background of a proven cultivar without disturbing their genetic constitution. Expression of undesirable genes can be blocked by the application of antisense gene technology and RNAi technology. Biotechnological interventions that could increase the efficiency of horticultural crop improvement are essential to generate plants with several desirable traits.

Standardization of Agrobacterium tumefaciens-mediated genetic transformation protocol in Punica granatum L. cv. Kandhari Kabuli

A protocol for Agrobacterium tumefaciens-mediated genetic transformation in Punica granatum cv. Kandhari Kabuli has been developed using cotyledon and hypocotyl explants via both direct and indirect organogenesis pathways. Agrobacterium tumefaciens strain LBA 4404 harboring gus and npt-II genes in a binary vector pBI 121 had been used for genetic transformation studies. Pre-culturing of explants for 48 h (2 days) on regeneration medium before agrobacterial infection significantly increased the transformation frequency. Co-cultivation period of 48 h (2 days) was found to be optimum for genetic transformation. Cefotaxime in a concentration of 500 mg/l was found optimal for controlling the excessive bacterial growth and transformed shoots were selected using 50 mg/l kanamycin. Only the transformed cells were able to grow on the selective regeneration medium. Transgenic shoots were rooted on half-strength MS medium supplemented with 0.10 mg/l NAA, 0.02% activated charcoal, 50 mg/l kanamycin and 500 mg/l cefotaxime and subsequently acclimatized to the culture room conditions. Stable transformation and expression were confirmed on the basis of GUS histochemical and spectrophotometric assays. This genetic transformation protocol will provide a platform for genetic manipulation of pomegranate for incorporation of genes governing various horticulturally important traits in future.

Agrobacterium-Mediated Genetic Transformation of Populus deltoides Marsh Clone G48 with gus and npt-II Genes

Agrobacterium-Mediated Genetic Transformation of Populus deltoides Marsh Clone G48 with gus and npt-II Genes The present investigation had been carried out to standardize a protocol for Agrobacterium- mediated gene transfer in Populus deltoides Marsh. Clone G48 using petiole explants. Reproducibility of already standardized regeneration protocol for clone G48 had been evaluated. High frequency shoot regeneration (72%) from petiole explants was obtained on MS medium supplemented with 0.5 mg l-1 BAP, 0.2 mg l-1 IAA and 15 mg l-1 adenine sulphate. Root regeneration (100%) in in vitro developed shoots was obtained on MS medium supplemented with 0.1 mg l-1 IAA. Increasing concentrations of kanamycin (10-50 mg l-1) were given to find out the minimum dose of kanamycin required for the selection of putative transformed cells during genetic transformation. It was observed that a doze of 50 mg l-1 kanamycin inhibited callus formation and shoot regeneration and the explants turned brown and started dieing. This concentration of kanamycin would be the most useful for selection of npt-II gene transformed petiole cells/ tissues. Disarmed Agrobacterium tumefaciens LBA 4404 strain containing a reporter β-glucuronidase (gus) gene in binary vector pBI 121 along with kanamycin resistance gene (npt-II) was used for genetic transformation experiments. Only the transformed cells/ shoots were able to grow on selective shoot regeneration medium containing 50 mg l-1 kanamycin, whereas control explants did not survive. Regenerated putative transgenic shoots were subjected to transient gene expression analysis using spectrophotometric GUS assay and were found positive. This genetic transformation protocol will provide a platform for genetic manipulation of P. deltoides clone G48 for incorporation of genes governing various silviculturally important traits in future....