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Articles by K. Nataraja
Total Records ( 9 ) for K. Nataraja
  Ravindra B. Malabadi and K. Nataraja
  This study for the first time was aimed at developing a protocol for the genetic transformation of embryogenic tissue derived from the vegetative shoot apices of mature trees of P. roxburghii. This was achieved via the introduction of a bar-GUS cassette under the control of the ubiquitin promoter, through biolistic transfer. Expression of positive histochemical GUS activity (31%) in the bombarded embryogenic tissue was observed. PCR analysis of bar transgenes (54%) transformation efficiency indicated successful genetic modifications of P. roxburghii embryogenic tissue by the pAHC25 plasmid. This is the first successful report of genetic transformation in P. roxburghii using embryogenic tissue derived from the mature trees indicating that Chir pine is amenable to gene transfer.
  Ravindra B. Malabadi and K. Nataraja
  This study for the first time highlights the influence of putrescine on somatic embryogenesis in a commercially important Indian pine Pinus gerardiana. Mature zygotic embryos produced the highest percentage of embryogenic tissue on half strength MSG basal medium supplemented with 9.0 μM 2, 4-D and 2.0 mg L-1 putrescine. However, the percentage of somatic embryogenesis was not similar in all the three genotypes of Chilgoza pine. The highest percentage of somatic embryogenesis (81.2±2.8a) was recorded in PG321 genotype. The lowest percentage of somatic embryogenesis (69.0±2.1a) was obtained in PG11 genotype. Incorporation of polyamine biosynthesis inhibitors in the initiation medium has inhibited somatic embryogenesis. Explants regained the embryogenic potential after the addition of the putrescine in the basal medium. These results support earlier findings of the role of polyamines in the process of somatic embryogenesis in several plant species including conifers.
  Ravindra B. Malabadi and K. Nataraja
  This study highlights the key role of peroxidases influencing somatic cells towards embryogenic pathway. Further the clonal identity of somatic seedlings was assessed using RAPD and ISSR markers. The lowest peroxidase activity was observed in embryogenic cultures on maintenance medium showing elongated cells with cleavage polyembryony as compared to control. Highest peroxidase activity was observed in non-embryogenic cultures showing round, globular and oval cells. Peroxidase activity is not only involved in reducing the accumulation of toxic amounts of H2O2 but might also influences the conversion of somatic cells into an embryogenic state and, therefore, can be used as a biochemical marker to differentiate embryogenic and non-embryogenic cultures. RAPD and ISSR analysis found no evidence of genetic variation either within or between the embryogenic lines established from three of these trees, or between these lines and the trees of origin, or between somatic embryos derived plantlets and the trees of origin.
  Ravindra B. Malabadi and K. Nataraja
  Present research highlights for the first time the expression of cDNA clones of genes involved in programming the apical meristem cells towards somatic embryogenic pathway influenced by external environmental stimulus like cold-pretreatment. Differential display was used to isolate the genes which are expressed specifically in embryogenic tissue induced by cold-pretreatment of thin sections of vegetative shoot apices of mature trees of Pinus roxburghii. We have developed a rapid method which employs magnetic beads to capture mRNA from cell lysates. Of the 56 cold-enhanced embryogenic-associated cDNAs identified, 20 were cloned. Nine of the 20 fragments which generated single bands on re-amplification were selected for cloning and further analysis. During reverse northern hybridization, all the 20 clones selected generated a positive signal when probed with labeled cDNA from cold-enhanced embryogenic tissue, but no signal when probed with cDNA from the non-embryogenic tissue (control treatment). All the 20 clones thus contained inserts that were specific to cold-enhanced somatic embryogenesis. This approach allows us to perform differential display and construction of cDNA libraries from the small amount of embryogenic tissue and outline a PCR-based method for confirming differential expression of large number of cloned bands in cases where RNA quantities are limiting.
  Ravindra B. Malabadi and K. Nataraja
  This investigation highlights for the first time the application of 24-epibrassinolide in the micropropagation of orchids. Successful initiation of protocorm-like bodies and in vitro regeneration of Cymbidium elegans was achieved using shoot tip sections and 24-epibrassinolide supplemented Mitra et al. (1976) basal medium. The highest percentage of explants (91.0%) producing PLBs (24.0±2.1) was recorded on 4.0 μM 24-epibrassinolide supplemented basal medium. All the newly formed protocorm-like-bodies survived and after nearly 12 weeks, small bud-like structures formed healthy shoots. Shoots produced roots when cultured on basal medium supplemented with 2.0 μM triacontanol. The well-rooted shoots were transferred to pots containing charcoal chips, coconut husk and broken tiles (2:2:1) and 100% survival rate was achieved. Our results for the first time demonstrate that 24-epibrassinolide provided compelling evidence that they can be effectively used in the micropropagation of orchids.
  Ravindra B. Malabadi and K. Nataraja
  This study highlights the positive role of TRIA in inducing the somatic embryogenesis using secondary needles of mature trees of P. roxburghii. Very few reports of induction of somatic embryogenesis using secondary needles as explants are available in conifers. Secondary needles of mature pines can be used as the best explant-source-material for the micropropagation of conifers. Explants cold-pretreated at 4°C and incubated for 3 days on 0.3% activated charcoal induced white-mucilaginous embryogenic tissue on DCR basal medium (initiation medium-I ) supplemented with 22.62 μM 2, 4-D, 26.85 μM NAA and 5 μM TRIA in all the 3 genotypes (PR17, PR100 and PR321) of Pinus roxburghii. Explants without cold pre-treatment induced non-embryogenic tissue on initiation medium supplemented with various concentrations of TRIA. Therefore, both the cold pre-treatment and the presence of TRIA in the initiation medium might have triggered the cells in programming towards the somatic embryogenesis. It was concluded that combination of stress induction by cold pre-treatment and inclusion of TRIA with DCR basal medium has a great potential of inducing embryogenic tissue in all the three genotypes tested. This has created a new possibility of micropropagation of pines for the current demands of commercial forestry.
  Ravindra B. Malabadi and K. Nataraja
  This study highlights for the first time the influence of smoke-saturated-water on somatic embryogenesis using vegetative shoot apices of mature trees of Pinus wallichiana, an important pine tree of Himalayan region. The addition of smoke-saturated -water derived from the local grasses has a significant effect on the different developmental stages of somatic embryogenesis of P. wallichiana. Smoke-saturated-water at a concentration of 10% in the DCR basal medium (Gupta and Durzan, 1985) resulted in the higher percentage of embryogenesis in all the three genotypes of P. wallichiana as compared against control. Lower concentrations of smoke-saturated water do not have any effect on somatic embryogenesis. On the other hand higher concentrations of smoke-saturated water inhibited embryogenesis in all the three genotypes of P. wallichiana. The presence of smoke-saturated-water in the maturation medium increased the rate of embryo development as evidenced by the occurrence of more number of well matured somatic embryos (PW-37; PW-34; PW120-39) recovered per gram fresh-weight of embryogenic tissue as compared against control. Maximum number of somatic embryos germinated successfully and resulted in the formation of vigorous seedlings compared against control. These observations suggest that the active ingredient (s) in smoke-saturated-water play a regulatory role in plant development. This plays an important role in commercial forestry.
  Ravindra B. Malabadi and K. Nataraja
  Pinus wallichiana A. B. Jacks (Himalayan blue pine or Bhutan pine) is one of the most recalcitrant species to in vitro propagation via somatic embryogenesis among all the Indian pines. Somatic embryogenesis is the only method that can enable us to produce large number of somatic seedlings in a short period of time to fulfill the urgent needs of afforestation programmes. Till today, no reports of somatic embryogenesis were available in Pinus wallichiana. To be commercially used, somatic embryogenesis technology must work with a variety of genetically diverse trees. This study highlights for the first time the successful brassinolide-mediated stimulation of embryogenesis in all the three genotypes of Pinus wallichian tested. 24-epiBrassinolide at 2.0 μ M with 9.0 μ M 2, 4-D enhanced the formation of embryogenic tissue from mature zygotic embryos on half-strength MSG basal medium. However, the frequency of somatic embryogenesis (PW145-87.4%; PW21-60.8%; PW106-91.5%) was not similar in all the three genotypes tested.
  Ravindra B. Malabadi and K. Nataraja
  A protocol is presented for genetically engineering Vanilla planifolia orchid using a routine transformation procedure via Agrobacterium tumefaciens. An expression vector containing nptII and GUS genes driven by the cauliflower mosaic virus (CaMV) 35S promoter was successfully introduced into the V. planifolia genome by A. tumefaciens. Protocorm-like bodies (PLB’s) derived from protocorms, were the target explants for transformation. The presence of the transformed gene in the transgenic orchid plants was confirmed by the PCR analysis; Southern blot and Northern blot analyses of the PCR product. Therefore, the foreign DNA was successfully integrated into the orchid genome and expressed transcriptionally and translationally in these orchid plants. The present transformation method reported is suitable for improving the V. planifollia orchid through genetic engineering.
 
 
 
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