Ayesh Gaur

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...

Morphogenic Response of Leaf and Petiole Explants of Broccoli Using Thidiazuron

Broccoli (Brassica oleracea var. italica) is an important nutritionally rich vegetable cole crop grown in the world. Environmental stress, pests, and diseases cause enormous yield losses because of a limited gene pool. Genetic manipulation is becoming an important method for broccoli improvement. The objective of present study was to evaluate the potency of thidiazuron (TDZ) as a plant growth regulator in evoking morphogenic responses in leaf and petiole explants of broccoli. An efficient, reproducible, and high frequency plant regeneration protocol has been standardized in broccoli cv. Solan green head. Leaf and petiole explants were cultured on Murashige-Skoog (MS) medium, supplemented with a wide range of TDZ concentrations. The following treatments were designed for efficient in vitro shoot regeneration: TDZ alone, TDZ with adenine, TDZ with naphthalene acetic acid (NAA), and TDZ with indole acetic acid (IAA). Among the 36 combinations of growth regulators used, the highest percentage of leaf explants producing shoot (89.25%) was recorded on MS medium containing 1.0 μM TDZ and 0.107 μM NAA. The multiple shoot regeneration response of petiole explant producing shoots (91.55%) was obtained on MS medium containing 2.0 μM TDZ and 0.107 μM NAA. Shoot multiplication and elongation were obtained on the same medium. For root regeneration in in vitro regenerated shoots, different concentrations of NAA were applied. High frequency (100%) root regeneration response with healthy and vigorous roots was observed on MS medium supplemented with 0.54 μM NAA. The regenerated plantlets with well-developed shoots and root system were transferred to pots containing cocopeat and successfully acclimatized. We recommend 1.0 μM TDZ with 0.107 μM NAA and 2.0 μM TDZ and 0.107 μM NAA combinations for adventitious shoot regeneration from leaf and petiole explants in broccoli cv. Solan green head respectively. This is the first report on high frequency organogenesis from leaf and petiole explants of broccoli cv. Solan green head using thidiazuron.

Genetic Engineering for Insect Resistance in Economically Important Vegetable Crops

Vegetables play a vital role in human nutrition and health by providing nutrients, vitamins, antioxidants, phytosterols, and dietary fiber. In the developing world, vegetable farming is a considerable part of the agricultural economy of different nations. Vegetable crop quality and quantity are seriously affected by various biotic and abiotic stresses, which destabilize rural economies in many countries. In the last many decades, conventional breeding has contributed significantly for the improvement of vegetable quality, yields, biotic and abiotic stress resistance, and postharvest management, but there are many constraints in conventional breeding, which can only be overcome by techniques of modern biology for genetic advancements.

Agrobacterium-Mediated Insect Resistance Gene (cry1Aa) Transfer Studies Pertaining to Antibiotic Sensitivity on Cultured Tissues of Broccoli

ABSTRACT Successful plant transformation requires an efficient regeneration protocol and a suitable selection method. A genetically engineered, disarmed Agrobacterium tumefaciens strain containing a binary vector pBin-1Aa with cry1Aa (insect resistance) and npt-II (neomycin phosphotransferase-II) genes was used for genetic transformation studies. Effects of antibiotics kanamycin and cefotaxime were studied to determine the aptness of kanamycin resistance as a selectable marker and for cefotaxime in controlling excessive bacterial growth during genetic transformation studies using cultured hypocotyl, cotyledon, leaf, and petiole tissues of Broccoli (Brassica oleracea L var. italica). Leaf and petiole explants exhibited decreased fresh weight as kanamycin concentration increased, resulting in full or partial inhibition of shoot regeneration. A significant or nonsignificant negative correlation occurred between kanamycin concentration and explant fresh weight over the time. Cefotaxime had little effect on regeneration potential. Growth of Agrobacterium cells was controlled at a concentration of 400 mg·L−1 cefotaxime in explants, and maximum putative transgenic shoot regeneration in explants of hypocotyls (44.88%) and cotyledons (36.29%) was obtained on MS (Murashige and Skoog)-selective shoot regeneration medium. Polymerase chain reaction analysis of genomic DNA using specifically designed primers detected the presence of the cryIAa and npt-II genes in kanamycin-resistant broccoli plantlets. Of five randomly selected putative transgenic shoots, three were positive for presence/integration of cryIAa and npt-II genes during T-DNA transfer and integration into the plant genome. Kanamycin and cefotaxime act as effective selective agents during genetic transformation pertaining to antibiotic sensitivity on cultured tissues of broccoli.

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....

Effect of a Potent Cytokinin Thidiazuron (TDH) on the Regener- ation Potential of Leaf and Petiole Explants of Cauliflower (Brassica oleracea L var. botrytis)

Plant regeneration studies were carried out using leaf and petiole explants on forty dif-ferent combinations and concentrations of TDZ i. e. TDZ, TDZ+IAA, TDZ+NAA and TDZ+Adenine. Among the forty combinations tried, the high percent shoot regeneration was observed in peti-ole explants (76%) on MS medium supplemented with 0.22mg/l TDZ whereas in case of leaf ex-plants only 44% shoot regeneration was achieved on MS medium supplemented with 0.77mg/l TDZ+ 0.087 mg/l IAA. For root regener-ation the in vitro regenerated shoots were cul-tured on MS medium containing different con-centration of IAA and NAA. Hundred percent root regeneration was observed on the MS medi-um supplemented with 0.05mg/l and 0.20mg/l NAA respectively. The regenerated plantlets were transferred to the pots containing cocopeat and acclimatized. The regenerated plantlets were morphologically similar. An efficient and repro-ducible plant regeneration protocol has been standardized in cauliflower cv. Pusa Snowball K-1 and is being used for Agrobacterium- mediated cry1Aa gene transfer studies.