Browsing by Author "SHARMA, MANJUSHA"
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ThesisItem Open Access ADVENTITIOUS SHOOT REGENERATION FROM LEAVES OF APPLE ROOTSTOCK MERTON I.793(2014) SHARMA, MANJUSHA; MODGIL, MANJUABSTRACT The present studies were aimed to develop an efficient and reproducible method for shoot regeneration from leaf explants of apple rootstock Merton I793.The influence of two cytokinins BA and TDZ with three auxins IAA, IBA and NAA in different combinations were evaluated for shoot induction using light and dark incubation. Direct and healthy shoots were obtained from leaves on MS medium with BA-NAA and BA-IAA combinations in light incubation and 100% regeneration was achieved on medium with 4 mg/l BA and 1 mg/l NAA . Both direct and indirect shoot regeneration was obtained on TDZ-NAA and TDZIBA. Maximum shoot regeneration was promoted on 0.8&0.6 mg/l TDZ with 1 mg/l NAA in light. Dark treated leaves showed low rates of regeneration with TDZ combinations. RAPD analysis of regenerants using 5 primers revealed that the shoots which originated directly from leaves were similar to mother plant due to monomorphism, while shoots originated through intervening callus revealed 61.2% polymorphism.ThesisItem Open Access CLONING OF ROL B GENE AND GENETIC TRANSFORMATION OF CLONAL APPLE ROOTSTOCK(UHF,NAUNI, 2018-02) SHARMA, MANJUSHA; MODGIL, MANJUABSTRACT Merton 793, an excellent apple rootstock adapted to a wider range of soil types and recommended for replantation in orchards, is one of those rootstocks which are difficult to root. Therefore, with the purpose of increasing its rooting ability, cloning of rol B gene from Agrobacterium rhizogenes strain SA79 and its transfer in leaves of apple rootstock Merton 793 using Agrobacterium tumefaciens mediated genetic transformation has been carried out. After screening of antibiotics, 500 mg/l cefotaxime combined with 35 mg/l kanamycin were chosen for selection of putative transgenic shoots in subsequent transformation experiments. Young leaf explants were infected and co-cultivated with Agrobacterium tumefaciens strain LBA4404 harboring the vector pCAMBIA2300 which contained the rol B and nptII genes both under the control of CaMV35S promoter. Highest putative shoot regeneration (22%) was obtained on 4 mg/l BA and 1 mg/l NAA with 1.25 average number of shoot per regenerating explant. Thirty putative transgenic lines /shoots were obtained from twelve events and maintained separately on shoot multiplication medium supplemented with 300 mg/l cef and 25 mg/l kan. Transformation was confirmed in five lines by polymerase chain reaction amplification and reverse transcriptase -PCR of the rol B and nptII genes using gene specific primers. These rol B transgenic, lines (E10(1), E10(2), E16(2), E23(1), E27(2)) showed significant increase in rooting percentage and root number compared to non transformed control. E27(2) resulted in 100% rooting on media without added IBA and 33 – 50% with 0.5 mg/l IBA (kept for brief period), while non transformed control shoots needed IBA for rooting. All transgenic lines resulted in 74-100% hardening success which is better than untransformed controls. Our results suggested that the rooting capacity in Merton 793 is correlated with the expression of rol B gene which in turn resulted in successful hardening. .