CLONING OF ROL B GENE AND GENETIC TRANSFORMATION OF CLONAL APPLE ROOTSTOCK
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Date
2018-02
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UHF,NAUNI
Abstract
ABSTRACT
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. .
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