Optimization of an in vitro regeneration and transformation protocol in Khasi Mandarin
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Date
2017-07
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Publisher
AAU, Jorhat
Abstract
Citrus species are the most widely grown fruit crops within the whole
world. India is the fourth largest producer of orange in the world. North-Eastern India
is considered as one of the centres of origin of many citrus species. Among them
Khasi Mandarin is the most widely grown citrus species. According to Ministry of
Agriculture and Irrigation, Govt. Of India, the yield of Khasi Mandarin is declining
day by day drastically due to biotic and abiotic stress. Conventional breeding for
overcoming this problem is limiting due to non-availability of resistant sources.
Recent advances in genetic engineering have made it possible to incorporate desirable
genes from alien sources to elite genotype mainly through Agrobacterium-mediated
genetic transformation. Citrus cultivars vary in their response to in
vitro organogenesis and genetic transformation. This results in need for a cultivarspecific
optimization of an in vitro regeneration and transformation protocol.
Most of the plant regeneration processes in citrus, through tissue culture,
involve use of Cotyledon, epicotyl segment, shoot-tip, internode, root meristem as
explants. A study was conducted to develop a regeneration and Agrobactetrium
mediated transformation protocol for Khasi Mandarin using zygotic seedling as
explants obtained from six-week-old in vitro grown seedlings. Modified MS media
containing 1 mg/l BAP, 0.5mg/l NAA and 0.4mg/l Kinetin shows the best result for
multiple shoot induction with an efficiency of 68%. The number of multiple shoots
developed was on an average 5. The modified MS medium containing containing 0.25
mg/l BAP,0.5 mg/l NAA, 0.5 mg/l IBA shows best result for rooting with an
efficiency of 82% with an average root length 2 cm. Zygotic explants with injured
shoot tip were used as explant for transformation with Agrobacterium strain AGL1,
harbouring plasmid pCAMBIA1301 containing hpt as selectable marker gene and gus
as a reporter gene. Modified MS media containing 100mM Acetosyringone was fund
to be most effective medium for co-cultivation. Regeneration and selection media
containing 1 mg/l BAP, 0.5mg/l NAA, 0.4mg/l Kinetin and 30mg/l hygromycin and
250mg/l timentin shows the best result. In vitro regenerated shoots that survived upto
3rd selection cycle were considered as putative transformants. Some of the putative
transformed shoots showed positive result for gus in PCR analysis.
The present investigation is a preliminary study on optimization of an in
vitro regeneration and transformation protocol in Khasi Mandarin. More and more
concentrated effort is needed to establish a most efficient regeneration and
transformation protocol considering various factors affecting genetic transformation
and regeneration efficiency.