Agrobacterium mediated genetic transformation of Citrus reticulata cv. Khasi mandarin
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Date
2020-02
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Abstract
Citrus is number one fruit of the world on accounts of its high nutritional value.
India is the fourth largest producer of Citrus in the world. The north-eastern region of
India is a rich treasure of various Citrus species. Khasi mandarin is the most
economically important one and plays a vital role in the socio-economic development of
the people in this region. Khasi mandarins are declining at a very high rate due to its
vulnerability to different pathogen and insect/ pest. Conventional breeding for
overcoming these problems are limited in Citrus and are directly associated with the
reproductive biology of Citrus. Recent advances in genetic engineering have made it
possible to incorporate desirable genes from elite genotype mainly through
Agrobacterium-mediated genetic transformation. Citrus species showed varied response
to in vitro regeneration and genetic transformation. Cultivar specific optimization of in
vitro regeneration and transformation protocol is very important. In the present
investigation, in vitro regeneration and Agrobacterium mediated genetic transformation
protocol for Khasi Mandarin was optimized using different explants like epicotyl,
hypocotyl, nodal and inter nodal segment obtained from six-week-old in vitro grown
zygotic seedling. Explants were transformed wih Agrobacterium strain LBA4404,
harbouring plasmid pBI121-AtSUC-GUS containing nptII as a selectable marker and
gus as a reporter gene. Hypocotyl was found to be the best explants for khasi mandarin
transformation and regeneration. MS medium supplemented with BAP (2mg/L), NAA
(0.5 mg/L), 2, 4-D (1mg/L), MES (0.5g/L), sucrose (30g/L) and acetosyringone
(100μM) was found to be best medium for co-cultivation. Modified MS medium
containing BAP (4mg/L), MES (0.5g/L), sucrose (30g/L), phytagel (4g/L), kanamycin
(50mg/L) and timentin (150mg/L) showed highest regeneration efficiency (18%).
Modified MS medium containing BAP (4mg/L), GA3 (0.5mg/L), MES (0.5g/L),
sucrose (30g/L), phytagel (4g/L), kanamycin (50mg/L) and timentin (150mg/L) showed
highest multiple shoot induction (6%). In vitro regenerated shoots that survived up to 3rd
selection cycle were subjected to GUS assay for confirmation of GUS expression in the
phloem tissues. Present investigation is a preliminary study for optimization of an in
vitro regeneration and genetic transformation protocol in Khasi Mandarin.