Agrobacterium mediated genetic transformation of pigeon pea (Cajanus cajan L. Millsp.) for resistance to spotted pod borer Maruca vitrata
Loading...
Date
2020
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Punjab Agricultural University, Ludhiana
Abstract
The present investigation dealing with introduction of cry1Ab gene for resistance to Maruca vitrata
into pigeonpea (Cajanus cajan L. Millsp.) through Agrobacterium-mediated in planta transformation
was carried out using three pigeonpea genotypes i.e. AL 15, AL 201 and PAU 881. Two methods of in
planta transformation were used for generating transgenic plants, including inoculation of pricked
embryo axes and floral dip transformation. Out of seven experiments on in planta transformation
method involving inoculation of pricked embryo axes, only two (OD600 of Agrobacterium broth = 0.6 -
0.7 containing 100 mM acetosyringone, 1 h dipping of 2-day old pricked seedlings) gave positive
results. A total of 5,022 half seeds were treated that gave rise to 4059 plants, out of which 15
(representing AL 15 and AL 201 genotypes) were PCR-positive with an overall transformation
frequency of 0.36 %. Out of 15 primary transformants, only 11 plants showed transcript accumulation
by semi-quantitative RT-PCR. The primary transformants were also analyzed for chimerism by
ELISA-based protein accumulation in selected branches. Out of a total of 182 branches of 10 RT –
PCR positive plants analyzed, 57, 43 and 49 branches had Cry1Ab protein content equal to or more
than 1.08 µg/g [Positive calibrator, (PC) 5.0 ppb], 0.58 µg/g (PC, 2.5 ppb) and 0.08 µg/g (PC, 0.5 ppb),
respectively. Besides, there were 33 such branches which did not show accumulation of Cry protein
indicating the presence of chimerism in primary transformants. The putative transgenics were advanced
to T1 generation, out of 904 plants analyzed, 97 were PCR-positive, thus exhibiting transformation
efficiency of 11.70 % (with genotype AL 201) and 10.24 % (with genotype AL 15). Twenty three T1
plants were randomly taken for determination of Cry1Ab protein content, out of which 12 showed high
protein content of more than 0.72 µg/g. These plants showed positive results in PCR and RT-PCR, and
further used for determining the efficacy of cry1Ab gene against second instar larva of Maruca vitrata
using insect bioassay. Both flowers and pods of these 12 T1 pigeonpea plants were used for bioassay
experiments. All 12 T1 plants showed restricted increase in larval weight as compared to nontransgenic
(control) plant. No insect mortality was observed, but larvae fed on two transgenic plants i.e.
201-344 and 15-537 showed no adult emergence as compared to other transgenic and control plants
where adult emergence was normally observed. Both of these two plants (201-344 and 15-537) had
high Cry protein content (0.88 µg/g) as compared to other transgenic plants, which established clearcut
positive correlation between amount of protein accumulated and inhibition of M. vitrata larval
growth. Further, T1 plants were advanced to T2 generation that were maintained in transgenic
glasshouse. In case of floral dip transformation, a total of 139 flower buds were treated and 454 seeds
were obtained. Ten out of 454 putative T1 plants were observed to be PCR-positive with a
transformation frequency of 2.2 %.
Description
Keywords
Citation
Manjinder Singh (2020). Agrobacterium mediated genetic transformation of pigeon pea (Cajanus cajan L. Millsp.) for resistance to spotted pod borer Maruca vitrata (Unpublished Ph.D. Dissertation). Punjab Agricultural University, Ludhiana, Punjab, India.