Agriculture Reference
In-Depth Information
hybridization is performed following the procedure used in Southern blot
analysis. Then subsequent methods are performed using protocol of Vashisht
et al. (
2005
).
(iv) Western blot analysis: Total soluble protein is obtained by homogenizing
young leaves and stems of transgenic rice and control plants in the extraction
buffer (200 mM Tris-HCl, pH 6.8, 20 % glycerol, 10 % 2-mercaptoethanol).
Then, 10 µg of protein from each preparation is separated by 10 % SDS-
PAGE, transferred to a hybond-C membrane (Amersham). Then the subse-
quent methods are performed using the protocol of Vashisht et al. (
2005
) and
Sambrook et al. (
1989
).
(v) Bioassay analysis: In order to assess the stress tolerant doubled haploid trans-
genic plants, some work needs to be done using the following methods:
a) Confirmation of transgenic plants by overexpression of targeted genes:
Selection, regeneration and growth of transgenic plants through overex-
pressing method is followed by the protocol of Tuteja (
2010
) and Mahajan
and Tuteja (
2005
).
b) Confirmation of transgenic plants by histochemical, ß-glucuronidase
(GUS) assay: This method is performed for screening the putative trans-
genic plants for the expression of GUS by following the protocol of Tuteja
(
2010
).
c) Screening of transformants under the stress conditions: Transgenic plants
along with wild type (WT) are evaluated for physical responses to drought
stress by assessing their transpiration response to water deficit using the
dry down techniques (Sinclair and Ludlow 1986).
d) Screening of transformants under the stress tolerance by leaf disk assay:
Transgenic plants (T
0
generation) are checked by leaf disk assay for stress
tolerance by following the protocol of Tuteja (
2010
).
Description of development of abiotic stress tolerant fertile transgenic plants using
genetic transformation and androgenetic techniques is mentioned here in the fol-
lowing paragraphs:
2.2.4 Use of Biotechnology to Improve Abiotically Stressed Crop Plants
Major stresses (abiotic and biotic) have a considerable impact on crop growth, de-
velopment, and productivity throughout the world (Zhao and Zhang
2007
). Now
a days, abiotic stresses have become a continuous hindrance in crop production.
Therefore, it is important to elaborate on the injury and tolerance mechanisms, and
improve the crop genotypes under abiotic stress conditions (Shariatpanahi et al.
2006
; Roy and Basu
2009
; Roy et al.
2011
). Abiotic stresses such as sub- and su-
pra optimal temperatures, excess salt levels, reduced water availability leading to
drought stress, excess water resulting in flooding stress and oxidative stress caused
by the combination of high light intensity, etc., showed adverse effects on almost
all major crop plants (Grover and Minhas
2000
; Gill and Tuteja
2010
; Fleury et al.
