LIST OF TABLES
6. IMPROVING AGRONOMIC TRAITS AND STRESS RESPONSE IN COWPEA BY VUNAC1/2-OVEREXPRESSION
6.2 METHODOLOGY
6.2.1 Construct preparation
The 888 bp open reading frames (ORF) sequences were cloned at the SfiI site in the pBEAB vector (provided from Gifu University, Japan) to generate the 35S:VuNAC1 and 35S:VuNAC2 constructs for overexpression (Fig. A4.1, Appendix)[372]. The VuNAC1/2-si constructs were prepared by cloning the 300 bp non-conserved gene region from the 3’-end were cloned at the BamH1 and KpnI sites in the pTRV2 vector (provided from NIPGR, India) for VIGS-based silencing (Fig. A4.1, Appendix 4). All the constructs were mobilized in the EHA105 Agrobacterium strain. The primers used were listed in Table A4.2, Appendix 4.
6.2.2 Genetic transformation
The healthy cowpea seeds of Pusa komal variety were sterilized in 0.2% HgCl2 (w/v) for 2 mins, followed by washing with deionized water (5X, 2 minutes each). After blot drying, the seeds were transferred to germination medium (1X MSB5 basal salt, 0.8 % Agar, 10.0 µM Thidiazuron, pH 5.8) and incubated at 27˚C in the dark for two days, followed by long-day
photoperiod condition (16 hr of light, 8 hr of dark) at 28°C, with white light illumination (110 µmol photons m-2s-1). 1-cm sections of cotyledonary nodes were excised from the four-day-old seedlings and dipped in LPGM medium (1X MSB5 basal salt, 1.0 µM BAP, 3% (w/v) sucrose, pH 5.5). The explants were injured at the node and dipped in the Agrobacterium strains carrying the 35S:VuNAC1/2 constructs (pre-cultured in AB minimal media + 50 µg/ml Kanamycin until the O.D.600 reaches up to 0.6) suspended in co-cultivation media (LPGM, with freshly added 100 µM Acetosyringone). The explants were infected with the empty vector carrying strains to generate wild type control plants, whereas explants without the infection served as a negative transformation control. The co-cultivation suspensions were shaken at 90 rpm for 30 minutes at 22˚C, blot dried, and co-cultivated further at 22˚C under dark conditions on Whatman paper soaked with the co-cultivation medium. After three days, the explants were washed in 500 mg/l Cefatoxime for 10 minutes, followed by washing in de-ionized water (5X, 2 minutes each).
After trimming, the explants were cultured in shoot induction media (1X MSB5 basal salt, 0.8%
Agar, 5.0 µM BAP, 0.5 µM Kinetin, 200 µg/ml Kanamycin, 500 µg/ml Cefatoxime, pH 5.8), at 27˚C for seven days. The regenerated explants were sub-cultured in shoot elongation media (1X MSB5 basal salt, 0.8 % Agar, 2.5 µM BAP, 0.1 µM Kinetin, 200 µg/ml Kanamycin, 500 µg/ml Cefatoxime, pH 5.8) for 15 days. The elongated shoots were transferred to the rooting medium (0.5 X MSB5 basal salt, 0.7 % Agar, 5.0 µM IBA, 500 µg/ml Cefatoxime, pH 5.7) and cultured until root induction. The seedlings were hardened in soilrite mix supplemented with
¼ MS medium for ten days and then grown in potting soil mixture in greenhouse conditions (photoperiod of 16/8 hr of light/dark, white light illumination of 110 µmol/m-2s-1, maintained at 28 °C).
6.2.3 VIGS assay
The EHA105 Agrobacterium strain harboring pTRV1, pTRV2, pTRV2-VuNAC1-si, and pTRV2-VuNAC2-si were grown in LB media containing 50 µg/ml Kanamycin and 25 µg/ml Rifampicin up to OD600=1.0. Equal volumes of cultures carrying pTRV1 and pTRV2 or its derivatives were mixed. The cells were harvested (3,000 x g for 5 min) and suspended in the agro-infiltration buffer (10 mM MgCl2, 10.0 mM MES-KOH, pH 5.6, and 100 µM Acetosyringone) and kept at RT for 4 hours. The sterilized cowpea seeds were imbibed in LPGM media for 24 hours in the dark to initiate sprouting. The healthy sprouts were co- cultivated with the pTRV1 and pTRV2 constructs by immersing in 10 ml of the agro- infiltration buffer and kept for shaking (150 rpm for 1 hour, 22 °C). The sprouts treated with empty pTRV1 and pTRV2 constructs served as wild type control plants. After soaking dry, the
sprouts were further co-cultivated at 22°C for 36 hrs in the dark and then cultured at 27°C with a 16/8 light/dark photoperiod. The germinated seedlings were then transferred to the soil pots and grown under greenhouse conditions.
6.2.4 Growth conditions for stress analysis 6.2.4.1 Seedling-stage study in hydroponic culture
Healthy seeds of wild type control and T2 generation transgenic lines were germinated for two days in the dark followed by 16/8 light/dark photoperiod conditions at 28°C. The four-day- old seedlings were grown in a gauzed hydroponics container supplied with ½ X modified Hoagland hydroponics media for approximately ten days until the first trifoliate leaves expanded [333]. The media was supplemented with 20% PEG 6000 (polyethylene glycol) and 200 mM NaCl to impose dehydration and salt stress. The trifoliate leaves from the stressed and the control plants were sampled at the indicated time intervals for RNA extraction using the RNeasy mini kit (Qiagen), followed by cDNA synthesis (Applied Biosystems, USA). The semi-quantitative and quantitative real-time PCR was performed in triplicates for each sample on Thermocycler Dice, Real-time system II (Takara, Japan). VuUbiquitin2 was used as a reference to determine the relative expression. The experiments were repeated three times with similar outcomes. To impose nutritional deficiency, the strength of the growth media was reduced to 1/10 fold, and the seedlings were grown for four weeks by replacing the media every three days.
6.2.4.2 Greenhouse study of soil plants
Four-day-old germinated seedlings were sown in soil pots and grown under greenhouse conditions for six weeks to acquire the mid-vegetative stage. For drought assay, the water was withdrawn for six weeks, followed by re-watering for recovery. The soil was supplemented with a gradient of NaCl solution (50 mM, 100 mM, 150 mM, and 200 mM, at an interval of two days) and then kept saturated with at 200 mM NaCl aqueous solution to impose salt stress for overall four weeks. The soil was drained with tap water to leach out deposited salt and maintained under normal greenhouse conditions to recover from the high salinity. For heat and cold stress, the six-week-old plants were transferred to growth chambers maintained at max/min temperature regime of 44°C/34°C and 18°C/ 12°C, to impose heat and cold stress for four weeks, while the plants maintained 27°C/ 20°C served as unstressed control. All the plants were moved to the normal green-house temperature regime (27°C/ 20°C) for recovery.
6.2.4.3 Study under lysimeter-operated conditions
The drought assay was carried out using Drought Simulator (Spura, India) to mimic the field environment in a poly-house. A field capacity (FC) of 95% was maintained throughout the vegetative growth of the plant. The FC was ramped down to 20% over eight days and maintained to impose drought stress throughout six weeks. For recovery, the FC was reset to 95%. A real-time monitoring computational server was used to determine the amount of water supplied and evapotranspiration. However, to assess the senescence phase, the plants were grown in fields, and their phenotype and biochemical parameters such as membrane damage and chlorophyll degradation were measured.
6.2.5 Measurement of physiological parameters and biochemical assays
6.2.5.1 Determination of photosynthetic parameters, relative water content, electrolyte leakage rate, and lipid peroxidation
The experiments were performed using the similar method described in sections 5.2.4.1, 5.2.4.2, 5.2.4.3, 5.2.4.4.
6.2.5.2 Determination of chlorophyll and carotenoid content
100 mg of each tissue sample was homogenized in 2 ml of ice-cold 80 % (v/v) acetone. The extract was centrifuged (3000 rpm, 10 min), and the supernatant was collected to measure the absorbance A646, A663, and A470. The chlorophyll and carotenoid content were estimated using the equations given by Lichtenthaler & Welburn (1983) [477]:
Chlorophyll a (μg/ml) = 12.21 (A663) - 2.81 (A646) Chlorophyll b (μg/ml) = 20.13 (A646) - 5.03 (A663) Carotenoids x+c (μg/ml) = (1000 A470-3.27Ca-104Cb) / 229 6.2.5.3 Determination of Na+ and K+ ion content
100 mg of oven-dried (75°C) leaf powder was digested in 1 N HCl at 90 °C water-bath for 30 minutes. The extract was cooled and centrifuged at 12000 rpm for 10 min to collect the top layer suspension. The extract was diluted 100 times using sterile de-ionized water. The ion content was estimated using Flame Photometer 128 (Systronics, India) in terms of ppm.
6.2.5.4 Histochemical Staining of H2O2 and O2-radicals
Fresh staining solutions of 1 mg/ml 3,3ʹ-Diaminobenzidine (DAB), pH 3.8) and 0.2% (w/v) Nitrotetrazolium blue chloride (NBT) solution in 50 mM sodium phosphate buffer, pH 7.5,