Supplementary material

Mature embryo-derived wheat transformation for tolerance to moisture stress

Kasirajan Lakshmi 1*, Kovilpillai Boomiraj , Bansal Chander Kailash 2

Materials and method

Plant tissue culture and Biolistic bombardment

Embryogenic calli were produced from mature seeds of the wheat (Triticum aestivum) cultivars HDR77 and the transformation procedure was followed as described in Kasirajan et al. (2013) (Fig 1).

PCR analysis

Putative transgenics along with the untransformed control plants were subjected to PCR analysis. Genomic

DNA was isolated from leaf tissues and a PCR reaction was carried out, using 100 ng of genomic DNA as template and with PAtRD29A forward and AtCBF3 reverse primers.

Response of Transgenic Plants to Water Deficit Stress

T1 plants of HRCB3#17 were grown in a 15 cm pot and watered daily. During the panicle initiation stage, irrigation was stopped for 15 days and the plants were photographed before and after stress. RWC of the plants was measured before and after moisture stress. The T1 plants as well as the wild type were recovered from stress by watering with Hoagland solution. Yield components like panicle number, grains per plant and seed weight per plant were recorded after the recovery. The yield advantage was calculated according to the formula,

Yield of transgenic – yield of WT plant Yield advantage (%) = ------X 100 Yield of the wild plant

Results

Screening of the T0 transformants for gene of interest (AtCBF3)

A total number of 595 calli of HDR77 were bombarded with the plasmid of the gene construct PAtRD29A::

AtDREB1A/CBF3::NOS, out of which 9 plants were PCR positive for the selection marker gene BAR. Out of 9, only two transformants showed positive amplification for their respective gene of interest (AtCBF3).

Genomic DNA of T0 transformants was isolated and a PCR reaction performed with the promoter-specific forward primer (AtRD29A) and cDNA-specific reverse primer (AtCBF3). HRCB3#17 and HRCB3#18 showed an amplicon of 1592 bp which were positive for their respective gene of interest (Fig. 2). The T 0 transformants were grown till the maturity and T1 seeds were harvested from both the individual T0 transformants. The seeds were collected and a part of these seed were taken for T1 analysis. The Summary of T0 putative transformants and the number of seeds harvested are shown in Table 1.

Analysis of T1 plants of HRCB3#17

Two hundred and eighty nine seeds were harvested from the T0 plants; out of which, 90 were chosen randomly for T1 segregation analysis. After 15-20 days of germination, genomic DNA was isolated from 100 mg leaf tissue and used for PCR analysis. Based on the PCR results, the T 1 plants were scored ‘+’ and ‘-’ for both the marker gene (bar) and the gene of interest (AtCBF3) (data not shown). The number of positive plants having both

BAR and CBF3, only CBF3 or BAR and neither BAR nor CBF3 is tabulated in Table 2. Four plants were marker- free and they were numbered HRCB3#17-1, 37, 52 and 59.

Evaluation of drought tolerance of transgenic plants

T1 plants of HRCB3#17 were subjected to water deficit stress for 15 days at the panicle initiation stage. The stress level in the plant was monitored by relative water content (RWC) according to which a T1 plant, which was negative for the presence of the CBF3 transgene, was included as vector control. RWC values of the T 1 transgenics lines of HRCB3#17 were 12-15% higher than the wild type plant during stress. Phenotypic difference between the wild type and T1 plants of HRCB3#17, before and after water deficit stress, and the seeds collected from the recovered wild type and T1 transgenic plants are shown in Fig. 3.

Yield components like panicle number, grains per plant and 100 seed weight were also recorded from the wild type, vector control and T1 plants. Yield advantage of the T1 transgenics of HRCB3#17-1, 37, and 59 over the wild type was calculated and tabulated (Table 3). As there was no seed set in HRCB3#17-52, we were not able to perform a yield component analysis. The data revealed that the transgenic lines HRCB3#17-1, 37, and 59 which were marker free gave a yield advantage of 8.5% on the wild type. Supplementary Table 1 Summary of T0 transformant and the number of seeds harvested

Plant Number Plants scored for Plants scored for Number of T1 No. of T1 seeds

the SMG BAR by the AtCBF3 by seeds harvested analysed

PCR PCR

HRCB3#17 + + 289 90 HRCB3#18 + + 137 36

Supplementary Table 2 Summary of PCR analysis for T1 plants of HRDCB3#17

No. of plants positive No. of plants positive No. of plants positive No. of plants negative

for both Bar and CBF3 for only Bar gene for only CBF3 gene for both Bar and CBF3 11 51 4 14

Supplementary Table 3 Yield advantage of the T1 transgenics of HRCB3#17

Yield component Panicle Grains per 100 seed wt (g) Seed wt per Yield

number plant plant (g) advantage over

WT (%) Plant Number WT (HDR77) 6 124 2.46 2.93 HRCB3#17-1 7 128 2.67 3.42 22.0 HRCB3#17-37 5 104 2.82 3.18 8.5 HRCB3#17-59 6 52 3.73 3.39 15.6

Supplementary Fig 1a) Embryos placed in callusing medium MS medium supplemented with 2 mg 2,4-D,/l 1 mg

ABA/l, 0.5 mg kinetin /1. B) Calli formed from the embryos after a period a month of incubation in dark and pooled in the centre of the petriplate to facilitate bombardment at a distance of 6 cm. C) Calli showing GUS histochemical localization at 48 h post-bombardment. D) Calli after bombardment transferred to selection medium MS medium supplemented with 0.5 mg IAA/l, 1 mg kinetin /l, 4 mg basta/l. E) Transformants growing under the second phase of selection. F) Shoots transferred to jam bottles for further development. G) Shoots transferred to half strength MS medium without growth hormones for root development. H) Developed plants transferred to 4” pot for hardening. I)

Hardened plants transferred to National Phytotron facilities for further development

Supplementary Fig 2 PCR screening of T0 putative transformants for the presence of the gene of interest. Genomic

DNA was isolated from leaf tissues of the putative transformants and a PCR reaction was carried out, using 100 ng genomic DNA as template and with PAtRD29A forward and AtCBF3 reverse primers. Plants HRCB3#17; HRCB3#18 and showed the amplicon of expected size 1592 bp (Lane 5, 6)

Supplementary Fig 3 Phenotypic differences between wild type (WT) and T1 plants of HRCB3#17 before and after

15 days of water deficit stress. Seeds collected from the recovered plants are also shown

Supplementary Fig 1

Supplementary Fig 2

Supplementary Fig 3