ICRISAT Archival Report 2006 - The seedlings of success in the ...

The transcription factor DREB1A, which regulates the gene expression via recognition of the DRE (Dehydration
Responsive Element) sequence was placed under the control of the stress inducible rd29A promoter, both isolated
from Arabidopsis thaliana, and introduced into the peanut variety, JL 24 through Agrobacterium tumifaciens
mediated gene transfer. Over 50 transgenic events were produced and advanced to the T 4 generation. The
transformants were screened by polymerase chain reaction (PCR), RT-PCR, and Southern analysis for the presence
and expression of DREB1A. Initial assessment of 14 transgenic events carried out by using the soil drying
experiments showed differences in their transpiration responses to soil drying. There was a significant variation
amongst the tested events under drought stress for transpiration efficiency (TE). A significant positive correlation
with SCMR(r = 0.7359) and a negative correlation with specific leaf area (SLA) (r = 0.8237) were obtained.
However, TE did not correlate significantly with Δ 13 C, thus suggesting a lack of relationship between TE and Δ 13 C.
Two events, RD2 and RD11 appeared to have higher TE than original cultivar JL 24.
Comparison of promising events RD2 and RD with high TE (JUG 24), and low TE (TAG 24) materials: The
purpose of this experiment was to confirm the superiority of these events and compare them to known germplasm
for high (JUG 24) and low (TAG 24) TE level. JL 24 had TE at the level of TAG 24, which was expected, lower
than TE in JUG 24. Results confirm that RD 2 has a higher TE than JL 24 under water deficit conditions. It also
showed that RD 2 had higher TE than the best reported genotype (JUG 24) for TE under water deficit. Event RD 11
had a TE that was only slightly above that of JL 24 and TAG 24. Therefore, only RD 2 event appeared to confirm its
previous superiority for TE.
Effect of DREB1A on the response to water deficit at different growth stages (early flowering, late-flowering,
and pod-filling): This experiment was carried out to: i) measure TE at different stages in two transgenic events (RD
2 and RD 11), control JL 24, and few breeding lines; and ii) test the effect of a water deficit applied at different
stages (early flowering, late-flowering, and pod-filling) on pod yield and pod number. We used the standard drydown
technique to apply the stress at different stages. At each stage, TE was measured so that sets of plants were
harvested before imposing the stress, and others (well-watered and water stress) harvested after water stressed plants
had depleted all the soil moisture. To measure and compare the effect of a drought spell at different stages on the
final pod yield, we applied the treatment (WS and WW) to two more sets of plants at each stage. After treatment
imposition, the WS were re-watered and kept until maturity along with the WW set. The WS set was re-watered
when their relative transpiration was between 10 - 20% of that of control. We based our analysis on pod numbers per
plants, rather than pod yield because of mite infection towards the end of crop maturation.
The TE measured at flowering stage was higher in RD 2 and RD 11 than in JL 24 under water stress conditions, but
was similar in all the lines under WW conditions. At the late flowering stage, TE of RD was superior to that of JL 24
only under WW conditions, whereas TE of RD 11 was similar to JL 24. Under water stress, TE was similar in all
three lines.
The effect of a water stress at mid-pod filling stage had no influence on the pod number relative to the control (pod
number was 90% of control across all genotypes), although a few genotypes such as ICR 48 and JUG 26 had a
relatively lower pod number under stress. By contrast, the relative pod number of plants exposed to stress at earlyand
late-flowering was 63 and 76 % of that of the control across all genotypes tested, with a fairly large variation
across genotypes (35 - 93% for early-flowering stage, and 49 - 98% for late-flowering stage). During the earlyflowering
stage, the drought tolerant genotype ICGV 91114 was able to maintain 93% of control pod number. RD 2
and RD 11 maintained 82 and 56% of control pod number, respectively, whereas JL 24 maintained only 63 % of
control pod number, indicating that RD 2 was able to maintain pod number when exposed to stress. When the stress
was applied at the late-flowering stage, there were no differences in relative pod number between RD 2 and JL 24
(61% in both cases). The breeding lines ICGV 91114, TAG 24, and ICGV 86031 had a relative pod number at about
90% of the control. This experiment showed that flowering is an extremely sensitive stage to water deficit, where
the differences between the genotypes were maximum.
Five more transgenic events of groundnut transformed with rd29::DREB1A were tested for TE. The positive plants
were tested at T 2 stage. Using the regular dry-down technique, a large number of replicates were tested for each
genotype. We included also RD2, the event showing consistently higher TE in previous experiments. Under WW
conditions, TE of RD was similar to that of JL 24. However, one event, RD33, had higher TE under WW than JL 24
(7.70 vs 4.80). Under water stress conditions, RD2 had higher TE than JL 24 (7.42 vs 4.89). Two events (RD33 and
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