Drought and salt are major abiotic stresses that cause severe
loss of Basmati rice production. During recent years, several genes which
are responsible for over-production of osmolytes and stress proteins have
been shown to confer increased tolerance to these stresses in transgenic
rice plants (Jain and Jain 2000). One particularly promising gene, HVA
1 from barley (Hong et al. 1988), encodes a group 3 late embryogenesis
abundant (LEA 3) protein. Previously, we demonstrated a positive correlation
between HVA I gene expression and drought and salt-stress tolerance
in a japonica variety of transgenic rice (Xu et al. 1996). However,
under normal environmental conditions, overproduction of stress proteins
using a constitutive promoter, requires extra cellular energy and building
blocks, and may hamper the normal growth of rice plants (Su et al.
1998). Therefore for this study, we generated transgenic Basmati rice plants
that synthesize a high level of LEA 3 protein only under stress conditions.
We previously transformed rice by particle bombardment, in which most plants
harbored two to five copies of integrated HVA I gene. Most plants
that harbored more than two copies of the transgene showed gene silencing
in later generations (Cheng et al. 1998). Consequently, for this
investigation we transformed rice through the Agrobacterium mediated
system, as this method produces transgenic plants with relatively low copy
number of transgene as compared to other transformation methods (Roy et
al. 2000).
To investigate the role of LEA 3 accumulation on salt and drought tolerance in Basmati
rice, we constructed two plasmids in pCambia 1200, both using the HVA I gene. One of the
plasmids, pRKJ 21, contains an ABA inducible promoter, and the other one, pRKJ 6, contains a
constitutive promoter. A third plasmid, pRKJ 5, was constructed in pCambia 1201 with the
HVA I gene driven by the rice actin I promoter. Since pCambia vectors
lack super-virulent genes and do not give higher transformation frequencies
in recalcitrant Basmati rice, we mobilized pCambia vector (harboring the
HVA I gene) into Agrobacterium tumefaciens strain LBA4404
containing pSB1 by triparental mating. pSB1 and pCambia plasmids belong
to different incompatibility groups.
For this study we used mature seed scutellum-derived embryogenic calli
as suggested by Jain et al. 2000. Calli were raised on MS medium
containing casein hydrolysate (300 mgL-1), 2, 4-D (2.5 mgL-1
), proline (560 mgL-1) and maltose (30 gL-1). Calli
were infected with Agrobacterium containing either pRKJ 5, pRKJ
6 or pRKJ 21. After co-cultivation (two days) and two or three selection
cycles (7-10 days each), hygromycin-resistant calli were transferred onto
MS medium plates containing maltose (30 gL-1), kinetin (2 mgL-1),
NAA (0.5 mgL-1)and agarose (10 gL-1) to regenerate
shoots. Regenerated shoots were then transferred to magenta boxes containing
half-strength MS medium plus sucrose (20 gL-1) and NAA (0.25
mgL-1) for efficient rooting of regenerated shoots. Both hygromycin
(50 mgL-1) and cefotaxime (250 mgL-1) were used
in all the media and at all stages to inhibit the growth of Agrobacterium
and reduce the number of escapes. Regenerated plants were then transplanted
in sterilized soil and grown in the green-house (30oC day and
24oC night, with a supplemental photoperiod of 10 hours). More
than one hundred transgenic Basmati rice plants of various lines have
been obtained from these transformation experiments. The presence of the
transgene was detected at several developmental stages in R1/R2 generations.
Transgenic expression was first detected by GUS activity in calli and
then in leaves and roots of transgenic plants (pRKJ5) through histochemical
assay with X-gluc (5-bromo-4-chloro-3-indolyl glucoronide) reagent incubated
at 37oC overnight. In the segregating population, the leaves
of individual plants were cut and dipped into solidified MS medium containing
hygromycin (100 mgL-1). One week later, the resistant and sensitive
phenotypes were scored. The leaves of resistant rice plants remained green,
but leaves from sensitive plants turned yellow and eventually brown (data
not shown).
Finally, genomic DNA from hygromycin-resistant transgenic rice plants
was analyzed by Southern blot hybridization. For this analysis 8-10 microg
of genomic DNA was digested with Sma I restriction enzyme (which cuts
once in all three plasmids) and analyzed by Southern blot analysis (Fig.
1) to determine the copy number of the transgene. The results revealed
that many hygromycin-resistant plants harbor a single copy of the HVA
I gene. The accumulation of barley LEA protein in a number of individual
R2 lines was determined by Western Blot analysis using LEA3 polyclonal
antibody. The presence of 22-Kd band in SDS-PAGE gel indicated that there
was barley LEA 3 protein accumulation in leaves of most of the transgenic
plants.
Our results have shown that low copy number of the HVAI gene is
integrated, stably inherited and highly expressed in Basmati rice genome.
More experiments are underway to test the transgenic Basmati rice plants
under different abiotic stress conditions.
This work was generously supported by the Rockefeller Foundation, USA.
References
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