Phase 2 (2006 Deadline)
Identification and Cloning of Drought-Related Genes in Wheat (T. aestivum)
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Transgenic wheat
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Sona Pandey (beginning July 2010)
Daniel Schachtman (prior to August 1, 2008) and Liming Xiong (August 1, 2008-July 2010),
Donald Danforth Plant Science Center, St. Louis, MO
Nasir Saeed, National Institute for Biotechnology and Genetic Engineering, Faisalabad
Pakistani Funding (MoST): $100,000
US Funding: $109,963
Project Dates on US Side: February 1, 2007 - November 30, 2011
Project Overview
Wheat is the staple food of millions of people around the world, and drought is the major limiting factor in its production. Pakistan is located in arid and semi-arid climate zones and is facing severe and growing shortages of irrigation water. The country’s average wheat yield is 2.5 metric tons per hectare, which is quite low compared with other countries like China (4.27), Egypt (6.07) and Mexico (4.62), and Pakistan has been a net importer of wheat grain in the last decade. To feed its growing population, there is a great need to focus on developing drought tolerant crops that can grow with limited water.
This project involves the use of microarray technology to identify and clone key genes involved in adaptation of wheat to drought stress. Candidate genes are being cloned into expression vectors for transformation and development of drought tolerant wheat. Wheat crop productivity and yield stability are affected by a number of factors (biotic and abiotic). In the past, attempts have been made to introduce drought tolerance genes by conventional breeding. Wheat and other crop genomes are being sequenced by a number of research teams, and many different genes have been tested under a range of conditions to determine whether they may contribute to drought tolerance. In this study, four drought tolerance enhancing genes (HVA1, WXP1, DREB1A and AtNCED3) and a stress responsive constitutive promoter rd29A have been chosen. Over-expression of these genes may enhance drought tolerance and agronomic value of wheat. Few studies have addressed the topic of drought tolerance in wheat using molecular tools; although breeding efforts and germplasm screens have been undertaken. In addition to using the above approach, microarray technology offers new ways to find gene expression changes and manipulate them for developing drought tolerant crops. It is particularly useful to examine where and when specific genes associated with water deficit stress are expressed. Full length cDNAs of a few candidate genes that are regulated by drought are being cloned into plant expression vectors for transformation, and the over-expression of these genes is being tested for their ability to increase wheat production under drought conditions.
Major Results
- Provided training to four PhD students who work in wheat biotechnology projects, three internship graduate students, and two laboratory technicians
- Transformed seven drought / salt tolerance enhancing genes into four wheat varieties / lines
- Presented results at various forums in Pakistan, which contributed to the development of collaborations with several government institutes
Quarterly Update
In 2011 the researchers need to test and prove that the wheat transformed from drought genes is useful for regions with less rainfall and irrigation water. This requires confirmation of cloned genes in coming generations, PCR and Southern blot analysis, and testing under drought and salt stress. This work is ongoing on, with several wheat varieties with these genes being tested. This activity will continue in hopes of obtaining good material for cultivation.
Progress Report Summaries
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2010 Show summary || Hide summary
On the US side, Dr. Liming Xiong left the Danforth Center but as of July 2010 he was replaced as principal investigator by another researcher at the Center, Dr. Sona Pandey. During 2010, the major objectives planned for the project on the U.S. side were accomplished except for a planned visit from co-PI Dr. Nasir Saeed in April 2011. On Pakistani side, seven drought/salt tolerance enhancing genes were transformed into four wheat varieties/lines. Rd29-HVA1 T2 transformed plants of Sehar 06, G-98 (NIA line), Ufaq and Bobwhite varieties were planted for field testing of their drought and salt tolerance. Transgenic wheat (T3 generation) containing AVP1 and AVP1-D genes were tested for drought tolerance under rain, no rain and irrigated conditions. Achievements of this project were presented at various forums in Pakistan.
Throughout the year, four PhD students have been working in wheat biotechnology projects. There were three internship graduate students from University of Agriculture and University of Arid Agriculture trained in wheat biotechnology. Two new laboratory technicians were inducted in the lab and trained in wheat tissue culture, DNA isolations, and PCR. At NIBGE, Dr. Saeed taught three M. Phil/PhD Biotechnology courses on abiotic stresses in plants that included developing drought and salt tolerant plants.
2009 Show summary || Hide summary
By December 2008, the team planted the second round of plants, and in the spring of 2009 they began conducting the respective physiological studies and then the microarray experiments with these new plants, including both drought-tolerant and drought-susceptible varieties. Subsequent transcriptome analysis of problems from the two cultivars showed that many predicted stress regulated genes exhibited changes in response to drought stress. Meantime, some novel genes were also found to be regulated by the stress. Several genes identified in the drought-tolertant cultivar would be good candidates for testing their potential roles in increasing wheat drought resistance in follow-up studies.
More important than the technical difficulties encountered, the major problem impacting this project was the non-payment of expected grant funds on the Pakistani side. Although Dr. Saeed eventually secured his US entry visa, unfortunately he was unable to obtain the required funds to make the trip to visit the Danforth Center in 2008 to participate in some of the experiments. He hoped to make the visit at some point in 2010, as soon as his grant funds arrive. Meanwhile, he and his colleagues and students persevered in their work on transformation of five drought tolerance enhancing genes into local Pakistani varieties of wheat. Dr. Saeed reports that as a result of the project they have been able to transform local wheat varieties by Agrobacterium and biolistic gun, the first time this has been accomplished in Pakistan. Five Pakistani PhD students were involved in the project, and Dr. Saeed also collaborated with researchers from the Sindh Agriculture Department and the Wheat Research Institute of the Arid Agriculture Research Institute, Faisalabad. He planned further transformation work in 2010 under a no-cost extension on the project.
2008 Show summary || Hide summary
The major focus of work in 2008 was to analyze the transcriptome of two wheat germplasms with contrasting drought resistance (one drought-resistant one and the other drought-sensitive). The purpose of this comparative transcriptome analysis was to identify candidate genes associated with drought resistance. For this strategy to work, it is important that the drought resistance of the cultivars be reliably determined. Ideally, the growth and development of the germplasms to be compared will be similar under normal growth conditions and their genetic backgrounds should be similar too. Dr. Saeed provided the seeds for four wheat germplasms that were previously compared under field conditions and were respectively referred as ether “drought resistant” or “drought susceptible.” Data on the drought resistance of these germsplasms, which has not yet been reported in the literature, showed that their growth and development appear to differ under normal growth conditions. Concerned with the suitability of these germplasms for in-depth transcriptome analyses, the researchers decided to subject them to additional physiological studies in parallel with the transcriptome analysis. These physiological studies will include the measurement of water relations and stomatal conductance of these germplasms under progressive drought stress. Drought resistance of the plants will also be compared under well-controlled laboratory conditions. These studies will determine whether these germplasms have altered drought resistance and which pair is better for comparison of their transcriptomes.
In August 2008, Dr. Liming Xiong of the Danforth Center took charge of the project on the US side after Dr. Schachtman left the center for a position in industry. Dr. Xiong began preparing the physiological and microarray analyses, and while conducting these experiments, he found that the seeds for two of the germplasms provided by his Pakistani partners were of very poor quality, perhaps due to long-term storage or quarantine treatments. The germination rate was very low, and the seedlings were not uniform in growth. This prevented the researchers from obtaining enough seedlings for any of the studies, although they planted four times the number of plants needed. In addition, these non-uniform seedlings also did not allow reliable comparison of their physiological response to drought stress. With this unexpected circumstance and the difficulty in obtaining more seeds from Pakistan, they had to grow the few germinated plants to maturity in order to get more seeds. This unexpected difficulty delayed the schedule for about five months.
2007 Show summary || Hide summary
Although some delays were encountered in 2007 in getting the project under way, Dr. Schachtman assisted Dr. Saeed by cloning genes for transformation into local wheat varieties while Dr. Saeed worked on transforming wheat. In the first stage of the project, one Arabidopsis gene DREB1A under the control of a constitutive promoter (FMV) and a drought inducible promoter rd29 were cloned, and the US side sent vectors to Pakistan containing AVP1 and AVP1D for transformation into wheat. Three more genes (HVA1, WXP1, and NCED3) were subsequently cloned, and each cDNA was cloned into plant transformation vectors containing a constitutive promoter and a drought inducible promoter. Dr. Schachtman sent all the clones to NIBGE by the end of November 2007 except rd29-HVA1. Those genes received before that date (rd29-DREB1A, rd29-WXP1, rd29-AtNCED3, FMV-DREB1A, FMV-WXP1, FMV-AtNCED3, FMV-HVA1, AVP1 and AVP1-D) were used in wheat transformations, which produced putative transgenic wheat plants of three wheat varieties (Sehar, Ufaq and Shafaq). These transgenic plants, which grew to maturity and produced seeds during the 2007-2008 wheat season, were confirmed by BAR selection, BASTA herbicide spray, and PCR amplifications of specific genes. When AVP1-D plants (To) were exposed to 14 days of drought stress in pots, the transgenic plants survived while the control plants died.
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