Successful Field Testing of Drought-Resistant Transgenic Rice

Two points of concern facing all who are focused on future food security are a growing population and the disruption of food production caused by the effects of climate change. One effect of climate change predicted is that of longer and dryer periods of drought. Drought stress has a significant impact on crop productivity, further impeding the ability to feed a growing population.

The quest to build tolerance to such stresses as drought into important crops is a significant area of study. We have previously written on developing crop tolerance to biotic and abiotic stresses and new studies in the area are regularly being reported on in biotechnology journals.

We write here about a new study in the Plant Biotechnology Journal (Open Access) which field trialled two modified species of rice, an extremely important crop, for increased drought resistance.

The Study

The article starts by citing a number of studies that have found that over-expressing particular genes has resulted in increased resistance to drought stress, although usually such tests are performed under laboratory conditions. The difference noted is that of improved grain yield in the transgenic crops compared to the non-transgenic control plants when subjected to drought stress.

The adaptation of plants to drought stress and cellular injury results from the accumulation of metabolites within the plant cells, a phenomenon identified by the accumulation of soluble sugars in response to such a stress. The raffinose family of oligosaccharides, which includes the raffinose and galactinol sugars, are one such family of sugars found to accumulate in plants under drought stress. The raffinose sugars are part of a metabolic pathway that is induced by a number of genes named the GolS genes. One of these genes, the GolS2, is induced only by water availability problems due to drought and salinity.

Using this knowledge, the researchers sought to over-express the GolS2 gene in rice plants grown under field conditions to assess whether the test plants performed better when stressed compared to their non-transgenic precursor.

Results

Creating the transgenic lines

After creating a gene construct containing the GolS2 gene taken from Arabidopsis thaliana and controlling its expression with the constitutive maize ubiquitin promoter, the constructs were transferred into two varieties of rice crop (Curinga and NERICA4) and plants confirmed to have been transformed with once copy of the gene construct were selected for study.

The modified plants were analysed for galactinol accumulation without any stress. The modified plants were shown to have considerably higher galactinol content than non-transformed plants. It was also tested whether markers for other drought inducible genes had seen an increase in transcription which would be an alternative explanation for the increase in galactinol content, but no inducement was found. Therefore, it appears likely that any difference in drought tolerance that may be seen would be related to the over-expression of the GolS2 gene.

Fig1 AtGolS2

Figure 1 from article. GolS2 transcription levels (top) and galactinol levels (bottom) in transgenic and non-transgenic lines (top).

Before beginning testing, any difference in agronomic quality between the transgenic and non-transgenic lines was also assessed with no obvious difference between them.

Testing the Transgenic Lines

Curinga lines were then tested with 3 weeks of drought stress at the vegetative stage of development (3 weeks old plants) with the following differences noted between the transgenic and non-transgenic lines:

  1. Transgenic lines maintained their height compared to the non-transgenic plants;
  2. Drought-induced leaf rolling occurred earlier in the non-transgenic plants;
  3. Dry biomass after re-watering were significantly higher in all but one line of the transgenic plants compared to the non-transgenic lines.

Testing was then carried by applying drought conditions to plants at the reproductive stage in field trials conducted in triplicate over 3 growing seasons. Grain yield in the transgenic lines was found to be significantly greater than in the non-transgenic lines. Two lines in particular were identified as consistently outperforming their non-transgenic cousins under severe drought conditions, including that they:

  1. Had a significantly greater number of panicles and of greater length;
  2. Had a lower incidence of leaf rolling;
  3. Recovered from the drought conditions faster;
  4. Flowered earlier; and
  5. Had greater grain fertility.

Following these tests five of the best performing transgenic lines were selected for further physiological and gene expression analysis. The analysis included testing of relative water content before and after stress, the photochemical efficiency of photosystem II and the chlorophyll content.

Relative water content between the test and control before drought showed no difference but after drought stress being imposed for a week the non-transgenic lines showed a decrease in relative water content with little change noted in the transgenic lines. Three weeks of drought resulted in transgenic lines losing 18% to 22% relative water content. The same length of drought reduced relative water content in non-transgenic lines by 30% in comparison.

Photochemical efficiency was tested by comparing the variable fluorescence of leaves with the maximum fluorescence. Unstressed and after one week of stress, the efficiency of both sets of crops were comparable. After three weeks of stress, transgenic lines retained greater photosynthetic efficiency compared to non-transgenic lines.

Chlorophyll content testing showed similar results; no difference between the two sets when unstressed, but chlorophyll content reduced significantly after three weeks of drought stress in the non-transgenic lines while such a drastic reduction was not seen in the non-transgenic lines.

Finally, the Curinga lines were tested in rain-fed water systems at a trial site in Columbia in three sets of tests between 2012 and 2015. Based on rainfall data taken over the previous 10 years, drought events occur at this site at a time the coincides with rice reproductive stages. When severe drought conditions arose during these trial periods, two transgenic lines in particular retained higher numbers of panicles and greater grain yield compared to non-transgenic lines.

Fig 6 AtGolS2

Figure 6 from article. Grain Yield in transgenic and non-transgenic lines in three experiments (a to c) and photo of transgenic line 2580 compared to non-transgenic Curina lines.

To test whether the positive effects of the gene construct on drought tolerance observed was transferable to a rice crop from a difference background, the NERICA4 lines were transformed and tested. Before drought conditions were imposed there was no phenotype difference between the two sets of crop. However, nine days of drought followed by a seven day recovery period resulted in most non-transgenic lines failing to recover (88.5%) compared to four of seven transgenic lines recovering with rates of survival being between 26.2 and 34.5%.

These lines were also tested for grain yield during the second and third field trials that the Curinga lines were tested in. The transgenic lines held higher grain yields than the non-transgenic lines.

The Correlation between Grain Yield and Galactinol Content

This section of the paper leaves something further to be researched.

The galactinol content of the plants increased in line with the increase in the transcription of the GolS2 gene. However, a dose effect of galactinol level on grain yield wasn’t always observed, leaving a question mark over the how the over-expression of the GolS2 and subsequent galactinol accumulation resulted in the observations reported by the researchers.

Conclusion

The study supports the proposition that over-expression of the GolS2 gene results in greater drought resistance and that such a result is seen in both laboratory and field testing. It also shows that over-expression of the gene doesn’t result in any unwanted phenotype changes in the crops that would affect their use in times of good water availability levels.

Why there was a lack of correlation between the galactinol levels and plant performance is a bit peculiar. It may be that there is no dose effect beyond a certain concentration of galactinol, but the question is worthy of further study.

Overall, this study reports an exciting development that could assist to secure our food supplies in areas increasingly affected by drought.

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