AIR3-CT94-2003 Sunflower Oil For Industrial Applications - SOFIA

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AIR Cluster IV - Oils and Fats : Biotechnology : Bulk Chemicals : Crops for Bulk Chemicals : Crops for Fine Chemicals : Fine Chemicals : Plant Genetics : Vegetable Oil/Fat

	Proposal No:	AIR3-CT94-2003
	Date Prepared:	September 1999
	Source:	Progress report December 1998

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Progress report December 1998

Summary

Introduction The overall aim of the project is the production of transgenic sunflower plants producing oil of modified fatty acid composition. In particular, it was intend to produce plants with high oleic acid content and reduced content of stearic acid as well as plants producing short- and medium- chain fatty acids. This was to be achieved by incorporating genetic constructs carrying the appropriate promoter and coding sequences.

Objectives The objective for the period covered by this report were to:

• analyse transgenic plants produced so far
• apply for and conduct field trials with the first fully characterised plants

Experiments designed to improve the transformation technology continued throughout the duration of the project.

Results First results with transgenic sunflowers had indicated that the effect of the ds10-C1FatB3 construct on the fatty acid composition was only minor. In order to very the validity of this observation, transgenic rapeseed plants have been produced harbouring the same construct. None of the 52 analysed transgenic rape seed lines showed the occurrence of capric acid when analysed by gas chromatography. The presence of the appropriate protein was also undetectable by Western analysis in 6 randomly chosen lines. In contrast, rape seed lines harbouring the C1FatB3 gene under the control of its own promoter resulted not only in the expression of the expected protein but also in accumulation of capric acid (C10:0) in seed storage oil.

A new chimeric ds10::GUS::ds10 construct has therefore been constructed that contains the GUS coding region cloned in the previously described expression cassette ds 10EC1. Transgenic tobacco plants containing the new chimeric gene have been produced. Analysis of 19 independent transgenic plants showed only a moderate reduction of GUS expression in immature (16dpa) embryos, compared to the previously characterised ds 10F2-delta plants. These results indicate that other genes cloned in ds10EC1 could also be efficiently expressed in transgenic plants, except for problems not directly related with the structure of the ds10EC1 cassette. This result makes it likely that the low levels of changes in the fatty acid profile observed in the transgenic sunflowers and rapeseed plants produced with the ds10-C1FatB3 construct are not due to differences in the ds10 sequences present in ds10EC1, compared to ds10F2-delta.

The observed absence of expression may find an explanation in the residual expression of the ds10 promoter in pollen grains. Accumulation of capric acid that is not immediately captured by storage tri-acylglycerides may prove deleterious for the developing pollen grain. This working hypothesis is currently being checked by a segregation analysis of the offspring of these plants.

With respect to the engineering of fatty acid diversity in the storage oil of sunflower seeds, the contribution of exon II of the FatB gene family has been investigated. Chimeric constructs combining the FATB3 gene with exon II sequences of divers origin and expression in E. coli led to the conclusion that exon II sequences are necessary but not sufficient for the chain length specificity of Cuphea FatB genes.

The binary vector pMH000-0 carrying both the sulfonamide and Basta resistance gene described previously proved to perform particularly well for rape seed transformation. Optimisation of the transformation protocol has continued. The routinely obtained overall efficiency to confirmed transgenic flowering plants is 0.4%. However, limitations still exist. For example type 2 plants proved to be sterile due to unfavourable environment (temperature too elevated during summer). Sunflower plants expressing the ClFatB3 gene under its own promoter are expected in the near future.

Field trials of transgenic plant type 5 have been performed with 22 previously selected lines (3 internal controls, 14 pure lines and 8 hybrids) at two locations. Seeds were harvested on an individual basis, and leaf samples were conserved for each plant in order to be able to establish a correlation between genotype and phenotype if necessary. Seed samples are currently being analysed.

Two selected 'high oleic' interspecific hybrid lines with good transformation potential were further multiplied in the field so that these genotypes will soon become available for transformation experiments. Two further interspecific hybrids with exceptionally high oleic acid contents have been field grown from selected half-seeds for the development of elite lines. This selection continues at present in winter sites in South Africa.

Conclusions Transgenic plants expressing the proposed genetic constructs have been produced and introduced into field trials (fifth generation) on schedule. Full characterisation of these plants, as well as of additional plants which are available in lower generations, is in progress and, once completed, will yield extensive information as to their genetic composition, the alterations of their physiology, the interactions between these levels, and their agronomic potential.

During the course of the production of the genetic constructs and the transgenic plants, a number of questions have arisen which required a faster answer than can be given using sunflower. In these cases, transgenic tobacco and rapeseed plants have been produced (as appropriate) in order to obtain the required information that can then be utilised for the choice of appropriate constructs for sunflower. In spite of the considerable progress made with the improvement of the transformation protocol, sunflower transformation still remains a heavy and time-consuming task.

To date, all proposed milestones have been reached and the performed experiments have yielded a number of interesting results but most investigations are not yet finally concluded. So far, the project has yielded one patent application. First manuscripts for publication in scientific journals are being prepared.