Besides rare practical developments emerging as commercialised transgenic plants numerous benefits acquired through plant genetic manipulation are due to availability of additional tools and new comprehensive insights in fundamental research. Much of this new information deal with various fundamental processes, such as molecular determinants in plant-pathogen interactions, physiological aspects in stress defences, expression regulation of genes implied in qualitative and quantitative control of food nutritional value. The in vitro generation of whole plants from selected transgenic tissues is a requirement for plant genetic engineering. As one of the most important food crop species, wheat has been extensively investigated with respect to plant regeneration from in vitro culture. Different explant sources have been evaluated for the induction of regenerable wheat tissue cultures: shoot tips, inflorescences, anthers, isolated microspores, and immature embryos. Immature embryos are regarded as the most efficient tissue source and thus until now are the material of choice for wheat genetic manipulation. Immature embryos or calluses initiated from immature embryos are the target tissues for direct or Agrobacterium-mediated DNA transfer. Transgenic plants are produced by somatic embryo induction in selected transformed tissues and shoot regeneration. Yet, immature embryos can only be harvested in the field over a very brief period of time each year or donor plants have to be grown under controlled environmental conditions that requires additional resources. Also it is currently assumed that the conditions under which donor plants are grown affect the capacity of the response of immature embryo to regeneration and transformation. Consequently the high production of immature embryos at the suitable stage and quality for successful in vitro culture is difficult to achieve. To circumvent these limitations, we propose an alternative technique applying the particle bombardment technology to calluses derived from mature embryos rather than immature tissues. Unlike their immature counterpart, mature embryos are available in large quantity throughout the year, being easily stored in the form of dried seeds and the physiological state of this starting material shows minimal variability
Objectives
The initial objective of this project was to develop a very straightforward transformation procedure applicable to fragmented mature zygotic embryos. The thin fragments (500µm mean diameter) obtained by grinding mature embryos are used as explants and plated on solid medium to initiate embryogenic calluses, the latter being the target tissues for particle bombardment DNA delivery. The core objective is thus to appraise whether or not calluses initiated from fragmented mature embryos constitute a suitable source of material for production of transformed tissues from which transgenic plants could be regenerated. Around this principal objective complementary studies are articulated: 1 The effect of growth regulator composition of the culture medium on the tissue fragment response and the morphogenic potential of induced calluses. 2 The development of a plant regeneration method and its performance assessment. 3 The receptiveness of bombarded calluses to particle penetration and consequent verification of their regeneration ability in spite of the mechanical lesions undergone by the target tissues. 4 The determination of the optimal duration of tissue preculture prior to bombardment and concurrently the significance of tissue selection 5. The demonstration of the transformation aptitude of calluses initiated from mature embryos.
Expected results
We have already developed an efficient transformation method based on mature embryo culture. Further studies are required to elucidate whether the recipient tissue variations mentioned in this report could be attributable to their proliferative potential, physiological state, or differences in DNA methylation and dynamics during tissue culture. Such additional advances in understanding biological determinants of the distinct transformation aptitudes and differences in the ability of each cell type to bring about gene silencing would be of considerable interest for any plant genetic manipulation.
Results obtained
We have already developed an efficient transformation method based on mature embryo culture. Further studies are required to elucidate whether the recipient tissue variations mentioned in this report could be attributable to their proliferative potential, physiological state, or differences in DNA methylation and dynamics during tissue culture. Such additional advances in understanding biological determinants of the distinct transformation aptitudes and differences in the ability of each cell type to bring about gene silencing would be of considerable interest for any plant genetic manipulation.
Partners
This work is the object of a Phd thesis, promoter Professor P. du Jardin, Gembloux Agricultural University. Studies applying the image analysis technique were conducted by Ir. O. Mostade, Walloon Agricultural Research Centre. Histology studies were performed with the help of Professor J. Bouharmont, UCL. Ir. V. Planchon and Dr. R. Oger, Walloon Agricultural Research Centre gave their contribution to statistical analyses.
