Development of an efficient B. rapa transformation system to facilitate studies on fruit development in a diploid Brassica oilseed crop.

GM technology (referred to here as transformation) is increasingly being exploited as a research tool for testing gene function. Put another way plant transformation can be exploited to over-express/ modify or silence plant genes in order to determine the function of a particular gene. The results of these studies are not to create GM crops, but to better understand the genetic control of these traits to facilitate conventional crop improvement strategies. For example, within the UK oilseed rape (Brassica napus) is grown widely for its seed oil. Compared to other crops, oilseed rape is a relatively young crop and one not yet fully optimized for efficient production (low harvest index of total: harvestable biomass), although profitable, there remains considerable scope for crop improvement. One such trait being early pod shatter (where seed is spilled in the field prior to, and during harvest) this accounts for >10% seed loss every year (valued at about £60 million per year). A better understanding of which genes are involved in early pod shatter, may lead to marker assisted breeding (non GM approach) to select breeding material with greater pod shatter resistance. The genetics of Brassica napus are further complicated because it is an amphidiploid species, meaning it contains the complete genome of the two diploid parental progenitors B. rapa (an oilseed and vegetable crop) and B. oleracea (a vegetable crop). The desire to understand the genetic control behind many fruit and oilseed traits is therefore best explored in the first instance in the genome of B. rapa. In the current proposal we aim to develop a robust and efficient transformation method in a rapid-cycling B. rapa oilseed crop. The availability of such a resource will be highly desirable within the research community for studying fruit and oilseed traits, and for which a diploid species is ultimately more desirable for genetic studies. The project is particularly timely with the near completion of the B. rapa genome sequence; and complementary to the B. rapa TILLING (Targeting Induced Local Lesions IN Genomes) a 'reverse genetics' resource, which has been produced in the same genetic background.

The current proposal aims to develop an efficient and robust transformation system in a rapid-cycling diploid Brassica oilseed crop (B. rapa). The availability of such a resource will be highly desirable within the research community for studying fruit and oilseed traits, and for which a diploid species is ultimately more desirable for genetic studies. The B. rapa variety R-o-18 has been chosen for this study as its plant architecture is similar to oilseed rape, B. napus, an amphidiploid Brassica. Moreover the genotype also complements the R-o-18 TILLING resource at JIC, and will enable researchers to confirm gene function in the TILLING mutants. Previous work by the group has shown R-o-18 to be a suitable candidate for transformation, and have developed a base transformation system for this genotype. However, the rate of transformation remains far from routine (between 0.2 and 1.0% of explants producing transgenic plants). It is estimated that the full economic cost to offer B. rapa transformation as a service at its current efficiency would be 5 times greater than that of B. oleracea (2010 fEC costs for B. oleracea being £4,163/ construct) and thus is not economically viable. The objectives of the study are therefore 1) To employ an empirical tissue culture approach to increase the number of cells targeted for transformed and the number of transgenic shoots initiated 2) To employ an empirical tissue culture approach to increase the successful transition of initiated transgenic shoots through to elongated viable shoots for subsequent rooting 3) In addition, evaluate a genetic approach to increase the number of cells targeted for transformed, by introducing the acdS gene from Pseudomonas putida UW4 onto the helper plasmid pSoup. This gene encodes for ACC deaminase; lowering of ACC the direct precursor to ethylene may increase explant susceptibility to Agrobacterium. 4) To evaluate the efficacy of the transformation by introducing an auxin reporter construct.

Who will benefit from this research and how? The output from the project will have a significant impact on research investigating the genetic control of fruit and seed development in Brassica. For example, the ability to use transformation as a research tool to better understand the genetics of fruit development may lead to marker assisted breeding (non GM approach) to select breeding material with greater pod shatter resistance. Early pod shatter (where seed is spilled in the field prior to, and during harvest) accounts for >10% seed loss every year, valued at about £60 million per year. Users will therefore be the scientific research community with beneficiaries being plant breeders, farmers and the public downstream. Economically the public would benefit from greater predictability of yields, through greater stability in production costs, which would impact on prices in the shops. Oilseed rape has emerged as the second largest oilseed crop with an annual worldwide production of 38 million tons of oil and demand is increasing. For this to be sustainable, seed yield needs to be dramatically increased through more efficient breeding programmes while at the same time minimising the amount of fertiliser input in order to protect the environment. The ability to determine gene function by exploitation of a transformation resource in a diploid rapid-cycling Brassica will therefore be a powerful research tool for those in the scientific research community engaged in addressing issues surrounding crop improvement and food security. Resources for B. rapa transformation will be made available to the research community through BRACT (www.bract.org) and through publication of results. What will be done to ensure that they have the opportunity to benefit from this research? 1) Communications and Engagement Both the PI and Co-I are engaged with discussions with the Brassica research community (UK and internationally) through collaborations and attendance at meetings. The UKBRC (UK Brassica Research Community) meet annually to share updates on current research and to identify future priorities, this research community also includes the UK Brassica plant breeders. Dissemination of results will be communicated through publication, presentations at conferences, as well as updates through the BRACT website. In addition we have access to a Brassica community emailing list (global) for efficient international dissemination. The PI and Co-I are also engaged in outreach activities through public lectures on the science behind GM, crop improvement and food security. Moreover, the Co-I has experience interacting with journalists and speaking on radio. JIC has specialist staff in its Communications Department of The Operation Centre (TOC) that handles web pages and user-friendly interfaces, outreach activities and press releases. 2) Collaboration Both the PI and Co-I have a strong track record of collaboration with other academics both within the plant science community and in interdisciplinary projects. These links will be further strengthened as we will keep the community informed about our progress. 3) Exploitation and Application B. rapa transformation will be added to the current resources offered by BRACT for Brassica transformation. These resources will be available to both UK and international research groups. Transformation services offered by BRACT currently work on a full economic cost recovery bases; by expanding and strengthening the BRACT portfolio we will continue to provide the UK research community with access to resources and facilities that play an important part in many research proposals.