WALNUT ROOTSTOCK TRANSFORMATION AND REGENERATION FROM VEGETATIVE TISSUE

The primary goal of this proposal is to develop novel regeneration technologies needed to engineer walnut rootstocks for pest and disease resistance. The productivity of the walnut industry of California is dependent the availability of superior rootstocks to combat soil disease and pest problems. Individual selections displaying resistance to either Phytophthora crown or root rots or to root lesion nematode, Pratylenchus vulnus, have been identified and can be clonally propagated but these selections are susceptible to crown gall. Rootstocks with multiple resistances are clearly desirable. Walnut transformation depends on the use of somatic embryos. Somatic embryos are readily obtained from immature, and therefore uncharacterized, seed but not from vegetative or other mature tissues. This precludes inserting any genes into a rootstock exhibiting another superior trait if the trait was identified in a seedling or mature tree. Walnuts have a long generation time and the most promising rootstocks, hybrids of English walnut x black walnut, are sterile or nearly sterile, factors that severely impede developing multiple resistances by breeding. The recent discoveries of genes that stimulate regeneration provide a unique avenue to develop cultivar independent transformation for walnut. Developing the capability to insert genes into elite walnut rootstock will provide a significant tool for improving walnut trees for the California walnut industry. OBJECTIVES 1. To test the efficacy of the XVE inducer system in walnut somatic embryos and track expression at various stages of the regenerated plant. 2. To determine the capability the ESR1 and PGA6 genes, under the control of a constitutive promoter and the XVE inducible system, to stimulate the production of transgenic somatic embryos or organogenic shoots for a range of rootstock germplasm. 3. To determine the regeneration capability of vegetative tissue collected from in vitro shoots established from embryos transformed with the ESR1 and PGA6 genes and determine if this capability to regenerate is maintained as the tree ages. 4. To determine the regeneration capability of vegetative tissues collected from in vitro shoots established from greenhouse and field grown elite trees when transformed with the ESR1 and PGA6 genes under the control of the XVE inducer system. PROCEDURES Establishing Agrobacterium cultures for transformation: Forty 40 μl of the glycerol stock of Agrobacterium strain EHA 105 containing the binary vector pER10-GFP, pDU03-PGA6 or pDU03-ERS1 (see below) was used to inoculate 20 ml of MGL medium (pH 7.0) containing 10 mg/l rifampicin, 5mg/l tetracycline and 20 mg/l spectinomycin sulfate, and incubated overnight