Analysis of critical components for Agrobacterium-mediated transformation of Rubus : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Lincoln University

Abstract

To progress towards stable integration of transgene(s) into any plant genome, a series of problems must be solved. When any part of a genetic transformation system is ineffective, the production of transgenic plants is not possible. Until the reasons for success or failure at each step are understood, the methods used for any particular plant species cannot be easily applied to other candidates for gene transfer. The efficiency of transformation is particularly important in woody species that are traditionally clonally propagated, because of the expected high frequencies of both somaclonal variants and low levels of expression of the transgene in transgenic lines produced. Therefore, the objectives of this thesis were to systematically reduce or eliminate barriers to the recovery of sufficient numbers of transgenic Rubus plants to develop a transformation system for Rubus crop genotypes.

Using a stepwise approach, reliable regeneration and Agrobacterium systems were developed for commercially important Rubus crop genotypes. These systems were combined with improved cocultivation and selection protocols, but few putatively transformed Rubus plants regenerated. The barriers to the recovery of large numbers of putatively transformed plants were investigated. Computer image analysis was used to quantify results of the histochemical GUS assay, in a study of early gene transfer events. Stable expression of the GUS reporter transgene was demonstrated in cocultivated Rubus explants. The frequency of gene transfer events in Rubus explants was comparable to the frequency achieved in Nicotiana plumbaginifolia, used as a positive control and known to be highly amenable to genetic transformation. Stable expression of GUS was also demonstrated in Rubus callus lines that did not regenerate. The survival and regeneration of transformed Rubus cells were identified as the factors limiting efficiency in recovery of transgenic Rubus plants. A novel approach to selection for transformation was developed. It involved extension of the nurse culture concept, in combination with information on the sites of highest competency for both regeneration and transformation in Rubus leaf explants. Large numbers of Rubus regenerated shoots survived with the new selection protocol. However, not one of the 466 regenerated plants (from 580 explants cocultivated) expressed GUS activity in leaves. Six regenerants remained green when rechallenged on medium containing kanamycin. Although molecular analyses proved the transgenic status of selected plants in the positive control species (N. plumbaginifolia), no transgenic Rubus plants were identified. The probability that some of the regenerants may have been chimeras of transformed and untransformed tissue is discussed.

Many problems encountered in this investigation of a transformation system for Rubus, have been solved, and reasons for success or failure at each step at least partially understood. The findings presented in this thesis on transformation of Rubus are equally applicable to many species and genotypes that are recalcitrant to transformation, or very hard to transform, especially other woody species.