IWBT 04-10 2005
The establishment of stable and synchronous embryogenic cell lines of grapevine rootstock cultivars for use in transformation systems.
Goussard, P G
University of Stellenbosch. Faculty of AgriSciences. Institute for Wine Biotechnology
The project focuses on the development of a fast and reliable protocol for the establishment of stable and synchronous embryogenic cell lines of six important rootstock cultivars for use in transformation with specific constructs. Further to being a prerequisite, the induction and maintenance of embryogenic callus are of critical importance for the establishment of the former. A high genotype-dependency was experienced in approaches to establish reliable and reproducible techniques for the induction and maintenance of embryogenic callus cell lines of important rootstock cultivars from immature anthers as explant material. During the 2000 to 2003 season several thousands of anthers were excised from Ramsey, Richter 99 and Richter 110 and incubated onto seven different media (PIV, Harst, Gray, Moszar, Goetz 1, Goetz 2 and Goetz 3) as previously used for rootstocks by other workers. Only the PIV medium gave rise to fair amounts of callus for all three cultivars, consisting mainly of watery and unorganised masses. Very few callus with embryogenic potential resulted. From the small amount of embryogenic callus, it was possible to select callus with good potential and to start to synchronise it for further multiplication. However, this selection process was hampered due to low multiplication rates despite regular transfers onto the PIV medium that was previously used for excellent maintenance and multiplication of embryogenic callus. During 2003 to 2008 multiplication rates of callus increased for Ramsey, Richter 99, Richter 110 and USVIT 8-7, especially when (i) incubated on a modified Gray medium in attempts to obtain larger biomass and to convert non-embryogenic calli into the embryogenic phase and (ii) subjected to suspension cultures. Synchronised embryogenic callus cultures of Ramsey, Richter 99, Richter 110 and USVIT 8-7 have been established and these cultures are now being propagated and maintained for use in a genetic improvement program. In addition, induction of somatic embryos from these cultures has been achieved successfully for Richter 110 prior to transformation thereof with reporter genes. Germination, shooting and establishment of transgenic plantlets have been obtained and are now being monitored.
The objectives are: 1. Optimisation of propagation and maintenance of embryogenic callus cultures of Ramsey, Richter 99 and Richter 110, which were established in project IWBT 4-10 2. Optimisation of somatic embryogenesis of rootstock cultivars 101-14 Mgt, Ruggeri and USVIT 8-7. The ultimate aim is to use these cultures for genetic transformation of all six rootstock cultivars.