5.3.19
USE OF REPETITIVE SOMATIC EMBRYO CULTURES TO ENGINEER DISEASE RESISTANCE IN PEANUT (ARACHIS HYPOGAEA)

EL LITTLE, ZV MAGBANUA and WA PARROTT

Department of Crop and Soil Sciences, University of Georgia, Athens, GA 30602, USA

Background and objectives
The long-term goal of our research is to genetically engineer desirable traits, such as disease resistance, into peanut (Arachis hypogaea) cultivars. As a first step, we are optimizing the efficiency of initiating repetitive somatic embryo cell lines of peanut for use in a particle bombardment transformation system. The advantages of using repetitive somatic embryo cell lines are the continual production of large amounts of transformable tissue, and the proliferation of transformed tissues for the regeneration of multiple plants. The development of an efficient transformation system would allow for the introduction of genetic sequences that are not normally accessible by conventional breeding methods. Tomato spotted wilt virus (TSWV), a negative-strand RNA virus, causes a ringspot disease on peanut that has resulted in serious production losses in the south-eastern USA and throughout the world. Resistance to TSWV has been engineered in other crops by transforming with the viral nucleocapsid (N) gene sequences [1], and the N gene has the potential to provide resistance in peanut cultivars.

Materials and methods
Somatic embryos were induced from mature peanut epicotyls on solid medium containing various auxins (including 2,4-D, picloram, dicamba and centrophenoxine) in concentrations ranging from 12.4-124 然. Repetitive embryos obtained after three to four transfers on solid auxin-containing medium were transferred to liquid medium with auxin levels ranging from 21-70 然 and sucrose levels ranging from 1-6%. Embryonic tissues from an established repetitive cell line were transformed with pANUH-1 using micro-projectile bombardment with a helium discharge particle gun. pANUH-1 consists of a sense-construct of the nucleocapsid (N) protein gene from a peanut isolate of the TSWV driven by the Actin-2 promoter. The construct also contains a gene for hygromycin resistance that is expressed by the Ubiquitin-3 promoter. The bombarded tissues were placed in liquid medium containing hygromycin to select for transformed repetitive embryos. Transformed lines were isolated, allowed to proliferate under selection, and tested for the presence of the N gene using PCR. Positive transformed lines were placed on auxin-free medium for conversion and regeneration.

Results and conclusions
Repetitive somatic embryo liquid cultures of peanut cultivar AT120 were established in liquid medium containing 23 然 2,4-D using embryos that were induced on solid medium containing 12.4 然 picloram. Cell line 120H was repetitive in liquid 7 months after induction and this line was transformable 1 month later. 25 flasks of tissue were bombarded, and 16 produced hygromycin-resistant tissues. 60 hygromycin-resistant lines that were positive for the N gene using PCR and specific primers were placed on conversion and regeneration medium. Although established repetitive cell lines were found to be highly transformable, the process of obtaining repetitive cell lines was highly inefficient. Further experiments were conducted to optimize components of the solid induction and liquid maintenance media. Induction of axes and transfer of embryos on solid medium containing higher concentrations of Picloram (83 or 124 然) produced highly repetitive embryos. Lowering the sucrose level in the liquid medium from 6 to 4% resulted in better growth of established cell lines. In order to maintain the highly repetitive state of the embryos when transferred from solid to liquid medium, experiments are ongoing to test increased levels of auxins in the liquid medium.

References


1. Pang SZ, Jan FJ, Gonzalves D, 1997. Proceedings of the National Academy of Sciences, USA 94, 8261-8266.