Fertile Transgenic Asparagus Plants Produced by Agrobacterium-mediated Transformation

Abstract

Transgenic plants of Asparagus officinalis cv. Mary Washington 500W were generated by Agrobacterium-mediated transformation. After induction of flowers by treatment with atrazine, calli were induced from male and female plants. Calli of male and female origin were co-cultivated with Agrobacterium EHA101 (pIG121Hm) and EHA101 (pARK5), respectively. About thirty shoots developed from female calli and three shoots developed from male calli within six weeks on selective medium supplemented with 100mg/l kanamycin and 500mg/l claforan. Most of the shoots did not form roots. Seven shoots from female calli formed roots and were successfully transferred to soil in a greenhouse. But shoots of male calli did not form any roots. Stable integration and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the R₀ and R₁ generations. In an analysis using the polymerase chain reaction (PCR), fragments of both the NPT II gene and the bar gene were amplified from five R₀ plant fragments. Southern analysis showed that both NPT II and bar probes hybridized to products of PCR from five R₀ plants. One of the five R₀ plants was crossed with a non-transgenic male plant. The segregation ratio of transformed to non-transformed R₁ plants was about 1:1. These results indicated that drug-resistant genes were transferred to Asparagus by Agrobacterium-mediated transformation.