Transgenic radish (Raphanus sativus L. longipinnatus Bailey) by floral-dip method--plant development and surfactant are important in optimizing transformation efficiency.

Transgenic radish (Raphanus sativus L. longipinnatus Bailey) plants were produced from the progeny of plants which were dipped into a suspension of Agrobacterium carrying both the beta-glucuronidase (gusA) gene and a gene for resistance to the herbicide Basta (bar) between T-DNA border sequences. The importance of development of the floral-dipped plant and presence of surfactant in the inoculation medium were evaluated in terms of transgenic plant production. Plants dipped at the primary bolt stage of growth, into a suspension of Agrobacterium containing 0.05% (v/v) Silwet L-77 resulted in optimum transformation efficiency, with 1.4% from 1110 seeds. The presence of Pluronic F-68 or Tween 20 in the inoculation medium was beneficial towards transgenic plant output compared to treatments without surfactant. Putative transformed T1 plants were efficiently selected by spraying with 0.03% (v/v) Basta and all herbicide-resistant plants tested positive for GUS activity when analysed both histochemically and fluorometrically. Southern analysis revealed that both the gusA and bar genes integrated into the genome of transformed plants and segregated as dominant Mendelian traits. These results demonstrate that radish can be genetically modified for the improvement of this important vegetable crop.

Induction of dwarfism in transgenic Solanum dulcamara by over-expression of a gibberellin 20-oxidase cDNA from pumpkin.

The gibberellin (GA) 20-oxidase (CmGA20ox1) from immature pumpkin seed produces predominantly inactive tricarboxylic acid GAs. We expressed CmGA20ox1 under the control of the CaMV 35S promoter in Solanum dulcamara to assess the usefulness of this gene for reducing GA content in transgenic plants. All transgenic plants obtained were semi-dwarfs with smaller, deep-green leaves and highly pigmented stems compared to the wild-type. Such transformants flowered earlier than the wild-type plants and produced more fruit and more seeds per fruit. The transgene was efficiently expressed, producing high levels of CmGA20ox1 transcript and protein. Furthermore, the concentration of GA(1) was reduced in leaves of the transformants to approximately 20% or less of that in the wild-type and to about 40% or less in stems. The concentrations of other 13-hydroxylated GAs were also reduced, except for the tricarboxylic acid, GA(17), which accumulated in the transformants due to 13-hydroxylation of GA(25). By contrast, the concentrations of non-13-hydroxylated GAs, GA(4) and GA(34), were not consistently reduced, indicating that the effect of expressing the pumpkin gene may not be predictable. Transcript abundance for a native GA 20-oxidase gene was higher in the leaves and stems of S. dulcamara transformed with the pumpkin gene than in wild-type, reflecting the feedback control of 20-oxidase gene expression that serves as a homeostatic mechanism for GAs.

Broad bean wilt virus Causes Necrotic Symptoms and Generates Defective RNAs in Capsicum annuum.

ABSTRACT A virus was isolated from hot pepper (Capsicum annuum cv. Hyang Chon) growing in Korea and displaying necrotic spots or streaks on leaves and stems followed by stunting and death of plants. Morphological and host range analyses of extracts from infected plants suggested that the causal agent of disease was a Broad bean wilt virus (BBWV), and the virus was tentatively named a Korean isolate of BBWV (BBWV-K). When the isolate was back-inoculated onto hot pepper plants, it induced symptoms similar to those of naturally infected hot pepper in the field. Two coat proteins (CPs) of 44 and 22 kDa, corresponding to a large CP and a small CP, respectively, were identified from the virus, and both reacted specifically with polyclonal antibody to BBWV 2. The complete nucleotide sequences of RNA 1 and RNA 2 of the isolate were determined from cDNA clones. The deduced amino acid sequence data from the putative proteins encoded by RNA 1 and 2 of the BBWV-K indicated a closer relationship with the isolates of BBWV 2 than BBWV 1. However, sequence comparison of the 5' noncoding regions of the viruses differentiates BBWV-K from other BBWV 2 isolates. Another distinctive feature of the BBWV-K is that it generates defective RNAs in hot pepper exhibiting necrotic symptoms, which is the first report of defective RNAs in the Fabavirus genera of BBWVs.

Shoot regeneration from cultured root explants of spinach (Spinacia oleracea L.): a system for Agrobacterium transformation.

A reliable plant regeneration system is described for the production of adventitious shoots from root explants of spinach. Explants from roots of axenic shoots and roots induced on cultured hypocotyl explants were used for adventitious shoot induction. Explants from apical, middle and basal root regions were incubated on Nitsch and Nitsch medium supplemented with α-naphthaleneacetic acid, gibberellic acid and kinetin. Optimum shoot regeneration was from explants of apical and middle root regions on medium with 20 µM α-naphthaleneacetic acid and 5.0 µM gibberellic acid. Shoots originated directly from root tissues without an intervening callus phase. Adventitious shoots were rooted and were grown to maturity in the glasshouse. This plant regeneration procedure has been exploited in preliminary studies of Agrobacterium-mediated transformation.