Genetic engineering of radish: current achievements and future goals.

Radish is a major root crop grown in the Far East and is especially important to some low-income countries where it is consumed on a daily basis. Developments in gene technology systems have helped to accelerate the production of useful germplasms, but progress has been slow, though achieved, via in planta methods and useful traits have been introduced. In the wake of the new Millennium, future goals in terms of improving transformation efficiency and selection of new traits for generating late-flowering radish are described. Furthermore, the techniques available for incorporating pharmaceutical proteins into radish to deliver edible proteins on-site are discussed. Finally, the concerns of releasing transgenic radish to the field in terms of pollen-mediated gene transfer are also reviewed. Such a report identifies key areas of research that is required to allow the crop satisfy the need of poor impoverished countries in the Far East.

Sugarcane DIRIGENT and O-methyltransferase promoters confer stem-regulated gene expression in diverse monocots.

Transcription profiling analysis identified Saccharum hybrid DIRIGENT (SHDIR16) and Omicron-Methyltransferase (SHOMT), putative defense and fiber biosynthesis-related genes that are highly expressed in the stem of sugarcane, a major sucrose accumulator and biomass producer. Promoters (Pro) of these genes were isolated and fused to the beta-glucuronidase (GUS) reporter gene. Transient and stable transgene expression analyses showed that both Pro( DIR16 ):GUS and Pro( OMT ):GUS retain the expression characteristics of their respective endogenous genes in sugarcane and function in orthologous monocot species, including rice, maize and sorghum. Furthermore, both promoters conferred stem-regulated expression, which was further enhanced in the stem and induced in the leaf and root by salicylic acid, jasmonic acid and methyl jasmonate, key regulators of biotic and abiotic stresses. Pro( DIR16 ) and Pro( OMT ) will enable functional gene analysis in monocots, and will facilitate engineering monocots for improved carbon metabolism, enhanced stress tolerance and bioenergy production.

Lettuce (Lactuca sativa L.).

Lettuce is a globally important leafy vegetable with the United States being the largest world producers. The crop is susceptible to a number of viruses that are aphid transmitted and also highly vulnerable to post harvest diseases. Although wild species of lettuce are an important source of disease resistance genes, their introgression into commercial lettuce has been limited owing to sexual incompatibilities. Hence, the development of a gene transfer system for lettuce would be extremely valuable both in improving the genetic diversity of the crop and also for the transfer of useful agronomic traits. This chapter describes an Agrobacterium-mediated gene delivery system that is highly adaptable for the production of transgenic plants using a wide range of lettuce germplasms. The system described, commonly referred to as the genotype-independent transformation system, has been used for the transfer of several agriculturally useful traits into commercial varieties of lettuce. In this case, A. tumefaciens strain LBA4404 carrying a binary vector with supervirulent pToK47 was used for infecting excised cotyledonary explants. The plant selectable marker gene neomycin phosphotransferase II (nptII) was used, and transformed plants were selected using kanamycin in the culture medium. The beta-glucuronidase gene with intron (gus-intron) was also used in the gene transfer study to confirm the transgenicity of regenerated plants further.

The noble radish: past, present and future.

Recent developments in plant biotechnology have revealed that radish can be genetically modified by a technique called "floral-dipping". This system has been used successfully to delay both bolting and flowering in radish by the co-suppression of the photoperiodic gene, GIGANTEA. Future research could use this system to improve the pharmaceutical value of the crop for global usage.

Production of transgenic crops by the floral-dip method.

The application of floral dipping toward the production of transformed plants has been rather limited. However, this procedure has enabled the successful production of transformed Medicago truncatula plants (a model plant for legume genetics) at efficiencies higher than those obtained by tissue culture methods. Indeed, this simple system, without requiring any knowledge of plant tissue culture, has been a breakthrough in the production of the first transgenic radish plants. This root crop is of major importance in the Far East, and the development of such a gene transfer system in radish has enabled agronomically important germplasms to be produced. Although the radish is closely related to Arabidopsis thaliana, it appears the two plants have different mechanisms of T-DNA transfer using floral dip. This chapter describes the simple system that has been adopted in the routine production of transgenic radish.

Modification of plant architecture through the expression of GA 2-oxidase under the control of an estrogen inducible promoter in Arabidopsis thaliana L.

The gibberellin (GA) 2-oxidase (PcGA2ox1) from bean catalyses the 2beta-hydroxylation of some precursor and bioactive GAs resulting in their inactivation. We have expressed PcGA2ox1 under the control of the estrogen receptor-based chemical-inducible system, XVE, to modify plant architecture and assess whether transgene expression is localised. Applications of estradiol to the shoot apical region of inducible PcGA2ox1 overexpressors exhibited delays in both bolting (maximum of 46 days) and times to anthesis (maximum of 62 days) compared to wildtype (36 and 41 days, respectively), without altering leaf area. Individual treated leaves showed signs of epinasty and became dark green; such estradiol-treated regions maintained these 'green-islands' well beyond the onset of leaf senescence. Northern blots revealed that the PcGA2ox1 transcript could be detected within 1 h of treatment. The level of PcGA2ox1 transcript appeared to peak 3-5 h after estradiol application in both high and semi expressors. Quantitative Reverse Transcription (QRT)-PCR data showed that GA down-regulated genes AtGA3ox1, AtGA20ox1 and SCARECROW-LIKE3 (SCL3) were up-regulated and the GA up-regulated genes AtGA2ox1 and AtExp1 were down-regulated in estradiol-treated leaves of inducible PcGA2ox1 overexpressors; neighbouring non-treated leaves showing no significant changes. Further molecular analyses revealed that expression of the transgene was confined to estradiol-treated leaves only. Expression profiles of GA down- and up-regulated genes in inducer-treated overexpressors appeared to be synchronised with changes in leaf phenotype. These observations suggest that PcGA2ox1 under the control of the XVE system can be used effectively to alter plant architecture in Arabidopsis by localised 2beta-hydroxylation of GAs at estradiol-treated sites.

Expression of an antisense GIGANTEA (GI) gene fragment in transgenic radish causes delayed bolting and flowering.

A late-flowering transgenic radish has been produced by the expression of an antisense GIGANTEA (GI) gene fragment using a floral-dip method. Twenty-five plants were dipped into a suspension of Agrobacterium carrying a 2.5 kb antisense GI gene fragment from Arabidopsis, along with the gusA and bar reporter genes, all under the control of a CaMV 35S promoter. From a total of 1462 seeds harvested from these floral-dipped plants, 16 Basta-resistant T1 plants were found to have GUS activity (transformation efficiency of 1.1%). Southern analysis confirmed the integration of one or two copies of the gusA gene in these herbicide-resistant plants. Expression of the GI gene in T1 plants was much reduced compared to both wildtype plants and plants transformed with pCAMBIA3301 (positive control). In the progenies of eleven T1 plants analysed (T2 generation), all lines showed a significant delay in both bolting and flowering times compared to wildtype and positive control plants, and that, the level of GI transcript was inversely proportional to the time of bolting and flowering. At a maximum, bolting and flowering times were delayed by 17 and 18 days respectively, compared to wildtype plants (in positive control plants, the delay was 23 and 26 days, respectively). Ten of the 11 lines exhibited a significant reduction in plant height compared to wildtype and positive control plants. This study provides evidence that down-regulation of the GI gene by co-suppression could delay bolting in a cold-sensitive long-day (LD) plant. Production of late-flowering germplasms of radish may allow this important crop to be cultivated over an extended period and also provide further food to the famine countries of S/E Asia.

Antisense and chemical suppression of the nonmevalonate pathway affects ent-kaurene biosynthesis in Arabidopsis.

Transgenic plants of Arabidopsis thaliana (L.) Heynh. (ecotype Columbia) expressing the antisense AtMECT gene, encoding 2- C-methyl- D-erythritol 4-phosphate cytidylyltransferase, were generated to elucidate the physiological role of the nonmevalonate pathway for production of ent-kaurene, the latter being the plastidic precursor of gibberellins. In transformed plants pigmentation and accumulation of ent-kaurene were reduced compared to wild-type plants. Fosmidomycin, an inhibitor of 1-deoxy- D-xylulose 5-phosphate reductoisomerase (DXR), caused a similar depletion of these compounds in transgenic plants. These observations suggest that both AtMECT and DXR are important in the synthesis of isopentenyl diphosphate and dimethylallyl diphosphate and that ent-kaurene is mainly produced through the nonmevalonate pathway in the plastid.