zki (1993) noted that genetic engineering in sugarcane would be a useful tool for reversing single flaws, such Genetic transformation of sugarcane (Saccharum spp.) holds promas disease susceptibility, in commercial cultivars. Sugarise for increasing yields and disease resistance. However, the tissue cane may benefit from genetic transformation because its culture and transformation process may produce undesirable field characteristics in transgenic sugarcane. The primary objective of this high ploidy level makes traditional breeding programs study was to evaluate variability in agronomic characteristics and difficult, while vegetative propagation of sugarcane allows field disease resistance of sugarcane transformed for resistance to for relatively stable transfer and multiplication of trans- Sugarcane mosaic virus (SCMV) strain E. One hundred plants derivedgenic materials (Gallo-Meagher and Irvine, 1996). from cultivars CP 84-1198 (n ϭ 82) and CP 80-1827 (n ϭ 18), consisting Birch (1996) predicted that new gene technologies of independent virus resistant lines VR 1 (n ϭ 14), VR 4 (n ϭ 24), would reshape the sugar industry, yet obstacles to their VR 14 (n ϭ 4), and VR 18 (n ϭ 58) were evaluated in Exp. 1. implementation remain. For example, somaclonal varia-Transgenics derived from CP 84-1198 had significantly greater tonnes tion caused by tissue culture procedures may produce of sucrose per hectare (TSH) and significantly lower SCMV disease undesirable field characteristics in genetically transincidence than those from CP 80-1827 in the plant-cane (PC), firstformed sugarcane that are not readily identifiable in the ratoon (1R), and second-ratoon (2R) crops. Plants from the VR 18 line had significantly greater economic indices and lower SCMV disease laboratory or greenhouse (Lourens and Martin, 1987; incidence than the VR 4 line in all three crops. Phenotypic variation Burner and Grisham, 1995; Oropeza and De Garcia, 1996; was high in Exp. 1, with tonnes of cane per hectare (TCH) ranging Sreenivasan and Jalaja, 1998; Arencibia et al., 1999). from 26 to 211 and TSH from 3.2 to 28.9 in the PC crop. Agronomic Burner and Grisham (1995) report significant somatrait variation decreased with increased selection pressure in Exp. 2, clonal variation in CP 74-383 sugarcane subjected to evaluating 30 VR 18 lines, with TCH ranging from 70 to 149 and TSH different propagation procedures. Normal variants infrom 8.5 to 19.0 in PC. The large variability in yield characteristics creased from the PC to 1R crops, but still totaled Ͻ22% and disease resistance encountered in this study demonstrates the of all plants. Lourens and Martin (1987) reported sonecessity of thorough field evaluation of transgenic sugarcane while maclonal variation in two sugarcane cultivars, CP 65-357 selecting genetically stable and agronomically acceptable material for and CP 72-356. The frequency of variants and suitability commercial use.of tissue culture treatments varied between cultivars, and they recommended that the effects of tissue culture on ...
For the first time, the phosphomannose isomerase (PMI, EC 5.3.1.8)/mannose-based "positive" selection system has been used to obtain genetically engineered sugarcane (Saccharum spp. hybrid var. CP72-2086) plants. Transgenic lines of sugarcane were obtained following biolistic transformation of embryogenic callus with an untranslatable sugarcane mosaic virus (SCMV) strain E coat protein (CP) gene and the Escherichia coli PMI gene manA, as the selectable marker gene. Postbombardment, transgenic callus was selectively proliferated on modified MS medium containing 13.6 microM 2,4-D, 20 g l(-1) sucrose and 3 g l(-1) mannose. Plant regeneration was obtained on MS basal medium with 2.5 microM TDZ under similar selection conditions, and the regenerants rooted on MS basal medium with 19.7 microM IBA, 20 g l(-1) sucrose, and 1.5 g l(-1) mannose. An increase in mannose concentration from permissive (1.5 g l(-1)) to selective (3 g l(-1)) conditions after 3 weeks improved the overall transformation efficiency by reducing the number of selection escapes. Thirty-four vigorously growing putative transgenic plants were successfully transplanted into the greenhouse. PCR and Southern blot analyses showed that 19 plants were manA-positive and 15 plants were CP-positive, while 13 independent transgenics contained both transgenes. Expression of manA in the transgenic plants was evaluated using a chlorophenol red assay and enzymatic analysis.
Efficient shoot regeneration of sugarcane (Saccharum spp. hybrid cv. CP84-1198) from embryogenic callus cultures has been obtained using thidiazuron (TDZ). Callus was placed on modified Murashige and Skoog (MS) medium containing 2.3 mM 2,4-dichlorophenoxyacetic acid (2,4-D), or 9.3 mM kinetin and 22.3 mM naphthaleneacetic acid (NAA) and compared with the same MS medium supplemented with 0.5, 1.0, 2.5, 5.0 or 10.0 mM TDZ. All TDZ treatments resulted in faster shoot regeneration than the kinetin/NAA treatment, and more shoot production than either the 2,4-D or kinetin/NAA treatments. Maximum response, as determined by total number of shoots (26 per explant) and number of shoots greater than 1 cm (4 per explant) 4 wk after initiation, was obtained with 1.0 mM TDZ. The shoots rooted efficiently on MS medium supplemented with 19.7 mM indole-3-butyric acid (IBA). These results indicate that TDZ effectively stimulates sugarcane plant regeneration from embryogenic callus, and may be suitable to use in genetic transformation studies to enhance regeneration of transgenic plants.
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