Miscanthus sinensis is a perennial C4-grass have tremendous potential as a forage and energy crop for cellulosic ethanol production. High biomass production, and least requirement of irrigation, fertilizer and pesticide make it attractive for large scale commercial cultivation. However, high lignin ...
Miscanthus sinensis is a perennial C4-grass have tremendous potential as a forage and energy crop for cellulosic ethanol production. High biomass production, and least requirement of irrigation, fertilizer and pesticide make it attractive for large scale commercial cultivation. However, high lignin content is a major constrain for animal digestibility and ethanol conversion. In order to develop low lignin transgenic Miscanthus sinensis, establishment of efficient tissue culture and transformation system is essential. We investigated the effects of different plant growth regulators, antioxidants on callus induction and plant regeneration using mature seeds as explant. Dehusked mature seeds were cultured on MS medium supplemented with 3.0 to 10.0 mg/L 2,4-D, dicamba or NAA, 30 g/L sucrose and 750 mg/L MgCl2·6H2O. A number of combinations of auxin and cytokinin (BA plus kinetin) were also used. MS medium containing 3.0 mg/L 2,4-D was found optimal for embryogenic callus induction (75.7%) from mature seed. The highest number of plants were regenerated (44.6%) upon transferring the embryogenic callus to MS medium supplemented with 1.0 mg/L 2,4-D plus 2.0 mg/L BA. Supplementation of the media with 5.0 mg/L AgNO3 and 10 mg/L L-cysteine enhanced the rate of plant regeneration. To develop an efficient Agrobacterium-mediated transformation system, seed-derived calli were infected and co-cultured with Agrobacterium tumefaciens carrying the binary vector pIG121Hm encoding the hygromycin phosphotransferase (HPT), neomycin phosphotransferase II (NPTII) and intron-containing β-glucuronidase (intron-GUS) gene in the T-DNA region. The effects of several factors on transformation and the expression of the GUS gene were investigated. Transient expression of transgene was influenced by the Agrobacterium tumefaciens strain, the duration of co-cultivation, duration of vacuum treatment, calli age, and the concentration of acetosyringone during inoculation and co-cultivation. Among the three strains tested, EHA105 was found most effective for transient GUS expression, followed by LBA4404 and GV3101. Co-cultivation periods for 3 days resulted in highest rate of transient GUS expression. Transformation frequency was increased when 2-month old calli were used. The inclusion of 200 µM acetosyringone in both the inoculation and co-cultivation media lead to an improvement in transient GUS expression. Vacuum treatment during infection of calli with Agrobacterium tumefaciens strains increased transformation efficiency. The transformation system developed in this study would be useful for transgenic Miscanthus plants with gene(s) of interest.
Miscanthus sinensis is a perennial C4-grass have tremendous potential as a forage and energy crop for cellulosic ethanol production. High biomass production, and least requirement of irrigation, fertilizer and pesticide make it attractive for large scale commercial cultivation. However, high lignin content is a major constrain for animal digestibility and ethanol conversion. In order to develop low lignin transgenic Miscanthus sinensis, establishment of efficient tissue culture and transformation system is essential. We investigated the effects of different plant growth regulators, antioxidants on callus induction and plant regeneration using mature seeds as explant. Dehusked mature seeds were cultured on MS medium supplemented with 3.0 to 10.0 mg/L 2,4-D, dicamba or NAA, 30 g/L sucrose and 750 mg/L MgCl2·6H2O. A number of combinations of auxin and cytokinin (BA plus kinetin) were also used. MS medium containing 3.0 mg/L 2,4-D was found optimal for embryogenic callus induction (75.7%) from mature seed. The highest number of plants were regenerated (44.6%) upon transferring the embryogenic callus to MS medium supplemented with 1.0 mg/L 2,4-D plus 2.0 mg/L BA. Supplementation of the media with 5.0 mg/L AgNO3 and 10 mg/L L-cysteine enhanced the rate of plant regeneration. To develop an efficient Agrobacterium-mediated transformation system, seed-derived calli were infected and co-cultured with Agrobacterium tumefaciens carrying the binary vector pIG121Hm encoding the hygromycin phosphotransferase (HPT), neomycin phosphotransferase II (NPTII) and intron-containing β-glucuronidase (intron-GUS) gene in the T-DNA region. The effects of several factors on transformation and the expression of the GUS gene were investigated. Transient expression of transgene was influenced by the Agrobacterium tumefaciens strain, the duration of co-cultivation, duration of vacuum treatment, calli age, and the concentration of acetosyringone during inoculation and co-cultivation. Among the three strains tested, EHA105 was found most effective for transient GUS expression, followed by LBA4404 and GV3101. Co-cultivation periods for 3 days resulted in highest rate of transient GUS expression. Transformation frequency was increased when 2-month old calli were used. The inclusion of 200 µM acetosyringone in both the inoculation and co-cultivation media lead to an improvement in transient GUS expression. Vacuum treatment during infection of calli with Agrobacterium tumefaciens strains increased transformation efficiency. The transformation system developed in this study would be useful for transgenic Miscanthus plants with gene(s) of interest.
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