To develop a transformation protocol of Rosa hybrida Samantha via Agrobacterium tumefaciens, the authors examined the effect of different factors on T-DNA transfer by measuring transient expression levels of an intron...To develop a transformation protocol of Rosa hybrida Samantha via Agrobacterium tumefaciens, the authors examined the effect of different factors on T-DNA transfer by measuring transient expression levels of an intron-containing -glucuronidase gene. The results indicate that explant, light condition, salt concentration and acetosyringone (AS) concentration in co-culture medium are the most important factors, and factors like co-culture temperature, co-culture period and bacteria density have a strong effect on the growth of bacteria and then T-DNA transfer. Optimized co-cultivation was performed by inoculation of embryogenic callus with bacteria at a density of OD600= 0.50.8 for 20 min and co-culture in darkness under 23 C on medium with 1/2 MS salts and 300 mol稬1 AS for 3 d.展开更多
Tall fescue （Festuca arundinacea Schreb.） is a cool-season turfgrass used on fairways in golf courses. The object of this study was to develop a more efficient, reliable, and repeatable approach in transforming the ...Tall fescue （Festuca arundinacea Schreb.） is a cool-season turfgrass used on fairways in golf courses. The object of this study was to develop a more efficient, reliable, and repeatable approach in transforming the grass using Agrobacterium （EHA105）, where β-glucuronidase gene （uidA） was used as a reporter and hygromycin phosphotransferase gene （hyg） as a selectable marker. An effective expression of transgene was observed in transforming 2-month-old calli derived from mature seeds （cv. Bingo） cultured on MS medium supplemented with 9 mg·L^-1 2, 4-D. A two-step solid medium selection with increasing hygromycin concentration （from 30 to 50 mg· L^-1） was used to obtain resistant calli. Transgenic plants have been produced from many independent transformed calli. The presence of functional β-glucuronidase gene （uidA） was detected in hygromycin-resistant calli. Transgenic plants were regenerated and PCR and Southern blot confirmed transgene integration in the tall fescue genome.展开更多
Canna being ornamental plants has a significant role in agriculture, medical, economy and food industry. Canna has a limited vase life due to the rapid loss of moisture from its perianth. For improving its market valu...Canna being ornamental plants has a significant role in agriculture, medical, economy and food industry. Canna has a limited vase life due to the rapid loss of moisture from its perianth. For improving its market value, cuticularisation of the perianth can be achieved by the expression of a heterologous cutin producing gene, using the tissue culture and transformation protocol developed in this study. Efficient, rapid and direct adventitious shoot regeneration was successfully established in Canna × generalis using recalcitrant rhizome explants. The explants were cultured on MS medium supplemented with 6-benzylaminopurine(6-BA), thidiazuron(TDZ), and kinetin. Among the four genotypes taken for tissue culture, the ‘Trinacria variegata' was the best responding cultivar. And 2 mg · L^(-1)6-BA or 1.5 mg · L^(-1) TDZ along with 0.1 mg · L^(-1) IAA was optimum for their regeneration. The highest regeneration was achieved in ‘Trinacria variegata'(36%) on 6-BA, 33% on TDZ while kinetin failed to evoke any regenerative responses. Regeneration was enhanced by supplementation of 100 mg · L^(-1) ascorbic acid(AsA), while, 100 mg · L^(-1) of l-cysteine or 100 mg · L^(-1) dithiothreitol(DTT), inhibited regeneration. Shoots were observed to develop 3–5 fibrous roots on MS medium supplemented with 0.5 mg · L^(-1) indole-3-butyric acid(IBA). The plantlets were transplanted into pots and acclimatised in glasshouse with 100%survival. For transformation of Canna, rhizome explants were co-cultivated for 60 min in Agrobacterium suspension. The explants were washed with 500 mg · L^(-1) cefotaxime solution, subjected to 100 mg · L^(-1) kanamycin selection followed by excision of the shoots and culturing them on IBA-supplemented media for root development. Transgene integration in the putative transformants was confirmed by PCR assay and copy number by Southern blot hybridisation analysis.展开更多
基金Supported by the National Natural Science Foundation of China (Grant No.30170666)
文摘To develop a transformation protocol of Rosa hybrida Samantha via Agrobacterium tumefaciens, the authors examined the effect of different factors on T-DNA transfer by measuring transient expression levels of an intron-containing -glucuronidase gene. The results indicate that explant, light condition, salt concentration and acetosyringone (AS) concentration in co-culture medium are the most important factors, and factors like co-culture temperature, co-culture period and bacteria density have a strong effect on the growth of bacteria and then T-DNA transfer. Optimized co-cultivation was performed by inoculation of embryogenic callus with bacteria at a density of OD600= 0.50.8 for 20 min and co-culture in darkness under 23 C on medium with 1/2 MS salts and 300 mol稬1 AS for 3 d.
基金Foundation project： This paper was supported by Zhejiang Provincial Science and Technology Plan of China （Grant No. 2003C30053） and Zhejiang Provincial Natural Science Foundation of China （Grant No.Y504076）.
文摘Tall fescue （Festuca arundinacea Schreb.） is a cool-season turfgrass used on fairways in golf courses. The object of this study was to develop a more efficient, reliable, and repeatable approach in transforming the grass using Agrobacterium （EHA105）, where β-glucuronidase gene （uidA） was used as a reporter and hygromycin phosphotransferase gene （hyg） as a selectable marker. An effective expression of transgene was observed in transforming 2-month-old calli derived from mature seeds （cv. Bingo） cultured on MS medium supplemented with 9 mg·L^-1 2, 4-D. A two-step solid medium selection with increasing hygromycin concentration （from 30 to 50 mg· L^-1） was used to obtain resistant calli. Transgenic plants have been produced from many independent transformed calli. The presence of functional β-glucuronidase gene （uidA） was detected in hygromycin-resistant calli. Transgenic plants were regenerated and PCR and Southern blot confirmed transgene integration in the tall fescue genome.
基金Financial support by UGC, New Delhi to RS and by CSIR
文摘Canna being ornamental plants has a significant role in agriculture, medical, economy and food industry. Canna has a limited vase life due to the rapid loss of moisture from its perianth. For improving its market value, cuticularisation of the perianth can be achieved by the expression of a heterologous cutin producing gene, using the tissue culture and transformation protocol developed in this study. Efficient, rapid and direct adventitious shoot regeneration was successfully established in Canna × generalis using recalcitrant rhizome explants. The explants were cultured on MS medium supplemented with 6-benzylaminopurine(6-BA), thidiazuron(TDZ), and kinetin. Among the four genotypes taken for tissue culture, the ‘Trinacria variegata' was the best responding cultivar. And 2 mg · L^(-1)6-BA or 1.5 mg · L^(-1) TDZ along with 0.1 mg · L^(-1) IAA was optimum for their regeneration. The highest regeneration was achieved in ‘Trinacria variegata'(36%) on 6-BA, 33% on TDZ while kinetin failed to evoke any regenerative responses. Regeneration was enhanced by supplementation of 100 mg · L^(-1) ascorbic acid(AsA), while, 100 mg · L^(-1) of l-cysteine or 100 mg · L^(-1) dithiothreitol(DTT), inhibited regeneration. Shoots were observed to develop 3–5 fibrous roots on MS medium supplemented with 0.5 mg · L^(-1) indole-3-butyric acid(IBA). The plantlets were transplanted into pots and acclimatised in glasshouse with 100%survival. For transformation of Canna, rhizome explants were co-cultivated for 60 min in Agrobacterium suspension. The explants were washed with 500 mg · L^(-1) cefotaxime solution, subjected to 100 mg · L^(-1) kanamycin selection followed by excision of the shoots and culturing them on IBA-supplemented media for root development. Transgene integration in the putative transformants was confirmed by PCR assay and copy number by Southern blot hybridisation analysis.
