Optimisation of Adventitious Shoot Regeneration and Agrobacterium-mediated Transformation in Canna × generalis(Canna Lily)
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摘要
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.
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.
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.
引文
Balachandran,S.M.,Bhatm,S.R.,Chandel,K.P.S.,1990.In vitro clonal multiplication of turmeric(Curcuma spp.)and ginger(Zingiber officinale Rosc.).Plant Cell Rep,8:521-524.
Behar,N.,Tiwari,K.L.,Jadhav,S.K.,2014.Effect of explant type in development of in vitro micropropagation of an endangered medicinal plant:Curcuma caesia Roxb.Biotechnology,13:22-27.
Bharalee,R.,Das,A.,Kalita,M.C.,2005.In vitro clonal propagation of Curcuma caesia Roxb.and Curcuma zedoaria Rose from rhizome bud explants.J Plant Biochem Biotechnol,14:61-63.
Faria,R.T.,Illg,R.D.,1995.Micropropagation of Zingiber spectabile Griff.Sci Hortic,62:135-137.
Faridah,Q.Z.,Abdelmageed,A.H.A.,Julia,A.A.,Nor Hafizah,R.,2011.Efficient in vitro regeneration of Zingiber zerumbet Smith(a valuable medicinal plant)plantlets from rhizome bud explants.S Afr J Bot,10:9303-9308.
Gill,S.S.,Tuteja,N.,2010.Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants.Plant Physiol Biochem,48:909-930.
Gupta,A.,Maurya,R.,Roy,R.K.,Sawant,S.V.,Yadav,H.K.,2013.AFLP based genetic relationship and population structure analysis of Canna-An ornamental plant.Sci Hort,154:1-7.
Hosoki,T.,Sasaki,H.,1991.In vitro propagation of Canna edulis Ker.by longitudinal shoot-split method.Plant Tissue Cult Lett,8:175-178.
Jefferson,R.A.,1987.Assaying chimeric genes in plants:the GUS gene fusion system.Plant Mol Biol Rep,5:387-405.
Khoshoo,T.N.,Mukherjee,I.,1970.Genetic-evolutionary studies on cultivated Cannas.VI.Origin and evolution of ornamental taxa.Theor Appl Genet,40:204-217.
Kromer,K.,Kukulczanka,K.,1985.In vitro cultures of meristem tips of Canna indica(L.).Acta Hort,167:279-285.
Kromer,K.,1979.Biological activity of endogenous and influence of exogenous growth regulators on Canna indica regeneration in vitro.Acta Hort,91:295-300.
Ming,O.C.,Wen,A.S.,Sinniah,U.,Xavier,R.,Subramaniam,S.,2007.Cysteine and acetosyringone are the two important parameters in Agrobacterium-mediated transformation of rose hybrid(Rosa hybrida L.)cv.Nikita.J Plant Sci,2:387-397.
Mishra,T.,Goyal,A.K.,Sen,A.,2015.Somatic embryogenesis and genetic fidelity study of the micropropagated medicinal species,Canna indica.Horticulturae,1:3-13.
Mun,S.C.,Mun,G.S.,2016.Development of an efficient callus proliferation system for Rheum coreanum Nakai,a rare medicinal plant growing in democratic People’s Republic of Korea.Saudi J Biol Sci,23:488-494.
Murashige,T.,Skoog,F.,1962.A revised medium for rapid growth and bioassays with tobacco tissue cultures.Physiol Plant,15:473-497.
Prakash,S.,Elangomathavan,R.,Seshadri,S.,Kathiravan,K.,Ignacimuthu,S.,2004.Efficient regeneration of Curcuma amada Roxb.plantlets from rhizome and leaf sheath explants.Plant Cell Tissue Org C,78:159-165.
Lv,Q.R.,Chen,C.S.,Xu,Y.J,Hu,S.K.,Wang,L.,Sun,K.,Chen,X.,Li,X.H.,2017.Optimization of Agrobacterium tumefaciens-mediated transformation systems in tea plant(Camellia sinensis).Hortic Plant J,3:105-109.
Rafeiro,E.,Barr,S.G.,Harrison,J.J.,Racz,W.J.,1994.Effects of N-acetylcysteine and dithiothreitol on glutathione and protein thiol replenishment during acetaminophen-induced toxicity in isolated mouse hepatocytes.Toxicology,93:209-224.
Raihana,R.,Faridah,Q.Z.,Julia,A.A.,Abdelmageed,A.H.A.,Mihdzar,A.K.,2011.In vitro culture of Curcuma mangga from rhizome bud.J Med Plant Res,5:6418-6422.
Sakai,T.,Imai,K.,2007.The influences of growth regulators and culture medium composition on shoot-tip cultures of edible Canna.Environ Contr Biol,45:155-163.
Sambrook,J.,Russell,D.,2001.Molecular Cloning:A Laboratory Manual,Vol.1-3,Third ed.Cold Spring Harbor Laboratory Press,New York.
Seyyedyousefi,S.R.,Kaviani,B.,Dehkaei,N.P.,2013.The effect of different concentrations of NAA and 6-BA on micropropagation of Alstroemeria.Eur J Exp Biol,3:133-136.
Smirnoff,N.,Wheeler,G.L.,2000.Ascorbic acid in plants:biosynthesis and function.Crit Rev Biochem Mol Biol,35:291-314.
Srivastava,J.,Vankar,P.S.,2010.Canna indica flower:new source of anthocyanins.Plant Physiol Biochem,48:1015-1019.
Tyagi,R.K.,Yusuf,A.,Dua,P.,Agrawal,A.,2004.In vitro plant regeneration and genotype conservation of eight wild species of Curcuma.Biol Plant,48:129-132.
Woradulayapinij,W.,Soonthornchareonnon,N.,Wiwat,C.,2005.In vitro HIV type 1 reverse transcriptase inhibitory activities of Thai medicinal plants and Canna indica L.rhizomes.J Ethnobot,101:84-89.
Zhang,J.,Wang,Z.W.,2011.Arabinoxylan from Canna edulis Ker by-product and its enzymatic activities.Carbohydr Polym,84:656-661.
Behar,N.,Tiwari,K.L.,Jadhav,S.K.,2014.Effect of explant type in development of in vitro micropropagation of an endangered medicinal plant:Curcuma caesia Roxb.Biotechnology,13:22-27.
Bharalee,R.,Das,A.,Kalita,M.C.,2005.In vitro clonal propagation of Curcuma caesia Roxb.and Curcuma zedoaria Rose from rhizome bud explants.J Plant Biochem Biotechnol,14:61-63.
Faria,R.T.,Illg,R.D.,1995.Micropropagation of Zingiber spectabile Griff.Sci Hortic,62:135-137.
Faridah,Q.Z.,Abdelmageed,A.H.A.,Julia,A.A.,Nor Hafizah,R.,2011.Efficient in vitro regeneration of Zingiber zerumbet Smith(a valuable medicinal plant)plantlets from rhizome bud explants.S Afr J Bot,10:9303-9308.
Gill,S.S.,Tuteja,N.,2010.Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants.Plant Physiol Biochem,48:909-930.
Gupta,A.,Maurya,R.,Roy,R.K.,Sawant,S.V.,Yadav,H.K.,2013.AFLP based genetic relationship and population structure analysis of Canna-An ornamental plant.Sci Hort,154:1-7.
Hosoki,T.,Sasaki,H.,1991.In vitro propagation of Canna edulis Ker.by longitudinal shoot-split method.Plant Tissue Cult Lett,8:175-178.
Jefferson,R.A.,1987.Assaying chimeric genes in plants:the GUS gene fusion system.Plant Mol Biol Rep,5:387-405.
Khoshoo,T.N.,Mukherjee,I.,1970.Genetic-evolutionary studies on cultivated Cannas.VI.Origin and evolution of ornamental taxa.Theor Appl Genet,40:204-217.
Kromer,K.,Kukulczanka,K.,1985.In vitro cultures of meristem tips of Canna indica(L.).Acta Hort,167:279-285.
Kromer,K.,1979.Biological activity of endogenous and influence of exogenous growth regulators on Canna indica regeneration in vitro.Acta Hort,91:295-300.
Ming,O.C.,Wen,A.S.,Sinniah,U.,Xavier,R.,Subramaniam,S.,2007.Cysteine and acetosyringone are the two important parameters in Agrobacterium-mediated transformation of rose hybrid(Rosa hybrida L.)cv.Nikita.J Plant Sci,2:387-397.
Mishra,T.,Goyal,A.K.,Sen,A.,2015.Somatic embryogenesis and genetic fidelity study of the micropropagated medicinal species,Canna indica.Horticulturae,1:3-13.
Mun,S.C.,Mun,G.S.,2016.Development of an efficient callus proliferation system for Rheum coreanum Nakai,a rare medicinal plant growing in democratic People’s Republic of Korea.Saudi J Biol Sci,23:488-494.
Murashige,T.,Skoog,F.,1962.A revised medium for rapid growth and bioassays with tobacco tissue cultures.Physiol Plant,15:473-497.
Prakash,S.,Elangomathavan,R.,Seshadri,S.,Kathiravan,K.,Ignacimuthu,S.,2004.Efficient regeneration of Curcuma amada Roxb.plantlets from rhizome and leaf sheath explants.Plant Cell Tissue Org C,78:159-165.
Lv,Q.R.,Chen,C.S.,Xu,Y.J,Hu,S.K.,Wang,L.,Sun,K.,Chen,X.,Li,X.H.,2017.Optimization of Agrobacterium tumefaciens-mediated transformation systems in tea plant(Camellia sinensis).Hortic Plant J,3:105-109.
Rafeiro,E.,Barr,S.G.,Harrison,J.J.,Racz,W.J.,1994.Effects of N-acetylcysteine and dithiothreitol on glutathione and protein thiol replenishment during acetaminophen-induced toxicity in isolated mouse hepatocytes.Toxicology,93:209-224.
Raihana,R.,Faridah,Q.Z.,Julia,A.A.,Abdelmageed,A.H.A.,Mihdzar,A.K.,2011.In vitro culture of Curcuma mangga from rhizome bud.J Med Plant Res,5:6418-6422.
Sakai,T.,Imai,K.,2007.The influences of growth regulators and culture medium composition on shoot-tip cultures of edible Canna.Environ Contr Biol,45:155-163.
Sambrook,J.,Russell,D.,2001.Molecular Cloning:A Laboratory Manual,Vol.1-3,Third ed.Cold Spring Harbor Laboratory Press,New York.
Seyyedyousefi,S.R.,Kaviani,B.,Dehkaei,N.P.,2013.The effect of different concentrations of NAA and 6-BA on micropropagation of Alstroemeria.Eur J Exp Biol,3:133-136.
Smirnoff,N.,Wheeler,G.L.,2000.Ascorbic acid in plants:biosynthesis and function.Crit Rev Biochem Mol Biol,35:291-314.
Srivastava,J.,Vankar,P.S.,2010.Canna indica flower:new source of anthocyanins.Plant Physiol Biochem,48:1015-1019.
Tyagi,R.K.,Yusuf,A.,Dua,P.,Agrawal,A.,2004.In vitro plant regeneration and genotype conservation of eight wild species of Curcuma.Biol Plant,48:129-132.
Woradulayapinij,W.,Soonthornchareonnon,N.,Wiwat,C.,2005.In vitro HIV type 1 reverse transcriptase inhibitory activities of Thai medicinal plants and Canna indica L.rhizomes.J Ethnobot,101:84-89.
Zhang,J.,Wang,Z.W.,2011.Arabinoxylan from Canna edulis Ker by-product and its enzymatic activities.Carbohydr Polym,84:656-661.