Yerba mate(Ilex paraguariensis A.St.-Hil.)is a species of great economic,social and environmental importance for the southern regions of Brazil,Uruguay and Argentina.Currently the most diverse products are obtained fr...Yerba mate(Ilex paraguariensis A.St.-Hil.)is a species of great economic,social and environmental importance for the southern regions of Brazil,Uruguay and Argentina.Currently the most diverse products are obtained from mate leaves,including mate tea.The objective of this study was to establish shoot meristem cultures(meristematic dome and a few primordia)of elite clones and identify the endophytic bacteria present in the explants.We tested the effect of clones(F1,F2,A03 and A07),culture media(MS,1/2MS,1/4MS and WPM),cytokinins(kinetin,BA and 2iP),activated charcoal(1,2 and 3 g L^-1),and disinfecting agent(sodium hypochlorite and mercuric chloride)on in vitro establishment.F1 and F2 clones were the most responsive for shoot meristem in vitro culture.WPM medium supplemented with 8.8 lM 2iP,0.2 lM NAA and 3 g L^-1 activated charcoal was the most suitable for the in vitro establishment of the F1 clone.No phytotoxic effect of the disinfectant was observed and some meristems sprouted.The isolated endophytic bacterium was identified for the first time in yerba mate as Agrobacterium larrymoorei.To conclude,we were able to establish in vitro culture of yerba mate using meristems as explants but the tissues were not free of endophytic microorganisms which could interfere with explant response.展开更多
Genetic transformation systems require protocols that allow regenerating transgenic plants from transformed tissues. This study aimed to establish a protocol for indirect organogenesis in leaf explants of a Eucalyptus...Genetic transformation systems require protocols that allow regenerating transgenic plants from transformed tissues. This study aimed to establish a protocol for indirect organogenesis in leaf explants of a Eucalyptus grandis x E. urophylla AEC 224 clone. During callogenesis stage, several concentrations of NAA and then NAA or 2,4-D combined with TDZ were tested in JADS culture medium for 30 days, followed by subculture of the explants in the regeneration medium, containing 5.0 μM BA and 0.5 μM NAA for another 30 days. In these media, the explant oxidation rate was high （95 %）. Thus, in order to reduce oxidation, different ;culture media were compared： WPM, MS, JADS and modified QL, followed by explant transfer onto regeneration medium. The highest percentage of regeneration and the lowest oxidation rate were achieved on WPM medium. Then, NAA and 2,4-D were tested in combination with TDZ and also TDZ and BA combined with NAA in WPM medium. The most efficient culture media in terms of shoot regeneration were WPM supplemented with 0.25 μM TDZ and 0.1μM NAA during 30 days for callus induction and then with 5.0 μM BA and 0.5 μM NAA for another 30 days. This protocol yielded a regeneration rate of 43 %, with a low oxidation of tissues. A rooting experiment was conducted using half strength MS medium and comparing three concentrations of IBA （2.46, 4.90 and 7.35μM）. The highest rooting percentage （35 %） was obtained on medium containing 2.46μM IBA. Once the shoots were rooted, acclimatization in a greenhouse was not challenging and plant survival reached 100 %.展开更多
Genetic transformation is becoming routine for engineering specific traits in important clones of recalcitrant species such as Eucalyptus;however,the efficiency is still low for most species,so many researchers still ...Genetic transformation is becoming routine for engineering specific traits in important clones of recalcitrant species such as Eucalyptus;however,the efficiency is still low for most species,so many researchers still use seeds instead of clones as initial explants.This work aimed to develop a genetic transformation protocol,based on a highly efficient in vitro organogenesis protocol,for an Eucalyptus urophylla clone selected in our breeding program.Plant growth regulators were evaluated for indirect organogenesis and rooting.In a two-step protocol,the combination of callus induction media supplemented with 0.5 μM thidiazuron+0.5 μM naphthaleneacetic acid(NAA)and shoot induction media supplemented with 5.0 μM benzylaminopurine+1.0 lM NAA allowed up to 85.6%shoot formation with more shoots per explants when compared with other concentrations.Transgenic plants expressing the uidA gene were obtained using Agrobacterium tumefaciens and selected for kanamycin resistance.A RAPD analysis was used to check for somaclonal variation.In tests using 11 RAPD primers,we did not observe somaclonal variation in the in vitro stages evaluated.展开更多
基金Brazilian Agricultural Research Corporation(EMBRAPA)Forestry
文摘Yerba mate(Ilex paraguariensis A.St.-Hil.)is a species of great economic,social and environmental importance for the southern regions of Brazil,Uruguay and Argentina.Currently the most diverse products are obtained from mate leaves,including mate tea.The objective of this study was to establish shoot meristem cultures(meristematic dome and a few primordia)of elite clones and identify the endophytic bacteria present in the explants.We tested the effect of clones(F1,F2,A03 and A07),culture media(MS,1/2MS,1/4MS and WPM),cytokinins(kinetin,BA and 2iP),activated charcoal(1,2 and 3 g L^-1),and disinfecting agent(sodium hypochlorite and mercuric chloride)on in vitro establishment.F1 and F2 clones were the most responsive for shoot meristem in vitro culture.WPM medium supplemented with 8.8 lM 2iP,0.2 lM NAA and 3 g L^-1 activated charcoal was the most suitable for the in vitro establishment of the F1 clone.No phytotoxic effect of the disinfectant was observed and some meristems sprouted.The isolated endophytic bacterium was identified for the first time in yerba mate as Agrobacterium larrymoorei.To conclude,we were able to establish in vitro culture of yerba mate using meristems as explants but the tissues were not free of endophytic microorganisms which could interfere with explant response.
基金financially supported by CAPES and Empresa Brasileira de Pesquisa Agropecuária(Embrapa,Brazil)
文摘Genetic transformation systems require protocols that allow regenerating transgenic plants from transformed tissues. This study aimed to establish a protocol for indirect organogenesis in leaf explants of a Eucalyptus grandis x E. urophylla AEC 224 clone. During callogenesis stage, several concentrations of NAA and then NAA or 2,4-D combined with TDZ were tested in JADS culture medium for 30 days, followed by subculture of the explants in the regeneration medium, containing 5.0 μM BA and 0.5 μM NAA for another 30 days. In these media, the explant oxidation rate was high （95 %）. Thus, in order to reduce oxidation, different ;culture media were compared： WPM, MS, JADS and modified QL, followed by explant transfer onto regeneration medium. The highest percentage of regeneration and the lowest oxidation rate were achieved on WPM medium. Then, NAA and 2,4-D were tested in combination with TDZ and also TDZ and BA combined with NAA in WPM medium. The most efficient culture media in terms of shoot regeneration were WPM supplemented with 0.25 μM TDZ and 0.1μM NAA during 30 days for callus induction and then with 5.0 μM BA and 0.5 μM NAA for another 30 days. This protocol yielded a regeneration rate of 43 %, with a low oxidation of tissues. A rooting experiment was conducted using half strength MS medium and comparing three concentrations of IBA （2.46, 4.90 and 7.35μM）. The highest rooting percentage （35 %） was obtained on medium containing 2.46μM IBA. Once the shoots were rooted, acclimatization in a greenhouse was not challenging and plant survival reached 100 %.
文摘Genetic transformation is becoming routine for engineering specific traits in important clones of recalcitrant species such as Eucalyptus;however,the efficiency is still low for most species,so many researchers still use seeds instead of clones as initial explants.This work aimed to develop a genetic transformation protocol,based on a highly efficient in vitro organogenesis protocol,for an Eucalyptus urophylla clone selected in our breeding program.Plant growth regulators were evaluated for indirect organogenesis and rooting.In a two-step protocol,the combination of callus induction media supplemented with 0.5 μM thidiazuron+0.5 μM naphthaleneacetic acid(NAA)and shoot induction media supplemented with 5.0 μM benzylaminopurine+1.0 lM NAA allowed up to 85.6%shoot formation with more shoots per explants when compared with other concentrations.Transgenic plants expressing the uidA gene were obtained using Agrobacterium tumefaciens and selected for kanamycin resistance.A RAPD analysis was used to check for somaclonal variation.In tests using 11 RAPD primers,we did not observe somaclonal variation in the in vitro stages evaluated.
