배발생 캘러스를 이용한 아그로박테리움 매개형질전환 장미 식물체 획득

Abstract

아그로박테리움 매개에 의한 형질전환 기술을 이용하여 국내에서 육성된 품종 'Sweet Yellow'로부터 유도된 체세포배 (배발생캘러스 포함)로 intron-GUS유전자가 전이된 식물체를 획득하기까지의 과정이 제시되었다. Intron-GUS 유전자를 포함하고 있는 Agrobacterium tumefaciens AgL1(O.D=0.7~1.6)에 30분 감염시켜 3일간 공동배양 한 후 <TEX>$4^{\circ}C$</TEX>에서 7일간의 저온처리를 거친 후 cefotaxim <TEX>$250\;mg{\cdot}L^{-1}$</TEX> 첨가 체세포배발아 배지에 배양된 체세포배 (배발생캘러스 포함)들 대부분으로 유전자가 전이된 것을 GUS transient assay에 의해 확인하였다. Intron-GUS유전자가 전이된 체세포배 (배발생캘러스 포함)로부터 신초원기를 유도한 후 신초를 재분화시켰고, 재분화된 신초로부터 다신초가 형성되도록 하였다. 다신초로부터 신초의 일부를 떼어 GUS transient assay 분석을 실시하여 intron-GUS 유전자의 발현을 확인한 후 발근시켜 순화 후 온실로 옮겼다. GUS transient assay에 의해 확인된 유전자 발현율은 100%였다. The process to acquire intron-GUS gene-expressed transformants from somatic embryos (including embryogenic calli) of Rosa hybrida cv. 'Sweet Yellow' using Agrobacterium-meditated transformation method was reported in this study. Somatic embryos including embryogenic calluses were infected with Agrobacterium tumefaciens AGL1 strain (O.D = 0.7~1.6) including intron-GUS gene for 30 min, and were co-cultured for 3 days. After co-cultivation, they were cultured on embryo germination medium (EGM) supplemented with <TEX>$250\;mg{\cdot}L^{-1}$</TEX> cefotaxim at <TEX>$4^{\circ}C$</TEX> for 7 days. Then, transient GUS gene expression was observed. Shoots were regenerated from the shoot primodia induced from the intron-GUS gene-transferred either somatic embryos or embryogenic calli cultured on EGM supplemented with both cefotaxim <TEX>$250\;mg{\cdot}L^{-1}$</TEX> and ppt <TEX>$2\;mg{\cdot}L^{-1}$</TEX>. Before induction of rooting from shoots cultured on shoot growing medium supplemented with both cefotaxim <TEX>$250\;mg{\cdot}L^{-1}$</TEX> and ppt <TEX>$2\;mg{\cdot}L^{-1}$</TEX>, the shoots were cultured on multi-shoot induction medium supplemented with both cefotaxim <TEX>$250\;mg{\cdot}L^{-1}$</TEX> and ppt <TEX>$2\;mg{\cdot}L^{-1}$</TEX> to induce multi-shoots. When expression of the gene from a part of the multi-shoots was identified by GUS transient assay, the putative transgenic multishoots were transferred to rooting medium supplemented with cefotaxim <TEX>$250\;mg{\cdot}L^{-1}$</TEX>. After the formation of healthy roots, transgenic plantlets were transferred to the greenhouse after acclimatization. The expression rate of the intron-GUS gene in the multi-shoots was 100%.