Agrobacterium LBA4404에 의한 국화 ′Shuho-no-chikara′에 LEAFY유전자의 도입

pSK109를 포함하고 있는 Agrobacterium LBA4004을 통하여 국화 'Shuho-no-chikara'에 LEAFY 유전자을 도입하였다. 엽절편체의 생존율은 kanamycin 10mg/L가 첨가된 배지에서 생존율 약 22%, 절편체의 재분화율이 10%정도였다. 그러나 kanamycin 20mg/L가 첨가된 배지에서는 엽절편은 약 5%정도 생존하였으나 전혀 재분화가 나타나지 않았다. 따라서 kanamycin 10mg/L를 1차 선발배지로, 20mg/L를 2차 선발배지로 선발하였다. Agrobacterium LBA4404와 국화 엽절편체를 3일간 공동배양 하는 것이 형질전환에 효과적이었다. LEAFY유전자가 삽입된 pSK109 vector를 포함한 Agrobacterium LBA4404의 형질전환 효율은 2차 선발까지 약 2.8% 였으며, Southern blot한 결과 0.13%만이 형질 전환체로 확인되었다. 형질 전환체 한계통은 포장에서 약 1주일 정도 조기개화하였고 화형은 정상이었다.

Several experiments were carried out to transfer LEAFY gene to Dendranthema grandiflora 'Shuho-no-chikara' by Agrobacterium LBA4404 carrying pSK109 encoding LEAFY gene. Kanamycin 10mg/L was used in first selection medium, and 20mg/L in the second one. Co-culture for 3 days was more effective in increasing transformation efficiency than that for 7 days. The transformation efficiency by Agrobacterium LBA4404 carrying pSK109 encoding LEAFY gene was about 2.8％ until the second selection, but only 0.13％ of shoots (two plants) was confirmed as a transgenic plants in Southern analysis. The escape of putative transformants was occured seriously in the process of selections, PCR analysis for confirming of neomycin phosphotransferaseII (npt II), and Southern analysis for LEAFY gene. One transgenic plant appeared 7 days'early flowering in field.