본 연구에서는 Agrobacterium tumefaciens 및 Ti-plasmid에 의한 감자 괴경세포의 형질전환 여부를 조사하여 다음과 같은 결과를 얻었다. Agrobacterium tumefaciens KU-12의 감염에 의하여 감자 괴경의 원반에 tumor가 형성되었으며, 이 원반을 호르몬이 없는 배지에서 배양한 결과 tumor에서 완전한 식물체가 재생되었다. 또한 tumor에서 재생된 식물체 내에는 lysopine dehydrogenase(LpDH) 활성이 있음을 확인하였다. 감자의 원형질체에 Ti-plasmid DNA(pTi-12)를 처리하여 호르몬이 결여된 배지에서 배양한 결과, 165개의 callus를 얻었으며, 그 중 일부에서는 뿌리가 형성되었다. 뿌리를 형성한 callus와 형성하지 않은 callus을 임의로 선택하여 LpDH 활성을 조사한 결과 둔 경우 모두 octopine을 합성함으로 형질전환체임을 확인하였다. 형질 전환된 식물의 genome내의 T-DNA 부위를 분석하기 위하여 DNA-DNA 혼성화실험을 성시한 결과, pTi-12 DNA내에 존재하는 T region내에는 Sma I fragment들 중 분자량이 134 kb, 37 kb 및 4.75 kb인 것이 완전히 내포되어 있으며, T region의 양쪽 끝은 분자량이 각각 9.7 kb와 4.55 kb인 Sma I fragment에 연관되어 있음을 알 수 있었고, 또한 T-DNA에 내포된 Sma I fragment들은 pTi-12 DNA 분자내에서 서로 연결된 상태로 존재하고 있음을 확인하였다.
In the present study, transformation of potato tuber cells by Agrobacterium tumefaciens and Ti plasmid treatment was studied. The results are as follows: Tumors were induced in potato tuber discs by infection with Agrobacterium tumefaciens KU-12. Whole plants were regenerated from the tumors on hormone-free medium. It was identified that the lysopine dehydrogenase (LpDH) activity was present in the tumor-derived plants. A total of 165 calli were obtained from Ti-plasmid DNA(pTi-12)-treated potato protoplasts on hormone-free medium and a few of them formed a root. Each of the non-rooted and rooted callus was randomly selected and tested for the presence of LpDH activity. Both of them were ocotopine positive, indicating that they were transformants. In order to analyze the T-DNA region maintained in transformed plant genome, the DNA-DNA hybridization experiment was performed. As a result, it was found that Sma I fragments of Ti plasmid (pTi-12) 13.4 kb, 3.7 kb, and 4.75 kb in size, were completely contained within T-region, and each fragment of 9.7kb and 4.55 kb was joined to opposite ends of T-region. Also, it was identified that Sma I fragments which were contained within T-DNA region were covalently linked together within the pTi-12 DNA molecule.
