In this study, plant regeneration through in vitro culture from plantlet stems of Yooja (C. junos Sieb.) and trifoliate orange (P. trifoliata Rafin.) was attempted to make mass-production system of virus-free plants having the same genotype with mother plant. In order to investigate physiological change depending on the developmental stage of plant regeneration, the changes of total protein, peroxidase and esterase activity and their isozyme patterns as well were examined in 1/2 MS medium. The results are as follows : 1. The MS medium for the optimal callus induction and shoot formation was utilized. The medium was supplemented either with 2,4-D and Kinetin or with BA and NAA. The optimal concentrations were the combination of 1.0mg/ 2,4-D +0.3mg/ Kinetin and 1.0mg BA +0.3mg NAA in callus induction and shoot formation, respectively. 2. For the plant regeneration from somatic embryos, 1/2 MS medium was used with supplements of growth regulators (free, 1.0mg/ IBA +1.0mg/ BA ,0.5mg/ IBA +0.5mg/ BA). Shooting and rooting were the best in the treatment of 0.5mg/ IBA and 0.5mg/ BA combination. 3. The total protein content has a tendency of increase with the developmental stage of embryo, but it was decreased at the plantlet. Also it was the highest at 8 and 6 weeks stage in C. junos Sieb. and P. trioliata Rafin, respectively. In the SDS-PAGE pattern of protein, C. junos Sieb. showed bands of 29.0 and 40kDa at 10 weeks. The 45,66 and 97.4 kDa bands at 10 weeks of culture were shown in P. trifoliata Rafin. 4. The highest esterase activity was shown at the 6 and 8 weeks of culture in C.junos Sieb. and P. trifoliata Rafin.., respectively. 5. Esterase isozyme patterns were shown difference according to the developmental stage. In C. junos Sieb. a new band was observed at pl 7.7 following 4 weeks culture. On the other hand, new bands in P. trifoliata Rafin. were observed at pl 7.5~6.5 following 4 and 6 weeks culture, respectively.

An efficient system of rice microspore culture could contribute to the production of genetically modified rice. The microspores were isolated by mechanical or shed methods. The number of microspores per 100 anthers isolated at uninucleate stage was higher than (or similar to) those at binucleate stage in isolation method with pestle or spatular, but microspore divisions were not easily observed on both stages. On the other hand, pollen division in shed pollen culture was observed more frequently at uninuclear than at binuclear stage. Cold pretreatment at 10~circC for 10 days resulted in the best multicellular division to produce microcalli at 12.5% efficiency in shed microspores. Heat shock at 33~circC for one hour before or after pollen shedding enhanced cell division and callus formation. Out of twelve green regenerants, two were haploids and ten were diploids based on the chromosome analysis of root tips. The size of stoma was 12m m in haploids and 15 ~mu~textrmm in diploids determined by scanning electron microscope (SEM).

본 연구에서는 헛개나무 조직 배양에서 체세포배 유도 및 식물체 재분화를 위한 적절한 배지, 생장조절물질, 탄소원 그리고 배양 환경을 조사하였으며 대량 번식 시스템을 위해 체세포 배로부터 식물체로 재분화 하기 전 세포 동조화와 건실한 유묘 생산에 최적인 배양 조건을 조사하였다. 강원도 양양 헛개나무 자생지에서 채취한 종자를 배배양하여 기내 발아시킴으로 기내 식물체를 얻었으며 이를 본 실험에 이용하였다. 기내 발아 후 7-14일 된 식물체의 자엽과 잎 절편을 NAA를 처리한 배지에 배양했을 때 callus 상태를 거치지 않고 직접 shoot가 유기되었는데 NAA 0.5mg/l에서 43.6%로 형성율이 가장 높았으며 절편체 당 shoot 의 수도 2.8개였다. NAA와 BA 조합처리하였을 때 BA 0.1mg/l+ NAA 1mg/l에서 38.1%의 형성율을 보였으며 절편체 당 shoot의 수는 4개 이상으로 유기되었다. 체세포배는 NAA와 2,4-D의 단독처리 또는 BA와의 조합처리에서 발생하였으며 직접 체세포배 또는 배발생캘러스가 형성하였다. BA 0.1mg/l+ 2,4-D 1mg/l 또는 BA 0.1mg/l+ NAA 1mg/l에서 발생한 배발생 캘러스의 생장과 성숙 및 발아에 최적 배양 조건을 조사하였을 때 GA3 1mg/l을 처리하여 배 발아를 촉진시켰으며 자엽은 발달하지 못하고 하배축이 신장하여 유근이 발생한 형태의 유묘를 생산하였다.

Transgenic plants from hypocotyl segments of buckwheat were produced with the Agrobacterium strain LBA4404 harboring the binary vector pBI121 containing chimeric genes of neomycin phosphotransferase II (npt II) and β -glucuronidase (gus). Two weeks after co-cultivation with Agrobacterium, most of the hypocotyl segments gradually became brown and died on the selection medium containing 100mg/~ell of kanamycin. Plants regenerated from the hypocotyl explants grown on selection medium were GUS-positive in the leaf, stem and vascular tissues by histochemical assay, and varied in gus activity (440-2568 pmol, 4-MU/mg protein) by fluorimetry. The plants showing GUS activity were confirmed of containing GUS and NPT-II genes by polymerase chain reaction (PCR). Within 3 months, transgenic buckwheat plants were able to obtained from the hypocotyl segments.