벼에 있어서 엽록체 small heat shock protein(small HSP)의 기능을 밝히기 위하여 Agrobacterium을 이용한 형질전환법을 이용하여 벼로부터 분리한 small HSP cDNA를 도입하였다. Agrobacterium의 감염에는 벼의 미성숙 배로부터 유도한 callus를 이용하였다. 형질전환 후의 재분화율은 약 30%였다. 형질전환을 통하여 얻어진 식물체의 genomic DNA로부터 PCR 분석과 Southern blot 분석으로 엽록체 small HSP 유전자의 도입을 확인하였다. 도입 유전자의 형질전환 벼에 있어서 유전자의 발현 양상을 northern blot 분석으로 조사하였다. 그 결과 도입된 유전자는 상온에서의 발현량이 서로 다르게 나타났으며 항상적으로 발현하고 있음을 확인하였다.

Plant molecular farming has attracted a lot of attention lately in the field of mass production of industrially valuable materials by extending application of the plant as a kind of factory concept. Among them, protein expression system using rice(Oryza sativa L.) callus is a technology capable of mass culture and industrialization because of a high expression rate of a target protein. This study was carried out to develop an Agrobacterium-mediated transformation system to increase the utilization of rice callus. The transformation efficiency was improved by using the hand when seeds were de-husked for callus induction. Furthermore, we were possible induction of callus from 6 years old seed smoothly. Selection of the callus contained the target gene was required a cultivation period of at least 3 weeks, and the most efficient selection period was after 6 weeks of culture including one passage. This selection was confirmed that the gene was stably inserted into the genomic DNA of the plant cell by the southern blot analysis and progeny test. Such an efficient selection system of rice callus that can be cultured in the long term will be contribute to the industrialization of useful recombinant proteins using rice.

Plant transformation systems have been developed using several different approaches, and the development of reliable and efficient transformation systems is still one of the most important breakthroughs in molecular biology and biotechnology of rice. However, there are many cultivars in rice and transformation efficiency is dependent on the cultivar. The conventional Agrobacterium-mediated rice transformation system using secondary calli requires 14-16 weeks for establishing transgenic plantlets, and it is thought that somaclonal variation through the Tos17 retrotransposon occurs during the tissue culture stage. Here, we modified an Agrobacterium-mediated transformation system in rice cultivar Dongjin, adding an endosperm removal step that is critical to transformation frequency and changing the Agrobacterium strain and media composition. This method increased transformation efficiency and stability. These results will provide valuable information for scientists to use a reliable and efficient rice transformation system.

Agrobacterium vector를 이용한 세포사멸 억제유전자인 AtBI-1(Arabidopsis thaliana Bax Inhibitor-1)을 벼에 도입하기 위하여, 벼품종별 식물체 재분화 능력과 AtBI-1 유전자가 도입된 형질전환체 벼에 관한 연구를 수행하여 얻어진 결과를 요약하면 다음과 같다. 벼의 품종별 식물체 재분화 능력을 조사한바 자포니카형 벼가 인디카형 및 통일형 벼에 비해 식물체 재분화능력이 높게 나타났다. 공시 품종 중에서 일품벼의 식물체 재분화율(7%)이 가장 높았다. 배양용기별 식물체 재분화 능력을 조사한바, petri-dish에서 보다 시험관에서 식물체 재분화율이 높았다. 4주동안 자란 ‘일품’ 유래의 callus를 Agrobacterium과 co-cultivation한 후 kanamycin과 carbenicillin이 첨가된 재분화 배지에 이식하였을 때 12.0%의 가장 높은 형질전환율을 나타내었다. 형질전환된 식물체의 genomic DNA를 이용한 PCR 결과는 Bin-AtBI-1-GFP 유전자는 1 kb 부근에 삽입된 것이 확인되었으며, Southern blot 분석에서도 확인이 되었다. 형질전환체의 CLSM(confocal laser scanning microscope) 분석에서 잎, 줄기, 뿌리에서 GFP가 발현되어 형광색을 띄고 있음을 확인 하였다.

Most indica rice varieties show a low efficiency of transformation because of difficulties in callus formation and low-regeneration frequencies in conventional culture such as MS16 or N6 medium. Recently, some improved methods were reported for Agrobacterium-mediated transformation using mature elite indica seeds however, these procedures take a long time (5–7 months) to obtain transgenic plants and still with significantly low efficiency. In this study, we provide additional improvements in the indica rice transformation protocol introducing new selection method by tosoflavin/tflA which was based on bacterial photosensitizer and its degrading enzyme pair. We introduced direct in planta transformation using early stage of germinating rice seeds instead of usual embryogenic callus. Methods that use embryos as a starting material for inoculation with Agrobacterium are also used for comparison with the new protocol using rice seeds. Transformed cells proliferated from rice seeds obtain toxoflavin resistance, and transgenic plants are eventually regenerated from those proliferated tissues. However, we found out that tissue proliferation from indica seeds and shooting and rooting are very sensitive to minor salt nutrients in the media and need to pay attention to use indica rice specific nutrient media. The use of naturally occurring photosensitizers such as toxoflavin as selection agents appears to give rapid and unambiguous selection results owing to their unique phytotoxic mode of action. In particular, the toxoflavin/tflA selection system might be useful for generating transgenic indica rice cultivar where high false-positive backgrounds with current selection marker systems are problematic.

Rice transformation method using A. tumefaciens has already been widely used to generate transgenic plants, the transformation rate is still low in most Korean elite cultivars. We made several modifications of the standard protocol especially in the co-cultivation step to improve the efficiency of the rice transformation. The co-culture medium was modified by the addition of three antioxidant compounds (10.5㎎/ℓ L-cysteine, 1mM sodium thiosulfate, 1mM dithiothreitol) and of Agrobacterium growth-inhibiting agent (5㎎/ℓ silver nitrate). Co-cultivation temperature (23. 5℃ for 1 day, 26.5℃ for 6 days) and duration (7 days) were also changed. The plasmid of pMJC-GB-GUS carrying the GUS reporter gene and the bar gene as the selectable marker was used to evaluate the efficiency of the transformation. After co-cultivation, a high level of GUS gene expression was observed in calli treated with the modified method. It is likely that those newly added compounds helped to minimize the damage due to oxidative bursts during plant cell-Agrobacterium interaction and to prevent necrosis of rice cells. And the transformation rate under the modified method was also remarkably increased approximately 8-fold in Heungnambyeo and 2-fold in Ilmibyeo as compared to the corresponding standard method. Furthermore, we could produce the transgenic plants stably from Ilpumbyeo which is a high-quality rice but its transformation rate is extremely low. Transformation and the copy number of transgenes were confirmed by PCR, bar strip and Southern blot analysis. The improved method would attribute reducing the effort and the time required to produce a large number of transgenic rice plants.

Rice transformation method using A. tumefaciens has already been widely used to generate transgenic plants, the transformation rate is still low in most Korean elite cultivars. We made several modifications of the standard protocol especially in the co-cultivation step to improve the efficiency of the rice transformation. The co-culture medium was modified by the addition of three antioxidant compounds (10.5㎎/ℓ L-cysteine, 1mM sodium thiosulfate, 1mM dithiothreitol) and of Agrobacterium growth-inhibiting agent (5㎎/ℓ silver nitrate). Co-cultivation temperature (23. 5℃ for 1 day, 26.5℃ for 6 days) and duration (7 days) were also changed. The plasmid of pMJC-GB-GUS carrying the GUS reporter gene and the bar gene as the selectable marker was used to evaluate the efficiency of the transformation. After co-cultivation, a high level of GUS gene expression was observed in calli treated with the modified method. It is likely that those newly added compounds helped to minimize the damage due to oxidative bursts during plant cell-Agrobacterium interaction and to prevent necrosis of rice cells. And the transformation rate under the modified method was also remarkably increased approximately 8-fold in Heungnambyeo and 2-fold in Ilmibyeo as compared to the corresponding standard method. Furthermore, we could produce the transgenic plants stably from Ilpumbyeo which is a high-quality rice but its transformation rate is extremely low. Transformation and the copy number of transgenes were confirmed by PCR, bar strip and Southern blot analysis. The improved method would attribute reducing the effort and the time required to produce a large number of transgenic rice plants.

In order to improve rice dough functionality, we cloned 4 kinds of high-molecular-weight glutenin subunit (HMW-GS) genes from bread wheat, ‘Jokyeong’. Among them, we first examined Dx5 gene to generate marker-free transgenic rice for advanced quality processing of bread and noodles. The GluB1 promoter was inserted into binary vector for seed specific expression of the Dx5 gene. Two expression cassettes comprised of separate DNA fragments containing only the high-molecular-weight glutein subunit (HMW-GS) protein (Dx5) and hygromycin phosphotransferase II (HPTII) resistance genes were introduced separately to tumefaciens EHA105 strain for co-infection. Each EHA105 strain harboring Dx5 or HPTII was infected to rice calli at 3: 1 ratio of Dx5 and HPTII, respectively. Then among 66 hygromycin-resistant transformants, we obtained two transgenic lines inserted both with Dx5 and HPTII gene to rice genome. We reconfirmed integration of the Dx5 and HPTII genes into the rice genome by Southern blot analysis. Wheat Dx5 transcriptsin rice seeds was examined with semi-quantitative RT-PCR. Finally, the marker-free plants containing only Dx5 gene were successfully screened at T1 generation. This result also provides that co-infection system with two expression cassettes could be efficient strategy to generate marker-free transgenic rice plants.

Eating quality is critical for consumers who take rice as staple food. Here we present the development and identification of high eating quality rice lines. The identification of positive transgenic lines, physicochemical properties of transgenic rice, mRNA expression and enzyme activity were analyzed. OsSbe1 was introduced into Gopumbyeo seeds using Agrobacterium-mediated transformation, and 1,005 out of 1,065 T1 plants were shown positive. The apparent amylose contents in T2 brown rice ranged from 11% to 25% in 890 favorable lines (Gopumbyeo was used as a reference with 18% of AAC). The activity of starch branching enzyme including three isoforms (SBE1, SBE3, and SBE4) in endosperms of T3 lines was higher than that of Gopumbyeo. Physicochemical properties related to eating quality for T3 polished rice were detected using 52 favorable lines out of 500 lines selected according to AAC. The Toyo taste meter value in 52 T3 lines ranged from 61.1 to 72.6, whereas 70.4 in Gopumbyeo. Of them, eleven lines displayed the higher palatability score than Gopumbyeo. Moreover, these elite lines produced higher yields (607.9~695.8 kg/10a) than Gopumbyeo (602.7 kg/10a). These results indicated the possibility of developing new high quality rice varieties in the future.

1. 본 연구에서는 공동배양 배지에 Agrobacterium 성장 억제물질인 silver nitrate를 첨가하고 변온과 여과지처리를 추가하여 공동배양 기간을 7일로 늘였으며, 또한 항산화 물질 3종을 공동배양 배지에 첨가하여 세포의 oxidative burst를 최소화함으로써 벼 형질전환효율을 높일 수 있었다. 또한 이 방법을 적용하여 형질전환이 어려운 품종을 대상으로도 형질전환 식물체를 작성할 수 있었다. 2. 벼 형질전환체의 70%에서 도입유전자

This study was conducted to produce the transgenic plant of rice. We obtained Agrobacterium AGL1 harbaring pCambial 300 vector with HPT gene. We carried out PCR analysis of 22 ea putative transgenic rice to investigate transformed lines. The 3 ea transgenic lines were detected insertion of HPT gene. Transgenic lines selected from PCR analysis were performed by Southern blot. From Southern blot, we obtained that two transgenic lines detected single band. We are going to study the method improving of cotransformation as well as transformation efficiency in rice.