Antibiotics marker-free with herbicide-resistant tall fescue plants were produced through Agrobacterium: mediated transformation system. Embryogenic calli derived calli were infected with Agrobacterium tumefaciens strain EHA105 harboring the pCAMBIA3300 vector containing the bar and the CP4-EPSPS genes. The PPT-resistant calli and plants were selected with 10 ㎎/L PPT, respectively. Soil-grown plants were obtained about 14-16 weeks after Agrobacterium-mediated transformation. Genetic transformation of the regenerated plants growing under selection was demonstrated by PCR, and Southern blot analysis revealed that one copies of the transgene were integrated into the plant genome of each transgenic plant. Expression of the bar and CP4-EPSPS genes in transgenic plants was confirmed by Northern blot analysis and CP4-EPSPS genes in transgenic plant ELISA uses antibody protein by ELISA Assays. Transgenic plants sprayed of two herbicides with glufosinate ammonium or glyphosate remained green and healthy. We therefore report here a successful and reliable Agrobacterium-mediated transformation of two herbicide-resistances and this method may be useful for routine transformation and has the potential to develop new varieties of tall fescue with several important genes.

The selectable marker-free rice plants containing mcry1Ac insecticidal gene isolated from Bacillus thuringiensis (Bt) were generated using a non-selection approach by Agrobacterium tumefaciens-mediated transformation. The nutritional composition of two lines of transgenic rice plants (RTB5 and RTB11) was compared with that of its non-transgenic counterpart. The results showed that, except for small differences in dietary fiber and some minerals, there was no significant difference between transgenic rice and conventional counterpart variety with respect to their nutrient composition. Most of measured levels of nutrients were within the range of values reported for other commercial cultivars, showing substantial equivalency. Therefore, the insertion of transgenes did not affect the nutritional composition of transgenic RTB5 and RTB11 rice grains.

Rice flour is used in many food products. However, dough made from rice lacks extensibility and elasticity, whereas that of wheat is suitable for many food products including breads. We have produced marker-free transgenic rice plants containing a wheat TaGlu-Ax1 gene encoding the HMG-GS from the Korean wheat cultivar ‘Jokyeong’ using the Agrobacteriummediated co-transformation method. The TaGlu-Bx7-own promoter was inserted into a binary vector for seed-specific expression of the TaGlu-Ax1 gene. Two expression cassettes comprised of separate DNA fragments containing only TaGlu-Ax1 and hygromycin phosphotransferase II (HPTII) resistance genes were introduced separately to the Agrobacterium tumefaciens EHA105 strain for co-infection. Each EHA105 strain harboring TaGlu-Ax1 or HPTII was infected to rice calli at a 3:1 ratio of TaGlu-Ax1 and HPTII, respectively. Then, among 210 hygromycin-resistant T0 plants, we obtained 20 transgenic lines with both TaGlu-Ax1 and HPTII genes inserted into the rice genome. We reconfirmed integration of the TaGlu-Ax1 gene into the rice genome by Southern blot analysis. Transcripts and proteins of the wheat TaGlu-Ax1 were stably expressed in the rice T1 seeds. Finally, the marker-free plants harboring only the TaGlu-Ax1 gene were successfully screened at the T1 generation.

본 연구는 Bacillus thuringiensis 유래의 살충성 mCry1Ac 유전자를 무선발 형질전환 방법으로 일미 벼에 도입하여 개 발된 마커프리 형질전환 Bt 벼 2계통의 일반성분 및 주요성분 (무기질, 아미노산) 함량을 확인하여 모본벼 및 다른 일반품 종과 함량차이를 비교 분석 함으로서 형질전환 벼의 영양성 분 동등성 여부를 확인하고자 수행되었다. 영양성분 분석결과 GM 벼 현미의 일반성분 조성 중 식이섬유 함량과 일부 무기 질 함량이 모본 벼인 일미와 비교하여 다소 유의적 차이가 있 었지만 일반품종에서 나타나는 함량범위 안에 포함되는 수치 이며, 아미노산 성분과 대부분의 일반성분 및 무기질의 함량 은 전반적으로 모본과 유의적 차이가 없었다. 따라서 형질전 환 벼에서 관찰된 일부 성분 차이는 Bt 유전자의 도입 효과가 아닌 재배 환경 및 토양성분의 차이에서 기인된 것으로 형질 전환에 의한 비의도적 영양성분 변화는 없는 것으로 판단된다.

Development of transgenic plant increasing crop yield or disease resistance is good way to solve the world food shortage. However, the persistence of marker genes in crops leads to serious public concerns about the safety of transgenic crops. In the present study, we developed marker-free transgenic rice inserted high molecular-weight glutenin subunit (HMW-GS) gene (Dx5) from the Korean wheat cultivar ‘Jokyeong’ using Agrobacterium-mediated co-transformation method. The Dx5’s own promoter was used for protein expression. Two expression cassettes comprised of separate DNA fragments containing only the Dx5 and hygromycin resistance (HPTII) genes were introduced separately into Agrobacterium tumefaciens EHA105 strain for co-infection. Each EHA105 strain harboring Dx5 or HPTII was infected into rice calli at a 3: 1 ratio of EHA105 with Dx5 gene and EHA105 with HPTII gene expressing cassette. Then, among 270 hygromycin-resistant transformants, we obtained 27 transgenic lines inserted with both the Dx5 and HPTII genes into the rice genome. We reconfirmed integration of the Dx5 gene into the rice genome by Southern blot analysis. Wheat Dx5 transcripts in T1 rice seeds were examined with semi-quantitative RT-PCR. Protein expression of the Dx5 was analyzed with Western blot using polyclonal antibody recognising x-type of glutenin subunits in T1 seeds. It was suggested that the protein-processing system was conserved between rice and wheat. Finally, the marker-free plants containing only the Dx5 gene were successfully screened at the T1 generation.

Until now many strategies have been used to produce marker-free transgenic plants such as co-transformation with negative selectable markers, site-specific recombination system, transposable elements mediated transformation, and etc. In this research, embryogenic calli induced from japonica rices, Ilmibyeo and Dongjinbyeo, were tranformed with the vector which simultaneously constructed with cre/loxP and argE genes in T-DNA. Transformation efficiencies were comparably lower than those of our previous studies, since the constructed genome size was relatively big (>10Kb). For eliminate the transformed tissues which contained positive selectable marker gene, tunicamycin was treated at regeneration and selection stages, since cre recombinase gene is expressed under the presence of this antibiotics. The plants were selected first under 50 mg․L-1 hygromycin at 28℃ for 2 weeks after the Agrobacterium-infection at 25℃ for 7 days. And then, the regeneration plants were successfully obtained on MS basal regeneration medium containing 0.1 mg․L-1 tunicamycin. The regenerated plants are now acclimatizing in greenhouse and molecular analysis are currently accomplished with these plants.