CAJ | 학술논문

为了躲避转基因技术对组织培养的过分依赖,本研究以小麦为材料尝试了一种对生长点直接进行转化的方法,并初步证明了这一方法的可行性.具体做法是:将种子萌芽,然后剥去胚芽鞘暴露出生长点,再用玻璃纤维制作的小刷子将生长点刺伤,最后用带有外源基因的农杆菌进行侵染处理.用携带hph-GUS基因的LBA4404农杆菌,侵染处理1～11日龄幼苗(生长点),共培养7 d后检测新生芽中GUS的瞬时表达情况;侵染1～2日龄幼苗(生长点),取长成植株的2～3朵小花进行GUS稳定表达检测.又用携带BADH和nptⅡ基因的AGL1农杆菌菌株侵染1日龄的幼苗(生长点),在含100 mg/L卡那霉素的蛭石中进行选择.结果表明,GUS的瞬时表达率随被处理幼苗的日龄增加而降低,以2日龄幼苗为最高(10.7%).用花序检测GUS的稳定表达,被侵染受体为1日龄幼苗时的表达率高于2日龄的幼苗.蔗糖的存在并不提高GUS基因在花序中表达的频率.不过花序表现为GUS阳性的植株后代,经PCR检测后并没有证实GUS基因存在.用AGL 1菌株进行转化,获得了13.6%的抗卡那霉素的绿色植株,但只有3株呈现PCR阳性,且只有1株结了实.
위료타피전기인기술대조직배양적과분의뢰,본연구이소맥위재료상시료일충대생장점직접진행전화적방법,병초보증명료저일방법적가행성.구체주법시:장충자맹아,연후박거배아초폭로출생장점,재용파리섬유제작적소쇄자장생장점자상,최후용대유외원기인적농간균진행침염처리.용휴대hph-GUS기인적LBA4404농간균,침염처리1～11일령유묘(생장점),공배양7 d후검측신생아중GUS적순시표체정황;침염1～2일령유묘(생장점),취장성식주적2～3타소화진행GUS은정표체검측.우용휴대BADH화nptⅡ기인적AGL1농간균균주침염1일령적유묘(생장점),재함100 mg/L잡나매소적질석중진행선택.결과표명,GUS적순시표체솔수피처리유묘적일령증가이강저,이2일령유묘위최고(10.7%).용화서검측GUS적은정표체,피침염수체위1일령유묘시적표체솔고우2일령적유묘.자당적존재병불제고GUS기인재화서중표체적빈솔.불과화서표현위GUS양성적식주후대,경PCR검측후병몰유증실GUS기인존재.용AGL 1균주진행전화,획득료13.6%적항잡나매소적록색식주,단지유3주정현PCR양성,차지유1주결료실.
Realizing the importance of in planta transformation for wheat to obviate the inconvenience of callus based transformation through Agrobacterium and particle bombardment an attempt was made for direct transformation of apical meristem. The study provides a positive clue for success of in planta transformation of wheat apical meristem. Coleoptiles of growing seedlings were cut and exposed meristems were wounded with fiberglass brush and inoculated with Agrobacterium. Apical meristems of 1 - 11 days old seedlings were inoculated with Agrobacterium tumefaciens LBA4404 containing hph-GUS genes in its plasmid and transient GUS expression was determined in regenerated shoots one week after co-cultivation. When apical meristems from 1-2 days old seedlings were inoculated then 2 to 3 florets from inflorescences were tested for stable GUS expression. One-day-old seedlings were inoculated with Agrobacterium tumefaciens AGL1 harboring nptⅡ and BADH genes in its plasmid and regenerated seedlings were selected on 100 mg/L kanamycin in vermiculite. Results indicate that transient GUS frequency decreased with increasing age of the seedlings with maximum (10.7 % ) in two days old seedlings. Numbers of GUS positive inflorescences were more when apical meristems from one-dayold seedlings were inoculated compared with 2 days old seedlings. Presence of glucose did not enhance the frequency of GUS expression in inflorescences. PCR test did not confirm the presence of GUS transgene in the progeny of plants with GUS positive inflorescences. Sixty-three kanamycin resistant green plants ( 13.6 % ) were obtained but only three plants were found PCR positive. Only one plant produced seeds.