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갯봄맞이(Glaux maritima L.)의 캘루스 배양을 통한 식물체 재분화
한증술,Han, Jeung-Sul 한국식물생명공학회 2017 식물생명공학회지 Vol.44 No.2
우리나라에서 멸종위기 해안식물로 지정된 갯봄맞이의 캘루스배양을 통한 재분화 프로토콜을 보고한다. 마디배양을 통해 육성한 기내 유식물체의 절편은 유전자형, 절편의 종류, 광조건 및 2,4-D 첨가농도에 따라 캘루스 형성에 상이한 반응을 보였다. 특히, 다른 처리와는 무관하게 연속 암배양이 절편으로부터 캘루스 유도에 결정적인 영향을 미쳤다. 2,4-D 0.1-3.0mg/L 농도 범위의 배지에서 유도된 캘루스를 동일한 조성의 배지로 이식하여 암배양했을 때왕성하게 캘루스가 증식하였다. 연속 암조건에서 zeatin 1.0mg/L 첨가배지에 'Pistachio' 유전자형의 캘루스를 배양했을 때만 유일하게 정상적인 신초가 재분화 되었고, 이 때 신초는 캘루스로부터 형성된 마디구조에서 발달하였으며 신초의 재분화율은 24.4%였다. 재분화 신초를 식물호르몬 무첨가 배지로 이식하여 명배양 했을 때 지상부 녹화와 더불어 뿌리가 발생하여 유식물체로 발달하였다. 기내 유식물체를 기 보고된 마디배양 프로토콜(Bae et al. 2016)에 따라 배양했을 때 효과적으로 유식물체를 증식시킬 수 있었다. 기외 순화시킨 유식물체는 내륙 자연 환경하에서 성숙식물체로 생육하였으며 이듬 해 4월에 개화하였다. 본 연구의 결과는 Bae et al. (2016)이 제안한 마디배양 프로토콜과 통합하여 운용될 수 있고, 통합된 프로토콜은 갯봄맞이의 기내 번식 시스템으로서 갯봄맞이 자생지 복원에 기여할 수 있을 것이다. A callus-mediated regeneration protocol for sea-milkwort, an endangered coastal plant species in South Korea, is reported here. The explants of in vitro-plantlets generated from a node culture revealed distinguishable responses in callus induction depending on genotype, explant source, light condition, and 2,4-D concentration. Especially, continuous darkness exclusively facilitated callus induction from explants prior to other treatments. The calli initiated on the media with 2,4-D ranging from 0.1 mg/L to 3.0 mg/L in the dark vigorously proliferated when subcultured on the same media in continuous darkness. Given 1.0 mg/L zeatin in addition to darkness to the calli of the 'Pistachio' genotype, normal adventitious shoots were only regenerated from nodular structures that formed earlier from the calli at the frequency of 24.4 percent. Regenerated shoots easily grew into plantlets with roots and green color on a phytohormone-free MS medium under lighted condition, that were used for node culture as plant materials. Node culture effectively multiplied plantlets in accordance with protocol by Bae et al. (2016). Acclimatized plantlet clusters developed mature plant clusters under inland environment, followed by flowering the following April. Results were merged with node culture protocol suggested by Bae et al. (2016), which, as an in vitro propagation system for sea-milkwort, may contribute to natural habitat restoration.
한증술(Jeung-Sul Han),김진화(Jin-Hwa Kim) 한국원예학회 2009 원예과학기술지 Vol.27 No.3
A regeneration protocol via direct somatic embryogenesis from the plumules of in vitro-sown seedlings was developed in hot pepper (Capsicum annuum L.). To induce somatic embryogenesis of hot pepper, the effects of various culture conditions and genotypes were evaluated. After mature seeds were directly sown on a modified Murashige and Skoog’s medium for pepper (MMSP) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BA), and high sucrose, the germination rate, formation of callus, and somatic embryogenesis were checked. The germinated seedlings of selected MC-12 and CCA323 among tested genotypes were transferred to plant growth regulator (PGR)-free MMSP, and then the frequency of somatic embryogenesis was investigated. The somatic embryogenesis from plumules of hot pepper seedlings was affected by cytokinins in particular genotypes: the frequency of somatic embryogeneis was decreased on the sowing media with more than 1 ㎎ㆍL?¹ BA and the transplanting medium with zeatin. Meanwhile, the partial substitution with D-mannitol for high sucrose did not influence the somatic embyrogeneis. Collectively, the highest rate (38.3%) of somatic embryo induction from plumule was obtained when the seeds of ‘MC-12’ inbred line were sown on the MMSP with 2 ㎎ㆍL?¹ 2,4-D, 1 ㎎ㆍL?¹ BA, and 80 gㆍL?¹ sucrose at 25℃ with illumination for 16 hours each day, and then transferred into the MMSP with no PGR and 30 gㆍL?¹ sucrose. Isolated somatic embryos germinated into normal plantlets after sub-culturing to PGR-free MMSP. The regenerants were easily acclimatized in a greenhouse and grew into mature plants. Most of the somatic embryo-derived plants were undistinguishable from the wild-type ones by their phenotypes except for only one somaclonal variant. In addition, all of the somatic embryo-derived mature plants normally set the fruits and generated the next progenies. We believe that the present results might be helpful for incorporating into a genetic engineering system in hot pepper.