• 현장농수산연구지
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• 제25권4호
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• pp.90-102
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• 2024

Haploid/double haploid plants developed from isolated microspores can significantly accelerate plant breeding. Haploid plants can naturally double their chromosomes to create a pure homozygous line of diploid plants. We present a method for producing embryos from isolated microspores of hot peppers (Capsicum annuumL.). We analyzed the polyploidization levels of the regenerated plants. The donor plants produced the optimal stage of microspores following short-term growth under low-intensity light, which resulted in high rates of embryogenesis and cotyledonary embryogenesis. To find an efficient culture method, liquid, doubled-layer, and 2-step cultures were tested. Liquid culture yielded the highest number of embryos, whereas the highest efficiency for cotyledonary embryogenesis was afforded by the doubled-layer culture. When normal cotyledonary embryos were transplanted onto a regeneration medium, they developed into complete plants. From these, 208 plants were tested via flow cytometric analysis, and 35.6% and 72.7% of the chromosomes from the Milyang-jare and LV2319 genotypes, respectively, were found to be spontaneous double haploids. These results are the same as those obtained on analyzing horticultural characteristics, including the size of leaves and the size and shape of fruits. The present study provides information on the practical application of isolated microspore culture of hot peppers, factors that affect embryogenesis, and methods for polyploidy testing.

• 식물조직배양학회지
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• 제27권3호
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• pp.227-232
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• 2000

당근 유식물체의 자엽을 절편으로 유도된 배형성능 세포를 여러가지 농도의 카드뮴 (0, 50. 100, 200. 500, 1000 $\mu$M)이 첨가된 MS 액체배지에 배양한 다음 MS 기본배지에 옮겨서 체세포배 발생을 유도했고 한편 카드뮴 처리구와 카드뮴이 처리되지 않은 대조구에서 체세포배를 얻었다. 현탁배양에서 정상적인 2개의 자엽을 갖는 체세포배 발생은 카드뮴 첨가 배지에서 감소하였으나 비정상적인 3개의 자엽을 갖는 체세포배가 많이 관찰되었다. 또한 비정상적인 1개, 3개 및 4개의 자엽을 갖는 체세포배보다 정상적인 2개의 자엽을 갖는 체세포배의 식물체 재생률이 가장 높았다. 카드뮴 처리구에서 발생된 체세포배의 발아율은 자엽수에 관계없이 대조구에서 발생된 체세포배의 발아율에 비해 감소하였다.

• Journal of Plant Biology
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• 제39권2호
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• pp.127-135
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• 1996

An efficient system of somatic embryogenesis was established for the red pepper plant (Capsicum annuum L. cv. Nokkwang) usign immature zygotic embryos. The size of the immature zygotic embryos and the concentrations of 2, 4-D and sucrose were found to be critical. Somatic embryos were induced via callus or directly from explants and regenerated into plantlets successfully. When zygotic embryos 1~2 mm long were cultured on the modified Murashige-Skoong (MS) medium supplemented with 2 mg/L 2, 4-D for 3 weeks in the dark, somatic embryos were induced directly from the apical region of zygotic embryos with the highest frequency being approximately 90%. To mature the somatic embryos, ABA and an ethylene inhibitor AgNO3 were used. The highest frequency of shoot regeneration (25% in each) resulted at 2$\mu$M ABA or 20$\mu$M AgNO3 treatment at rates 3.7 and 1.6 times control, respectively. Shoots developed mainly from the cotyledonary node on CoCl2-containing medium, and from the upper side of cotyledon on medium containing AgNO3 while the embryos on the control medium produced shoots from both the cotyledonary node and the upper region of cotyledons both at frequencies of 50%. Indirect somatic embryogenesis via callus was induced at an efficiency of approximately 10% with zygotic embryos 3~4 mm long cultured on MS medium containing 5~10 mg/L, 2, 4-D for 5~7 weeks under a continuous light condition. The plants regenerated from the somatic embryos were morphologically normal. Using scanning electron microscopy, the direct and indirect somatic embryogeneses were observed to follow the globular, heart and torpedo stages, similar to zygotic embryogenesis. Also, suspensors appeared in the early globular and ovoid-shaped late globular embryos during indirect somatic embryogenesis.

• 식물조직배양학회지
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• 제27권2호
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• pp.149-154
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• 2000

땅두릅 미성숙 화기절편을 이용하여 1 mg/L 2,4-D의 고체배지에서 배발생 캘러스를 유도하였고 동일조건의 액체배지에서 증식시켰다. 배발생 세포를 270$\mu\textrm{m}$의 걸름체로 거른 다음 배발생 세포는 싸이토카이닌이 포함된 MS 액체배지에서 2주 동안 배양하였고 그후 생장조절물질이 없는 MS 액체배지에서 5주간 배양하여 1차 체세포배가 유도되었다. 이들중 어뢰형과 자엽형 시기의 배를 생장조절물질이 없는 MS고체 배지에 치상하였을 때 캘러스의 형성 없이 2차배가 직접 유도되었다. 2차배를 유도할 때 2mg/L kinetin에서 얻어진 1차 배에서 가장 높은 빈도의 2차배가 발생되었다. 식물체 재생은 1 mg/L kinetin 혹은 1 mg/L zeatin을 처리하여 얻어진 1차 자엽형배로부터 유도된 2차 자엽형배를 생장조절물질이 없는 MS 고체배지에 치상하였을 때 높게 나타났다 (각각 25.4%, 28.6%). 2차배로부터의 3차배 발생과 식물체 재생과의 상관 관계를 볼 때 식물체의 재생에 있어서 3차배의 배발생은 저해적인 영향을 주었다.

• Journal of Plant Biotechnology
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• 제6권2호
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• pp.113-118
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• 2004

Somatic embryos were formed from calli obtained from axillary shoots (raised from nodal segments of glasshouse-grown plants under aseptic conditions), internodal segments (from in vitro-raised plants), and root and coty-ledonary leaf segments (from in vitro-raised seedlings) after 8 weeks of initial culture. Embryo formation was the highest (97.33%) from cotyledonary leaf callus on Mura-shige and Skoog's (MS) medium containing kinetin (KN) (3 mg/L). Somatic embryo induction was lesser with different combinations of auxins while it increased to 100% in internodal segment and cotyledonary leaf calli with 6-benzyladenine (BA) (2mg/L) along with 2,3,5-triiodobenzoic acid (TIBA) (2mg/L). The shoots were induced from somatic embryos raised from root, coty-ledonary leaf and internodal segment calli grown on MS medium containing BA in combination with indole-3-acetic acid (IAA). Maximum of 66.67% cultures formed shoots on MS medium containing BA (1mg/L) in combination with IAA (2mg/L). The shoots raised from somatic embryos were rooted on MS medium supplemented with indole-3-butyric acid (IBA) (2mg/L). The plantlets transferred to the field showed 70% survival rate after one year.

• Journal of Ginseng Research
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• 제23권3호
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• pp.148-163
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• 1999

The efficacy of three auxins, viz. 2,4-0, NAA and dicamba, were compared for the induction of somatic embryogenesis in American ginseng (Panax quinquefolium L.). Somatic embryos (SEs) formed on ginseng cotyledonary, zygotic embryo and shoot explants after 8 weeks of induction by the auxin stimuli. Significantly more somatic embryos were induced by culture of any of the ginseng explants on media supplemented with $5{\mu}M$ 2,4-0 than any other auxin treatment. Shoots derived from somatic embryos had the greatest regenerative potential and zygotic embryos the least. Explants generated from green (unstratified) seeds gave similar or higher frequency of embryogenesis as the explants derived from stratified seeds. Histological and SEM studies confirmed that the regenerimts were somatic embryos. Somatic embryos germinated and developed into normal plants in $3\~6$ months. About $10\%$ of plantlets from second generation SEs formed flowers within 10 weeks, particularly on media supplemented with $GA_3$ The development of a regeneration system for ginseng through somatic embryogenesis is a necessary first step for mass propagation and genetic improvement of American ginseng.

• Journal of Plant Biotechnology
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• 제7권4호
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• pp.259-265
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• 2005

Indian citrus ringspot virus (ICRSV)-free plants of Kinnow mandarin (Citrus nobilis Lour x C. deliciosa Tenora) were raised from virus-infected plants using unfertilised ovules as explants. Plants were tested by indirect ELISA and RT-PCR before using their explant. An amplified product of 539 bp was obtained by RT- PCR in ICRSV infected plants. Unfertilized ovules were excised from unopened flower buds of plants tested postive for virus and were cultured on Murashige and Skoog's (MS) basal medium supplemented with various concentrations of kinetin (KN) or malt extract (ME). Maximum induction (31.94%) of embryogenic callus was observed on MS medium supplemented with KN ($9.29\;{\mu}M$). Transfer of embryogenic calli to similar media composition resulted in somatic embryogenesis in all cultures, with an average number of 60.36 globular, 17.39 heart and 7.71 cotyledonary-shaped somatic embryos per culture. All cotyledonary shaped embryos developed into complete plantlets within 60 days on transfer to similar medium. Embryogenic callus induction, somatic embryo formation, maturation, germination and plantlet formation were achieved on MS medium supplemented with KN ($9.29\;{\mu}M$) alone. The plantlets derived from somatic embryos were transferred to sterilized soil, sand and vermiculite (3:1:1) mixture. After acclimatization, the plantlets were transferred to screen house and were indexed for ICRSV employing indirect ELISA and RT-PCR and found free of virus. A distinct feature of this study is the induction of somatic embryogenesis from unfertilised ovules to produce virus-free plants.

• Plant Biotechnology Reports
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• 제4권4호
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• pp.261-267
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• 2010

We describe culture conditions for a high-efficiency in vitro regeneration system of Papaver nudicaule through somatic embryogenesis and secondary somatic embryogenesis. The embryogenic callus induction rate was highest when petiole explants were cultured on Murashige and Skoog (MS) medium containing 1.0 mg $1^{-1}$ ${\alpha}$-naphthaleneacetic acid (NAA) and 0.1 mg $1^{-1}$ 6-benzyladenine (BA) (36.7%). When transferred to plant growth regulator (PGR)-free medium, 430 somatic embryos formed asynchronously from 90 mg of embryogenic callus in each 100-ml flask. Early-stage somatic embryos were transferred to MS medium containing 1.0 mg $1^{-1}$ BA and 1.0 mg $1^{-1}$ NAA to germinate at high frequency (97.6%). One-third-strength MS medium with 1.0% sucrose and 1.0 mg $1^{-1}$ $GA_3$ had the highest frequency of plantlet conversion from somatic embryos (91.2%). Over 90% of regenerated plantlets were successfully acclimated in the greenhouse. Secondary somatic embryos were frequently induced directly when the excised hypocotyls of the primary somatic embryos were cultured on MS medium without PGRs. Sucrose concentration significantly affected the induction of secondary embryos. The highest induction rate (89.5) and number of secondary somatic embryos per explant (9.3) were obtained by 1% sucrose. Most secondary embryos (87.2-94.3%) developed into the cotyledonary stage on induction medium. All cotyledonary secondary embryos were converted into plantlets both in liquid and on semisolid 1/3-strength MS medium with 1.0% sucrose.

• 식물조직배양학회지
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• 제26권3호
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• pp.143-148
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• 1999

당근의 배양세포로부터 체세포배의 발생과정에 미치는 아스콜빈산 및 dehydroascorbic acid의 영향을 밝히기 위하여 본 실험을 시도하였다. 비배발생세포의 배양에 처리된 아스콜빈산은 세포증식을 촉진시켰을 뿐인데 dehydroascorbic acid는 세포증식을 억제시키면서 배발생세포로 전환시킨 효과가 있었다. 배발생세포의 배양에 처리된 아스콜빈산은 체세포배 발생을 억제시켰지만 dehydroascorbic acid는 체세포배발생을 촉진시켰다. 그러나 이와 같은 발생촉진은 구상배에서 중단되므로 성숙에는 오히려 저해적이었다. 이상의 결과로부터 당근의 캘러스배양에 dehydroascorbic acid를 처리하여 빠른 시일내에 배발생캘러스를 확보한 다음 dehydroascorbic acid 첨가 배발생 배지에서 초기배발생기간 배양 후 MS 기본배지로 옮겨 배양하면 고빈도의 체세포배생산 실험계가 확립될 것으로 판단된다.

• 식물조직배양학회지
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• 제27권2호
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• pp.137-142
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• 2000

천궁 (Cnidium officinale)의 잎, 엽병 및 화기 외식편을 재료로 하여 2,4-D가 첨가된 배지에 배양하였을때 캘러스 형성은 어느 외식편에서나 비슷한 수준으로 유기되었지만 배형성능 캘러스는 화기유래 캘러스에서만 형성되었다. 체세포배발생의 유도는 2mg/L 2,4-D를 첨가한 MS배지에서 잘 일어났으며 2,4-D제거배지에서 계대배양하면 외식편당 78개의 체세포배가 형성되었다. 체세포배의 자엽수에 있어서 변이가 빈번하게 관찰되었으나 배지에 1% 활성탄을 첨가하면 비정상적인 자엽수를 갖는 체세포배의 발생률이 감소되는 효과를 얻을 수 있었다. 자엽기 체세포배는 MS 기본배지에서 저조한 발아율을 보이는 반면에 활성탄 첨가배지에서는 현저한 발아 향상을 보였다.