• 한국자원식물학회지
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• 제32권1호
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• pp.19-28
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• 2019

An efficient shoot regeneration condition for pea cv. 'Sparkle' was developed by using optimum explant, plant growth regulator concentrations, and pretreatment of BA onto explant. The average shoot number per explant showed the highest on two kinds of shoot induction media (MSB5 media containing 2 mg/L BA and a combination of 2 mg/L BA and 1 mg/L TDZ) when cotyledonary node explants were cultured. Moreover, the pretreatment of explant in 200 mg/L BA solution was found to be more effective in shoot induction than that of non-pretreatment. By histological study, cell division and proto-meristem were formed near the surface of the sub-epidermal and epidermal cell layers of cotyledonary node in earlier than 3 days after culture. The analysis of genetic stability of regenerants by using thirteen ISSR markers showed that in vitro regenerated plants showed polymorphism with 8.3% compared with their mother plants.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2019년도 추계학술대회
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• pp.30-30
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• 2019

Pea (Pisum sativum) is one of important legume crops in the world. It is commonly used as a protein source for animal and human diet, and also used as a natural nitrogen source which is produced by a symbiotic bacterium in their root nodule and helpful for terrestrial ecosystem. The successful in vitro manipulation is depended on three main factors including physiology of plant donor, in vitro manipulation approach, and stress physiology during plant cultivation. Moreover, genotype is an important for plant manipulation; different genotype gives the different response to regeneration efficiency. An efficient condition of shoot induction for pea (Pisum sativum cv. 'Sparkle') was developed by using optimum explant, plant growth regulator concentrations, and pretreatment of BA onto explant. The average shoot number per explant showed the highest on two kinds of shoot induction media (MSB5 media containing 2 mg/L BA and a combination of 2 mg/L BA and 1 mg/L TDZ) with cotyledonary node explants culture. Moreover, the pretreatment of explant in 200 mg/L BA solution was found to be more effective in shoot induction than that of non-pretreatment. The analysis of genetic stability of regenerants by using 13 ISSR markers presented that in vitro regenerated plants showed polymorphism with 8.3% compared with their mother plants.

• Journal of Plant Biotechnology
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• 제32권4호
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• pp.257-262
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• 2005

Agrobacterium과 자엽절 공동배양으로 대두 형질전환체를 생산하였다. 멜론 (슈피VIP품종)의 자엽절 절편은 선발 마커로서 bar와 reporter로서 gus유전자가 포함된 pPTN289 또는 선발마커로서 nptII유전자와 reporter로서 gus유전자로 제작된 pPTN290벡터를 LBA4401, GV3101, EHA101에 각각 형질전환하여 공동 배양하였다. 최대 형질전환빈도(0.16%)는 EHA101 (pPTN289)균주로 공동배양한 자엽절 절편을 glufosinate가 첨가된 선발배지에서 얻을 수 있었으며, 최종적으로 glufosinate저항성과 잎 ($T_0$), 화기 ($T_0$), 종자 ($T_1$) 및 유식물체 ($T_1$)에서 GUS양성반응을 나타내는 5개체를 얻었다. Southern분석에 의하여 GUS유전자가 멜론 genomic DNA에 도입되어 있음을 확인하였다.

• Journal of Plant Biotechnology
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• 제3권2호
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• pp.95-100
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• 2001

To select the section with shoot formation ability, the calli and shoot formation from three sections (first leaf including cotyledonary node, hypocotyl and cotyledon explants) of 5-days-seedlings of soybean were induced on MS medium supplemented with 1.0 mg/L BAP, 3% sucrose, and 0.3% gelrite for one month. The first leaf section exhibited the highest shoot formation rate (51%), followed the hypocotyl section (10%) and the cotyledon section (0%). The shoot formation rates and shoot number of the four excised sections (whole first leaf, a half of the first leaf, a third of the first leaf and only node) of the first leaf were also investigated on the same medium. A half of the first leaf explant and the third of the first leaf explant had higher shoot formation rates (76-80%) and numbers (3-4 / explants) than those in other two explants. Effects of six cytokinins (kinetin, zeatin, BAP, 2iP, PBA, and TDZ) on shoot formation were determined, using the half of the first leaf explants. Zeatin (1.0 mg/L) exhibited the highest in shoot formation rate (94%) and numbers (8 / explant). In addition, the combined effects of three cytokinins (zeatin, BAP, and TDZ; 0.5, 1.0, 2.0 mg/L, respectively) and an auxin (IAA; 0.0, 0.5, 1.0, 2.0 mg/L) were determined. The combination (1:1, v/v) of zeatin (1.0 mg/L) and IAA (1.0 mg/L) exhibited the highest in shoot formation rate (96%) and numbers (16 / explant), twice more than zeatin (1.0 mg/L) alone. The shoot cuttings were transferred and cultivated on the rooting media supplemented with only auxin, IBA at various concentrations. The highest root formation (8 / shoot) was achieved on the medium supplemented with 1.5 mg/L. After 4 weeks of cultivation, the plantlets with an extensive root system were transplanted in pots with a soil mixture of vermiculite and fine sand. Transferred to field, about 75% of the plantlets survived.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2017년도 9th Asian Crop Science Association conference
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• pp.176-176
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• 2017

Hydropriming had positive effects on the time for germination to reach 50%, the germination index, the time to final germination percentage, and the number of uniform seedlings with enlarged cotyledons in in vitro germination of small watermelon. In addition, the highest shoot initiation was obtained from hydroprimed cotyledonary nodes ($95{\pm}6%$), followed by non-primed cotyledonary nodes ($78{\pm}6%$), hydroprimed cotyledons ($72{\pm}4%$), and non-primed cotyledons ($48{\pm}4%$). Meanwhile, no shoots were initiated from hypocotyls. The total number of shoots that initiated from cotyledonary nodes and cotyledon explants was insignificant, indicating that both cotyledons and cotyledonary node were good sources for the in vitro culture. Choosing explant sources that favor tetraploidy should be the key for producing higher polyploidy plants; a total of 10.5% of tetraploid regenerants were entirely identified from cotyledon explants. Cotyledons with highly differentiated cells might show higher variations than cotyledonary nodes with more preexisting meristematic cells. Cells of cotyledon tissue might undergo changes in ploidy level during differentiation of the culture, or it might be that some of the variations were already present in the tissues of the donor plant. Morphological changes in fruit length of tetraploid regenerants are genotype-dependent.

• Journal of Plant Biotechnology
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• 제43권3호
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• pp.341-346
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• 2016

The objectives of this study were to 1) evaluate the efficiency of the protocol of Agrobacterium-mediated transformation of cucumber to introduce phytoene synthase-2a carotene desaturase (PAC genes); 2) demonstrate the integration of PAC genes into the genome of putative transgenic cucumber based on growth on selection medium, PCR and Southern analysis; 3) evaluate the expression of PAC genes in transgenic cucumber based on the analysis of RT-PCR and Northern blot hybridization. Out of 5,945 cotyledonary-node explants inoculated with Agrobacterium, 65 (1.1%) explants produced 238 shoots. Integration of PAC genes into the genome of the cucumber was demonstrated based on the analysis of gDNA-PCR, 21 out of the 238 plants regenerated; while 6 plants proved positive for Southern blot hybridization. Transgene expression was demonstrated based on analysis of RT-PCR, 6 plants proved positive out of the 6 plants analyzed; while 4 plants out of 6 proved positive during Northern blot hybridization. This study successfully demonstrated the production of transgenic cucumber, integration, and expression of the PAC gene in cucumber.

• 식물조직배양학회지
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• 제22권5호
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• pp.291-298
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• 1995

시호(Bupleurum falcatum L.)의 잎절편으로부터 체세포배형성을 통한 식물체 재생과 체세포배 발생의 이상을 일으키는 2,4-D의 영향에 대하여 또한 체세포배의 자엽수와 발아와의 관계에 대하여 기술하였다. 배발생능 캘러스는 1mg/L 2,4-D가 첨가된 MS 고형기본배지에서 배양된 캘러스로부터 선발하였다. 2,4-D 첨가 MS 기본배지에서 6주간 배양후 3주간 2,4-D가 제거된 배지에서 배양하여 체세포배 발생과 비정상적인 체세포배의 자엽발생을 관찰하였다. 이상자엽의 빈도는 정상인 2개 자엽에서 65% 인데 비하여 1개의 자엽에서 7%, 3개의 자엽을 갖는 체세포배에서는 25%, 4개 자엽에서는 5%, 5개 자엽에서 2% 및 나팔형 자엽배는 3% 이었다. 2개의 정상적인 자엽을 갖는 체세포배의 발아율이 80%인 것에 비하여 1, 3 4, 5개의 자엽을 갖는 체세포배에서는 25%, 58%, 38% 및 20%로 발아율이 낮았다. 그러나 나팔형의 비정상 체세포배는 전혀 발아되지 않았다. 다자엽배의 구조는 체세포배의 뿌리 또는 배축에서 원통상전형성층을 나타내었지만 자엽절에서는 자엽수와 동일한수의 전형성층속으로 분포되었다. 체세포배의 자엽수 변이는 전형성층의 수와 밀접한 관계가 있다.

• Plant Biotechnology Reports
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• 제3권3호
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• pp.205-211
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• 2009

An efficient in vitro regeneration protocol for moth bean [Vigna aconitifolia (Jacq.) Marechal] via somatic embryogenesis has been developed. Embryogenic callus cultures were established from the cotyledonary node as explant on semi-solid Murashige and Skoog (MS) medium supplemented with $0.75mg\;1^{-1}$ 2,4-dichlorophenoxyacetic acid (2,4-D) and $1.5mg\;1^{-1}$ 6-benzylaminopurine (BA) and with various additives ($50mg\;1^{-1}$ ascorbic acid and $25mg\;1^{-1}$ each of adenine sulphate, citric acid and $_L-arginine$). Numerous somatic embryos differentiated on MS basal nutrient medium supplemented with $0.25mg\;1^{-1}$ 2,4-D and $0.5mg\;1^{-1}$ of kinetin (Kin). Sustained cell division resulted in the formation of cell aggregates, which progressed to the globular- and heart-shaped somatic embryos and then, if they differentiated properly, to the torpedo shape and cotyledonary stages. The transfer of embryos onto fresh MS basal medium containing $0.2mg\;1^{-1}$ BA and $2.0mg\;1^{-1}$ gibberellic acid enabled the embryos to achieve complete maturation and germination. More than 80% of somatic embryos were converted into true-to-type fertile plants. In vitro-regenerated plantlets with well-developed roots were successfully hardened in a greenhouse and established in soil.