• Plant Biotechnology Reports
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• v.2 no.3
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• pp.213-218
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• 2008

Swertia chirata is an endangered gentian species that prefers to grow at higher altitudes. This ethnomedicinal herb is known primarily for its bitter taste caused by the presence of important phytochemicals that are directly associated with human health benefits. Due to a continuous loss of habitat and inherent problems of seed viability and seed germination, alternative strategies for propagation and conservation are urgently required to prevent the possible extinction of this species. We have formulated a reproducible protocol for the rapid propagation and conservation of this plant using leaves taken from in vitro shoot cultures. Direct induction of more than seven shoot buds per explant was achieved for the first time when the explants were placed on MS medium supplemented with $2.22{\mu}M$ N-6-benzyladenine, $11.6{\mu}M$ kinetin, and $0.5{\mu}M$ ${\alpha}-naphthalene$ acetic acid. Direct organogenesis was noted exclusively from the adaxial surface of the basal segments of leaves. Leaves closer to the apical meristem were more responsive than those farther away from the meristem. Plants raised through direct organogenesis were evaluated for their clonal fidelity by chromosomal analysis and DNA fingerprinting. Complete plants were successfully transferred to the field condition and produced viable seeds. Given the enormous potential of this age-old medicinal plant in terms of potential health-benefitting drugs, this protocol can be used for commercial propagation purposes and to initiate future genetic improvement studies.

• Korean Journal of Plant Resources
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• v.32 no.3
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• pp.237-243
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• 2019

Soybean is the one of recalcitrant legume species for shoot induction. Shoot regeneration via direct organogenesis was investigated in five soybean cultivars, 'Dawon', 'Pungsan', 'Daewon', 'Taekwang' and 'Chongdoo 1' by using cotyledonary node explants. Out of 5 soybean cultivars, an efficient shoot regeneration condition was developed in the two soybean cultivars, 'Dawon' and 'Pungsan'. When various kinds of plant growth regulators with different concentration were estimated, the optimum medium condition for shoot induction in both soybean cultivars was MS + B5 vitamin supplemented with BA at concentration 2 mg/L. In addition, shoot formation efficiency was increased with 97.09% and 93.88% by the pretreatment of BA onto the explants before in vitro culture in both cultivars. Shoot induction in 'Dawon' cultivar was originated from epidermal tissue and sub-epidermal layers when histological changes were investigated under shoot regeneration after culturing cotyledonary node segments on shoot induction medium for 0 to 21 days. Especially, cell dedifferentiation was observed from parenchyma cells to meristematic cell in 3-day cultured segments.

• Journal of Plant Biotechnology
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• v.8 no.1
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• pp.43-50
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• 2006

The goal of this research was to develop an efficient cryopreservation protocol for germplasms of yam (Diosorea batatas), that were cultivated in Korea. Comparative studies with four other cryogenic techniques and subsequent experiments for shoot regrowth were conducted. in vitro-grown shoot-apices of the D. batatas were successfully cryopreserved by encapsulation-dehydration. The maximum survival of shoot-apices could be achieved when the precultured (with 0.3 M of sucrose for one day) and encapsulated (with a 3%(w/v) Na-alginate solution) apices were dehydrated for $3.5{\sim}4\;h$ prior to direct immersion in LN (liquid nitrogen). The frequency of regrowth rate of cryopreserved apices was not decreased during 3-month storage period. The thawing method markedly affected survival of the cryopreserved apices, and thawing at $40^{\circ}C$ for 3 min produced the best results. When cryopreserved apices were post-cultured on the post-culture medium (MS), supplemented with $0.2mgl^{-1}$ of BA ($N_6$-benzyladenine) and $0.2mgl^{-1}$ of kinetin, they showed direct shooting without callusing.

• Korean Journal of Agricultural Science
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• v.46 no.1
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• pp.151-161
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• 2019

Ashwagandha (Withania sominifera Duanal) is an important medicinal herb with increased demand after discovering its anti-stress and sex stimulating properties that are attributed to the presence of biologically active alkaloid compounds. The aim of this study was to elucidate a proper agro technological package that ensures the optimum growth of Ashwagandha to obtain the finest quality without degrading the pharmacologically active constituents. Mixtures of organic and inorganic fertilizers were combined with direct seeding and transplanted as four different treatments in this study. The fresh and dry weights of the tubers were recorded up to 12 months starting from two months after sowing (MAS) while the shoot height, root length, number of leaves, fresh and dry weights of the shoot and the root with a shoot ratio of up to 6 MAS were determined. The results revealed that the growth of Ashwagandha was not affected significantly by the method of planting, type of fertilizer or their combinations during most of the harvests. However, tubers harvested at 6 MAS had the highest recorded dry tuber weight per plant in all four treatments compared to the early harvests where two direct seeded treatments had the best results. Comparison of the phytochemical compounds showed that direct seeding with organic fertilizer had the highest recorded values for alkaloid and withaferine A contents with a lower percentage of fiber compared to the treatments with inorganic fertilizer. In conclusion, direct seeding with organic fertilizer and tubers harvested at 6 MAS are recommended as the best cultivation conditions and harvesting stage to obtain high quality tubers of Ashwagandha, respectively.

• Plant Biotechnology Reports
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• v.5 no.2
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• pp.187-195
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• 2011

The gesneriaceous perennial plant Titanotrichum oldhamii has beautiful foliage and attractive bright yellow flowers. However, breeding of T. oldhamii by conventional sexual hybridization may be difficult because sexual reproduction of this species is very rare. In the present study, plant regeneration systems via both direct and indirect formation of adventitious shoots from leaf explants were established as the first step toward breeding T. oldhamii by using biotechnological techniques. Adventitious shoots were formed efficiently on medium containing $0.1mg\;l^{-1}$ benzyladenine. Histological observation showed that shoot formation on this medium occurred directly from leaf epidermal cells without callus formation. On the other hand, leaf explants formed calluses on medium containing $0.1mg\;l^{-1}$ 2,4-dichlorophenoxyacetic acid. The calluses could be maintained by monthly subculturing to fresh medium of the same composition. When the calluses were transferred to plant growth regulator-free medium, they formed adventitious shoots. Directly and indirectly formed shoots rooted well on medium containing $0.1mg\;l^{-1}$ indole-3-butyric acid. Plantlets thus obtained were successfully acclimatized and grew vigorously in the greenhouse. Flow cytometry analysis indicated that no variation in the ploidy level was observed in plants regenerated via direct shoot formation, whereas chromosome doubling occurred in several plants regenerated via indirect shoot formation. Regenerated plants with the same ploidy level as the mother plants showed almost the same phenotype as the mother plants, whereas chromosome-doubled plants showed apparent morphological alterations: they had small and crispate flowers, and round and deep green leaves.

• Journal of Plant Biotechnology
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• v.2 no.1
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• pp.51-54
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• 2000

Leaf explants of the cucumber (Cucumis sativus L.) were cultured on Murashige and Skoog's (MS) medium supplemented with various concentrations of $\alpha$-naphthalene acetic acid (NAA) and 6-benzylaminopurine (BAP). Direct shoot orgnogenesis as well as callus formation with somatic embryos and multiple shoots was observed from leaf explants of cvs. Shinhukjinju and Chungjang. The highest frequency of shoot formation 80% was observed on MS medium supplemented with NAA/BAP (5.0 ${\mu}{\textrm}{m}$/2.5 ${\mu}{\textrm}{m}$), with explants forming 3-7 shoots. Shoots formation occured within 3 to 4 weeks. Only one subculture of calli was required for plant regeneration on normal growth regulator-free medium. Plantlets transferred to soil developed into plants of normal appearance, which flowered and set fruits.

• Korean Journal of Weed Science
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• v.17 no.1
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• pp.24-30
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• 1997

The influence of nutrients on the phytotoxicity of herbicides (bensulfuron-methyl, pyrazosulfuron-ethyl, imazosulfuron, dimepiperate, and molinate) was investigated in controlled-environment growth chamber with direct-seeded rice (Oryza sativa L. cv. Dongjin). The phytotoxicity of bensulfuron-methyl, pyrazosulfuron-ethyl, and imazosulfuron for rice was greater in nutrient culture than in no-nutrient condition. The root growth of rice applied with these herbicides was more inhibited than the shoot growth. The most severe inhibition was obtained with pyrazosulfuron-ethyl application. The growth inhibition of rice applied by dimepiperate was increased under no-nutrient culture condition. Dimepiperate suppressed more remarkably shoot growth than root. Especially the shoot elongation was much more inhibited than the others. The shoot growth inhibition in rice applied by molinate was severer than the root. The shoot growth was reduced under nutrient culture condition, while the root growth was reduced under no-nutrient culture.

• Journal of Plant Biotechnology
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• v.36 no.3
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• pp.275-280
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• 2009

Direct shoot regeneration from leaf or internode or petiole segments was conducted in 33 cultivars of chrysanthemum. Shoot regeneration rates varied according to cultivars, culture media, and explant types. The high shoot regeneration rate of more than 70% in 15 cultivars (‘Pink Pangpang’, ‘Orange Memory’, ‘Relance’, ‘Zinba’, ‘Beakma’, ‘Innocence’, ‘Sunny Pangpang’, ‘Euro Yellow’, ‘Dublin’, ‘Boramae’, ‘Peak’, ‘Euro White’, ‘Vesuvio White’, ‘Linneker Salmon’ and ‘Pink Pride’) and 2 ones (‘Forward’ and ‘Agason’) was obtained from the segments of leaves and internodes, respectively, cultured on MS medium containing 1.0 mg-$L^{-1}$ BAP, 0.5 mg-$L^{-1}$ IAA and 30 g-$L^{-1}$ sucrose. That in 6 cultivars (‘Shuhonochikara’, ‘Hakunosen’, ‘Whitney Pangpang’, ‘Plaisir D’Amour’, ‘Grace’ and ‘Kumsu’) was observed from the segments of leaves or internodes cultured on 1/2 MS medium 1.0 mg-$L^{-1}$ BAP, 0.5 mg-$L^{-1}$ IAA and 15 g-$L^{-1}$ sucrose In case of 3 cultivars (‘Ilweol’, ‘Puma White’ and ‘Sharon’), when internode explants excised from mother plants, which were pre-cultured on MS medium containing 2 g-$L^{-1}$ activated charcoal and 30 g-$L^{-1}$ sucrose for two months in the dark, and cultured on MS medium containing 1.0 mg-$L^{-1}$ BAP, 0.5 mg-$L^{-1}$ IAA and 30 g-$L^{-1}$ sucrose, that was shown. Seven cultivars including ‘Puma Yellow’, ‘Argus’, ‘Yes Morning’, ‘Whiparam’, ‘Hakunohikari’, ‘Charming Eye’ and ‘Moon light’ requires more improved culture conditions. Tissues with the highest shoot regeneration rate were in descending order, leaf, petiole, and internode segments.

• Korean Journal of Plant Resources
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• v.31 no.6
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• pp.684-694
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• 2018

This study describes the successful establishment of a cryopreservation protocol for Citrus limon cultivars: 'Frost Eureka limon' and 'Cook Eureka limon', using a droplet-vitrification method. The shoot tips that were excised from in vitro grown seedlings of the two cultivars were preserved in liquid nitrogen (LN) and successfully regenerated into whole plants. Excised shoot tips were pre-cultured for 1 or 2 days in 0.3 M and 0.5 M sucrose solutions at $25^{\circ}C$ and incubated in a loading solution (LS) composed of 17.5% glycerol + 17.5% sucrose in Murashige and Skoog (MS) medium for 40 min at $25^{\circ}C$. Prior to direct immersion in LN for 1 h, the shoot tips were dehydrated with plant vitrification solution 2 (PVS2) at $0^{\circ}C$ or PVS3 at $25^{\circ}C$. The frozen shoot tips were re-warmed and unloaded with 1.2 M sucrose in $\text\tiny{^1/_2}$ MS for 30 min at $25^{\circ}C$. Shoot tips were post-cultured overnight on survival medium and then micrografted onto 'trifoliate orange' (Poncirus trifoliate (L.) Raf. seedling rootstocks for recovery and to produce whole plants. The highest regrowth rates were 53.5% and 50.3% for cryopreserved shoot tips of 'Frost Eureka limon' and 'Cook Eureka limon', respectively, when pre-cultured in 0.3 M and 0.5 M sucrose concentrations in a sequencing manner, with LS and treated with PVS2 for 60 min at $0^{\circ}C$. We also investigated whether the ammonium ion concentration on post-culture medium affected the viability of the cryopreserved Citrus shoot tips. The viability of cooled samples, following culturing on woody plant media (WPM) containing $\text\tiny{^1/_4}$ ammonium nitrate overnight before micrografting, was the highest (70.3%) in 'Frost Eureka limon'. The study described here is a cost-effective and safe method to conserve Citrus fruit cultivars, for the improvement and large-scale multiplication of fruit plants and for breeding disease resistance.

• Korean Journal of Plant Resources
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• v.36 no.6
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• pp.562-570
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• 2023

The droplet-vitrification technique for cryopreservation has proven successful across a diverse range of germplasm, ensuring safe and effective long term preservation. In this study, we investigate an effective cryopreservation protocol using the droplet-vitrification technique for shoot tips of Freesia hybrida cultivars 'Sunny Gold' and 'Sweet Lemon'. To determine optimal conditions for Freesia cryopreservation, we employed a carefully selected standard procedure along with additional treatments and alternative solutions. For 'Sunny Gold', the highest regrowth rate of 24% was achieved when shoot tips underwent dehydration with PVS3 solution for 120 minutes before direct immersion in liquid nitrogen (LN) for 1 hour, coupled with a standard protocol involving a two-step preculture with 0.3 M - 0.5 M sucrose, loading with C4 for 40 minutes, and unloading with 0.8 M sucrose for 40 minutes. In the case of 'Sweet Lemon,' regrowth of cryopreserved shoot tips was observed with dehydration treatments, including PVS2 (A3) for 60 minutes and PVS3 (B1) for 60 minutes, as well as longer exposure. The results reflect the distinct sensitivity of shoot tips to chemical toxicity and osmotic stress in these two genotypes. This study provides valuable evidence to consistently enhance the effectiveness of cryopreservation methods for the long-term conservation of Freesia germplasm.