• Korean Journal of Medicinal Crop Science
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• v.15 no.2
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• pp.73-81
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• 2007

An efficient regeneration system was developed using leaf, petiole, and internode explants. Highly embryogenic callus was obtained following cultivation on MS basal nutrient supplemented with 2 $mg/{\ell}$ 2,4-D. Globular, heart, torpedo and cotyledon shaped somatic embryo were produced from the surface of embryogenic callus. Direct shoot regeneration without intermediate callus formation has been achieved on MS medium supplemented NAA and BAP. The percentage of response varies with different concentration of auxin and cytokinin treated individually or in combination. The best shoot regeneration response (54.28%) and number of shoot per explant (12.67) were achieved on the medium supplemented with 0.1 $mg/{\ell}$ NAA and 1 $mg/{\ell}$ BAP. The regenerated shoot transformed into young plant when cultured into elongation and root induction medium. More than 90% of in vitro propagated plants could survive when transferred to the greenhouse for acclimation. This optimized regeneration system can be used for rapid shoot proliferation and genetic transformation.

• Plant Biotechnology Reports
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• v.3 no.4
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• pp.333-340
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• 2009

Developmental anomalies in the plumule meristem of peanut (Arachis hypogaea L.) somatic embryos resulted in poor shoot differentiation and reduced plant recovery. Existing meristems with caulogenic potential have never been tested for embryogenesis in peanut. The present experiment was designed to test the mature zygotic embryo axis derived plumule with three meristems for somatic embryogenesis. Embryogenic masses and embryos developed from the caulogenic meristems in the axils. Exposure of 2 weeks in primary medium with $90.5{\mu}M$ 2,4-D suppressed the shoot tip differentiation temporarily which then regained the ability to form the shoot on withdrawal of 2,4-D. Exposure of 4 weeks in primary medium with $90.5{\mu}M$ 2,4-D suppressed the shoot tip differentiation irreversibly. No shoot formation was noted from the tips in any of the cultures which were in secondary medium with $13.6{\mu}M$ 2,4-D. Development of somatic embryos directly from axillary meristems was confirmed histologically. Conversion frequency of these embryos was 11%. Thus, in this report, we describe a method to obtain somatic embryos from the determined organogenic buds of the axillary meristem, by culturing the nodal explant vertically on embryo induction medium. It also displays the possibility of obtaining both embryogenic and organogenic potential in two parts of the same explant simultaneously. The possibility of extending this approach for genetic transformation in in vivo system through direct DNA delivery or Agrobacterium injection in meristems can also be explored. Using Agrobacterium rhizogenes, we have demonstrated the possibility of gene transfer in the axillary meristems of seed-derived plumule explant.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2003.04a
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• pp.131-132
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• 2003

Leaf discs and apical meristems were cultured in Murashige and Skoog (MS) medium supplemented with cytokinin and auxin at different concentrations. Callus production was observed in all tested media after six days of incubation. Callus produced in the presence of high concentration of NAA (2.0mg/1) was fragile in texture and yellow in colour. Highest callus formation was observed from leaf discs in the medium supplemented with 1.0mg/1 NAA and 0.5 mg/l BAP in dark at $25{\pm}1{\circ}C$. Percentage of callus formation was 95% and mean callus fresh weight was 654.88 43.53 mg. Shoots were induced from the callus after 4 weeks in 1/2MS medium supplemented with BAP and kinetin both at 0.5mg/1. When elongated shoots were separated and transferred into multiplication medium (MS+0.5mg/1 BAP+0.5mg/1 kinetin) multiplication rate was 6.4 after 6 weeks. Higher concentrations of BAP caused callus production at the base. Direct shoot induction was observed from apical meristems in MS medium in the presence of 0.175 mg/1 IAA + 2.25mg/1 BAP and 0.175 mg/1 IAA + 3.0 mg/1 BAP in 16 hour day at $25{\pm}1{\circ}C$. Explants (apical meristems) elongated to form a single shoot forming a callus at the base. Adventitious buds were sprouted out from the base. Percentage explants which producing shoots was 28.57 and 65.5 respectively. Multiple shoot induction was also observed in the same media. Highest multiple shoot production was observed in the presence of 0.175 mg/l IAA and 3.0mg/l BAP, Mean number of shoots per explant was 5.36 and the mean shoot length was $16.66{\pm}4.15$mm. Shoots (20 30m length) were tested for root induction. Excised shoots were transferred into rooting media, which contains different concentrations of NAA and IAA. Best rooting performance was observed in 1/2MS medium supplemented with 0.1mg/1 NAA after 10 days of incubation in 16 hr photoperiod at $25{\pm}1{\circ}C$. Mean number of roots per shoot was 6 and the mean root length was 252mm. Rooted plantlets were transferred into sterile coir dust:sand (1:4) mixture and maintained in a humid chamber for two weeks, They were gradually exposed to the natural environment. After three weeks they were transferred to pots containing coir dust:sand (1:2) mixture for further development where the 90% survival was observed.

• Korean Journal of Plant Resources
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• v.17 no.1
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• pp.1-6
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• 2004

Apical meristems tissue were cultured on LS medium with different zeatin and NAA concentrations to compare their potential to regenerate shoots, roots and bulblet formation. Shoot regeneration from apical meristem was effective on LS medium added with zeatin 0.1, 0.5, 1.0 mg/L alone treatment or zeatin 0.5 mg/L and NAA 1.0 mg/L combination treatment. A high frequency of root regeneration was obtained on LS medium supplemented with zeatin 0.5 mg/ and NAA 1.0 combination treatment. Linsmaier and Skoog(LS) medium with NAA 3.0 mg/L and zeatin 1.0 mg/L combination treatment gave the best results for normal bulblet formation#KW=Allium wakegi ; plant regeneration ; bulblet formation

• Plant Biotechnology Reports
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• v.3 no.3
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• pp.175-182
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• 2009

Simple, reproducible, high frequency, improved plant regeneration protocol in Eastern Cottonwood (Populus deltoides) clones, WIMCO199 and L34, has been reported. Initially, aseptic cultures established from axillary buds of nodal segments from mature plus trees on MS liquid medium supplemented with $0.25mg\;1^{-1}$ KIN and $0.25mg\;1^{-1}$ IAA. Nodal and internodal segments were found to be extra-prolific over shoot apices during course of aseptic culture establishment, while $0.25mg\;1^{-1}$ KIN concentration played a stimulatory role in high frequency plant regeneration. Diverse explants, such as various leaf segments, internodes, and roots from in vitro raised cultures, were employed. Direct plant regeneration was at high frequency of 92% in internodes, 88% in leaf segments, and 43% in root segments. This led to the formation of multiple shoot clusters on established culture media with rapid proliferation rates. Many-fold enhanced shoot elongation and growth of the clusters could be achieved on liquid MS medium supplemented with borosilicate glass beads, which offer physical support for proliferating shoots leading to faster growth in comparison to semi-solid agar or direct liquid medium. SEM examination of initial cultures confirmed direct plant regeneration events without intervening calli. In vitro regenerated plants induced roots on half-strength MS medium with $0.15mg\;1^{-1}$ IAA. Rooted 5- to 6-week-old in vitro regenerated plants were transferred into a transgenic greenhouse in pots containing 1:1 mixture of vermicompost and soil at $27{\pm}2^{\circ}C$ for hardening and acclimatization. 14- to 15-week-old well-established hardened plants were transplanted to the field and grown to maturity. The mature in vitro raised poplar trees exhibited a high survival rate of 85%; 4-year-old healthy trees attained an average height of 8 m and an average trunk diameter of 25 cm and have performed well under field conditions. The regeneration protocol presented here will be very useful for undertaking genetic manipulation, providing a value addition to Eastern Cottonwood propagation in future.

• Korean Journal of Plant Resources
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• v.18 no.3
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• pp.382-389
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• 2005

This study was conducted to establish the system of effective in vitro propagation by various explant sources culture of Bippeastrum hybridum Hort, 'Dazzler'. We tested the effects of optimal explant source, plant growth regulators on bulblet formation and plant regeneration. Callus was readily produced on the different tissues excised from floral buds whereas, bulbs and shoots were formed only on pedicel explants as compared with anthers, styles and ovaries. Pedicel is the best optimal explant for in vitro propagation. Two distinct pathways, organogenesis through callus and direct bulblet formation, could be recognized in pedicel culture. Up to the $80-100\%$ of bulblet formation and shoot organogenesis from the pedicel in fifteen days before anthesis were effectively induced by MS medium supplemented with 0.5 mg/L NAA and 1.0 mg/L BA. Plantlet regeneration was successfully achieved from pedicel-derived callus, via shoot bud induction or direct bulblet formation. The bulblets with blooming flower were produced within 2 years.

• Korean Journal of Plant Resources
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• v.19 no.3
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• pp.385-391
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• 2006

In vitro-grown axillary buds of Melia aredarach were successfully cryopreserved by vitrification. On the MS medium supplemented with BA 1 mg/L, multiple shoots were developed within $4{\sim}5$ weeks. Plantlets of Melia azedarach were cold-hardened at $10^{\circ}C$ for a 16-hr photo-period for 6 weeks. Excised axillary shoot-tips from hardened plantlets were precultured on a solidified Murashige & Skoog agar medium (MS) supplemented with 0.7 M sucrose for 1 day at $25^{\circ}C$. Axillary shoot-tip meristems wert dehydrated using a highly concentrated vitrification solution (PVS2) for 60 min at $0^{\circ}C$ prior to a direct plunge into liquid nitrogen (LN). The PVS2 vitrification solution consisted of 30% glycerol (w/v), 15% ethylene glycol (w/v), 15% DMSO (w/v) in MS medium containing 0.4M sucrose. After short-term warming in a water bath at $40^{\circ}C$, the meristems were transferred into 2 ml of MS medium containing 1.2M sucrose for 15 min and then planted on solidified MS culture medium. Successfully vitrified and warmed meristems resumed growth within 2 weeks and directly developed shoots without intermediary callus formation. The survival rate of cold-hardened plantlets for 3 and 4 weeks was 90%. We did not find any difference in PCR-band patterns between control and cryopreserved plants. This method appears to be a promising technique for cryopreserving axillary shoot-tips from in vitro-grown plantlets of Medicinal plants.

• Korean Journal of Medicinal Crop Science
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• v.7 no.2
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• pp.94-100
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• 1999

To establish the mass propagation system of Lavandula spica cv. Marino, shoot tip, node, internode and leaf segment cultures were carried out. RAPD was applied to detect the somaclonal variation. Callus induction was very high in the medium supplemented with 1 mg/l 2.4-D, 2 mg/l NAA. especially and combined with 0.05 mg/l BAP from leaves. Shoot formation was high with $2{\sim}4\;mg/l$ BAP or 4 mg/l BAP + 0.2 mg/l NAA from shoot tip. Shoot proliferation was 9.1 times in the $B_{5}$ medium with 0.5 mg/l BAP and 0.01 mg/l NAA. Root formation was improved in NAA, which was the concentration of 0.1 to 1 mg/l and 1 mg/l IAA. Nursery survival rate was enhanced over 90% and growth was looked good in the acclimation soil consisting of peatmoss : vermiculite : perlite (1:1:1, v:v:v). Randomly amplified polymorphic DNA banding patterns based on polymerase chain reaction (PCR) were used to assess the genetic variation in plants regenerated from in vitro culture.

• Journal of Korean Society of Forest Science
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• v.96 no.1
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• pp.83-88
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• 2007

Micropropagation of Ulmus davidiana Planch was established via adventitious shoot formation from the segments of adventitious roots. Adventitious roots were produced directly from root segments of seedlings on a 1/2 SH medium plus various concentrations of IBA. The maximum growth of adventitious roots was observed in the presence of 2.0 mg/L IBA. After the segments of adventitious roots were cultured on various cytokinins (zeatin, 2-iP, BA, kinetin) and cytokinins plus auxin (IBA), formation of adventitious shoot was investigated. Among cytokinin treated, kinetin was the most effective on both adventitious shoot induction and number of shoots. Especially, 2.0 mg/L kinetin was the best to increase adventitious shoot induction (95.8%) and a number of shoots (8.4). Adventitious shoots were rooted on 1/2 WPM medium and the plantlets were acclimated 100% on composed soil (peatmoss : vermiculite 1 : 1).

• Plant Biotechnology Reports
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• v.4 no.2
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• pp.157-163
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• 2010

A protocol for in vitro propagation from pseudobulb sections of Lycaste armomatica (Graham ex Hook) Lindl., an ornamental and fragrant orchid, was developed. The effect of four cytokinins: kinetin (K), metatopolin (mT), $N^6$-benzyladenine (BA), and thidiazuron (TDZ), in equimolar concentrations, was investigated. Shoot formation from apical and basal pseudobulb sections was obtained in all treatments. A few medial sections cultured in media supplemented with BA formed protocorm-like bodies. Shoot formation was greater from the basal section than the apical, and mainly occurred in explants cultured in media containing TDZ. The highest average numbers of shoots per explant were achieved from basal sections cultured in media supplemented with TDZ at 4.4, 8.87 and 2.2 ${\mu}M$, forming on average 9.9, 8.6 and 7.3 shoots per explant, respectively. Since the medial pseudobulb section was the worst explant for propagation of L. aromatica, we recommend that pseudobulbs be divided into two sections; the basal half should be cultured in MS medium supplemented with TDZ at 4.4 ${\mu}M$ and the apical half with TDZ at 2.2 ${\mu}M$. Subculturing individual shoots in MS medium without plant growth regulators allows further development and rooting. A survival rate of more than 90% under greenhouse conditions was achieved. This research represents a direct contribution to the conservation and sustainable use of this valuable natural resource.