• Current Research on Agriculture and Life Sciences
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• v.18
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• pp.1-7
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• 2000

This study was carried out to determine the effects of plant growth regulators on organogenesis from Cymbidium kanran and Cymbidium hybrida. Optimal rhizome formation from Cymbidium kanran was obtained on MS medium with 10 ppm kinetin+2 ppm NAA. and optimal protocorm formation from Cymbidium hybrida was obtained on MS medium with 10 ppm kinetin+0.05 ppm NAA. However, in this study the optimal media for the callus induction from both explants was not identified. Optimal shoot induction from rhizome of Cymbidium kanran was obtained on MS medium with 10 ppm BA+2 ppm NAA and 5 ppm BA+2 ppm NAA. Optimal shoot induction from protocorm of Cymbidium hybrida was obtained on MS medium with 10 ppm kinetin+2 ppm NAA.

• Journal of Plant Biotechnology
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• v.42 no.3
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• pp.239-244
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• 2015

Calanthe striata f. sieboldii Decne. ex Regel is a terrestrial orchid with beautiful flowers arranged in racemose inflorescences. This species is threatened due to over-collection and loss of suitable habitats. Asymbiotic germination is useful in the conservation efforts to re-establish plants in the wild, and for commercial propagation. In this study, we investigate the effects of sodium hypochlorite (NaOCl), three types of culture media (Phytomax Orchid Maintenance - POM, Seed Germination Maintenance - SGM, and Murashige and Skoog 1962 - MS), and plant growth regulators on embryo swelling, protocorm formation, and embryo diameter of C. striata f. sieboldii. Treatment with 1% NaOCl for 30 min greatly enhanced embryo swelling (28.3%), embryo diameter ($205.8{\mu}m$), and embryo protocorm formation (54.8%) compared to seeds without NaOCl treatment (embryo swelling 8.5%, embryo diameter $14.6{\mu}m$, and protocorm formation 13.4%) on POM medium. Protocorm formation on POM medium supplemented with 1.0 mg/L N6-benzyladenine (BA) (95.6%) was better than the control (54.5%). Additionally, the effects of activated charcoal (AC) and sucrose on seedling growth in in vitro culture were examined. The protocorm converted into healthy plants with well-developed shoot primordia on the POM medium with AC and sucrose. The most suitable conditions for seedling growth after 10 weeks of culture were the POM medium with AC or sucrose. These results show effective asymbiotic seed germination and in vitro seedling growth of C. striata f. sieboldii.

• Journal of Plant Biology
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• v.11 no.3
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• pp.23-30
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• 1968

Using several varieties of Cymbidium, investigations were carried out to make clear how the protocormic tissue develops from the cultured explant. Explant to be cultured were prepared in several ways: exclusively apical meristem, apical meristem dissected out with the basal part attached, axillary bud primordia in their initial stage of development, or apical or axillary bud dissected out as a whole etc. It was observed that protocorms or protocormic tissues were developed from the explant's meristematic tissues regardless of where these tissues were located. Apical meristem, leaf primordia, leaf axil, or internodal part of young bud turned easily protocormic, while the scaly leaves of axillary bud or stem tissue of mother shoot turned quickly brwonish and died away. Both in axillary and apical bud explant alike, whether they were cultured whole or divided, some took quickly green color while others were slower, and some developed protocorms easily while others remained unchanged for months. Varietal difference as well as environmental factors seemed to be responsible for it. Further details should be clarified by histogenetical investigations.

• Journal of Plant Biotechnology
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• v.44 no.3
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• pp.343-348
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• 2017

Cymbidium aloifolium (L). Sw. is an exquisite epiphytic orchid of the Kolli Hills (Eastern Ghats) of Tamil Nadu in Southern India. It is fast disappearing from its natural habitats due to deforestation and low germination rate in natural habitat. In the present study, an attempt was made to germinate the seeds from un-dehisced capsule of Cymbidium aloifolium (L). Sw under in vitro condition. The seed germination and protocorm development were recorded in three different well known media namely Knudson C (KC), Half strength Murashige & Skoog (1/2 MS) and Vacin & Went (VW) media. The highest seed germination of 90% was observed KC basal media after $30^{th}$ days whereas germination percentages were 40% and 30% on 1/2 MS and VW media respectively. The well-developed protocorm were transferred to KC media supplemented with 6-Benzyl Amino Purine (BAP) and Naphthalene acetic acid (NAA) where BAP (1.0 mg/l) and NAA (1.0 mg/l) together were found to be optimum for the highest shoot formation. About 90% of the shoots found to be well rooted after transfer to the KC medium differently supplemented with 1.5 mg/l Indole-3-acetic acid (IAA) and 1.0 mg/l Indole-3-butyric acid (IBA). Though rooting also took place in the two basic media but the duration was longer when compared with the hormone-supplemented media. The rooted plantlets were hardened and kept under greenhouse conditions which can be relocated in natural habitats.

• Journal of Life Science
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• v.19 no.3
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• pp.378-383
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• 2009

Agrobacterium tumefaciens-mediated transformation procedure for the phalaenopsis orchid, established by using Protocorm-like bodies (PLBs), was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. PLBs obtained from the axillary bud of a peduncle were maintained on a hyponex medium supplemented with 1 g/l of activated charcoal, 30 g/l of sucrose and 0.1 mg/l thiamine. The multiplication rate of PLBs was about 90% in case of subculture PLBs to be cut transversely into 1/3 part from top position. The PLBs were inoculated with Agrobacterium strain EHA105 harboring both $\beta$-glucuronidase (GUS) and hygromycin-resistant genes for 20 minutes after dipping treatment. Transformation efficiency was the highest with a Agrobacterium culture medium and dipping treatment of O.D. 0.8. Newly induced PLBs were put on selection medium containing 1 mg/l hygromycin for 2 months. Hygromycin-resistant phalaenopsis plants that regenerated after the selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by PCR and Southern blot using GUS specific primers and probe.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.173-173
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• 2017

Phalaenopsis orchid is one of the most important flowers in flower industry. We conducted an experiment to find out the response of different concentration of chlorocholine chloride (CCC) for the in vitro regeneration of protocorm like bodies (PLBs) of Phalaenopsis 'Fmk02010'. We used five different concentrations of CCC and these were 0 (control), 0.01, 0.1, 1 and $10mgL^{-1}$ in modified MS medium and cultured for 42 days. We added two major salts ammonium nitrate ($412.5mgL^{-1}$) and potassium nitrate ($950.0mgL^{-1}$) to the MS medium for the modification. Maximum numbers of PLBs were found from media with $0.01mgL^{-1}$ of CCC (15.667) and maximum fresh weight (0.211 g) as well. The 100% PLB formation rate was also found from $0.01mgL^{-1}$ of CCC. We found 58.83% variation in number of PLBs ($R^2=0.5883$) and 47.44% variation in fresh weight ($R^2=0.4744$) to the different CCC concentrations. Our study suggested that increase in the CCC concentration negatively affect the PLBs organogenesis of Phalaenopsis. We can suggest that the addition of very low concentration of CCC in plant culture medium can increase the number, formation rate and fresh weight of PLBs of Phalaenopsis.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.177-177
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• 2017

Phalaenopsis orchid is one of the most important flowers in flower industry. We conducted an experiment to find out the response of different concentration of chlorocholine chloride (CCC) for the in vitro regeneration of protocorm like bodies (PLBs) of Phalaenopsis 'Fmk02010'. We used five different concentrations of CCC and these were 0 (control), 0.01, 0.1, 1 and $10mgL^{-1}$ in modified MS medium and cultured for 42 days. We added two major salts ammonium nitrate ($412.5mgL^{-1}$) and potassium nitrate ($950.0mgL^{-1}$) to the MS medium for the modification. Maximum numbers of PLBs were found from media with $0.01mgL^{-1}$ of CCC (15.667) and maximum fresh weight (0.211 g) as well. The 100% PLB formation rate was also found from $0.01mgL^{-1}$ of CCC. We found 58.83% variation in number of PLBs ($R^2=0.5883$) and 47.44% variation in fresh weight ($R^2=0.4744$) to the different CCC concentrations. Our study suggested that increase in the CCC concentration negatively affect the PLBs organogenesis of Phalaenopsis. We can suggest that the addition of very low concentration of CCC in plant culture medium can increase the number, formation rate and fresh weight of PLBs of Phalaenopsis.

• Journal of Plant Biotechnology
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• v.50
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• pp.108-114
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• 2023

In vitro conservation is one of the most effective strategies for rare plant protection, especially for orchid species. To maximize the success rates of in vitro explant establishment (stage I) in conservation programs, the application of tissue culture additives such as Plant Preservative Mixture^TM (PPM^TM) should be emphasized. In this study, we used Dendrobium thyrsiflorum Rchb.f. (1875) seeds and seedlings as a model for the evaluation of PPM^TM's phytotoxicity in the meristematic tissues of epiphytic orchids. PPM^TM had no observable inhibitory effect on protocorm, shoot, or root development when it was supplemented at 0.1%. PPM^TM supplementation caused adverse effects on D. thyrsiflorum explants at concentrations > 0.2%. At high concentrations, young in vitro seedlings showed damage, especially at the root tissue level. Based on this model, supplementation of 0.1-0.2% PPM^TM to culture media was successfully implemented to establish in vitro cultures of other rare orchid species in our conservation program.

• Journal of Plant Biotechnology
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• v.41 no.4
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• pp.223-228
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• 2014

To establish an efficient proliferation and regeneration of PLBs (protocorm-like bodies) of Phalaenopsis plants, a variety of propagation medium types, various concentraions of sucrose as well as liquid and solid type were tested in this study. Further, activated charcoal, citric acid and ascorbic acid were compared whether these agents are suppose to reduce the browning in culture process using PLBs of Phalanopsis plants. With regard to the proper propagation medium, VW medium showed 1.3 ~ 2 times highr than those of other medium in an index of increasing for fresh weight and 50% higher than those of other medium in the frequency of shoot regeneration. However, regarding liquid and solid types of culture, there were no significant differences in the proliferation of PLBs and regeneration of shoots from PLBs. In the experiment for a variety of sucrose concentrations (0 ~ 50 g/l), 10 g of sucrose showed 30 ~ 50% higher than other concentrations in increasing index and 10 ~ 50% higher in the regeneration of shoots from PLBs. Regarding the reduction of browning in tissue culture via PLBs of Phalaenopsis plants, 1 g of activated charcoal showed only 1.5% browning of PLBs cultured. Whereas, other treatments including citric acid and ascorbic acid showed 6 ~ 16% of browning of PLBs. Therefore, activated charcoal was selected as an efficient anti-browning agents for the culture of PLBs in Phalaenopsis plants. Using above-described results can be contibuted to the establishment of mass propagation system using PLBs of Phalaenopsis plants in the future.

• Korean Journal of Plant Resources
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• v.31 no.4
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• pp.372-377
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• 2018

This study was investigated the effect of immature rhizome production according to harvest times-based treatment method for seed production of Gastrodia elata. The results revealed that when the tuber weight of G. elata harvested in spring (GEHS) was ${\geq}100g$, the rate of artificial fertilization, protocorm formation, and immature rhizome formation was 90.9%-94.8%, 3.1%-5.4%, and 10.1%-15.3%, respectively. When G. elata harvested in fall (GEHF) was treated at a low temperature for 4 weeks or more, the rate of artificial fertilization, protocorm formation, and immature rhizome formation was 70.4%-87.6%, 2.2%-2.6%, 8.7%-9.5%, respectively. Therefore, to produce seeds and immature rhizomes, GEHS must have tubers of more than 100 g, whereas GEHF requires breaking dormancy by low-temperature treatment for 4 weeks or more. Compared with those of GEHS, the rate of artificial fertilization, protocorm formation, and immature rhizome formation was lower in GEHF; however, it was higher than those in the natural germination state. Thus, it can be expected that G. elata can be produced throughout the year by ensuring that the seeds and immature rhizomes of G. elata are produced using a constant tuber weight and by breaking dormancy with low temperature treatment.