Asymbiotic Germination and Low-temperature in Vitro Conservation of Cymbidium Dayanum

[Objective] The aim of the research was to establish asymbiotic germination and low-temperature in vitro conservation technique system of Cymbidium dayanum by using plant tissue culture technique to realize its rapid propagation and long-term conservation in vitro. [Method] With mature seeds of C. dayanum as explants, different media were selected to establish asymbiotic germination technique system. With protocorms as materials, conservation, resumptive proliferation and plant regeneration conditions were selected to establish low-temperature in vitro conservation technique system preliminarily. [Result] Mature seeds of C. dayanum could germinate after cultured 90 days on MS media as well as ＂Hyponex 1＂ media. The germination rate reached more than 98%. Protocorms inoculated in ＂Hyponex 1＂ media could be conserved continuously at 5 ℃ in dark for more than 18 months and the survival rate could reach 90%. Conserved protocorms could realize resumptive preliferation culture both on 1/2 MS and ＂Hyponex 1＂ media. The seed- ling-strengthening and rooting media were 1/2 MS media. [Conclusion] This research provided practical basis for in vitro conservation and rapid propagation of C. dayanum germplasm resource.

Optimization of Culture Conditions for Chromium-enriched Protocorm of Dendrobium candidum

[Objective] To optimize the techniques for culturing Cr-enriched Dendrobi-um candidum protocorm. [Method] By adopting Plackett-Burman test, the effects of time, temperature, Cr-content in medium, NAA content, KT content and light intensi-ty on Cr-enriched protocorm were researched. Three factors of time, Cr-content in medium and light intensity had statistical y significant effects; then steepest ascent procedures were applied to define optimal response region of these three factors; fi-nal y the optimal factors were determined by RSM analysis. [Result] The results showed that the optimal conditions for Cr-enrichment were chromium concentration at 0.37 mg/L, culture time of 59 d and il umination intensity of 1 822.22 lx. The predicted value of Cr-content in protocorm was 5.08 mg/kg. [Conclusion] RSM can optimize the techniques for culturing Cr-enriched protocorm of Dendrobium can-didum, provide the optimum process parameters and lay the foundation for further research.

In vitro germination and propagation of a threatened medicinal orchid,Cymbidium aloifolium(L.) Sw.through artificial seed

Objective:To study the in vitro germination and plantlet regeneration from artificial seeds of Cymbidium aloifolium(C.aloifolium),a highly threatened medicinal orchid of Nepal.Methods:Artificial seeds were produced in vitro by encapsulation of protocorms with 4%sodium alginate and 0.2 mol/L calcium chloride solution.In vitro germination and plantlet regeneration of the artificial seeds were tested by culturing them on different strength of Murashige and Skoog(MS) liquid media(0.25,0.5 and 1.0) and MS liquid medium supplemented with 0.5 mg/L benzyl amino purine and 0.5 mg/L naphthalene acetic acid.Freshly produced artificial seeds were stored up to 28 d at 4 ℃.In order to check the viability,storage artificial seeds were treated with five different sterilization techniques(T_1 T_2,T_3 T_4,T_5) and inoculated on full strength(1.0) of MS liquid medium after each 7 d of interval upto28 th days.Results:The highest percentage of germination(100%) of artificial seed was obtained on quarter(0.25),half(0.5) and full(1.0) strength of MS liquid medium.Experimentally,full strength of MS liquid medium was more effective for earlier seedling development of C.aloifolium.Artificial seeds were successfully stored at 4 ℃ till 28 th days.Treatments T_1 and T_2showed 97.5%viability of storage artificial seeds and hence considered as the most effective sterilization techniques to recover the plant from storage artificial seeds.Plantlets developed from artificial seeds were successfully acclimatized in potting mixture containing cocopeat,litter and sphagnum moss with 85%survival rate.Conclusions:The present study revealed that artificial seeds are the good alternative explants for in vitro mass propagation and short term conservation of C.aloifolium.

Efficient Plant Regeneration of Cymbidium aloifolium (L.) Sw., a Threatened Orchid of Nepal through Artificial Seed Technology

Artificial seed technology is a method of considerable potential for mass propagation and conservation of rare, endangered and threatened species. In the present investigation, artificial seeds were obtained through encapsulation of three weeks old protocorm (3.0 ± 1.0 mm diameter) of Cymbidium aloifolium with calcium alginate. Artificial seeds were cultured on liquid Murashige and Skoog (MS) or Knudson (Kn C) medium at different strength (×1.0, 0.5, 0.25) and full strength (1.0) of both media supplemented with 0.5 mg/l 6-benzyl aminopurine (BAP) and 0.5 mg/l α-naphthalene acetic acid (NAA). Full strength of MS medium without plant growth regulators was found to be the most favourable condition for efficient plantlet regeneration of C. aloifolium (9.83 shoot and 2.66 roots per culture). The storage potential of artificial seed was tested at 4°C and room temperature (RT, 21°C ± 2°C) for up to 90 days on both media and found 83.33% viability at 4°C storage on MS media. Eighty five percent of plantlets regenerated from artificial seeds culture were successfully hardened in a potting mixture of cocopeat, clay and sphagnum moss (2:1:1). Hence, the methodology can be used for propagation and conservation of C. aloifolium through artificial seed system.

<i>In Vitro</i>Mass Scale Propagation of Wild <i>Cymbidium lowianum</i>with a Rare and Endangered Plant

The wild Cymbidium lowianum, a national-level rare and endangered species of Orchid, is an excellent garden plant with ornamental flowers with striking, deep red lips in a V-shaped formation. The objective of this study was to establish a micropropagation protocol system via immature seeds of wild Cymbidium lowianum, evaluate the Murashige and Skoog (MS), half-strength Murashige and Skoog (1/2 MS) medium, 6-benzylaminopurine (BA), a-napthaleneacetic acid (NAA), organic additions activated charcoal (AC) and banana pulp (BP) effects on the different morphogenesis (seed germination, multiple shoot and rooting) in vitro. The optimal combination for the germination of seed was 1.0 mg·L-1 BA with 0.5 mg·L-1 NAA in 1/2 MS, and addition 0.3%AC, which resulted in 95% seed germination in 90 days. The best formulation for multiple shoot was 1/2 MS medium containing 2.5 mg·L-1 BA, 0.5 mg·L-1 NAA and addition 8% BP in which produced 19.8 shoots per protocorm in 60 days. Multiple shoots were cut and rooted in 1/2 MS supplemented with 1.5 mg·L-1 NAA,0.1 mg·L-1 BA and 0.3% AC, roots initiated 20 days after culture, the rooting percentages reached to 100%, in which 4.7 per shoot produced roots in 60 days. The survival rate of plantlet was up to 92% in moss after 30 days. This finding reveals that it is possible to obtain in vitro culture of Cymbidium lowianum using immature seeds in asymbiotic culture.

Rapid Propagation of Rhynchostylis retusa in Vitro

An efficient regeneration system of Rhynchostylis retusa was established to provide technical reference for the application of tissue culture tube seedlings in production.The mixtures of callus and protocorm from aseptic germination were used as explants.The optimal media of each stage was selected for callus proliferation,protocorm occurrence and growth,rejuvenation and rooting via a single,complete combination and orthogonal experiment.The results showed that the optimal medium for callus proliferation,protocorms occurrence and growth was 1/2 Murashige and Skoog(MS)medium adding 50 g·L^(−1) banana puree,0.1 mg·L^(−1)α-naphthaleneacetic acid(NAA),1.5 mg·L^(−1)6-benzylaminopurine(6-BA)and 1.0 mg·L^(−1) kinetin(KT)with 17.33 proliferation coefficient of callus and 19.63 occurrence coefficient of buds after 90 days.Then the buds occurred from protocorm were cultured on 1/2 MS medium including 100 g·L^(−1) banana puree,1.0 mg·L^(−1) NAA,2.0 mg·L^(−1)6-BA and 0.05 mg·L^(−1) KT,in which the proliferation coefficient of callus was 10.32 and occurrence coefficient of buds reached 17.87.In the further subculture,the same medium was simultaneously used for callus proliferation,protocorm occurrence and bud growth.The plantlets developed roots in 1/2 MS medium containing 70 mL·L^(−1) coconut water and 1.5 mg·L^(−1) NAA with 100%rooting rates after 90 days.The survival rate was more than 90%after domestication and transplantation.This regeneration protocol will provide technique foundation for protecting wild resource and developing artificial cultivation.

Tissue Culture and Rapid Propagation Techniques of New Variety in Cymbidium hybridum

[Objectives]This study was conducted to select media suitable for proliferation,differentiation and rooting of Cymbidium hybridum"Huangjinjia".[Methods]The lateral buds and protocorms of the new variety C.hybridum"Huangjinjia"were used as materials to explore the effects of different concentrations of 6-BA and NAA on protocorm proliferation and rooting.[Results]The optimal medium for protocorm propagation was 1/2 MS+6-BA 1.0 mg/L+NAA0.5 mg/L+potato 50 g/L+sucrose 20 g/L,in which the protocorms multiplied easily and grew rapidly.The optimal medium for inducing adventitious buds was1/2 MS+6-BA 1.5 mg/L+NAA 0.3 mg/L+sucrose 30 g/L+banana 25 g/L+apple 25 g/L+activated carbon 1.0 g/L,in which the induction rate of adventitious buds reached 335%.The optimal medium for rooting was 1/2 MS+NAA 0.5 mg/L+sucrose 25 g/L+banana 75 g/L+apple 25 g/L+activated carbon1.0 g/L,in which the average root length was 3.0 cm,the average number of roots was 2.6,and plantlets had green leaves,thick roots and suitable plant height.[Conclusions]This study provides a theoretical basis and reference for the establishment of a rapid propagation system using lateral buds.