Plant regeneration from protocorm-derived callus of Cypripedium formosanum

In Vitro Cellular & Developmental Biology - Plant (Impact Factor: 0.98). 09/2003; 39(5):475-479. DOI: 10.1079/IVP2003450


Totipotent callus of Cypripedium formosanum, an endangered slipper orchid species, was induced from seed-derived protocorm segments on a quarter-strength Murashige and
Skoog medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid and 4.54 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (thidiazuron). This callus proliferated well and was maintained by subculturing on
the same medium. On average, 13 protocorm-like bodies could be obtained from a piece of 4 mm callus after being transferred
to the medium with 4.44 μM N6-benzyladenine after 8 wk of culture. The regenerated protocorm-like bodies formed shoots and roots on medium containing 1
g l−1 activated charcoal and 20 g l−1 potato homogenate. After 24 wk of culture on this medium, well-developed plantlets ready for potting were established.


Available from: Yung-I Lee
  • Source
    • "In hybrid Cymbidium, VW basal medium used for PLBs formation effectively while 1/2x MS basal medium used for callus induction and formation (Teixeira, 2012). Callus of Vanda coerulea was successfully maintained for a month on 1/4x strength of media (Lang & Hang, 2006) and callus of Cymbidium ensifolium was also effectively maintained on 1/2x MS media (Lee & Lee, 2003). There was no callus maintain observed on KC basal medium. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Vanda dearei is an endemic orchid of Borneo and has been listed as an endangered orchid in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Vanda dearei has beautiful pale yellow-flowers, large petals and strongly scented. Therefore, in vitro micropropagation has been applied in order to develop a novel micropropagation method to mass produce this species. Through callus culture techniques, orchids with limited resources can be mass propagated in a shorter period. However, callus culture in orchid is hardly maintain due somatic embryogenesis properties and easily regenerated to plantlets. Thus, this study aims to develop an efficient protocol for callus cultures of V. dearei by manipulating basal media strengths and carbon sources. Callus induced from the leaf segments of V. dearei were used as explants and were cultured on KC, Mitra, MS and VW basal medium at different nutrient strengths (1/4, 1/2, 1 and 2x) added with 1.0:0.1 mg/l TDZ:NAA and 1 to 4% (w/v) of sucrose, glucose or fructose, respectively. All cultures were incubated in the dark with temperature of 25±2°C. Results showed that callus growth has improved with decreased nutrients strength of basal media. Quarter strength of Mitra medium promotes the best condition for callus maintenance to approximately 8.00±17.89% at 8 weeks of culture. This is followed by the ½ strength of MS and ¼ strength of VW with 8.00±10.95% and 5.00±10.00%, respectively. Callus grown on the other basal strengths are mainly differentiated and developed into protocorm like bodies (PLBs), especially at double strengths (100±0% explants turn into PLBs). In addition, low percentage of necrosis (less than 28%) was also observed on Mitra basal medium compared to the other media (more than 36%). Sucrose has been identified as the best carbon source to support callus growth followed by glucose and fructose. Addition of 1% (w/v) sucrose increased callus maintenance up to 32±17.9%, promote cell differentiation and increased average size of callus (1.52±0.63 callus score). This treatment also support the longest retention time of explant maintained in callus for 5 weeks and has the lowest percentage of callus necrosis (20±24.5%).
  • Source
    • "Rev. Ceres, Viçosa, v. 59, n.2, p. 185-191, mar/abr, 2012 Diferentes respostas foram observadas no meio de cultura e da concentração de carvão ativado empregada , dependendo da espécie utilizada. Lee & Lee (2003) obtiveram resultados positivos na regeneração de brotos e raízes a partir de protocormoides de Cypripedium formosanum Hayata adicionando 1 g L -1 de carvão ativado ao meio MS com ¼ da concentração de sais. Ket et al. (2004) observaram que a adição de 1 g L -1 ao meio Hyponex suplementado de 0,1 g L -1 de benziladenina e 1-2 mg L -1 de thidiazuron promovia a formação múltipla de brotos em segmentos nodais de Anoectochilus formosanus Hayata, entretanto o crescimento era lento e os caules não se alongavam. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Orchids are over-exploited in their natural environment due to their ornamental importance, which is leading some species to extinction. In vitro culture is an alternative for ex-situ conservation. Our objective was to determine an efficient medium for in vitro seed germination and initial seedling growth of Cattleya forbesii and Cattleya harrisoniana. Seeds of Cattleya forbesii were incubated in Murashige & Skoog (MS) medium (T1) and MS added with 2,5 g L-1 of activated charcoal (T2). For the last, plants with 1 ± 0,2 cm of height were submitted to the following treatments: T1, T2, MS reduced to the half strenght (T3) and MS reduced to the half strenght supplemented with 1,25 g L-1 of activated charcoal (T4). Thirty days after seed inoculation of C. forbesii, it was verified 45% and 90% of germination in T1 and T2, respectively. The addition of activated charcoal also promoted an increase in the height of the C. forbesii seedlings after 180 days of in vitro cultivation. After 240 days of culture of C. harrisoniana shoot height, total fresh mass, number of roots and leaves, length of the largest root and diameter of the shoots were significantly stimulated in T2 compared to the other treatments. We suggest the use of MS medium added with 2,5 g L-1 of activated charcoal because is significantly favorable for seed germination and initial growth for both species.
    Revista Ceres 04/2012; 59(2):185-191. DOI:10.1590/S0034-737X2012000200006

  • Voenno-medit͡sinskiĭ zhurnal 04/1961; 3:62-5.
Show more