Rapid in vitro propagation of Dendrobium hybrids through direct shoot formation from foliar explants, and protocorm-like bodies

http://images.wikia.com/orchids/en/images/d/d8/Callus_formation_and_plant_regeneration_from_callus_through_somatic_embryo_structures_in_Cymbidium_orchid.pdf

Callus formation and plant regeneration from callus through somatic embryo structures in Cymbidium orchid

In vitro production of phenolic compounds and antioxidant activity in callus suspension cultures of Habenaria edgeworthii: A rare Himalayan medicinal orchid

Leaf explants of Paphiopedilum phiIippinense hybrids (hybrid PH59 and PH60) directly formed adventitious shoots from wound regions within 1 month, when cultured on modified Murashige and Skoog medium (1/2-strength macro- and full-strength micro-elements) free of plant growth regulator in darkness. The combinations of 2,4-dichlorophenoxyacetic acid ((2,4-D) acid (0, 4.52 and 45.25 μM) and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ) (0, 0.45, 4.54 and 22.71 μM) were used to test their effects on direct shoot bud formation from two types of explants (1.5-cm long intact leaf explants and 0.5-cm long leaf segment explants). In hybrid PH59, 4.54 μM TDZ increased mean numbers of shoots per explant with leaf segment explants. In hybrid PH60, 4.52 μM 2,4-D plus 0.45 μM TDZ promoted direct shoot bud formation from leaf segment explants. In addition, three treatments (4.52 μM 2,4-D, 22.71 μM TDZ, 4.52 μM 2,4-D plus 4.54 μM TDZ) gave a higher response than control on mean numbers of shoots per explant with intact leaf explants. Healthy plantlets each with one to three roots were obtained from leaf-derived shoots after transfer onto a hormone-free medium for 22 months. These plantlets were acclimatized in a greenhouse and grew well with 100% survival rate.

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Plant regeneration through direct shoot bud formation from leaf cultures of Paphiopedilum orchids

Paphiopedilum orchids are among the world’s most popular orchid due to their impressively beautiful flowers. Propagation of these orchid genera has been hampered by the naturally slow growth rate of the plant, which renders it very difficult to be propagated through conventional methods. In vitro culture techniques have provided a useful alternative technology for propagating this recalcitrant species. In this study, the propagation of P. rothschildianum was achieved through the in vitro formation of secondary protocorm-like bodies (PLBs) from the primary PLB that developed from stem-derived callus. The PLBs were cultured on half-strength MS medium supplemented with different concentrations (1.0, 2.0, 3.0, and 4.0 μM) of 6-benzyladenine (BA) and kinetin for the induction of secondary PLBs. The highest number of secondary PLBs formed was obtained on half-strength MS medium supplemented with 4.0 μM kinetin, with an average of 4.1 PLBs per explant after 8 weeks of culture. The secondary PLBs continued to proliferate further and formed 9.5–12.1 new PLBs per secondary PLB after being subcultured onto half-strength plant growth regulator-free MS medium supplemented with 60 g/L banana homogenate (BH). These tertiary PLBs were subcultured onto media containing different organic additives, such as BH, coconut water, potato homogenate, and tomato homogenate, for plantlet regeneration. Among the organic additives tested, the addition of 20% CW to half-strength MS medium resulted in the best average plantlet regeneration percentage from the PLBs, 67.9%, after 8 weeks of culture.

In vitro propagation of Paphiopedilum orchid through formation of protocorm-like bodies

Totipotent calli of a Paphiopedilum hybrid (Paphiopedilum callosum ‘Oakhi’ × Paph. lawrenceanum ‘Tradition’) were induced from seed-derived protocorms on a 1/2 strength Murashige–Skoog medium plus 1–10 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1–1 mg l−1 1-phenyl-3-(1.2.3-thiadiazol-5-yl)urea (TDZ). These calli grew well when subcultured on the same medium, but proliferated more on 1/2 MS medium plus 5 mg l−1 2,4-D and 1 mg l−1 TDZ. Calli developed further along a route of production of protocorm-like bodies and eventually formed plantlets that could be transplanted to pots and grew well.

Yung-Haw Lin

Papers

Plant regeneration from callus culture of a Paphiopedilum hybrid