Agrobacterium-mediated transformation of the wild orchid Cattieya maxima Lindi / Transformación mediada por Agrobacterium de la orquídea silvestre Cattleya maxima Lindl / Transformação da orquídea silvestre Cattleya maxima Lindl mediada por Agrobacterium

Abstract Protocorms are unique anatomical structures; they are akin to rhizoids and are formed by young orchid seedlings under physiological conditions. Explanted orchid tissues produce similar structures called protocorm-like bodies (PLBs) when exposed to appropriate in vitro growing conditions. Both the propagative nature of PLBs and the easiness by which they can be generated, make these structures an attractive alternative to seed-mediated production for growing large numbers of plants. To increase somatic embryogenesis and optimize the procedure, PLBs of Cattleya maxima were transformed using the Agrobacterium tumefaciens method. The T-DNA carried a Hygromycin-resistance gene, a visible marker (GFP5-GUSA) and a rice gene encoding the Somatic Embryogenesis Receptor Kinase, deemed to be important for somatic embryogenesis. Treated PLBs generated somatic embryos developing Hygromycin-resistant plantlets. The insertion of T-DNA was confirmed by PCR, and GFP expression was observed using a fluorescent stereomicroscope. Transformed Cattleya maxima PLBs were more efficient in forming somatic embryos (60 - 80%) than untransformed controls (45 - 57%), and this contrast was maximized in hormone-free, Murashige and Skoog (MS) medium (80% of the transformed plants compared to 57% of the untransformed ones). This finding supports the notion that SERK plays an important role in Orchid embryogenesis.

Resumen Los protocormos son estructuras anatómicas únicas: son similares a los rizoides y se forman por vástagos jóvenes de orquídeas bajo condiciones fisiológicas. Los tejidos explantados de orquídeas producen estructuras llamadas Cuerpos Similares a Protocormos (PLBs) cuando están expuestos a condiciones apropiadas de crecimiento in vitro. Tanto la naturaleza propagativa de los PLBs como la facilidad con que se generan, hacen de estas estructuras una alternativa atractiva, frente a la mediada por semillas, para la producción de gran número de plantas en crecimiento. Para aumentar la embriogénesis somática y optimizar el procedimiento, se transformaron PLBs de Cattleya maxima usando el método de Agrobacterium tumefaciens. El T-DNA portaba un gen de resistencia a la Higromicina, un marcador visible (GFP5-GUSA) y un gen de arroz que codificaba para el receptor tipo quinasa de embriogénesis somática (SERK), considerado importante en la embriogénesis somática. Los PLBs tratados generaron embriones somáticos y desarrollaron plántulas resistentes a la Higromicina. La inserción del T-DNA se confirmó por PCR, y la expresión de GFP se observó usando un estereomicroscopio fluorescente. Los PLBs transformados de Cattleya maxima fueron más eficientes en desarrollar embriones somáticos (60-80%) que los controles no transformados (45-57%) y este contraste se maximizó en medio Murashige y Skoog (MS) libre de hormonas (80% de las plantas transformadas en comparación con 57% de las no transformadas). Estos hallazgos apoyan la noción de que SERK juega un papel importante en la embriogénesis de orquídeas.

Resumo Os protocormos são estruturas anatômicas únicas: são similares aos rizoides e se formam por hastes jovens de orquídeas sob condições fisiológicas. Os tecidos explantados de orquídeas produzem estruturas chamadas Corpos Similares a Protocormos (PLBs) quando estão expostos a condições apropriadas de crescimento in vitro. Tanto a natureza propagativa dos PLBs como a facilidade com que se generam, fazem com que estas estruturas sejam uma alternativa atrativa, comparativamente a mediada por sementes, para a produção de grandes números de plantas em crescimento. Para aumentar a embriogênesis somática e otimizar o procedimento, se transformaram PLBs de Cattleya maxima utilizando o método de Agrobacterium tumefaciens. O T-DNA carregava um gen de resistencia a Higromicina, um marcador visível (GFP5-GUSA) e um gen de arroz que codificava para o receptor tipo quinasa de embriogênesis somática (SERK), considerado importante na embriogênesis somática. Os PLBs tratados geraram embriões somáticos e desenvolveram plântulas resistentes a Higromicina. A inserção do T-DNA se confirmou por PCR, e a expressão de GFP se observou utilizando um estereomicroscópio de fluorescência. Os PLBs transformados de Cattleya maxima foram mais eficientes em desenvolver embriões somáticos (60-80%) que os controles não transformados (45-57%) e este contraste se potencializou em meio Murashige y Skoog (MS) livre de hormônios (80% das plantas transformadas em comparação com 57% das não-transformadas). Estes resultados apoiam a noção de que SERK desempenha um papel importante na embriogênesis de orquídeas.

In vitro regeneration and ploidy level analysis of Eulophia ochreata Lindl.

Various parameters including explant-type, medium compositions, use of phytohormones and additives were optimized for direct and indirect regeneration of E. ochreata, a medicinal orchid under threat. Protocorm-like-bodies (PLBs) proved to be the best explants for shoot initiation, proliferation and callus induction. Murashige and Skoog’s (MS) medium containing 2.5 mg L-1 6-benzylaminopurine (BAP), 1.0 mg L-1 kinetin (Kin) and additives (adenine sulfate, arginine, citric acid, 30 mg L-1 each and 50 mg L-1 ascorbic acid) was optimal for shoot multiplication (12.1 shoots and 7.1 PLBs per explant with synchronized growth), which also produced callus. Shoot number was further increased with three successive subcultures on same media and ~40 shoots per explant were achieved after 3 cycles of 30 days each. Additives and casein hydrolysate (CH) showed advantageous effects on indirect shoot regeneration via protocorm-derived callus. Optimum indirect regeneration was achieved on MS containing additives, 500 mg L-1 CH, 2.5 mg L-1 BAP and 1.0 mg L-1 Kin with 30 PLBs and 6 shoots per callus mass (~5 mm size). The shoots were rooted (70% frequency) on one by fourth-MS medium containing 2.0 mg L-1 indole-3-butyric acid, 200 mg L-1 activated charcoal and additives. The rooted plantlets were hardened and transferred to greenhouse with 63% survival rate. Flow-cytometry based DNA content analysis revealed that the ploidy levels were maintained in in vitro regenerated plants. This is the first report for in vitro plant regeneration in E. ochreata.

Mass propagation of Cymbidium giganteum Wall. ex Lindl. using in vitro seedlings.

In vitro seedlings were used as explants for protocorm like bodies (PLBs) production which in turn were used for regeneration purpose. PLBs were induced from the base of seedlings (1.0-1.5 cm in size) in MS + BAP (8.88 µM). After 90 days of inoculation, PLBs production rate started declining and most of the PLBs turned into plantlets. Preculture of seedlings in 1.0 µM thidiazuron (TDZ) for 7 days and transfer to BAP supplemented medium resulted in production of 16 PLBs per seedling within 90 days of culture. Increase of TDZ concentration to 2.5 µM and preculture time 15 days, resulted in induction of highest number of PLBs (19 PLBs per seedling) in the basal medium. The results emphasized the importance of thidiazuron (TDZ) concentration and preculture time for PLBs proliferation from the base of seedlings. The PLBs thus produced were used for regeneration studies. Irrespective of single, segmented or clumps of PLBs, the regeneration response was 100% in 2,4-D (4.52 µM) and KN (4.64 µM) but when KN was replaced by BAP (8.88 µM), response was observed only in clumps of PLBs, whereas in single and segmented ones it was 99 and 97%, respectively. Regenerants developed stout root system in half strength M medium supplemented with 2.84 µM of IAA and transferred to greenhouse with 90% survival. The present study holds tremendous potential as the mother plant is not destroyed and PLBs are produced as a continuous system.

In vitro regeneration of Coelogyne nervosa A.Rich. and Eria pseudoclavicaulis Blatt., threatened orchids of Western Ghats, India.

The seeds of C. nervosa and E. pseudoclavicaulis were germinated asymbiotically on Knudson C (KC) and Schenk and Hildebrandt basal medium (SH). Growth regulators such as 2,4-Dichlorophenoxyacetic acid (2,4-D) individually and in combinations with benzyladenine (BA) and kinetin were used for callus induction from the protocorm like bodies. Coelogyne nervosa showed maximum (90%) callus induction in Knudson C medium supplemented with 2,4-D (2.26 µM) and Eria pseudoclavicaulis showed 60% callus induction in Schenk and Hildebrandt medium supplemented with 2,4-D (2.26 µM). Calli developed a route of production of protocorm-like bodies and eventually developed into plantlets on transfer to growth regulator free half strength basal medium. The well rooted plants were hardened successfully in the potting mixture containing coconut husk, charcoal, and brick pieces in the ratio 2:1:1.

Effect of different culture conditions on protocorm-like bodies of Dendrobium candidum in bioreactor culture / 中草药

Objective: To explore the effect of different culture conditions on protocorm-like bodies (PLBs) of Dendrobium candidum in bioreactor culture. Methods: Inoculation quantity, ventilation, sucrose concentration, and light intensity were taken to carry out the single factor experiment design. Biomass was determined by drying method, polysaccharide content by phenol-sulfuric acid method, and SPSS16.0 software was used for data analysis. Results: Under the bioreactor culture, the nutrient medium components for the PLBs proliferation of D. candidum and polysaccharide accumulation were 1/2 MS medium supplemented with 1.0 mg/L NAA, 5% coconutmilk, and 3% sucrose, pH value was 6.0. The culture conditions, such as inoculation with 40 g/L, 15 μm pore porous nozzle, aeration volumes by 1.0 and 0.5 L/min used alternately, and photon flux of 2000 lx could improve the proliferation coefficient and polysaccharide contents of PLBs. Conclusion: Different culture conditions have the significant effect on the growth of PLBs and the accumulation of polysaccharide. Suitable culture conditions have the very important practic significance to the biomass of PLBs and main medicinal ingredient production of PLBs.

Research and application of Dendrobium officinale protocorm/protocorm-like bodies / 中国药学杂志

OBJECTIVE: To review the research progress of Dendrobium officinale protocorni/protocorm-like bodies according to relevant references at home and abroad in recent len years, and to explore its development prospect. METHODS: Based on the searched literatures, the research progress in the aspects of culture techniques, chemical constituents and pharmacological effects were summarized. RESULTS AND CONCLUSION: There had been a lot of researches on D. officinale protocorm/PLBs culture techniques, and D. officinale protocorm/PLBs could be used for resource regeneration and production of active polysaccharides, but its material basis and pharmacological activity still need to be further studied.