Chong-Jin Goh

An in vitro method for rapid regeneration of a monopodial orchid hybrid Aranda Deborah using thin section culture

A thin section culture system for rapid regeneration of the monopodial orchid hybrid Aranda Deborah has been developed. Thin sections (0.6–0.7mm thick) obtained by transverse sectioning of a single shoot tip (6–7mm), when cultured in Vacin and Went medium enriched with coconut water (20% v/v), produced an average 13.6 protocorm-like bodies (PLB) after 45 days, compared to 2.7 PLB formed by a single 6–7 mm long shoot tip under same culture condition. Addition of α-naphthaleneacetic acid to Vacin and Went medium enriched with coconut water further increased PLB production by thin sections. PLB developed into plantlets on solid Vacin and Went medium containing 10% (v/v) coconut water and 0.5 g l−1 activated charcoal. With this procedure, more than 80,000 plantlets could be produced from thin sections obtained from a single shoot tip in a year as compared to nearly 11,000 plantlets produced by the conventional shoot tip method.

Micropropagation of citrus mitis blanco-multiple bud formation from shoot and root explants in the presence of 6-Benzylaminopurine

Different vegetative parts of Citrus mitis seedlings and mature plants were used as explants in culture. Shoots were readily produced from epicotyl, leaf, shoot tip and nodal segments of seedling explants. A high frequency (66–100%) of shoot regeneration was obtained from shoot tip and nodal stem segments of mature plants cultured on medium with or without benzylaminopurine (BAP). Bud induction from roots was examined in 3 systems: (a) intact roots on whole seedlings; (b) intact roots on decapitated (shootless) seedlings; (c) excised roots. The highest yield of buds was obtained from intact roots on whole seedlingx cultured in medium with 0.5 mg 1−1 BAP, from which 275 shoots were obtained from 13 cultures in 9 weeks. Regenerated shoots were rooted in basal medium and the plantlets were successfully transplanted to soil. The role of growth substances on bud formation from roots is discussed.

A Method for Isolation of Total RNA from Fruit Tissues of Banana

We describe a rapid and efficient method for isolation of total RNA from banana fruit tissues. The RNA was extracted with a high ionic strength buffer at room temperature. The proteins, genomic DNA and secondary metabolites in the extract were then removed by precipitation with pre-cooled potassium acetate and repeated phenol/chloroform/isoamyl alcohol extractions. The RNA was recovered by ethanol precipitation. It was relatively free of ribonucleases and was suitable for RT-PCR and northern blot analysis. The procedure can be completed in less than 4 hours.

High frequency direct shoot bud regeneration from excised leaves of mangosteen (Garcinia mangostana L.)

Abstract An improved procedure for direct shoot bud regeneration from young leaves of mangosteen (Garcinia mangostana L.) has been developed. Regeneration was achieved by culturing 3-mm transverse sections of about 10-day-old leaves on woody plant medium enriched with 20 μM benzyladenine (BA), 20 g/l sucrose and 2.5 g/l Phytagel. A wound response in the presence of BA at the time of culture was found to be essential for shoot bud induction. Explant size, concentration of BA in the medium, timing of addition of BA to the medium as well as the time of wounding of explant significantly influenced shoot bud regeneration. Leaves from field-grown seedlings produced an average of 45 shoot buds per leaf after 50 days of culture, compared with 8 shoot buds per leaf from in vitro shoots. Regenerated shoots (5–6 mm tall) excised from the explants required BA (5 μM) for further growth. Rooting was induced with indolebutyric acid (IBA) when the shoots reached 10–15 mm and were established in vermiculite/sand mixture in pots.

Plantlet regeneration through different morphogenic pathways in pommelo tissue culture

Pommelo (Citrus grandis Osbeck) plantlets were regenerated through different morphogenic pathways in culture. Multiple shoot regeneration through de novo organogenesis was obtained with epicotyl segments and root cultures. Shoot regeneration was observed in 84% of the midtal epicotyl segments cultured in Murashige and Skoogs medium (MS) with 2.2 μM benzyladenine (BA) and 83% of the middle and proximal epicotyl segments cultured on basal medium. Isolated root segments cultured on medium containing 0.089 μM BA showed best shoot regeneration at 71% with an average of 3.3 shoots per segment. Callus tissues derived from cotyledon and leaf explants regenerated shoots on BA-enriched medium. Shoots were also obtained at high frequencies from shoot-tip and nodal explants. Roots developed when regenerated shoots were excised and cultured on half strength MS medium with 2.5 μM indolebutyric acid.

An efficient in vitro method for mass propagation of a woody ornamentalIxora coccinea L.

Various factors that affect culture establishment, shoot growth, proliferation and rooting ofIxora coccinea L., a woody shrub, were studied. Stem cuttings (decapitated shoot, three nodes) were the most suitable explants for multiple-shoot proliferation, and when cultured on a woody plant medium (WPM) containing 2.5 μM BA produced axillary shoots which branched repeatedly, yielding an average of 27 shoots per explant after 6 weeks in culture. Kinetin, 2-iP, zeatin and thidiazuron all induced multiple-shoot formation, but were less effective than BA. While the presence of IAA in the multiplication medium was detrimental to shoot proliferation, shoot growth was not affected by IAA. The production of large amounts of basal callus and vitrification of shoots were the major problems to be avoided in proliferating shoot cultures. Addition of TIBA to the multiplication medium markedly reduced basal callusing, while sealing the culture vessels with a fluorocarbon polymer (tetrafluoroethyleneperfluoroalkyl vinyl ether) film (Neoflon PFA film) almost completely eliminated vitrification. A reduction in the number of vitrified shoots was also achieved with AVG treatment. Following this protocol of using BA-supplemented WPM and Neoflon film, it would be possible to produce more than 100,000 plants from a single stem cutting in 1 year.

Differential gene expression during floral transition in an orchid hybrid Dendrobium Madame Thong-In

Abstract We have developed an in vitro orchid (Dendrobium grex Madame Thong-In) system as a model for investigating gene expression during floral transition. Total RNA was isolated from vegetative shoot apical meristems (VSAM) and transitional shoot apical meristems (TSAM). Using the mRNA differential display method, we identified 53 cDNA clones differentially expressed in VSAMs and 16 cDNA fragments specifically expressed in TSAMs. Northern blot analysis confirmed that, on the basis of their transcripts, 12 cDNAs decreased and 8 cDNAs increased during floral transition. Sequence analysis suggested that 5 clones are likely to be transcription factors, including one MADS-box gene of the AGL2 subfamily, one class 1 knox gene and one homolog of the Drosophila seven-up gene. Other clones showed homology with those coding for Arabidopsis SOD copper chaperone, CKI and the tobacco 21D7 protein.

The isolation, molecular characterization and expression of dihydroflavonol 4-reductase cDNA in the orchid, Bromheadia finlaysoniana

Abstract The cDNA, ODFR, encoding dihydroflavonol 4-reductase (DFR) was isolated from a cDNA library constructed from Bromheadia finlaysoniana flowers using a homologous probe generated via PCR. Southern blot analysis suggests that DFR is represented by a single gene in this orchid while Northern blot and reverse transcriptase-PCR analysis showed that DFR is expressed in all purple colored tissues. A phylogenetic tree constructed based on multiple alignments among DFR sequences showed that the DFR genes isolated from barley, maize and B. finlaysoniana, which are all monocotyledons, are more similar to each other than to DFR sequences from dicotyledonous plants.

Direct somatic embryogenesis from protoplasts of Citrus mitis Blanco

Protoplasts isolated from embryogenic suspension cultures of Citrus mitis were cultured in a medium without any plant growth substances. Somatic embryos developed directly from protoplasts without an obvious intervening callus phase. As many as 1,800 somatic embryos developed from 4 ml of protoplast suspension (density 2×106/ml) cultured for 35 days. Upon transferring the embryoids to medium with 1 mgl−1 GA3, they developed into plant-lets. Rooted plantlets were obtained in 3 months after protoplast isolation.

Cloning and characterization of full-length cDNA clones encoding chalcone synthase from the orchid Bromheadia finlaysoniana

Abstract cDNA clones encoding chalcone synthase (CHS) (EC 2.3.1.74), a key enzyme involved in flavonoid and anthocyanin biosynthesis were isolated from a cDNA library constructed from flowers of the orchid, Bromheadia finlaysoniana (Lindl.) Rchb.f using a homologous probe generated via PCR. The complete nucleotide sequences of the 3 clones, OCHS3 , OCHS4 and OCHS8 were determined. The lengths of OCHS3 , OCHS4 and OCHS8 are 1445, 1 382 and 1 439 bp, respectively. All the cDNAs contained a single open reading frame of 1 185 bp, encoding a polypeptide of 394 amino acids with a calculated molecular mass of 42.9 kDa. The nucleotide sequences of OCHS3 , OCHS4 and OCHS8 showed that they were not identical but exhibited a high degree of similarity (> 97 %). The deduced amino acid sequence showed 76–82 % homology, whereas the nucleotide sequence showed 59–68 % homology to CHS of other plants. Southern blot analysis indicated that CHS is possibly encoded by a small multigene family in the genome of B. finlaysoniana . Northern blot analysis revealed that CHS was expressed at very high levels in young leaves which are flushed with anthocyanin, whereas it was expressed at much lower levels in flowers which are faintly coloured and completely undetectable in other organs. Within different floral organs, CHS was found to be expressed at the highest levels in sepals, followed by stalks and columns whereas it was hardly detectable in lips and petals. However, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, a significantly more sensitive method compared to northern hybridization, demonstrated that CHS transcripts could be amplified from all parts of the plant and all floral organs examined.