Chiang-Shiong Loh

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.

Cerium and lanthanum promote floral initiation and reproductive growth of Arabidopsis thaliana

The effects of cerium and lanthanum on the vegetative growth, floral initiation and reproductive growth of Arabidopsis thaliana were studied. Addition of cerium nitrate (0.5-10 µM) or lanthanum nitrate (0.5-50 µM) to the culture medium significantly increased the lengths of primary roots, but had no significant effects on the number of rosette leaves produced per plant, plant heights and dry weights during the vegetative growth stage (17 days after seed germination). The percentage of plants bolted was significantly increased with the addition of 0.5-10.0 µM cerium nitrate or lanthanum nitrate. The combination of 0.5 µM cerium nitrate and 0.5 µM lanthanum nitrate was found to be most effective on the induction of floral initiation. The height, dry weight and average number of flower numbers of 35-day-old plants growing in media containing cerium nitrate or/and lanthanum nitrate (0.5-10.0 µM) were found to be significantly higher than those in the control medium. The endogenous levels of cytokinins (zeatin riboside, dihydrozeatin riboside and isopentenyl adenosine) and carbohydrates (sucrose, glucose and fructose) in leaf and root tissues of plants growing in the medium supplemented with 0.5 µM cerium nitrate and 0.5 µM lanthanum nitrate were not significantly different from those of plants in the control medium. Application of 0.5 µM cerium nitrate and 0.5 µM lanthanum nitrate enhanced the effects of 10(-6) M IPA on root growth, plant height and flowering. The role of cerium and lanthanum in promoting floral initiation and reproductive growth and the possibility of developing non-hormonal flowering promoting agents are discussed.

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.

High frequency early in vitro flowering of Dendrobium Madame Thong-In (Orchidaceae)

We have successfully developed a method to induce early in vitro flowering of the self-pollinated seedlings of a tropical orchid hybrid, Dendrobium Madame Thong-In. Transition of vegetative shoot apical meristem to inflorescence meristem was observed when young protocorms were cultured in modified KC liquid medium. In contrast, protocorms cultured on Gelrite-solidified medium only produced axillary shoots and roots. CW was required to trigger the transitional shoot apical meristem and BA enhanced inflorescence stalk initiation and flower bud formation. However, normal flower development was deformed in liquid medium but developed fully upon transferring to two-layered (liquid over Gelrite-solidified) medium. Under optimal condition, in vitro flowering was observed about 5 months after seed sowing. Segregation of flower colours was observed in these seedlings and seedpods formed upon artificial pollination of the in vitro flowers.

Direct somatic embryogenesis, plant regeneration and in vitro flowering in rapid-cycling Brassica napus

Abstract A simple method to induce somatic embryogenesis from seeds of rapid-cycling Brassica napus is described. Seedlings cultured on Murashige and Skoog (MS) basal medium produced somatic embryos directly on hypocotyls and cotyledons after 2 to 3 subcultures onto the same medium. A low pH of the medium (3.5–5) was more conducive to somatic embryogenesis than a higher pH (6 and 7). Embryogenic potential of the seeds was inversely correlated to seed age: about 41–68% of immature seeds between the ages of 14 and 28 days after pollination (DAP) formed somatic embryos compared to 0–11% of the seeds obtained 29–37 DAP. About 54% of the somatic embryos produced secondary embryos after subculturing onto the same medium. The embryogenic potential of the cultures has been maintained on MS basal medium for 2 years (12 generations) without diminution. Up to 75% of the secondary embryos developed into plantlets on MS medium enriched with 10–6 M zeatin, and 40% of these produced flowers when transferred to an optimised flower-induction medium. Viable seeds were produced in self-pollinated in vitro flowers.

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.

Direct shoot bud formation from leaf explants of seedlings and mature mangosteen (Garcinia mangostana L.) trees

Abstract This paper reports the direct regeneration of shoot buds from leaf segments of mangosteen (Garcinia mangostana L.) cultured in vitro. Explants were taken from 1–3-year-old seedlings and 16 year-old mature fruiting trees. The optimal level of benzylaminopurine (BAP) for shoot bud development from seedling leaf explants was 5 mg l−1. Higher concentrations were also effective but shoot buds were clustered and stunted. Very young red leaves, 2–3 cm long, from mature trees also produced shoot buds on this medium. When leaves from mature trees that had turned from red to green were used, the frequency of shoot bud formation decreased. The number of vegetative buds was higher in red leaves from seedlings compared with young red leaves from mature trees. Secondary shoot buds also developed on leaves of cultured epiphyllous shoots from both seedlings and mature trees. Shoots from leaves of seedlings and mature trees, measuring more than 2 cm were rooted with an acute auxin treatment. Plantlets from seedling leaf explants were successfully acclimatised and established in soil.

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.

Clonal propagation of guava (Psidium guajava L.) from seedlings and grafted plants and adventitious shoot formation in vitro

Abstract Different parts of guava ( Psidium guajava L.) seedlings and the nodal segments of grafted plants were used as explants for in vitro culture. A high frequency (75–100%) of shoot regeneration was obtained from seedling hypocotyl, shoot tip and nodal segments cultured in MS medium with or without BA. The optimal BA Level for shoot tips and nodal segments is 0.1 mg l −1 . Nodal segments form regenerated shoots were used for further multiplication; an average of 3.2 shoots segment −1 could be obtained after a period of 8 weeks in medium with 0.5 mg l −1 BA. Shoots were also regenerated from leaf explants. Regenerated shoots were rooted in basal medium with 100% rooting frequency and more than 90% of the plantlets were successfully established in soil.