VW Medium Research Articles

In vitro encapsulation technique for conservation ofDendrobium friedericksianum

Efficient artificial seed protocol for the orchid Dendrobium friedericksianum Rchb.f. was developed for in vitro conservation study. Dendrobium friedericksianum Rchb.f. is a beautiful native Thai wild orchid found in the eastern provinces of Thailand especially in Chanthaburi province. Currently, illegal forest harvests are impacting this orchid. Meanwhile, environmental changes due to wood-cutting and the decreasing rate of natural breeding cause decreasing numbers of this orchid and lead to a higher risk of extinction. For the reasons mentioned, we have been trying to either keep or preserve the orchid strains. The present study was to develop an effective and applicable protocol for short term in vitro storage of protocorm-like bodies (PLBs) of this orchid species. PLBs were encapsulated in 3% sodium alginate (Na-alginate) and 100 mM calcium chloride (CaCl2.2H2O). The efficiency of survival declined with both increasing storage duration and storage temperature. The encapsulated PLBs were stored at 4 and 25°C for 30, 60, 90, 150 and 180 days. After storage, PLBs were transferred to modified VW medium to determine the time taken for germination, the germination percentage, and the morphological categorization of regenerated PLBs. Encapsulated PLBs stored at 4°C had rapid deterioration and complete death within 150 days, while those stored at 25°C were more tolerant to storage. Encapsulated PLBs survived longer when stored at 25°C compared to 4°C. All plantlets survived after acclimatization when transferred to greenhouse.

INDUCTION OF SPOROPHYTIC DIVISION IN ORCHIDS MICROSPORES BY STRESS

<p>Orchid is one of the important ornamental plants in Indonesia this plant generally propagated by seed. Enhancing quality of this plant through breeding technology by various plant tissue culture methods and biotechnology, including doubled haploid technology are necessary. The most efficient method in creating doubled haploids plant is via microspore embryogenesis. We have develop new, innovative doubled haploid technology using the technique of isolated microspore culture. The goals are to obtain data on the male gametophyte development, viable embryogenic microspores, microspores derived embryos and double haploid plants of Orchid. <br />Development of male gametophytes were analysed by isolation of microspores and pollen at various stages and staining with DAPI. Isolated orchid buds of Dendrobium hybrid 1, Vanda tricolor and Spathoglotis plicata were subjected to cold temperatures (4oC) for 7 days, microspores were then isolated by crushing the pollinia using glass rod and cultured them in embryogenesis A2, NP, MS and VW medium, viability of the microspores were determine by using Flourescein diacetate (FDA). Isolated Orchid pollinia were cultured in starvation medium B at various temperatures and duration of time to evaluate embryogenic response, isolated microspores then were cultured further in the basic embryogenesis medium and incubated at 25 oC in the darkness. <br />The result showed that floral characteristics for the late-uninucleate stage of the microspores were different for every orchid spesies. Ovulum lenght was used for Vanda, while in Dendrobium, Phalaenopsis, Arachnis, Spathoglottis plicata and Cattleya, varied length of flower bud was used. Isolated microspores of Dendrobium hybrid 1, Vanda tricolor and Spathoglotis plicata at 7th days of culture in different media formulation showing different respond of viability. Medium A2 keeping viability of Dendrobium hybrid 1 microspores better than any other medium, while in Vanda tricolor and Spathoglotis plicata embryogenesis NP medium was superior. Incubation of orchid pollinia at 4 and 25 oC were successfully maintain viability of the microspores during starvation periods but not able to block gametophytic development. In contrast starved pollinia at 33oC were succesfully block gametophytic development, percentage of embryogenic microspores after starvation of isolated pollinia at 33°C for 4 days was superior compare to any other treatments. Symmetrical divisions and some multicellular structures were observed, which were clear indication for the sporophytic development of microspore-derived embryos, they had developed and after a few weeks they degenerated and died.</p><p><br /><strong>Keywords</strong>: flower bud-pollinia-microspore-stress-embriogenic-embryo-Orchid</p>

Kandungan Klorofil Daun Planlet Anggrek Bulan (Phalaenopsis amabilis (L.) Bl.) Hasil Seleksi dengan Asam Salisilat secara In vitro

Phalaenopsis amabilis is one of Indonesias national flowers. P.amabilis has beautifulflowers and exciting and high economic value. However, there are some constraints in its development, one of which is Fusarium wilt caused byFusarium oxysporum, for this is not maximized in handling. One of effective procedure to control the disease is to use salicylic acid as a selective agent resistance to the disease. Study of the effect of the addition of salicylic acid(concentrations 15, 30, 45 and 60 ppm) have been studied in vitro in mediumVW(Vacint and Went) on the content ofchlorophyll a, b, and total leaf plantlets P.amabilis. The research was implemented in tissue culture laboratory, Department of Biology, Faculty of MIPA, University of Lampung from February to March 2015. This study used a completely randomized design with 3 replications. Analysis of variance and LSD test performed at 5% significance level. Calculation and extraction of chlorophyll was conducted by Harbourne method(1987). The absorbance was measured with a (Shimadzu UV 800)spectrophotometer at wavelength of 663 and 646 nm. The results showed that the content of chlorophyll a, b, and total plantlets of P. amabilis leaves significantly increased at the concentration of salicylic acid in the medium VW 15, 30, 45 and 60 ppm compared with control(0 ppm).Key words : Phalaenopsis amabilis plantlets, chlolophyll, salicylic acid, VW medium.

IN VITRO PROPAGATION OF NATIVE ORCHID DENDROBIUM SPECTABILE (BLUME) MIQ.

The forests of Papua and New Guinea have a rich variety of flowering plants. One orchid species from Papua and New Guinea is the Dendrobium spectabile. The D. spectabile has unique characteristics in which the flowers are slightly different in a florescent light. The flowers of D. spectabile are dissimilar with most of the other Dendrobium sp. Unlike the common Dendrobium, the D. spectabile will not flower without much higher levels of light. They will even take full sunlight for longer periods of time. The D. spectabile will start flowering when it reaches 4-5 years old. Recently, many people exploit this species, causing it to become very rare. Therefore, it is important to study the propagation method in order to conserve this species. In this study, in vitro propagation using different culture media were explored. The aim of this study is to compare two different media, i.e. medium VW with modification and medium W3, which has been known to give highest respond for growth of D. spectabile. The experiment was carried out at Tissue Culture Laboratory, CV Ayu Orchid Bali, from April-August 2012. The experiment employs Completely Randomized Design (CRD) design with 20 replications for each media. The result of the experiment showed that the highest respond for growth of D. spectabile was VW medium. This was indicated by higher number of plantlets (average 306.75), number of leaves (average 3.45) and plant height (average 2.65 cm). However, the leaves of plantlets using medium W3 showed dark green and the explants were stronger than explants in medium VW.

다양한 배지종류, sucrose 농도 및 갈변억제물질 처리에 의한 팔레놉시스 PLB 증식 및 재분화 체계확립

Abstract To establish an efficient proliferation and rege-neration of PLBs (protocorm-like bodies) of Phalaenopsis plants, a variety of propagation medium types, various concentraions of sucrose as well as liquid and solid type were tested in this study. Further, activated charcoal, citric acid and ascorbic acid were compared whether these agents are suppose to reduce the browning in culture process using PLBs of Phalanopsis plants. With regard to the proper propagation medium, VW medium showed 1.3 ~ 2 times highr than those of other medium in an index of increasing for fresh weight and 50% higher than those of other medium in the frequency of shoot regeneration. However, regarding liquid and solid types of culture, there were no significant differences in the proliferation of PLBs and regeneration of shoots from PLBs. In the experiment for a variety of sucrose concentrations (0 ~ 50 g/l), 10 g of sucrose showed 30 ~ 50% higher than other concentrations in increasing index and 10 ~ 50% higher in the regeneration of shoots from PLBs. Regarding the reduction of browning in tissue culture via PLBs of

Chitosan specificity for the in vitro seed germination of two Dendrobium orchids (Asparagales: Orchidaceae)

The effects of different types of chitosan on seed germination and protocorm development were determined for two orchid species, Dendrobium bigibbum var. compactum and Dendrobium formosum. Six chitosan types derived from polymer or oligomer chitosan each with 70, 80 or 90% levels of deacetylation (P70, P80, P90, O70, O80 and O90, respectively), were evaluated as direct medium supplements at 0, 10, 20, 40 or 80 mg/L in modified VW medium by following seed germination and protocorm growth for 12 weeks. Chitosan of all six tested types and four concentrations were found to significantly enhance the proportion of D. formosum seeds that germinated, when compared to these germinated without chitosan. In contrast, chitosan caused no enhanced germination rate was noted for D. bigibbum var. compactum with all tested chitosans and doses tested. However, almost all types of chitosan at 10 mg/L, except O90, were able to significantly improve the growth of D. bigibbum var. compactum protocorms, whilst 10 or 20 mg/L of P70 chitosan was the best formula to enhance the growth of D. formosum protocorms. It is concluded that chitosan responses in seed germination and protocorm development were somewhat species and developmental stage dependent. Therefore, the appropriate chitosan application for each plant species should be evaluated first before use.

Estudo da germinação e crescimento in vitro de Hadrolaelia tenebrosa (Rolfe) Chiron & V.P. Castro (Orchidaceae), uma espécie da flora brasileira ameaçada de extinção

As orquídeas são espécies seriamente ameaçadas de extinção. Por isso, estudos sobre a propagação e desenvolvimento dessas plantas são extremamente importantes. Assim, este trabalho procurou estudar a influência do meio de cultura na germinação in vitro de sementes de Hadrolaelia tenebrosa, no desenvolvimento inicial dos protocormos até a formação das plântulas, bem como no crescimento dessas durante o primeiro ano de cultivo in vitro. Os resultados demonstraram que o método ideal para a multiplicação de Hadrolaelia tenebrosa é a utilização do meio Knudson C para a germinação das sementes. Após seis meses as plântulas devem ser transferidas para o meio VW e nele mantidas até o início da aclimatização. Ressalta-se o fato de que para essa espécie o meio de cultura mais eficaz para a germinação, não foi o mais adequado para o desenvolvimento das plantas. Estas informações são de grande importância para a otimização do processo de propagação de espécies de orquídeas.

EFFECT OF PLANT GROWTH REGULATORS ON CLONAL PRODUCTION THROUGH BASAL STEM EXPLANT CULTURE OF PHALAENOPSIS HYBRID

This study was conducted to develop the clonal propagation technique through in vitro culture using basal stem explants in Phalaenopsis hybrid grown in vitro. The highest frequency of protocorm-like body (PLB) formation was obtained when basal stem explants were cultured on VW medium containing 30 g/L sucrose, 500 mg/L activated charcoal, 150 ml/L coconut water, 1 mg/L NAA, 5 mg/L 2iP and 2.5 g/L gelrite. PLBs transferred to Hyponex medium were regenerated to plantlets. Plantlets transferred to plastic pots containing spagnum moss were developed and successfully acclimatized under greenhouse. Flowers opened in plants regenerated from basal stem explants. The flower was not different from both mother plant and plant induced through clonal propagation of Phalaenopsis hybrid.

IN VITRO SHOOT AND LEAF PROLIFERATION OF ENCYCLIA TAMPENSIS (LIND.) ORCHIDS

Encyclia is a genus of orchids with four members native to Florida that are Endangered or commercially exploited. If a commercial orchid tissue culture media was found that could be used to propagate Encyclia, the resulting plants could be introduced into protected habitats to increase the wild populations or could be grown commercially to lessen the demand for collected plants. Six month Encyclia tampensis (Lindl.) seedlings were grown in Orchid Maintenance/Replate Medium w/Banana and Charcoal with 6 g·L–1 agar (C), ½ strength Orchid Multiplication Medium with 6 g·L–1 agar and 10 g·L–1 charcoal (OM), Knudson C Orchid Medium – Morel Modification with 6 g·L–1 agar and 10 g·L–1 charcoal (K), or Vacin and Went Modified Orchid Medium w/agar and sucrose with 10 g·L–1 charcoal (VW) for seven weeks. Plants grown in OM and VW media both produced more shoots and roots (7.56 leaves, 1.92 shoots and 6.67 leaves, 1.76 shoots respectively) than the control medium (5.87 leaves, 1.70 shoots) with OM producing the highest averages. Plants grown in the K medium averaged significantly less shoots and leaves than the other three media (4.94 leaves, 1.35 shoots).