Germplasm Preservation: Storage of Seed, Pollen and Tissue of Selected Malaysian Orchid Species

In an attempt to develop methods for cheap, long-term preservation of orchid germplasm, seed, pollen and tissue of four local species were subjected to various storage conditions and subsequently determined of their effects on viability. The resultant quality of stored materials varied over the range of conditions and species investigated. Storage of seeds of Arundina graminifolia, Cymbidium finlaysonianum and Spathoglottis plicata at seed moisture content of less than 5 percent and at storage temperature of less than 5°C gave percentage viability that ranged between 60 and 90 percent for a storage duration of more than twelve weeks. Storage in liquid nitrogen (-196°C) gave the highest mean among the temperatures tested, confirming the orthodox nature of the seed

Etiology of bacterial soft rot of Orchids.

Symptoms of bacterial soft rot were observed on the Phalaenopsis sp. and Dendrobium sp. orchids. The disease caused death in many plants, especially those of the Phalaenopsis sp. at the seedling stage and ofyoung plants. Bacteria were consistently isolated on diseased plants. Tests proved the pathogenicity of the isolates on orchids. Steps were camed out to complete Koch's postulate. Based on the cultural, physiological and biochemical properties the pathogen was identified as Erwinia chrysanthemi Burk., McFadden and Dimock 1953

Callus induction in pineapple (Ananas comosus L.) cv. Moris and Josapine.

The induction of callus from Meristemic Globular Bodies (MGB) of two pineapple cultivars, namely Moris and Josapine, under six concentration levels of auxin NAA and six concentration levels of 2,4-D in Murashige and Skoog solid media, was investigated. 2,4-D auxin treatments failed to induce calli in both cultivars. However, 53.71, 75.19 and 85.93 μM levels of auxin NAA caused calli induction in Moris while levels 32.22, 53.71 and 75.19 μM also induced calli Josapine. The percentage of MGB calli formation increased with increasing time of culturing. At 6 weeks of culturing, 83% of Moris MGB explants formed calli on 85.93 μM NAA, while 50% of Josapine MGB explants formed calli on 53.71 μM NAA. Calli cultures have been an essential tool in the in vitro selection of desirable plants under manipulated conditions and from in vitro mutations via somaclonal variation. More importantly, calli are increasingly used for the application of cellular level genetic modification techniques such as the Agrobacterium-mediated transformation, particle bombardment and protoplast isolation and fusion. In this study, auxin NAA successfully initiated and proliferated calli in Moris and Josapine globular meristemic cultures

Genetic stability of in vitro multiplied Phalaenopsis gigantea protocorm-like bodies as affected by chitosan

Chitosan is a carbohydrate polymer derivative of chitin which presents in shell of crustaceans. This biopolymer is a non toxic and environmentally friendly, considered as a plant growth stimulator in some plant species. The present study investigates the effects of chitosan and media types on multiplication and genetic stability of Phalaenopsis gigantea protocorm-like bodies (PLBs). PLBs were inoculated in liquid New Dogashima Medium (NDM) and Vacin and Went (VW) supplemented with various concentrations of chitosan (0, 5, 10, 15, 20 and 25 mg/L). The highest PLB multiplication was observed on VW and NDM supplemented with 10 mg/L chitosan with mean number of PLBs 177 and 147, respectively. Chitosan promoted the formation of juvenile leaves and the highest number was observed in NDM supplemented with 20 mg/L chitosan with mean number of 66 leaves after 8 weeks of culture. Genetic stability was assessed among mother plant and secondary PLBs after 2, 4, 6, and 8 weeks of culture in liquid media. 8 out of 10 ISSR markers produced a total of 275 clear and reproducible bands with mean of 6.9 bands per primer. The secondary PLBs produced during sub-culturing process of chitosan treated liquid culture were genetically uniform and similar to mother plant

Molecular characterization and phylogenetic relationships among and within species of Phalaenopsis (Epidendroideae: Orchidaceae) based on RAPD analysis

Random amplified polymorphic DNA (RAPD) analysis for 20 species of Phalaenopsis was conducted to determine their genetic distances and relationships. Among 20 different primers used for RAPD analysis, 10 primers showed polymorphism, and according to the primer type, 26 to 54 DNA fragments were amplified. A total of 414 polymorphic fragments were generated by 10 primers and used for correlation group analysis. The highest value of Similarity index was 0.28 between Ph. violacea malaysia and Ph. violacea witte. The dendrogram resulting from UPGMA (Unweighted Pair Group Method using Arithmetic average) hierarchical cluster analysis separated the original species into three groups: The first group had five species of Ph. violacea blue, Ph. belina, Ph. violacea malaysia, Ph. violacea witte, and Ph. gigantea; the second group included Ph. lamelligera, Ph. amabilis, Ph. parishii, Ph. labbi nepal, Ph. speciosa, Ph. lobbi yellow, Ph. venosa, Ph. hieroglyphica, and Ph. maculata; the third group consisted of Ph. minho princess, Ph. leopard prince, Ph. mannii, Ph. modesta, Ph. cornucervi and Ph. pantherina. RAPD markers can thus be successfully applied in this economically important group of orchids for the study of molecular characterization and relationships. The data acquired from this study could be used for identification and classification of other orchid genera and oriental Phalaenopsis

Effects of auxin and cytokinin on callus induction in Catharanthus roseus (L.) G. Don

The study was conducted to observe the effect of different concentration and combination of auxin and cytokinin towards the callus induction of C. roseus. Explants comprising of basal leaf with petioles of Catharanthus roseus were cultured onto MS media supplemented with different types and concentrations of auxins (naphthalene acetic acid (NAA) and 2,4-dichlorophenoxyacetic (2,4- D)) and cytokinins (benzyl amino purine (BAP), and kinetin). Calli produced from explants showed differences in response in each of the treatment combinations. Treatments with kinetin and NAA, BAP with 2,4-D (Experiment B) did not differ significantly. Treatment with 3.0 mg L−1 BAP + 3.0 mg L−1 NAA (Experiment C) gave the highest dry weight (2.776 g) suggesting an optimum level of combination for callus induction

In vitro mass multiplication of Artocarpus heterophyllus Lam var. Tekam yellow

A protocol for rapid micropropagation of Artocarpus heterophyllus from seeds of a single fruit was established. The seeds were successfully sterilised using 40% Clorox (20 min) + 20% Clorox (15 min) and 50% Clorox (20 min) + 20% Clorox (15 min). The survivability percentage was 44.44%, while the contamination percentage was 14.81%. Experiments to assess the effect of shoot tip and different node positions on shoot induction, and to test the effect of decapitation on shoot proliferation were performed. The explants used for both experiments were derived from 8-week-old seedlings grown in half-strength MS basal media supplemented with 2.5 mg/L BAP. There was no significant difference in terms of percentage of explants regenerating shoots and mean shoot number produced per explants. However, node 2 significantly produced the highest mean shoot length (2.53 cm). In the decapitation experiment, there was no significant difference in terms of percentage of explants regenerating shoots and mean shoot length. Nevertheless, decapitated shoots significantly produced the highest mean shoot number per explant (18.33). 2.5 mg/L BAP was chosen as the best treatment for shoot induction from seed with a mean shoot number of 7.33 and mean shoot length of 2.95 cm. For shoot multiplication, 1.0 mg/L BAP significantly produced the highest mean shoot number (17.13), while 5.0 mg/L BAP significantly produced the highest shoot length (2.95 cm). For rooting, IBA at 2.5 mg/L and 5.0 mg/L produced the highest mean root number at 18.73 and 17.27 respectively. The highest mean root length (3.37 cm) was significantly obtained in the control treatment. The plantlets were successfully acclimatised in a potting mixture consisting of top soil and organic soil (1:1) with 88.89% survival rate

Cryopreservation of protocorm-like bodies (PLBs) of Phalaenopsis bellina (Rchb.f.) christenson by encapsulation-dehydration

Protocorm-like bodies (PLBs) of Phalaenopsis bellina were successfully cryopreserved by the encapsulation-dehydration approach. Various stages in obtaining successful cryopreservation using this method were optimized. Encapsulated PLBs precultured in half-strength MS medium supplemented with 0.75 M sucrose for 3 days exhibited the highest viability in terms of 2,3,5-triphenyltetrazoliumchloride (TTC) reduction. The amount of sucrose in the PLBs after incubation in different concentrations of sucrose for different periods of time determined by HPLC. The highest sucrose concentration was 7 mg/g of PLBs for the PLBs treated with 0.75 M sucrose for 3 days as compared to the control which had only 1 mg/g sucrose. After sucrose preculture, the PLBs were subjected to desiccation using one of two methods. Desiccation using silica gel was more efficient in reducing PLBs moisture content. After 6 h of desiccation, PLBs desiccated using laminar air flow had 43.5% moisture content while for those desiccated using silica gel had 32% moisture content. PLBs desiccated to different moisture contents were plunged into LN. After storage in LN the encapsulated PLBs were re-warmed. Two weeks after re-warming PLBs viability was determined by TTC reduction and re-growth assessed. Encapsulated PLBs precultured with 0.75 M sucrose for 3 days followed by desiccated using silica gel for 5 h resulting in a moisture content of 39% lead to the highest post re-warming viability in terms of TTC reduction (46.6% of control PLBs) and 30% re-growth