Hybrid Cymbidium Research Articles

Hyaluronic Acid Can be a New Plant Growth Regulator for Hybrid Cymbidium Micropropagation

Present authors investigated the effect of two biopolymer hyaluronic acid (HA9) and chitosan (Chitosan H), and N6-benzyladenine (BA) supplemented with modified MS on organogenesis in protocorm-like bodies (PLBs) of a Cymbidium cultivar. The percentages of PLB formation from cultures showed 100 % in all treatments except for control (82.7%), 0.1 mg/l HA9 (93.3%) and 10 mg/l Chitosan H (93.3%) treatments. Addition of 0.1 mg/l HA9 to culture medium was highly efficient for PLB multiplication. Both 10 mg/l HA9 and 1.0 mg/l BA significantly increased the number of shoots in cultures. The maximum percentage of shoot (100%) was obtained at 1.0 mg/l chitosan H. Increase in fresh weight showed higher values at BA treatments. Information gained from this study shows that hyaluronic acid (HA9) which is new additive for plant tissue culture act as growth regulator for hybrid Cymbidium micropropagation. DOI: http://dx.doi.org/10.3329/ptcb.v22i1.11261Plant Tissue Cult. & Biotech. 22(1): 59-64, 2012 (June)

Symbiosis between fungi and the hybrid Cymbidium and its mycorrhizal microstructures

Tissue culture seedlings of the hybrid Cymbidium were inoculated with six different fungal strains, isolated from the roots of different wild terrestrial orchids. About three months later, the average increment of fresh weight of seedlings inoculated with strains CF1, CF3 and CF12 were respectively 130.26%, 345.65% and 153.34% while that of the control was only 88.40%. The dif- ferences between the three treatments and the control were statistically significant (α = 0.05), highlighting the treatment with strain CF3 (α = 0.01). In addition, the three strains were obtained by re-isolating. Pelotons, regarded as typical structures of orchid my- corrhiza, were also found in the inoculating roots under a microscope. It seems that the strains of CF1, CF3, and CF12 are associated with the hybrid Cymbidium and supplied the orchid with nutrition. It can be confirmed that the three strains are beneficial for the seedlings of this hybrid.

Analysis of the Germplasm Resources and Genetic Relationships Among Hybrid Cymbidium Cultivars and Native Species with RAPD Markers

Abstract The random amplified polymorphic DNA (RAPD) marker was assessed to detect the genetic relationships among 48 hybrid Cymbidium cultivars from Japan, Korea, China, and USA, and 2 species of native Cymbidium. Twenty primers were screened from 100 random decamer primers, and a total of 258 DNA bands were amplified, 253 of which (98.1%) were polymorphic. The average number of polymorphic DNA bands amplified by each primer was 12.6. All cultivars were distinguishable when a number of primers were considered. Genetic similarities among the cultivars and species were estimated based on the amount of band sharing ranging from 0.364-0.817 with an average of 0.581. According to the data, a dendrogram of genetic relationship, which was constructed using the UPGMA method, showed that all the tested cultivars and native species were classified into five cluster groups with the similarity coefficient of 0.592. It revealed that the genetic relationships among tested accessions were to some extent related with their origin, flower colour, branch type, and genealogy. It further indicated that the RAPD technique is a useful tool for studying the genetic relationships among hybrid Cymbidium cultivars.

Multiple Regeneration Pathways via Thin Cell Layers in Hybrid Cymbidium (Orchidaceae)

Protocorm-like body (PLB) and subsequent shoot development in hybrid Cymbidium Twilight Moon ‘Day Light’ can be established in vitro via 3 pathways: PLBs, PLB thin cell layers (TCLs), or embryogenic callus (EC). Traditionally Cymbidium hybrids are mass-produced commercially through the neo-formation of secondary PLBs (2° PLB) from initial or primary PLBs (1° PLB) or PLB segments, or from PLB TCLs, resulting in a moderate number of 2° PLBs (average 4.46 2° PLBs/1° bisected PLB, or 1.12 2° PLBs/ PLB TCL). This study shows that EC can be induced from 1° PLBs or PLB TCLs. Thereafter, resulting 2° PLBs (average 22.1 2° PLBs/EC cluster derived from 1° PLB) form directly from the EC on the same medium or following the transfer of EC onto PGR-free medium. By flow cytometry and PCR-RAPD analysis, the cytogenetic stability of 1° PLBs, of resulting 2° PLBs and EC, and plants derived therefrom was demonstrated.

Priming abiotic factors for optimal hybrid Cymbidium (Orchidaceae) PLB and callus induction, plantlet formation, and their subsequent cytogenetic stability analysis

Abstract Abiotic factors affect the induction of PLBs and callus in hybrid Cymbidium Twilight Moon ‘Day Light’. The initiation and proliferation of new PLBs and callus could be achieved on NAA and kinetin, supplemented at 0.1 mg l −1 each, respectively, both within 45–60 days. Bacto agar was found to be the most suitable solidifying agent for PLB induction, although a higher shoot fresh weight was obtained on Gelrite; a pH 5.3 was optimal while pH 4.5 caused 100% explant necrosis; coconut water, when supplied at 10–20% (v/v) resulted in a significant increase in the number of PLBs formed per PLB segment (23.1 versus 14.6 in controls) while a massive (almost four-fold) increase in fresh top weight occurred when PLB explants were placed in liquid culture, as a result of hyperhydricity; Fe-EDTA (1 mg l −1 ) and activated charcoal (1 g l −1 ) stimulated total fresh weight and PLB formation in the presence of PGRs; PLB formation decreased but total fresh shoot weight increased with the addition of niacin or myo -inositol, both vitamins. Dark-grown PLB-induced plants were etiolated and had longer internodes and higher fresh weight than light-grown control plants at 45 μmol m −2 s −1 ; at 15 μmol m −2 s −1 shoots were slightly etiolated, fragile, and PLB formation was scarce. RAPD and mtDNA analysis of all resultant PLBs, callus or plants showed them to be genetically identical, with comparable chlorophyll contents. Despite the detection of cytological variation between different plant parts, little variation resulted from abiotic factor treatment.

Priming biotic factors for optimal protocorm-like body and callus induction in hybrid Cymbidium (Orchidaceae), and assessment of cytogenetic stability in regenerated plantlets

Protocorm-like bodies (PLBs) and callus were induced in epiphytic hybrid Cymbidium Twilight Moon ‘Day Light’, where induction capacity was strongly explant dependent. Following the use of various explant sources (PLB, leaf tip or base, root tip or base, cell and tissue ‘suspension’), highest PLB formation and callus induction occurred when we used whole PLBs, PLB segments or PLB transverse thin cell layers (tTCLs) or longitudinal TCLs (lTCLs). Plantlet growth and photosynthetic state from whole or bisected PLBs, as well as from tTCLs were not significantly different, after analysis of chlorophyll content. However plantlets generated from lTCLs showed lower values for growth and photosynthetic parameters. All resultant plants were shown to be cytogenetically identical using RAPD and mtDNA analysis despite cytological variation and endopolyploidy occuring between different plant parts. Acclimatization and survival rate was shown to be 100% in the generated plants.