Growth Of Protocorm-like Bodies Research Articles

Use of Light Spectra for Efficient Production of PLBs in Temperate Terrestrial Orchids

Wild orchids, especially the terrestrial temperate ones are endangered species due to challenges in their natural habitats. Therefore, there is an urgent need to introduce efficient propagation methods to overcome the natural reproduction problems of these orchids. In this study, the effects of different light spectrums, explant types, wounding, and combinations of different plant growth regulators (PGRs) on direct somatic embryogenesis (DSE) of two species of these endangered orchids listed in the conservation category, were studied. The highest percentages of DSE formation and embryo germination were observed in Dactylorhiza umberosa protocorm explants exposed to white light (400–730 nm) and in Epipactis veratifolia protocorm explants exposed to a combination of red and far-red spectra (R: FR = 70:30). This occurred while red (610–700) alone and in combination with far-red (710–730 nm) spectrum induced embryogenesis more than the blue spectrum and dark condition in E. veratifolia. Thidiazuron (TDZ, 3 mg L−1), produced the highest percentage of protocorm-like bodies (PLBs) on protocorm explants in both orchids. Kinetin (Kin, 2 mg L−1) and Benzyladenine (BA 3 mg L−1) had the most effect on the survival and growth of PLBs, respectively, in D. umberosa and E. veratifolia. Species did not show similar embryogenesis responses under light spectrums. In a medium containing 3 mg L−1 TDZ, white light and R-FR spectra produced the most PLBs on wounded protocorm explants of D. umberosa and E. veratifolia respectively. The developmental stage of apical meristem of PLBs in both species was more advanced under R-B spectra, compared to others.

Protocorm Like Bodies (PLBs) dendrobium orchid “Gatton Sunray”

The Dendrobium Orchid Gatton Sunray is a hybrid orchid of Dendrobium pulchellum × Dendrobium illustre. A study was aimed to find the best type of medium and type of banana for the multiplication of Protocorm-Like Bodies (PLBs) of this orchid. This research was conducted from February to September 2020 at the Plant Tissue Culture Laboratory, Faculty of Agriculture, The University of Bengkulu. Treatments included type of media and cultivar of ripe banana fruit, added as source of organic complex, consisted of 4 cultivars, namely Ambon Curup, Kepok, Tanduk and Mas. Ripe bananas were mashed and added to the in vitro medium as much as 50 g.L-1. Types of media consist of Murashige and Skoog (MS), ½ Doses of Murashige and Skoog (MS) media, and Complex of fertilizer media. The results showed that the ½ MS medium added with 50 g.L-1 cv. Kepok fruit produced the greatest multiplication (11.8 PLBs per jar), the best growth of PLBs, including the greatest PLBs number (28.5 PLBs per jar), the heaviest fresh weight (1.30 g per jar) and the longest PLBs (2.56 cm).

OPTIMIZATION PARAMETER FOR PRODUCTION OF PROTOCORM-LIKE BODY AND GROWTH OF GRAMMATOPHYLLUM SPECIOSUM

Micropropagation of orchids are often through the production of Protocorm Like Bodies (PLBs) and this research conducted a study to discover the optimal condition for the growth of Protocorm Like Bodies (PLBs) and also plantlets regeneration of Grammatophyllum speciosum Several aspects such as different carbon sources (sucrose, maltose) and the presence of charcoal alongside with the variety of concentration of agar were taken into account for the optimization. This study discovered that the 30 g/L of sucrose in 1 mg/L of BAP was superior in producing heavy PLBs. However, the trend differs in the number of plantlets where maltose (5 g/L) is seen to produce a higher number of plantlets compared to sucrose. Charcoal plays an important role in the growth of PLBs where the heaviest PLBs were recorded in the medium supplemented with 0.20 % of charcoal along with 4.5 g of agar. Vice versa relationship was recorded for the number of plantlets where without charcoal in 4.5 g of agar produce the highest number of plantlets.

Efficient Chromosome Doubling of an Interspecific Hybrid Dendrobium Stardust ‘Fire Bird’ by Treatment of Amiprofos-methyl to Protocorm-Like Body

Dendrobium Stardust ‘Fire Bird’ is an interspecific hybrid between D. unicum and D. Ukon ‘Arai’ and is known to be sterile and prevents further breeding. Chromosome doubling is an essential method to increase the size of plant organs, such as flowers, and restore fertility of interspecific hybrids, which enables further breeding for ornamental traits. To induce chromosome doubling, the protocorm-like bodies (PLBs) of a given variety were treated in vitro for 6 to 48 h with 10 to 160 mg L−1 amiprofos-methyl (APM). Higher APM concentrations resulted in lower rates of survival of treated PLBs. Furthermore, subsequent growth of PLBs was hampered with prolonged APM treatment, although 10 and 20 mg L−1 APM was optimal for doubling chromosomes. Plantlets with a swollen and shorter stem were observed after the APM treatment and these plants were also chromosome-doubled and became a distinctive morphological marker for the primary selection of chromosome-doubled plants. Chromosome-doubled plants of D. Stardust ‘Fire Bird’ will be utilized for the future breeding of unique Dendrobium cultivars.

Effect of two bio-polysaccharides on organogenesis of protocorm-like bodies in Phalaenopsis cultured in vitro

Different types of bio-polysaccharide play a vital role in the growth of PLBs cultured in vitro. In this study, to we investigated the potential impacts of two bio-polymers,: hyaluronic acid (HA9) and sodium alginate (ALG) on the organogenesis of protocorm-like bodies (PLBs) in Phalaenopsis under white LED lights. PLBs of Phalaenopsis ‘Fmk02010’ were explanted on modified MS medium with different concentrations of HA and (ALG). The highest average number of PLBs per explant (24.6) was recorded for ALG alone at a concentration of 0.01mg/L, and the fresh weight was also highest at the same concentration. The combination of 0.01mg/L ALG and 0.01mg/L HA also resulted in a large number of PLBs (23.8) and high fresh weight. As opposed to, the highest number of shoots /explant (3.6) was observed at the treatment of the combination of 1mg/L ALG and 10mg/L HA. This study shows that the application of ALG and HA alone, and in combination, at low concentrations, increased the average number of PLBs and the amount of fresh weight, but shoot formation was higher at a high concentration compared with control.

Optimization of genetic transformation of orchid (Dendrobium Sonia) mediated by Agrobacterium tumefaciens

Dendrobium Sonia is a popular orchid and used for many purposes in Vietnam. In this study, the transformation is set on protocorm like bodies (PLBs) mediated by Agrobacterium tumefaciens containing vector with GUS gene and kanamycin resistance gene. The main factors influencing the transformation including medium for regeneration and growth of PLBs, acetosyringone (AS) concentration, the co-cultivation time, Agrobacterium strain, bactericidal antibiotic concentrations and screening of transformed PLBs were surveyed. Results showed that MS medium supplemented with 1.0 mg/l NAA in combination with 0.5 mg/l or 0.3 mg/l BA was suitable for PLBs regeneration from stem tissue slices. The highest transformation efficiency (percentage of PLBs with GUS gene expression) was obtained when PLBs were infected and co-cultivated with Agrobacterium on the medium containing 100 µM AS for 3 days (with strain EHA105 and C58) or 4 days (with strain LBA4404). Medium containing 300 mg/l cefotaxime and 7-15 mg/l hygromycin was suitable for the disinfection and screening of transformed PLBs.

Short-term storage of alginate-encapsulated protocorm-like bodies of Dendrobium nobile Lindl.: an endangered medicinal orchid from North-east India.

Synthetic seed technology is an exciting and rapidly growing area of research as deals with conservation and storage of rare, endangered and desirable genotypes along with its easy handling and transportation. As propagation of many ornamental and medicinally important plant species is labour intensive, application of different growth retardants and osmotica in simple artificial seed system would dramatically reduce labour requirement by storing the germplasm in vitro. Moreover, the primary aim of developing in vitro storage methods is to reduce the frequent demands of subculturing and preserving the unique genetic constituent of the germplasm. Dendrobium nobile is a pharmaceutically important orchid mostly used in the Chinese herbal drug industry for its medicinal property. Commercial exploitation of this species has considerably depleted their population in wild. Hence, for conserving this valuable germplasm, short term in vitro storage of Protocorm-Like Bodies (PLBs) of D. nobile was carried out using different osmotica (sucrose and mannitol). It was observed that incorporation of low sucrose and mannitol (3 and 5 %) in the encapsulating matrix showed almost similar results with that of control. In all these cases, more than half of PLBs burst out from the matrix thus making these concentrations of sucrose and mannitol along with control not suitable for storage studies. However, with the increase in concentration to 7.5 and 12.5 % in the encapsulating matrix, no outburst of encapsulated PLBs was recorded till 60 days of storage; hence it can be concluded that these concentrations play an important role in minimizing the growth of PLBs during storage condition.

Propagation and Establishment of Three Endangered Mexican Orchids from Protocorms

Protocols for in vitro propagation from protocorms of Mormodes tuxtlensis Salazar, Cuitlauzina pendula La Llave & Lex., and Lycaste skinneri (Batem. Ex. Lind.) Lind., three endangered species distributed in Mexico and highly appreciated as ornamentals, were developed. The effect of two different culture media, Murashige and Skoog (MS) and modified Knudson (KCm), combined with varying concentrations of N6-benzyladenine (0, 2.2, 4.4, 8.9, and 13.3 μM) and α-naphthaleneacetic acid (0, 0.5 and 2.7 μM), were investigated. Shoot formation and development of protocorm-like bodies were observed. For all three species, cultures in MS produced more shoots per explant than those in KCm, and those shoots were longer and more robust in appearance. Maximum number of shoots for M. tuxtlensis (1.5) and C. pendula (24.3) were obtained in media supplemented with 13.3 μM and 2.2 μM N6-benzyladenine, respectively. Conversely, for L. skinneri the greatest shoot production (16.4) was achieved in medium supplemented with 2.7 μM α-naphthaleneacetic acid. Subculturing explants in MS basal medium allowed further development and rooting of the shoots as well as growth of protocorm-like bodies. The effect of different potting mixes on ex vitro survival plantlets was also investigated; pine bark:oak charcoal:pumice (3:1:1) allowed the highest survival rates in all three species.

Micropropagation of Cypripedium macranthos var. rebunense through Protocorm-like Bodies Derived from Mature Seeds

Cypripedium macranthos var. rebunense is the most famous terrestrial orchid in Japan, since the variety has large beautiful yellowish-white flowers and is threatened with extinction. Establishment of an efficient method for micropropagation is urgently needed. When imbibed mature seeds of the orchid, that had been pre-chilled at 4°C for 3 months, were sown onto Hyponex-peptone medium that contained both α-naphthaleneacetic acid (NAA) and cytokinin, protocorm-like bodies (PLBs) were formed from germinated seeds. Although the growth of PLBs was very slow, plantlets were easily regenerated from the PLBs on hormone-free medium. The PLBs were subcultured eight times along 2 years without loss of ability to regenerate plantlets, and one aggregate of PLBs (ca. 5 mm in diameter) produced ca. 10 plants within a year. A reduction of commercial value through a large-scale micropropagation by this method will be able to prevent illegal collection from the wild populations.

Effects of dividing on the growth and organogenesis of protocorm-like bodies in Doritaenopsis

Protocorm-like bodies (PLB) obtained through in vitro culture of seedling leaf segment of Phalaenopsis cultivar ‘Surfrider’ × ( Phalaenopsis cultivar ‘Joseph Hampton’ × Doritaenopsis cultivar ‘Kaala Gleam’) were cultured on media containing peptone or tryptone at various concentrations. The addition of peptone and tryptone to the culture medium caused promoted growth of PLB. Maximum stimulation was obtained when 2 g l −1 of tryptone was added. The effects of dividing and size of PLB on the growth and organogenesis of PLB were examined. Segments from dorsal parts of PLB had a tendency to form plantlets, while segments from basal parts produced no plantlets, but a larger number of PLB than dosal segments. From the results of the growth and the PLB production rate, 1 4 B segments are recommended for use in proliferation of PLB.