Efficient Method for Direct Embryogenesis in Phalaenopsis Orchid

Acta Physiologiae Plantarum, 2009

The influence of light regime, explant position and orientation on direct embryo formation from leaf explants of two Phalaenopsis, P. amabilis and P. Nebula, were investigated to optimize the protocol for regenerating of this orchid. When explants were cultured in light, direct embryogenesis was retarded in both species. Embryos showed whitish to pale green in color and larger size than those cultured in darkness. Furthermore, light regime induced explant browning, embryo necrosis and eventually low plantlet conversion rate. Sixty days of culture in darkness is the most suitable duration for direct embryo induction. Explant orientation also significantly affected direct embryo formation, and explants placed adaxial-side-up on culture medium had higher embryogenic response than abaxial-side-up orientation. In both species, the cut end had highest embryogenic competence than other parts of the explant. Moreover, when the leaf explant was cut transversely into two segments, the leaf basal segment had higher embryogenic competence than the leaf tip segment.

HAYATI Journal of Biosciences

Phalaenopsis amabilis is an important national flower of Indonesia as a parent for orchid breeding, so that needs a good strategy to produce high number of plants. The objective of this research is to analyze the use of thidiazuron (TDZ) for producing high number of plantlets, through directly induction of somatic embryos (SEs) from various explants. The method was used 20 each of protocorms, leaves, stems and roots as explants. The explants were dissected transversely, then put on various culture media: New Phalaenopsis (NP) and NP + (1, 2, 3) mgL À1 TDZ. Cultures were maintained at 25 C with continous white light. The formation of SEs was observed every week for 8 weeks. The results showed that SEs formation increased inline with the addition of TDZ concentration to the NP medium, for both velocity and amount of SEs formation. In NP0, SEs were formed at (26.07 ± 0.73) days after inoculation of protocorm, whereas on NP + (1, 2, and 3 mgL À1) TDZ, SEs were formed at (17.85 ± 0.67) days, (15 ± 0.64) days, and (11 ± 0.64) days, respectively. All types of explants formed SEs on NP + TDZ (1e3 mgL À1), whereas only 14 of 20 protocorms produced SEs (70%), and 8 of 20 stems formed SEs (40%) in NP0. In roots, SEs was formed on NP + 2 mgL À1 TDZ and NP + 3 mgL À1 TDZ. For stems, the highest amount of SEs (28.25 ± 1.07) was reached on NP + 3 mgL À1 TDZ, followed by protocorm (23.30 ± 1.13) SEs and roots (8.25 ± 0.68) SEs. In contrast, in NP0, the amount of SEs was very low (1.25 ± 0.46) from stem and (1.50 ± 0.65) from protocorms, there was no evidence of SEs formation in the leaves and roots.

Plant Tissue Culture and Biotechnology

An efficient in vitro regeneration protocol through somatic embryogenesis was established from longitudinally bisected protocorm (lTCL) of an endangered orchid Dendrobium ovatum. The efficiency of EC and SEs from the protocorms significantly relied on the concentration of PGRs. MS medium supplemented with TDZ (1.0 mg/l) induced optimum of EC (31.8%) and SEs (28.1/explant). Similarly, ZEA (0.5 mg/l) induced EC (27.6%) and SEs (17.1/explant). The combined treatment of TDZ (1.0 mg/l) and NAA (0.5 mg/l) resulted in the maximum induction of EC (58.6%) and SEs (39.8/explant) in an upright incubated explant. In another combined effect of ZEA (1.0 mg/l) and NAA (0.5 mg/l) induced EC (43.8%) and SEs (24.3/explant), whereas the combination of BAP (0.5 mg/l) and NAA (0.5 mg/l) produced EC (34.4%) and SEs (16.8/explants). The induced EC and SEs where healthier on medium containing TDZ + NAA than on the medium containing ZEA+NAA and BAP+NAA. The orientation of lTCL explants seemed to interact wi...

2014

Most temperate terrestrial orchids are endangered species. Attempts to produce plantlets from plants of the genus Epipactis by asexual methods have totally failed. This study was conducted using somatic embryogenesis as a rapid vegetative propagation technique for conservation of E. veratrifolia. For these purposes, effects of different types of plant growth regulators (PGRs), different types of explants, light and dark conditions, and the effect of gibberellic acid (GA 3 ) and Paclobutrazol (PBZ) were investigated on somatic embryogenesis induction. Abscisic acid (ABA) pre-treatment effectiveness on inducting somatic embryogenesis and increasing the number of embryos per explant were investigated. Subsequently, 16 media were tested to find the best medium for plant regeneration and shoot and root proliferation. BA (3 mg L −1 ) resulted in a better response than the other PGRs by supporting the development of 17 embryos per protocorm. PBZ, which resulted in 11 embryos per explant, was better than GA 3 . FAST medium supplemented with organic substances was recognized as the best medium for plant regeneration and shoot and root proliferation. ABA pre-treatment had positive effect on somatic embryogenesis initiation. This study, for the first time, succeeded in finding a rapid and suitable protocol for propagation and genetic resource conservation of E. veratrifolia.

American Journal of Plant Sciences, 2011

A newly efficient protocol has been established for high frequency somatic embryogenesis through callus culture of Coelogyne cristata. The best frequency of callusing was obtained from leaf segments (3 -5 mm) cultured on the MS medium supplemented with 2 mg·L -1 2,4-D and 2 mg·L -1 BA combination. A negative effect of coconut water was observed on the callus induction medium. When callus lines No. 4, 6 and 8 induced from leaf segments were sub-cultured separately on 1/2 MS and MS media containing AC (1 -3 g·L -1 ), formation of somatic embryos was found. However, percentages of embryo formation and the number of embryos per explants were strongly affected by media and callus lines used. The effect of 1/2 MS media is definitely better than MS medium for somatic embryogenesis from the selected lines of leaf derived callus. Among the callus lines, line no. 4 is the best for somatic embryogenesis followed by line no. 6 and 8. The somatic embryos converted into healthy plants with well developed shoots on the same media. The plantlets were transferred to 1/2 MS medium containing 1 g·L -1 AC for plant regeneration until 8 weeks of culture and successfully acclimatized in the greenhouse.

Journal of Horticultural Science & Biotechnology, 2016

Most temperate terrestrial orchids are endangered species. Attempts to produce plantlets from plants of the genus Epipactis by asexual methods have totally failed. This study was conducted using somatic embryogenesis as a rapid vegetative propagation technique for conservation of E. veratrifolia. For these purposes, effects of different types of plant growth regulators (PGRs), different types of explants, light and dark conditions, and the effect of gibberellic acid (GA 3 ) and Paclobutrazol (PBZ) were investigated on somatic embryogenesis induction. Abscisic acid (ABA) pre-treatment effectiveness on inducting somatic embryogenesis and increasing the number of embryos per explant were investigated. Subsequently, 16 media were tested to find the best medium for plant regeneration and shoot and root proliferation. BA (3 mg L -1 ) resulted in a better response than the other PGRs by supporting the development of 17 embryos per protocorm. PBZ, which resulted in 11 embryos per explant, was better than GA 3 . FAST medium supplemented with organic substances was recognized as the best medium for plant regeneration and shoot and root proliferation. ABA pre-treatment had positive effect on somatic embryogenesis initiation. This study, for the first time, succeeded in finding a rapid and suitable protocol for propagation and genetic resource conservation of E. veratrifolia.

Taylor & Francis, 2017

Most temperate terrestrial orchids are endangered species. Attempts to produce plantlets from plants of the genus Epipactis by asexual methods have totally failed. This study was conducted using somatic embryogenesis as a rapid vegetative propagation technique for conservation of E. veratrifolia. For these purposes, effects of different types of plant growth regulators (PGRs), different types of explants, light and dark conditions, and the effect of gibberellic acid (GA 3) and Paclobutrazol (PBZ) were investigated on somatic embryogenesis induction. Abscisic acid (ABA) pre-treatment effectiveness on inducting somatic embryogenesis and increasing the number of embryos per explant were investigated. Subsequently, 16 media were tested to find the best medium for plant regeneration and shoot and root proliferation. BA (3 mg L −1) resulted in a better response than the other PGRs by supporting the development of 17 embryos per protocorm. PBZ, which resulted in 11 embryos per explant, was better than GA 3. FAST medium supplemented with organic substances was recognized as the best medium for plant regeneration and shoot and root proliferation. ABA pre-treatment had positive effect on somatic embryogenesis initiation. This study, for the first time, succeeded in finding a rapid and suitable protocol for propagation and genetic resource conservation of E. veratrifolia.

In Vitro Cellular & Developmental Biology - Plant, 2003

The influences of various carbohydrate sources, dried yeast (DY), and 6-benzylaminopurine (BA) were estimated on growth and development of shoot tip-derived suspension cells of phalaenopsis orchid. Among the carbohydrates tested on Doriataenopsis cultured on gelled medium, glucose at 58.4 mM gave the highest efficiency of protocorm-like body (PLB) formation. Maltose and sorbitol only induced PLB formation without callus proliferation. Sucrose induced comparable callus proliferation to glucose but without PLB formation. In contrast, fructose resulted in half the amount of callus proliferation as occurred with glucose. Lactose was an inadequate carbon source as neither PLB formation nor callus proliferation occurred. DY enhanced cell proliferation at 0.1-1 g l 21 but inhibited both cell proliferation and PLB formation at 10 g l 21. Low BA (0.4 mM) slightly increased callus proliferation but inhibited PLB formation. Only one treatment, sucrose and 1 g l 21 DY, yielded a small number of plants. For suspension cultures of Phalaenopsis Snow Parade and P. Wedding Promenade, PLB formation was most efficiently induced by sucrose at 29.2 mM for P. Snow Parade and 14.6 mM glucose for P. Wedding Promenade. Histological observation revealed that cells in suspension culture developed into plants through the same developmental process as germinating seeds.

Ciência Rural

ABSTRACT: The aim of the present study was to induce the formation of somatic embryos in protocorms from Phalaenopsis Classic Spotted Pink hybrids at two physiological maturation stages, namely: 80 and 120 days after seed inoculation (DASI). Protocorms were inoculated in ½ MS medium supplemented with 0.1 mg L-1 ANA and 3 mg L-1 TDZ. Protocorms were inoculated 120 days after sowing were more developed at the 15th cultivation day due to the formation of pro-embryogenic structures. It was possible seeing the formation of globular- and torpedo-stage somatic embryos at the 30th day of cultivation in somatic embryogenesis (SE) induction medium. The protocorms inoculated at the 80th DASI did not formed somatic embryos; they oxidized 20 days after cultivation in SE-induction medium. The formation of somatic embryos happened directly on the explant, thus characterizing a direct somatic embryogenesis. The embryos converted into plants when the somatic embryos were transferred to the nutrient ...

Acta Physiologiae Plantarum, 2008

Leaf explants of two Phalaenopsis, P. amabilis and P. Nebula, were used to test the effects of auxins (2,4-D, IAA, IBA, NAA), cytokinins (2iP, BA, kinetin, TDZ, zeatin), GA 3 , ancymidol, polyamines (putrescine, spermine, spermidine), ACC, AgNO 3 and CoCl 2 on the amount of direct embryo formation on different leaf locations (the cut end, the adaxial side, the abaxial side and the leaf tip). The results showed that there was a genotypic effect on direct embryo formation induced by cytokinins that 13.32 lM BA and 4.92 lM 2iP was the most effective in P. amabilis and P. Nebula, respectively. Besides, explant position highly affected embryogenic competence of leaf cells in both species that the cut end showed highest embryogenic response, the adaxial side was the second, and then the abaxial side and the leaf tip. Altogether, cytokinins tested were all effective in both species, and ACC at 20 lM had 35% of embryogenic response in P. amabilis. However, auxins, GA 3 , ancymidol and polyamines were inhibitory in both species.