Induction Of Protocorm-like Bodies Research Articles

Induction of Protocorm-Like Bodies (PLBs) Phalaenopsis spp. Hybrids Mutation through Ultraviolet Irradiation (UV254) and Ethyl Methane Sulfonate (EMS)

Phalaenopsis sp. is the most-produced orchid species in Indonesia. Compared to conventional breeding, mutation induction by using mutagens, such as Ultraviolet Light-C (λ = 254 nm) (UV254) and Ethyl Methane Sulfonate (EMS), could probably result in new superior orchid variants. This research aims to get some mutants with phenotypes that have visual differences in the Phalaenopsis spp. hybrids wild type. There were 4 durations of UV254 irradiation: 5’ on, 85’ off; 10’ on, 80’ off (1 day and 7 days for each treatment); 4 concentrations of EMS used in this research: 0.05%; 0.06%; 0.07%; 0.08% for 6 hours of immersion; selected UV254 irradiation (5’ on, 85’ off (7 days)) combined with these concentrations. UV254 irradiation treatment (5’ on, 85’ off (1 day and 7 days); 10’ on, 80’ off (7 days)) resulted in some mutants with leaf phenotypes that were visually different from the wild type; 0.05% EMS treatment resulted in PLBs mutant with a visually larger size than the wild type; 0.08% EMS treatment and combination treatments (for EMS 0.05% and 0.08% for each treatment) resulted in non-growing albino PLBs. Hence, mutation induction using UV254 and EMS in this research produced several most likely mutants having visual differences that may be more desirable than the wild type.

Leaf-based induction of protocorm-like bodies, their encapsulation, storage and post-storage germination with genetic fidelity in Mokara Sayan × Ascocenda Wangsa gold

• First report on direct induction of PLBs Mokara Sayan × Ascocenda Wangsa gold. • Earliest and maximum PLB induction occurred on MS medium fortified with 3 mg L –1 TDZ. • Synthetic seeds were developed encapsulating PLBs with 3 % Na-alginate and 75 mM CaCl 2 . • Better storage of synthetic seeds up to 180 days was attained at 25°C in comparison to 4°C. • RAPD marker-based analysis ensured genetic integrity of synthetic seed-derived plantlets. An innovative and accelerated in vitro mass propagation protocol via direct induction of protocorm-like bodies (PLBs) from leaf sections of orchid hybrid Mokara Sayan × Ascocenda Wangsa gold was developed for the first time. Initially, leaf sections were transferred into Murashige and Skoog (MS) medium supplemented with two cytokinin sources namely, thidiazuron (TDZ) and zeatin with variable concentrations. The earliest (∼8 days) induction of PLBs with maximum number (34 PLBs cm –2 leaf section) and induction frequency (82.8 %) were recorded in MS medium fortified with 3 mg L –1 TDZ. The same medium formulation resulted in the highest growth rate (∼93.7 mg day –1 ) with maximum diameter of PLBs (3.4 mm) when compared with other treatments. Observations on proliferating PLBs under variable pressure scanning electron microscopy illustrated their distinctive growth phases wherein proliferating PLBs exhibited globular shape, typical epidermal texture with a rough surface that steadily became more wrinkled, consisting of basal absorbing hair-bodies and the apical zone forming shoot primordia. Stereo microscopic observations revealed gradual maturation of PLBs, initiation of leaf and shoot primordia. PLBs were encapsulated with calcium-alginate for short-term storage and germplasm exchange. Lower concentrations of sodium alginate (1 and 2 %) produced synthetic seeds that were soft to grip and extremely irregular in shape, whereas higher concentration (4 %) resulted in stiffness of synthetic seeds and reduced germination frequency. The most suitable gel matrix to form uniform spherical beads was optimized at 3 % sodium alginate and 75 mM calcium chloride. Synthetic seeds were effectively stored at 25 °C for up to 180 days. The germination efficiency of synthetic seeds gradually declined after storage at both 4 °C and 25 °C, with synthetic seeds stored at 25 °C being more tolerant than those stored at 4 °C. Even after 180 days of storage, the synthetic seeds that were stored at 25 °C exhibited 71.2 % germination frequency and ∼14 days to germination. Synthetic seeds stored at 4 °C deteriorated rapidly and degenerated completely within 180 days. Random amplified polymorphic DNA marker-based analysis carried out with randomly selected synthetic seed-derived plantlets showed 100 % monomorphism with a total 512 bands of molecular size ranging between 130—2100 bp, ensuring post-storage genetic fidelity.

Cattleya tigrina (Orchidaceae) in vitro regeneration: Main factors for optimal protocorm-like body induction and multiplication, plantlet regeneration, and cytogenetic stability

The induction and subsequent regeneration of protocorm-like bodies (PLB) is the most common morphogenetic pathway for orchid micropropagation. The regeneration of plants from PLB can be a bottleneck for some species due to asynchronicity. Genetic fidelity is another issue, and the cytogenetic stability of regenerated plantlets deserves special attention due to the common occurrence of endopolyploidy in orchids. In the present work, we defined optimal conditions for PLB induction and plant regeneration for Cattleya tigrina, a threatened orchid from the Brazilian Atlantic Rain Forest. We estimated the optimum thidiazuron (TDZ) concentration for PLB induction from leaf explants, while also comparing whole leaves vs thin cell layer (TCL) as explants. For plantlet regeneration from PLB clusters, we investigated the effects of different concentrations of GA3 and sucrose, as well as the dehydration of PLBs and the use of temporary vs continuous immersion systems. Furthermore, the cytogenetic stability of regenerated plantlets was analyzed using flow cytometry, and the differences in stomatal morphology were studied. Exogenous TDZ (25 µM) was the most effective concentration for PLB induction, with 17.5% induction rate. Leaf basal regions, either in whole leaves or in TCL, were the most responsive for PLB induction. The dehydration of PLBs for 120 min increased the fresh weight increment (FWI) in plantlet regeneration, but at the expense of decreased shoot number and increased mortality. Temporary-immersion bioreactors were found to be the most suitable system for PLB multiplication compared to continuous immersion system. Shoot development was challenging due to previously acquired habituation to TDZ. Sucrose and GA3 showed an interactive effect on shoot growth. GA3 improved shoot formation, and maximum shoot production was estimated to occur at 10.3 and 9.7 µM GA3 on media containing 1.5% and 2% sucrose, respectively, resulting in larger number of developed plantlets (∼220) per culture flask. The flow cytometry analysis showed that 26.5% of plants regenerated from PLBs had doubled ploidy levels. The ploidy alterations were reflected in changes on leaf epidermis, such as increased stomatal density and number.

Ploidy mosaics: does endopolyploidy in explants affect the cytogenetic stability of orchids regenerated from PLBs?

The induction and regeneration of protocorm-like bodies (PLBs) is a morphogenetic pathway widely used for orchid micropropagation. As endopolyploidy, i.e., the coexistence of cells with different ploidy levels, is a common feature in orchid tissues, a natural question arises when using somatic tissues as explants for orchid micropropagation: does endopolyploidy in explants affect the cytogenetic stability of regenerated plantlets? To answer this question, Epidendrum fulgens was used as a model plant, and flow cytometry was used to analyze endopolyploidy in pollinia, petals, labella, leaf bases, leaf tips, root tips, and protocorm bases and apices, which were subsequently used as explants for PLB induction and plant regeneration. Ploidy screenings showed contrasting ploidy patterns in samples, endopolyploidy being detected in all tissues, with C-values ranging from 1 to 16C. Protocorm bases and root tips presented the highest proportion of endopolyploidy, while petals and protocorm apices showed the lowest proportion. Flower parts exhibited high oxidation for PLB induction and pollinia failed to produce PLB or callus. The highest induction rate occurred at 10 µM TDZ, with 92%, 22%, and 0.92% for protocorm bases, leaves, and root tips, respectively. Plantlets were more easily regenerated from PLBs induced from protocorm bases than from leaves and roots. Doubled ploidy levels were registered in a proportion of 11% and 33% for PLB-regenerated plantlets obtained from protocorm bases and leaf bases, respectively, which was not directly associated with the proportion of endopolyploid cells or cycle value of explants. Explants have different endopolyploid patterns and cycle values. Polyploid regenerants were obtained from leaves and protocorms, although in rates not associated with the proportion of endopolyploid cells in the explants.

An Overview of Orchid Protocorm-Like Bodies: Mass Propagation, Biotechnology, Molecular Aspects, and Breeding

The process through induction, proliferation and regeneration of protocorm-like bodies (PLBs) is one of the most advantageous methods for mass propagation of orchids which applied to the world floricultural market. In addition, this method has been used as a tool to identify genes of interest associated with the production of PLBs, and also in breeding techniques that use biotechnology to produce new cultivars, such as to obtain transgenic plants. Most of the molecular studies developed have used model plants as species of Phalaenopsis, and interestingly, despite similarities to somatic embryogenesis, some molecular differences do not yet allow to characterize that PLB induction is in fact a type of somatic embryogenesis. Despite the importance of species for conservation and collection purposes, the flower market is supported by hybrid cultivars, usually polyploid, which makes more detailed molecular evaluations difficult. Studies on the effect of plant growth regulators on induction, proliferation, and regeneration of PLBs are the most numerous. However, studies of other factors and new technologies affecting PLB production such as the use of temporary immersion bioreactors and the use of lighting-emitting diodes have emerged as new tools for advancing the technique with increasing PLB production efficiency. In addition, recent studies on Phalaenopsis equestris genome sequencing have enabled more detailed molecular studies and the molecular characterization of plantlets obtained from this technique currently allow the technique to be evaluated in a more comprehensive way regarding its real applications and main limitations aiming at mass propagation, such as somaclonal variation.

In vitro tetraploid induction from multigenotype protocorms and tetraploid regeneration in Dendrobium officinale

In vitro mitotic polyploidization using anti-microtubule agents has been commonly used for polyploid production. The present study was an attempt to develop an in vitro oryzalin chromosome doubling and stable tetraploid regenerating protocol for Dendrobium officinale. In this study, we successfully induced tetraploids by treating protocorms developed from the seeds of multiple genotypes. The highest frequency of polyploidy was 37.40%, achieved with 14.4 µM oryzalin treatment for 24 h. Among the obtained polyploid seedlings, 72 solid tetraploid, 33 mixoploid and 3 octoploid genotypes were identified via screening using flow cytometry (FCM). Three peaks were observed in the histograms of the diploid, tetraploid, and octoploid leaves, but four peaks were observed only in mixoploid leaves with FCM, indicating the existence of endopolyploid cells and the occurrence of conventional endoreduplication in the leaves of all ploidy levels. Recurrent ploidy identification of various tetraploid genotype regenerated plantlets obtained via protocorm-like bodies (PLBs) derived from axenic stem-node segments maintained stable tetraploid levels. Comparisons of phenotypic characteristics revealed that relative to the diploid plantlets, the tetraploid plantlets exhibited increased stem diameter, root diameter, labellum width and gynostemium width. Furthermore, the tetraploid plantlets showed lower plant height, leaf length and root length than the diploid plantlets. This efficient polyploid induction and ploidy-stable regeneration protocol can be used for the mass production of tetraploid D. officinale. The tetraploid genotypes regenerated in this study might be useful for nobile Dendrobium breeding in the future. In vitro induction of various tetraploid genotypes in D. officinale and subsequent rapid micropropagation through induction of PLBs from axenic nodal segments were performed to obtain ploidy-stable regenerated plantlets

The effect of light quality on protocorm-like body induction, cell division, and endopolyploidy occurrence in Phalaenopsis ‘Spring Dancer’

The effect of light quality on protocorm-like body induction, cell division, and endopolyploidy occurrence in <i>Phalaenopsis</i> ‘Spring Dancer’

Changes in Morphological Characteristics, Regeneration Ability, and Polysaccharide Content in Tetraploid Dendrobium officinale

Dendrobium officinale Kimura et Migo is a famous traditional Chinese medicinal plant. It produces various phytochemicals, particularly polysaccharides, which have nutraceutical and pharmaceutical values. To increase its biomass production and polysaccharide content, our breeding program has generated a series of polyploid cultivars through colchicine treatment of protocorm-like bodies (PLBs). The present study compared two tetraploid cultivars, 201-1-T1 and 201-1-T2, with their diploid parental cultivar, 201-1, in an established in vitro culture system. Tetraploid ‘201-1-T1’ and ‘201-1-T2’ had shorter leaves and shorter and thicker stems and roots, and they produced higher biomass compared with the diploid cultivar. The length and width of stomata significantly increased, but stomatal density decreased in tetraploid cultivars. The PLB induction rates from the stem node explants of the tetraploid cultivars were significantly higher than those of diploid. However, the PLB proliferation of tetraploids was lower than that of the diploid. The mean number of plantlets regenerated from tetraploid PLBs was also lower than that of the diploid after 4 months of culture. Polysaccharide contents in stems, leaves, and roots of 6-month-old tetraploid plantlets were significantly higher than those of diploids. The polysaccharide content in the stem of ‘201-1-T1’ was 12.70%, which was a 2-fold increase compared with the diploid cultivar. Our results showed that chromosome doubling could be a viable way of improving D. officinale in biomass and polysaccharide production.

A Combinational Phytomolecular-Mediated Assessment in Micropropagated Plantlets of Coelogyne ovalis Lindl.: A Horticultural and Medicinal Orchid

Coelogyne ovalis Lindl. is an evergreen epiphytic, sympodial, ornamental orchid having medicinal properties due to the presence of bioactive compounds such as flavidin, flavidinin, coelogin. In the present study, nodal buds of Coelogyne ovalis, when cultured in Knudson C medium supplemented with meta-Topolin (mT), 6-benzyl aminopurine (BAP), kinetin (Kn) and α-naphthalene acetic acid (NAA), responded to the induction of protocorm-like bodies (PLBs) or shoot buds. The highest numbers of PLBs (22.73 ± 0.47) and shoot (14.53 ± 0.27) per explant were augmented in the medium containing a combination of mT (10 µM) and NAA (0.5 µM). Meta-Topolin in the medium was found to be superior in enhancing the response of explants as compared to BAP and Kn. The best rooting of the shoots was observed in the medium supplemented with 10 µM indole-3-acetic acid. Well-developed plantlets obtained after 20 weeks of culture were acclimatized and transferred to the net house. Genetic stability of the acclimatized plantlets was evaluated and contrasted with the mother plant using two molecular markers, viz. start codon targeted and inter-simple sequence repeats, wherein 5.15% clonal variability was observed. Phytochemical screenings revealed that the micropropagated tissues exhibited higher contents of secondary metabolites, antioxidant potentials and chlorophyll contents as compared to the mother plant. The present study could be of great significance for the conservation and commercial utilization of C. ovalis, an orchid facing threat due to its overexploitation from nature.

Micropropagation of Tolumnia Orchids through Induction of Protocorm-like Bodies from Leaf Segments

A protocol for plant regeneration via direct induction of protocorm-like bodies (PLBs) from leaf segments of Tolumnia Snow Fairy was developed as a basis for mass production. Ten-month-old, in vitro–grown donor plantlets were obtained by inducing shoots from buds on the flower stalk. Leaf segments harvested from plantlets of different heights and from expanding leaves at different positions were compared, as were two BA concentrations with 0.5 mg·L−1 NAA. The greatest rate of PLB induction (16.7%) was observed when leaf segments taken from 1- to 2-cm height plants were cultured in Murashige and Skoog (MS) basal medium supplemented with 2 mg·L−1 BA and 0.5 mg·L−1 NAA after 16 weeks of culture. When using leaf explants, only inner, expanding leaves cultured on MS basal medium supplemented with 4 mg·L−1 BA and 0.5 mg·L−1 NAA resulted in PLB induction, at an average rate of 25.5 PLBs per explant. After 16 weeks of culture, histological and scanning electron microscopy (SEM) observations revealed that PLBs originated from epidermal cells of leaf explants. PLBs of 1 to ≤2 mm in diameter continued to proliferate after 4 weeks of culture. These secondary PLBs could be produced from either whole PLBs or the upper side of PLBs. Finally, PLBs were regenerated into plantlets. After ≈14 months of culture, fully developed plants exhibiting well-developed roots and shoots were acclimatized. These plants grew well, with 1-year survival rates of nearly 73%, for plants originating as explants taken from 1- to 2-cm tall plants, and of 79%, for plants originating as explants taken from inner leaves. Some mature plants flowered 1 year after transplantation. This study presents a simple system that can provide a large number of PLBs for mass propagation in a short time that can be converted into plants and also used for the new cultivars of Tolumnia orchids.

Strategies for the Regeneration of Paphiopedilum callosum through Internode Tissue Cultures Using Dark–light Cycles

Paphiopedilum spp. is one of the most commercially popular orchids because of its variety of shapes, sizes, and colors. However, it is at risk for extinction because of its exploitation. Regeneration of orchid plants using internode segments is extremely difficult. In this study, young P. callosum plants (1.5 cm) were exposed to eight dark–light cycles (14 days of dark and 1 day of light) for stem elongation to increase the number of nodes to obtain internode tissues. After 75 days of culture, the highest callogenesis (31.25%) was achieved when internode tissue was cultured on liquid Schenk and Hildebrandt (SH) medium containing 30 g·L−1 sucrose, 1.0 mg·L−1 Thidiazuron (TDZ), 1.0 mg·L−1 2,4-Dichlorophenoxyacetic acid (2,4-D), and cotton wool as the support matrix. The optimal media for induction of protocorm-like bodies (PLBs) were the same compositions as previously mentioned and were supplemented with 9 g·L−1 Bacto agar as the gelling agent. PLB clumps (5–6 PLBs/clump) produced the best shoots on medium containing 0.5 mg·L−1 α-Naphthaleneacetic acid (NAA) and 0.3 mg·L−1 TDZ. Among the organic substances tested, 200 g·L−1 potato homogenate (PH) added to Hyponex N016 medium supplemented with 1.0 mg·L−1 NAA, 30 g·L−1 sucrose, 170 mg·L−1 NaH2PO4, 1.0 g·L−1 peptone, and 9 g·L−1 Bacto agar resulted in the best rooting. The rooted plantlets with four to five leaves were acclimatized and had a 100% survival rate. The method presented in this research provides a strategy for the development of highly effective propagation of Paphiopedilum species using ex vitro explants for both conservation and horticultural purposes.

PLBs induction and clonal plantlet regeneration from leaf segment of commercial hybrids of Phalaenopsis

ABSTRACTInduction of Protocorm-like bodies (PLBs) from leaf segments is a clonally propagation technology that used somatic embryogenesis regeneration from in vitro buds of floral stalks, and is a potential alternative for replacement actual low-yield and high-cost clonal propagation of Phalaenopsis orchids. The objective of this work was to evaluate induction of PLBs in leaf segments of two commercial hybrids of Phalaenopsis, arranged in culture media with different combinations of plant growth regulators. The leaf segments (0.4–0.5 cm2) used for PLBs inductions were obtained from young in vitro-induced shoots from inflorescence stalks. They are cultivated in NDM culture media, supplemented with combinations of plant growth regulators Naphthaleneacetic Acid, Thidiazuron and Benzyladenine. The flasks were kept for 60-d in darkness and then for cold white light with photon flux density of 35–40 μmol m−2 s−1. The cultivar ‘Ph908’ showed a higher percentage of leaf segments with PLBs (45%) and number of PLBs (25 per leaf segment), in medium containing 0.25 mg L−1 of TDZ, whereas ‘RP3’ showed only 10% containing PLBs and 2 PLBs per leaf segment in NDM with 1.0 mg L−1 NAA, 20 mg L−1 BA and 0.125 mg L−1 TDZ.

Clonal Fidelity of Micro-propagated Phalaenopsis Plantlets Based on Assessment Using Eighteen Ph-Pto SNAP Marker Loci

Phalaenopsis amabilis is an Indonesia native orchid species having large, white flowers with yellow labellum coloration. This studies aimed to develop Phal. amabilis micropropagation methods and evaluate the regenerated plantlet fidelity. Media supplemented with Thidiazuron (TDZ) and Polyvinylpyrrolidone (PVP) and medium pH adjustment effects to induce protocorm-like bodies (PLBs) from leaf explants and proliferate secondary PLBs were investigated. Clonal fidelity among regenerated plantlets was evaluated using eighteen SNAP marker loci. The results showed that the ½ MS medium supplemented with 3 mg L-1 TDZ and 0.5 g L-1 PVP was the best for PLB induction while the ½ MS medium supplemented with 0.5 mg L-1 TDZ was the best for PLB proliferation. For PLB induction, the media pH was adjusted into pH=7 for efficient PLB regeneration. Based on the assessment using 18 SNAP marker loci, four variant alleles in three loci (11.8%) out of a total 34 plantlets were detected. The mutation frequency at the evaluated SNAP marker loci was 2.5 x 103 (0.25%). Changes in SNP alleles may not always result in phenotype changes and allele variant occurrences may not affect phenotype fidelity of micro-propagated Phal. amabilis plantlets. Therefore, further studies about the phenotype fidelity among plantlets are necessary.

&lt;b&gt;Determination of endogenous IAA and carbohydrates during the induction and development of protocorm-like bodies of &lt;i&gt;Cattleya tigrina&lt;/i&gt; A. Richard

Tissue culture techniques have been employed for orchid mass propagation by means of the morphogenetic route of protocorm like-bodies (PLBs). This study aimed to analyze and compare Indole-acetic acid (IAA) and sugar endogenous levels in protocorm-like bodies (PLBs) induction and development in Cattleya tigrina, in order to better understand this process and to optimize micropropagation procedure protocols. Leaves grown on MS (Murashige and Skoog) culture medium, suplemented with 9 μM TDZ for PLBs induction and development were collected after 0, 2, 7, 14, 30, 60 and 100 days of cultivation, for further analysis. Increase of IAA and reduction of sugar levels are strongly related to morphogenetic response, that is, PLBs formation over the preexisting ones and leaf primordia formation. Sucrose, fructose and glucose presence in this study is related to cell signaling. Thus, hormonal signals and carbohydrates alter metabolism, triggering PLBs initiation and development in C. tigrina.

&lt;b&gt;Dynamics in global DNA methylation and endogenous polyamine levels during protocorm-like bodies induction of &lt;i&gt;Cattleya tigrina&lt;/i&gt; A. Richard

Cattleya tigrina is endemic to the Atlantic forest biome and classified as vulnerable in the Red Book of Brazilian Flora. In vitro techniques comprise valuable tools for the conservation of endangered plant species. The aim of the present study was to evaluate the morphological features, global DNA methylation levels and free polyamines during protocorm-like bodies (PLBs) induction of C. tigrina . Along with that, an efficient protocol for in vitro propagation of this species is proposed. The first evidences of PLBs induction in C. tigrina occurred at seven days in culture, starting from the basal portion of the leaf abaxial surface. A hypomethylation marked the beginning of cell differentiation, followed by an increased global DNA methylation at 35 days in culture, coinciding with a subtle change in the structures morphogenetic development. During PLBs induction, putrescine exhibited higher levels as compared to spermidine and spermine, and apparently presents a major role during the PLBs induction in C. tigrina . Due to the apparent secondary PLBs formation, this protocol can represent a highly efficient method for in vitro propagation of this species.

In vitro culture of Chloraea gavilu Lindl., an endemic terrestrial orchid from Chile

Orchids are the second most diverse plant family, recognized for their importance as ornamental species; this has driven research development in propagation. One of the most common culture methodologies is in vitro asymbiotic germination, in which the nutritional conditions that provide orchids with a fungal partner are emulated. Although Chile possesses more than 60 terrestrial orchid species, in vitro cultivation protocols have only been developed for Chloraea crispa. In Southern Chile, Chloraea gavilu stands out due to its floral characteristics. We evaluate different explants and cultivation conditions for C. gavilu. The best germination and development results were achieved in the MM medium +0.1% yeast extract +1% sucrose +0.454 µmol l−1 TDZ, using immature seeds of 24–30 days after pollination, which we cultivated into seedlings in order to be acclimatized and mycorrhized. In addition, induction of protocorm-like bodies was achieved from germinated seeds, using the same culture media as in the germination and development of immature C. gavilu seeds. This resulted in the successful asymbiotic germination of immature seeds, along with the micropropagation of a terrestrial, temperate orchid. We hope to use our protocol in the commercial production of Chilean orchid species as well as to propagate threatened species.

Thidiazuron Triggers Morphogenesis in Rosa canina L. Protocorm-Like Bodies by Changing Incipient Cell Fate.

Thidiazuron (N-phenyl-N′-1,2,3-thiadiazol-5-ylurea; TDZ) is an artificial plant growth regulator that is widely used in plant tissue culture. Protocorm-like bodies (PLBs) induced by TDZ serve as an efficient and rapid in vitro regeneration system in Rosa species. Despite this, the mechanism of PLB induction remains relatively unclear. TDZ, which can affect the level of endogenous auxins and cytokinins, converts the cell fate of rhizoid tips and triggers PLB formation and plantlet regeneration in Rosa canina L. In callus-rhizoids, which are rhizoids that co-develop from callus, auxin and a Z-type cytokinin accumulated after applying TDZ, and transcription of the auxin transporter gene RcPIN1 was repressed. The expression of RcARF4, RcRR1, RcCKX2, RcCKX3, and RcLOG1 increased in callus-rhizoids and rhizoid tips while the transcription of an auxin response factor (RcARF1) and auxin transport proteins (RcPIN2, RcPIN3) decreased in callus-rhizoids but increased in rhizoid tips. In situ hybridization of rhizoids showed that RcWUS and RcSERK1 were highly expressed in columella cells and root stem cells resulting in the conversion of cell fate into shoot apical meristems or embryogenic callus. In addition, transgenic XVE::RcWUS lines showed repressed RcWUS overexpression while RcWUS had no effect on PLB morphogenesis. Furthermore, higher expression of the root stem cell marker RcWOX5 and root stem cell maintenance regulator genes RcPLT1 and RcPLT2 indicated the presence of a dedifferentiation developmental pathway in the stem cell niche of rhizoids. Viewed together, our results indicate that different cells in rhizoid tips acquired regeneration competence after induction by TDZ. A novel developmental pathway containing different cell types during PLB formation was identified by analyzing the endogenous auxin and cytokinin content. This study also provides a deeper understanding of the mechanisms underlying in vitro regeneration in Rosa.

Induksi PLB Anggrek Vanda sumatrana Schltr. Liar Pada Media MS dengan Penambahan BAP dan NAA serta Ploidisasi dengan Kolkisin

The study about PLB induction of wild Vanda sumatrana Schltr. on MS media suplement with BAP and NAA and ploidisation by colchicine treatment was conducted from December 2014 until November 2015 at the Laboratory of Genetics and Cell Biology and Laboratory of Plant Physiology and Tissue Culture, Biology department, Faculty of Mathematic and Natural Science, Andalas University, Padang. The study aimed to 1) knowing the best concentration of 6-Benzyl amino purin (BAP) and α-Naphtalene acetic acid (NAA) for Protocorm Like Bodies (PLB) induction from shoot tip of V. sumatrana, 2) knowing the PLB response of V. sumatrana to concentrations and soak period of colchicine and 3) find the effective concentrations and soak period of colchicine to induce tetraploid on PLB of V. sumatrana. Shoot tips from in-vitro cultured of V. sumatrana were subcultured on Murashinge and Skoog (MS) medium supplement with 3 mg/l BAP + 0,5 mg/l NAA, 3 mg/l BAP and 1,5 mg/l BAP. PLB of diploid V. sumatrana from the best treatment were soaked in 0.05% and 0.1% colchicine for 24 and 48 hours respectively in MS liquid medium, as control were set PLB without colchicine treatment. The results showed that MS medium supplemented with 1.5 mg/l BAP was the best formula to induce PLB. The highest percentage of survival rate of PLB and percentage of survived PLB regenerated shoot was obtained from 0.05% colchicine with 24 hours soak period treatment. The effective treatment to induce tetraploid on PLB of V. sumatrana Schltr. was obtained from 0.05% colchicine solution for 24 hours soak period.

Plant regeneration via callus culture and subsequent in vitro flowering of Dendrobium huoshanense

A protocol for regenerating and subsequent in vitro flowering of an economical important and endangered medicinal orchid, Dendrobium huoshanense, was established mainly via indirect protocorm-like body (PLB) formation. A four-step method was developed to induce successful plant regeneration on 1/2 MS medium supplemented with suitable plant growth regulators (PGRs). Step 1 (callus induction): the root tip explants (1 cm long) were cultured at 1 mg l−1 2,4-D + 1 mg l−1 TDZ for 3 months. Step 2 (callus proliferation): the calli were subcultured with a 1-month interval at 1 mg l−1 2,4-D + 1 mg l−1 TDZ. Step 3 (PLB induction): the calli were cultured at 2 mg l−1 NAA + 1 mg l−1 BA for 2 months. Step 4 (plantlet conversion): the 2-month-old PLBs were cultured at 0.1 mg l−1 IBA for 4 months. It took at least 6 months to produce well-rooted regenerated plantlets with an average of 3.2 roots and 3.6 leaves from the initial callus. The 6-month-old rooted plantlets were transferred onto PGR-free 1/2 MS medium for 6 months, and then potted with Sphagnum moss for acclimatization. After 2 month of culture, the survival rate was 100 %. The in vitro flowers were obtained on the 8-month-old plantlets at 1 mg l−1 IBA, 5 mg l−1 IBA and 0.1 mg l−1 NAA, but the flowers showed a lack of the gynandrium. The abnormity was overcome by the aid of 5 mg l−1 TDZ, and subsequently, the capsules formed without artificial pollination. This protocol provides the basis for further investigation on cell suspension, micropropagation, in vitro flowering and breeding programs in Dendrobium huoshanense.

Genetic stability and phytochemical analysis of the in vitro regenerated plants of Dendrobium nobile Lindl., an endangered medicinal orchid

An efficient genetically stable regeneration protocol with increased phytochemical production has been established for Dendrobium nobile, a highly prized orchid for its economic and medicinal importance. Protocorm like bodies (PLBs) were induced from the pseudostem segments using thidiazuron (TDZ; 1.5mg/l), by-passing the conventional auxin–cytokinin complement approach for plant regeneration. Although, PLB induction was observed at higher concentrations of TDZ, plantlet regeneration from those PLBs was affected adversely. The best rooting (5.41 roots/shoot) was achieved in MS medium with 1.5mg/l TDZ and 0.25% activated charcoal. Plantlets were successfully transferred to a greenhouse with a survival rate of 84.3%, exhibiting normal development. Genetic stability of the regenerated plants was investigated using randomly amplified polymorphic DNA (RAPD) and start codon targeted (SCoT) polymorphism markers which detected 97% of genetic fidelity among the regenerants. The PIC values of RAPD and SCoT primers were recorded to be 0.92 and 0.76 and their Rp values ranged between 3.66 and 10, and 4 and 12 respectively. The amplification products of the regenerated plants showed similar banding patterns to that of the mother plant thus demonstrating the homogeneity of the micropropagated plants. A comparative phytochemical analysis among the mother and the micropropagated plants showed a higher yield of secondary metabolites. The regeneration protocol developed in this study provides a basis for ex-situ germplasm conservation and also harnesses the various secondary metabolite compounds of medicinal importance present in D. nobile.