Protocorm-like Bodies Formation Research Articles

Invitro and Mass Multiplication of Cymbidium mastersii an Ornamental Orchid through Protocorm-like Body Formation

The epiphytic orchid species known as Cymbidium mastersii can only be found in the region of India known as Northeast India. The preservation of wild populations faces a significant obstacle in the form of a significant threat when it comes to the necessity of obtaining an additional source of the material required to fulfil commercial demands. The objective of this research was to build an in vitro system for the bulk multiplication of C. mastersii, and it was successful in that endeavour. This required the capacity to control the growth of the seedlings through the development of the primary protocorm and germination from a limited number of fruit capsules. Also necessary was the ability to limit the amount of fruit capsules that could be used. Each protocorm resulted in the production of a number of robust plantlings that, once they had been separated from their mother plant, flourished in the conditions of the greenhouse. The child plants, in terms of both their size and the development they underwent, were genetically identical to their parent plant. The streamlined process will make it possible to cultivate Cymbidium mastersii for use in the commercial sector, which will reduce the negative influence on remaining viable wild populations. We offer here for the very first time a method of producing C. mastersii on a commercial scale that makes use of bioreactor technology.

In vitro Regeneration of an Endangered Medicinal Orchid Dendrobium crepidatum Lindl. & Paxton through Protocorm like bodies.

A protocol for regeneration of an endangered medicinal orchid Dendrobium crepidatum was established mainly via indirect protocorm like body (PLBs) formation. The PLBs procured from in vitro growth cultures was assessed in ½ MS medium alone and in combinations with 6-Benzyl amino purine (BAP), Kinetin (Kn) and α-Naphthalene acetic acid (NAA). The calli were induced from longitudinally bisected segments of PLBs. The optimum percentage of callus was obtained in ½ MS medium supplemented with BAP (1.0 mg/l) and combination of BAP (1.0 mg/l) with NAA (0.5 mg/l), where 64.3% and 83.3% of callus was formed respectively. PLB formation was achieved when callus was transferred onto the respective combinations in ½ MS basal medium. The optimum percentage (91.6%) of PLBs were regenerated in the combination of BAP with NAA. These PLBs differentiated into intact plantlets about 3 cm in height and with 4-5 roots were transplanted into potting mixture with highest rate of 93.3% survival rate in a green house.

Protocorm-like body formation, stem elongation, and enhanced growth of Anthurium andraeanum ‘Tropical’ plantlet on medium containing silver nanoparticles

Protocorm-like body formation, stem elongation, and enhanced growth of Anthurium andraeanum ‘Tropical’ plantlet on medium containing silver nanoparticles

Effect of microalgae Messastrum gracile and Chlorella vulgaris on the in vitro propagation of orchid Cattleya labiata

The aim of this study was to evaluate the effect of two green microalgae (Messastrum gracile and Chlorella vulgaris) in comparison with plant growth regulators (6-benzylaminopurine, BAP; thidiazuron, TDZ; zeatin, ZEA) on the in vitro propagation of Cattleya labiata, an endangered orchid, using the thin cell layer (TCL) technique from protocorms. TCL sections were cultivated in MS/2 medium containing M. gracile extract (EM) and biomass (BM); C. vulgaris extract (EC) and biomass (BC); and BAP, TDZ, and ZEA in different concentrations. Subsequently, the explants were grown in MS/2 medium, with 2 g L−1 of activated charcoal, to induce elongation and roots formation. For acclimatization, plants were transplanted in trays using Sphagnum sp. as substrate. TCL explants showed a higher formation of protocorm-like bodies (PLBs) than entire protocorms. Explants cultivated in media supplemented with BM showed a high rate of PLB regeneration (59%) and high mean number of PLBs (4 per explant) and 85% survival after acclimatization of the plants. Supplementation with BAP stimulated similar morphogenic responses to those observed with BM and superior results obtained with ZEA and TDZ. Mass propagation of C. labiata plants was successfully achieved using TCL, and it is recommended to supplement the MS/2 medium with 4 g L−1 of BM or 4 mg L−1 of BAP. Microalgae extracts and biomasses are effective alternatives for in vitro propagation of C. labiata that can replace plant growth regulators, as they favored the formation of PLBs and plants.

In vitro plant regeneration through protocorm formation in orchid Calanthe odora

The regeneration of plants through formation of protocorm and protocorm-like bodies was achieved by in vitro asymbiotic immature seed culture of Calanthe odora. This is the first report on in vitro regeneration of plantlets of Calanthe odora Griff., the IUCN red listed threatened species. The immature seeds as explants were most regenerative than mature seeds. The MS media fortified with different concentrations of Kinetin alone or in combination with NAA were tested to sort out as the best regeneration media. From the experimental results the best medium was sorted as MS medium supplemented with 1.0 mg/l Kinetin and 1.0 mg/l NAA for germination and formation of protocorm-like bodies from immature seed explants followed by transfer to MS media fortified with 1.5 mg/l Kinetin and 1.0 mg/l NAA for development of protocorm to plantlets. The plantlets after hardening in in vitro were transferred to polyhouse where 86.7% survival rate was recorded on vermiculite potting media.

Organic Compounds: Contents and Their Role in Improving Seed Germination and Protocorm Development in Orchids

In nature, orchid seed germination is obligatory following infection by mycorrhizal fungi, which supplies the developing embryo with water, carbohydrates, vitamins, and minerals, causing the seeds to germinate relatively slowly and at a low germination rate. The nonsymbiotic germination of orchid seeds found in 1922 is applicable toin vitropropagation. The success of seed germinationin vitrois influenced by supplementation with organic compounds. Here, we review the scientific literature in terms of the contents and role of organic supplements in promoting seed germination, protocorm development, and seedling growth in orchids. We systematically collected information from scientific literature databases including Scopus, Google Scholar, and ProQuest, as well as published books and conference proceedings. Various organic compounds, i.e., coconut water (CW), peptone (P), banana homogenate (BH), potato homogenate (PH), chitosan (CHT), tomato juice (TJ), and yeast extract (YE), can promote seed germination and growth and development of various orchids. They also stimulate seedling development, formation of protocorm-like bodies (PLBs), plantlet growth, and multiple shoot formation. The addition of organic compounds to culture media, individually or in combination, accelerates seed germination and seedling development. Different types and concentrations of organic nutrients are needed for the success ofin vitrocultures, depending on the species and genotype.

In vitro studies on Dendrobium fimbriatum Hk.F – An endangered orchid

A protocol for in vitro propagation from protocorms of Dendrobium fimbriatum a distributed in all over world and highly appreciated as an ornamental, was developed. Two different explants, entire protocorms and longitudinal halves of protocorms, were used. In addition, the effect of two different culture media, Murashige and Skoog (MS) and modified Knudson (KCm), supplemented with N6- benzyladenine (BA) (0, 0.5, 1, 2, 3, and 5 mg_L–1) and/or a-naphthalene acetic acid (NAA) at 0, 0.1, and 0.5 mg_L–1 was investigated. Adventitious shoot formation by direct organogenesis was obtained in all treatments; in some cases, the formation of protocorm like bodies was induced. Shoot formation was greater for entire protocorms; the best treatment was MS medium containing at BA 1 to 2 mg_L–1 in combination with at NAA 0.1 mg_L–1. The average height of shoots was three times greater in MS medium than in KC m medium. Sub culturing individual shoots in MS medium without plant growth regulators, but with 1 g_L–1 activated charcoal, allowed further development and rooting. An ex vitro survival rate of almost 100% was achieved. This study represents a comprehensive application for propagation, conservation, and sustainable use of this valuable natural resource.

Effect of plant growth regulators and carbon sources on proliferation and shoot formation of PLBs in Dendrobium candidum

Dendrobium candidum Wallich ex Lindley is a traditional Chinese medicine plant and has been widely used for medicinal and ornamental purposes. In this study, several different factors affecting micro propagation of protocormlike bodies (PLBs) such as basal media, plant growth regulators, and carbon sources. The proliferation PLB derived from seeds was the best in H3P4 basal medium containing 0.1 mg · L-1 NAA and 0.1 mg · L-1 Kinetin. PLB growth was the best when 10 g · L-1 sucrose was added to the carbon atoms in the medium. The rate of shoot formation from the propagated PLB was the highest in 1/4 MS or H1P2 medium containing 10 g · L -1 sucrose, and the shoot length was longer than the others.

Molecular spectrum of somaclonal variation in PLB-regenerated Oncidium revealed by SLAF-seq

Protocorm-like body (PLB) formation is an efficient method that has been widely used in the propagation of orchids. There is little information regarding the genome-wide molecular spectrum of somaclonal variation in PLB-regenerated orchids. We used specific-locus amplified fragment sequencing (SLAF-seq) to investigate the variations of single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) in somaclones of Oncidium ‘Milliongolds’, which was regenerated via PLB formation after in vitro culturing for 10 years. A high average variation rate (1.00 × 10− 3) was detected in 11 somaclones, and the most abundant variations were line specific. The average Ti/Tv ratio was 2.17, and G:C > C:G was the rarest type of SNP. The SNPs adjacent to adenine and thymine were more frequent than those adjacent to guanine and cytosine. The most prominent InDels were mononucleotide InDels. More than half of the InDels occurred within or adjacent to homopolymeric or polynucleotide repeats, and a majority of the repeats were thymine or adenine homopolymers. This study expands the current understanding of the somaclonal variation that occurs in PLB regeneration systems and long-term subcultures, which will be useful for improving the use of PLB formation in the propagation and breeding of orchids. We also confirmed that SLAF-seq is an efficient technology for genome-wide analysis of somaclonal variations for most species without reference genome sequences. We expanded the understanding of somaclonal variation in PLB regeneration and long-term subcultures of orchids, and confirmed SLAF-seq is efficient for genome-wide analysis of somaclonal variations without reference genome sequences.

Micropropagation of dendrobium heterocarpum lindl.

Dendrobium heterocarpum Lindl. is an endangered species which is currently used as ornamental pot plant for its beautiful flowers. An increase in collection for trade or any other purposes may lead to a dramatic decrease in the population of this species, thus becoming rare or endangered species in the near future. In this study, effects of plant growth regulators (BA, NAA, IAA, IBA, TDZ) and natural supplements (carrot, potato, and banana extracts) on protocorm like bodies (PLBs) formation; growth and development of shoot; and root regeneration of D. heterocarpum Lindl. as well as type of substrates on acclimatization and growth of seedlings were investigated. The results showed that PLBs formation was optimal on MS medium supplemented with 2.0 mg/L BA and 1.0 mg/L NAA (7.11 PLBs/explant; PLBs formation percentage of 68.9%) or MS medium supplemented with 1 mg/L TDZ and 0.5 mg/L NAA (7.29 PLBs/explant; PLBs formation percentage of 75.53%). For subculture, MS medium supplemented 1.5 mg/L BA and 60 g/L banana extract (22.40 shoots/explant; shoot length of 2 cm) was the most suitable for shoot regeneration and growth. Additionally, root formation was the most suitable on ½ MS medium supplemented with 1 mg/L NAA (4.4 roots/shoot; root length of 3.12 cm; root formation of 95.56%). Finally, the sufficiently rooted plantlets were transferred to greenhouse for hardening. After 60 days, coconut fiber substrate was the most suitable for seedling growth and development (with survival rate of 97.78%, root number of 5 and shoot length of 3.4 cm). The results of propagation in vitro Dendrobium heterocarpum Lindl. contribute to conservation and sustainable development as well as towards the rapid multiplication of seedlings for commercial commercialization of this wild orchid species.

Efficient Plant Regeneration Through Protocorm-like Bodies Derived from Shoot Tip Cultures of Vanilla planifolia Andr.

A protocol for obtaining a ready source of protocorm-like bodies (PLBs) was developed in Vanilla planifolia. MS containing BA 1 mg/l, IBA 0.5 mg/l, tryptone/peptone 2 g/l and 2% sucrose was used to induce PLBs from axillary bud explants. PLB formation from the shoot/root tips involved the direct conversion of the apices without an intermediate callus stage. PLBs either directly developed into plantlet or produced secondary protocorm like bodies. Thin cell layer culture of protocorm like bodies can be used as a commercial application of tissue culture technology for mass micro-propagation. The details of production of PLBs and their transformations in culture are also discussed. The plantlets derived from PLB were rooted in culture and established in the field after hardening.Plant Tissue Cult. & Biotech. 28(1): 91-98, 2018 (June)

<b>Determination of endogenous IAA and carbohydrates during the induction and development of protocorm-like bodies of <i>Cattleya tigrina</i> 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.

<b>Dynamics in global DNA methylation and endogenous polyamine levels during protocorm-like bodies induction of <i>Cattleya tigrina</i> 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.

A MADS-box gene associated with protocorm-like body formation in Rosa canina alters floral organ development in Arabidopsis

We isolated a gene encoding a putative MADS-box protein designated RcAGL15 from a protocorm-like body (PLB), a somatic embryogenetic structure) of Rosa canina L. RcAGL15 encodes a predicted protein of 251 amino acid residues with a molecular weight of 28 kDa and a theoretical isoelectric point of 7.5. Reverse-transcription polymerase chain reaction shows that RcAGL15 is mainly expressed in PLB and regenerated PLB, but not in leaves of aseptic seedlings, incubated leaflets before callus formation, calli, rhizoids, or leaves of regenerated shoots. Transgenic, ectopic expression of RcAGL15 in Arabidopsis changes numbers of floral organs and reduces content of indole-3-acetic acid, but increases levels of abscisic acid. Thus, RcAGL15 appears to be a new MADS-box gene that plays regulatory roles in morphological development and the number of flower organs, as well as somatic embryogenesis.

Effect of Light Quality, Sucrose and Trehalose on In Vitro Organogenesis of Cymbidium devonianum (Lindl.)

The present study investigated the effect of sucrose, trehalose and combination of sucrose and trehalose with modified MS medium on in vitro regulation of protocorm-like bodies (PLBs) of Cymbidium devonianum under different quality of lights (white fluorescent tube, green, red and blue LED). As a result of this study, new PLB and shoots were successfully regenerated on modified MS medium under different quality of lights. The highest PLB formation rate (100%) and the highest shoot formation rate (85%) were observed amongst explants cultured on medium supplemented with 10 g/l sucrose + 10 g/l trehalose under green LED. The maximum fresh weight of PLBs, the highest average number of PLBs and shoots were recorded on medium containing 10 g/l sucrose + 10 g/l trehalose under green LED. For plant tissue culture, sucrose is considered an indisputably important carbon and energy source and biosynthesis of trehalose is similar to that of sucrose. The hereby study concluded that the contribution of LED lights, sucrose and trehalose (combined) can induce PLB and shoot formation of Cymbidium devonianum tissue culture without the use of any other plant growth regulator, whereas the green light showed the best formation rate compare with the other studied qualities of lights.

In vitro Propagation and Forest Reestablishment of Cymbidium finlaysonianum Lindl., an Endangered Medicinal Orchid

An in vitro plant regeneration protocol was successfully established for Cymbidium finlaysonianum Lindl., an epiphytic endangered medicinal orchid, by culturing seeds from 4-month-old green self-pollinated fruits. Tetrazolium (TZ) viability test indicated a mean embryo viability of 97.80 %. Three asymbiotic orchid seed germination media, namely Knudson C medium (KC), New Dogashima medium (ND), and Vacin and Went medium (VW), were studied to select a suitable medium for seed germination. The highest seed germination percentage was 83.8 % on VW medium. Protocorm-like bodies (PLBs) of C. finlaysonianum Lindl. induced from protocorm segments cultured in VW liquid medium supplemented with 8.84 µM 6-benzylaminopurine (BAP) gave the highest % PLB formation and number of PLBs per explant, at 85.7 and 5.2, respectively. VW medium supplemented with 0.2 % (w/v) activated charcoal (AC) gave the highest number of roots per plantlet and root length, at 6.4 roots and 5.2 cm, respectively. The highest percentage of plantlet survival recorded at 24 months after reestablishment in the forest was 71.6 %.

Application of Chondroitin Sulfate on Organogenesis of Two <i>Cymbidium</i> spp. under Different Sources of Lights

The aim of this study was to present chondroitin sulfate as a plant growth regulator and to give an overview about light effects on PLBs (protocorm like bodies) culture of Cymbidium dayanum and Cymbidium finlaysonianum cultured in vitro. Chondroitin sulfate is a sulfated glycosaminoglycan (GAG) composed of a chain of alternating sugars N-acetylgalactosamine and glucuronic acid. It is widely used as a material for food ingredients, cosmetics and medicine. PLBs were cultured on modified MS medium containing different concentration of chondroitin sulfate (0, 0.1, 1 and 10 mg/l), under four sources of lights: conventional white fluorescent tube, red LED, green LED and blue LED. In C. dayanum, 100% PLBs formation rate was observed at 0.1 mg/l chondroitin sulfate with modified MS medium under green LED and 1 mg/l chondroitin sulfate under blue LED; the maximum shoots and roots formation were observed under green LEDs (93% and 80% respectively) when media contained 0.1 mg/l chondroitin sulfate. In C. finlaysonianum, every concentrations of chondroitin sulfate enhanced the growth rate of PLBs when compared to control treatment, under all four sources of lights. The highest values were recorded with 0.1 mg/l chondroitin sulfate which induced 100% PLBs formation under blue LED, while 10 mg/l chondroitin sulfate had induced 100% PLBs formation under green LED. The highest percentage of shoots (73%) was initiated in the medium containing 10 mg/l chondroitin sulfate under green LED. Plant development was strongly influenced by the light quality and plant growth regulator functions as chemical messengers for intercellular communication of plant. The results demonstrated that low concentrations of chondroitin sulfate could promote PLBs, shoots and roots formation of Cymbidium spp. under green and blue LED.

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.

Proliferation of Protocorm-Like Bodies of Dendrobium mannii and Dendrobium mirbelianum in Chitosan-containing Media In Vitro

Dendrobium mannii and Dendrobium mirbelianum were rare and endangered orchid species. In vitro micro propagation could be used to conserve and proliferate these species. The aims of this study were to determine the effects of chitosan on proliferation of the protocorm-like-bodies (PLBs) of in vitro grown Dendrobium mannii and D. mirbelianum. Chitosan has been reported as an important component to promote orchid growth in vitro. In this study chitosan was included in the in-vitro media at the concentrations of 0, 1, 2, 3, and 4 mg.L-1. The highest PLBs formation, clump diameter, and fresh weight on D. mannii were obtained from explants cultured on medium supplemented with 1 mg.L-1 chitosan. After 14 weeks of culture, fresh weight, clump diameter and average number of PLBs of D.mannii were 29.52 %, 33.00 % and 35.88% greater at chitosan (1 mg.L-1) compared to control media. D. mirbelianum treated with low chitosan concentrations (1 and 2 mg.L-1) had 65.71% more leaves than those without chitosan treatment. Chitosan, however, had an adverse effects on growth of both species at 4 mg.L-1. Medium contain 4 mg.L-1 chitosan induced PLBs proliferation of D.mannii, but inhibited PLBs proliferation of D.mirbelianum. These results suggested that chitosan had important roles in PLB proliferation and could be used for the propagation of D. mannii PLBs in vitro. Keywords: rare orchid species, in-vitro

Biochemical characterization of embryogenic calli of Vanilla planifolia in response to two years of thidiazuron treatment

Vanilla planifolia embryogenic calli were cultured for two years on a medium containing thidiazuron (TDZ). Due to the presence of TDZ, these calli were under permanent chemical treatment and the differentiation of adventitious shoots from protocorm-like-bodies (PLBs) was blocked. When embryogenic calli were transferred onto a medium without TDZ, shoot organogenesis and plantlet regeneration occurred. To gain better knowledge about the biochemical and molecular processes involved in the morphoregulatory role of TDZ, hormonal and metabolomic analyses were performed. Our results indicate that in the presence of TDZ, embryogenic calli contained a high amount of abscisic acid (ABA) essentially metabolized into abscisic acid glucosyl ester (ABAGE) and phaseic acid (PA), which was the most abundant. When transferred onto a medium without TDZ, shoot regeneration and development take place in four stages that include: embryogenic calli growth, differentiation of PLBs from meristmatic cells zones (MCZ), shoot organogenesis from PLBs and the elongation of well-formed shoots.From a hormonal perspective, the significant reduction in ABA metabolism and its readjustment in the ABAGE pathway triggered PLBs formation. However, this first morphogenesis was stimulated by a strong reduction in IAA metabolism. The organogenesis of PLBs into shoots is associated with an increase in ABA catabolism and a gradual shift in cellular metabolism towards shoot differentiation. Thus, the initiation of the elongation process in shoots is correlated with an alteration in metabolite composition, including an increase in energy reserves (sucrose/starch) and a rapid decrease in alanine content. Our data highlighted the relationship between endogenous hormone signalling, carbohydrate metabolism and shoot organogenesis in Orchid plants.