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Standardization of Tissue Culture Techniques in Phalaenopsis Orchids
Journal of Plant Biochemistry & Physiology

Journal of Plant Biochemistry & Physiology
Open Access

ISSN: 2329-9029

+44 20 3868 9735

Research Article - (2022)Volume 10, Issue 3

Standardization of Tissue Culture Techniques in Phalaenopsis Orchids

S Parvathy*
 
*Correspondence: S Parvathy, Department of Agriculrural Sciences, Savitribai Phule Pune University, Pune, India, Tel: 6238930675, Email:

Author info »

Abstract

Every year, hundreds of new species with diverse floral traits, lifestyles, habitat distributions, and trophic patterns are discovered and evolved in the Orchidaceae, the biggest and most fascinating family of flowering plants with over 800 genera and roughly 30,000–35,000 specie. Due to their distinctive use as cut flowers and pot plants, orchids are arguably the most popular decorative crop species in the world. It is a vast and diverse family of flowering plants with important ornamental, medicinal, conservation, and evolutionary research applications. Since ancient times, their ubiquitous beauty has captivated humans. Because of their exotic beauty and long shelf life, orchids are commonly grown as ornamental cut flowers.

Orchid cultivation has really been prominent in tropical and subtropical regions, with many large, mechanized, and well-managed greenhouses. Phalaenopsis is the world's most significant potted orchid, with enormous breeding and in vitro propagation technology achievements in countries such as Belgium, the Netherlands, Taiwan, and Thailand. Another reason for its popularity is the simplicity with which floral induction may be regulated for year-round output. Cut orchids such as Cymbidium, Dendrobium, Oncidium, and Vanda are widely used as cut flowers all around the world. Orchids have become increasingly popular around the world market as a result of their long shelf life, diverse colour palette, and other desirable traits that purchasers want based on their location or culture.

Keywords

Monopodial orchids; Phalaenopsis; Sympodial orchids

Introduction

Orchids are classified into two main categories depending on their development habits: monopodial and sympodial. Monopodial orchids, such as Phalaenopsis and Vanda, have the main stem that grows year after year, but sympodial orchids, such as Cattleya and Cymbidium, have a main stem that stops growing at the end of each season. Due to the vast spectrum of floral character uniqueness and the access to a large number of varieties and hybrids, monopodials have gained in popularity around the world. The monopodial orchids Phalaenopsis, Aranda, Mokara, and Vanda are the most popular [1-20].

Phalaenopsis, often known as moth orchid, is among the most recognized orchid species because of its beautiful and long-lasting flowers and simplicity of growth in artificial environments. The name Phalaenopsis comes from the Greek terms ‘phalaina’, which means ‘moth’, and ‘opsis’, which means ‘look-alike’, and refers to the flowers' resemblance to a fluttering moth. The deep, damp, and warm woods of India, Indonesia, and the Philippines are habitats to about 60 species of this genus. They can be found between 200 and 400 metres above sea level. These orchids have a great commercial value as cut flowers on the worldwide flower market. Phalaenopsis orchids have long fibrous roots, short leafy shoots, and long-lasting flat blooms grouped in a floral stalk that frequently bends at the terminal. Phalaenopsis is usually propagated by cutting or dividing off-shoots as it is a monopodia plant. These methods, however, have a low multiplication rate and impede the growth of the mother plant, making them ineffective for large-scale production. Vegetative propagation is difficult, and seedling quality varies [21-28].

Based on their floral characteristics, Phalaenopsis orchids can be divided into two categories: grandiflora (cut flower) and multiflora (pot plant). Multiflora varieties have short, multiple inflorescences with many tiny blooms, and grandiflora types have long, bowing inflorescence with huge flowers. Long coarse roots, short leafy stems, and long-lasting flat blooms organized in a flowering stem that frequently branches at the end characterizes Phalaenopsis orchids [29-36]. Since Phalaenopsis is a monopodial plant, it is traditionally propagated by cutting or dividing off-shoots. However, these methods result in a low multiplication rate and slow the mother plant's growth, making them ineffective for large-scale production. As a result, vegetative propagation is difficult, and seedling qualities vary. Flowering takes at least three years in a greenhouse, which is one of the major challenges in commercial Phalaenopsis production. As a result, tissue culture may be a viable alternative for propagating this orchid species.

Cross-pollination involving selected orchid plants has increased the quality of commercial orchids. The hybrid will then be rebuilt through seed germination [37-45]. The hybrid will exhibit a wide range of phenotypes; therefore this method has various disadvantages. Although a considerable amount of time is required to further select hybrids with favourable characteristics. In vitro culture, also known as micropropagation, can be used to regenerate specific orchids in order to produce uniform plants in a short period of time. In most cases, the propagule exhibits similar features of its parent [46-54].

A number of researchers have attempted to rejuvenate Phalaenopsis orchids. The same orchid genus has been successfully propagated using PLBs. Various plant hormones and explant sources have been used to induce PLBs [55-67]. Utilizing leaf cells from propagules, PLB generation on MS media with BAP and NAA or TDZ has been described. 5.4 M NAA and 4.5 M TDZ were used to produce PLBs and plantlets from Doritaenopsis orchid flower stalks.

In light of the foregoing, the goal of this work was to establish a procedure for in vitro mass multiplication of superior Phalaenopsis genotype.

Materials and Methods

The study entitled “Morphological studies and standardization of tissue culture technique in Phalaenopsis orchids” was conducted in the Department of Plant Biotechnology and Department of Plant Breeding and Genetics, College of Agriculture, Vellayani, Thiruvananthapuram, during 2020-2021. The objectives of this research were to standardize tissue culture technique in Phalaenopsis genotype. The following are the most important studies that were undertaken.

  • Identification of the best explant from Phalaenopsis genotype for propagation
  • Standardization of tissue culture media for Phalaenopsis

Experiment: Phalaenopsis genotype with high flower yield attributing quantitative vegetative and floral characters and commercially valuable qualitative floral traits were chosen for in vitro mass multiplication studies. This study aimed to identify the best explant from the selected genotype for the standardization of tissue culture protocol for the same. The materials and methods used in this experimental are described in detail below [68-87].

Materials

Source of explants: Phalaenopsis genotype with good cut flower quality selected based on statistical analysis of morphological traits from eight commercially available moth orchids maintained in the green house of Department of Plant Biotechnology and Department of Plant Breeding and Genetics, College of Agriculture, Vellayani was used as explants source. These are monopodia orchids that produce fleshy and conspicuous leaves. From the selected genotype, inflorescence stalk with axillary bud, inflorescence stalk and intermodal segment of inflorescence stalk were taken as explants for in vitro propagation of orchid.

Design: CRD, Treatments: 3, Replications: 5, Treatments (explants), Inflorescence stalk with axillary bud, Inflorescence stalk, and Inter nodal segment of inflorescence stalk.

Composition of media

In this investigation, plant tissue culture was performed using ½ MS medium supplemented with several growth regulators. The MS medium's composition is listed.

Experimental method

Fully developed inflorescence stalk, intermodal segment of inflorescence stalk and axillary bud with inflorescence stalk before flowering were taken as explants. The procedure used to prepare explants for inoculation differed depending on the type of explant [88-96].

Inflorescence stalk

Individual inflorescence stalks were gathered from mother plants by cutting them at the base using a sharp knife, leaving the basal node on the stalk for future inflorescence formation. The stalks were cleansed with cotton soaked in 70% alcohol before being cut into nodal sections and cleaned in distilled water. The sections were surface sterilized after the bracts protecting the dormant buds were removed.

Intermodal segment of inflorescence stalk

Individual inflorescence stalks were harvested from mother plants by cutting them at the base with a sharp knife, leaving the basal node for later inflorescence production. The stalks were cleaned with cotton soaked in 70% alcohol before being trimmed into intermodal sections and cleaned in distilled water. The sections were then surface sterilized.

Axillary bud with inflorescence stalk

Shoot tips (Axillary bud) with inflorescence stalk was collected from the parent plant under aseptic condition using sterile blades and cleaned with cotton soaked in 70% alcohol followed by surface sterilization before removing the leaf sheath.

Standardization of surface sterilization

Explants were surface sterilized to ensure that they were free of microbial contamination. Prior to surface sterilization, explants were exposed to the various pretreatments. Explants were carefully cleaned under running tap water. They were then soaked in a 0.1 percent baiting solution with two drops of pill along with 0.1 per cent streptomycin for 40 minutes followed by repeated washing with distilled water before surface sterilization [97-117].

Surface sterilization of explants was performed inside a laminar air flow chamber. The explants were washed thoroughly in sterile double distilled water before applying treatments. The inflorescence stalk explants were then cut into smaller pieces of 4 cm-5 cm with a sharp blade for easy handling during sterilization. The cut explants were treated with surface sterilization agents with continuous shaking for different durations as per (Table 1). Each treatment was replicated five times. After each surface sterilization, the explants were rinsed thrice with sterile double distilled water and 1 cm-2 cm length explants were inoculated on half strength MS medium to study the explant survival. Axillary bud with inflorescence stalk was rinsed thoroughly with double distilled water after separating them from leaf sheath. 0.1 percent Mercuric chloride (HgCl2) followed by alcohol (70 %) were employed with all the three explants for varied time durations (5 min, 8 min, 10 min, and 12 min) in order to design an effective surface sterilization approach [118-120].

Treatment No. Treatments Duration (minutes) Explant used
ST1 0.1% HgCl2 5 IS, IIS, ABIS
70% alcohol 5
ST2 0.1% HgCl2 8 IS, IIS, ABIS
70% alcohol 8
ST3 0.1% HgCl2 10 IS, IIS, ABIS
70% alcohol 10
ST4 0.1% HgCl2 12 IS, IIS, ABIS
70% alcohol 12
ST-Surface sterilization Treatment; IS-Inflorescence stalk; IIS-Intermodal segment of inflorescence stalk; ABIS-Axillary bud with inflorescence stalk

Table 1: Treatments to evaluate the effectiveness of surface sterilization.

Following mercuric chloride treatment, explants were washed three to four times with sterile double distilled water inside the laminar air flow cabinet to remove any traces of HgCl2. The end portions of the inflorescence (nodal and intermodal) segments on both sides were removed and cut into a 2 cm long piece.

Axillary bud with flower stalk will be separated from leaf sheath under aseptic condition using sterile blade and trimmed to 2 cm long piece.

Observations recorded

  • Percentage survival of explants at culture initiation stage
  • Percentage propagation of explants
  • Days required for response

Preparation

Plant tissue culture media used for the study was half strength Murashige and Skoog medium preparation. Major and minor nutrient stock solutions were made by dissolving the needed amount of chemicals in a precise volume of double glass-distilled water. Plant growth regulators were first dissolved in 1N NaOH or 95 percent ethanol, then double glass-distilled water was added to make up the volume, and the stock solutions were kept refrigerated at 4 degrees Celsius [121-134].

The stock solutions of plant growth chemicals were only kept for one week, but the other stock solutions were kept for up to one month. Six stock solutions for macro nutrients, micro nutrients and organic supplements (A, B, C, D, E, and F) were prepared and kept chilled in pre-cleaned glass bottles. The stock solutions of various growth regulators were kept in the refrigerator in order to make the media preparation procedure easier. Plant growth substance stock solutions could only be kept for a week, although other stock solutions could be kept for up to a month. All of the glassware used in the culture media preparation were washed with tap water containing a few drops of lanoline and then rinsed with double glass-distilled water.

All stock solutions were pipetted into a 1000 ml beaker in proportionate volume. Sucrose was added and dissolved immediately. Other chemicals were added directly to the media while it was being prepared for certain treatments. Then, using double glass-distilled water, the volume was increased to 950 ml. After adding all of the medium components except the agar, the pH was adjusted using an electronic pH metre between 5.5 and 5.7 using 0.1 N NaOH and 0.1 N HCl. The medium was then added with 8.0 g L-1 agar, and the final volume was increased to 1000 ml using double glass-distilled water. The beaker with media was then placed in a microwave oven to boil, with intermittent stirring with a glass rod, until the agar melted and a clear solution was obtained. Then 15 ml-20 ml of hot medium was transferred into pre-sterilized glass culture tubes measuring 25 mm x 150 mm with proper labels, which were then closed with non-absorbent cotton plugs and autoclaved [135-142].

Inoculation of explants

Inoculation of explants was carried out in Laminar Air Flow Chamber as it ensures aseptic condition for safe transfer of plant material to sterile medium. Swabs with 70% alcohol were used to sanitize the work table of the laminar air flow cabinet. Prior to operating in the laminar air flow cabinet, the UV lamp was turned on for 20 minutes to create an aseptic atmosphere within the cabinet. Wash the glassware (Petri dishes and beakers) and tools (blades, scalpels, and tweezers) required for inoculation thoroughly, rinse with double glass distilled water, wrap with plastic film and store in a polypropylene cover that can be autoclaved, and autoclaved at 121Ë?C and 1.06 Kg cm-2 pressure for 40 minutes. After surface sterilization treatments, the trimmed explants were carefully transferred to culture medium with the help of sterile forceps. The culture vessels should be opened and closed in front of the spirit lamp flame during explant inoculation for ensuring sterile condition and thereby minimizing the chance of contamination. Explants were inoculated in such a way that their cut end had as much contact with the medium for maximum utilization of media components. Inoculated culture vessels were then incubated in culture room.

Culture conditions

In an air-conditioned culture room with a 16 hours photoperiod, the cultures were kept. To meet the explants' light requirements (2000 lux), white fluorescent tubes were used. The humidity in the culture chamber ranged from 60 to 80 per cent. Sub culturing was done at two week intervals. The culture vessels and racks were wiped with 70 per cent ethanol at an interval of three days to avoid the entry of contaminants [143-148].

Identification of best explant

The explants were selected based on certain criteria such as maximum percentage survival at culture initiation stage observed one week after inoculation, fastest response in basal medium (½ MS) with different hormonal combinations (2-3 mg/L BAP and 0.5-1 mg/L NAA) with maximum percentage propagation(Table 2). Explants selected from this experiment were used for further in vitro micro propagation studies of selected superior Phalaenopsis genotype.

Sl. No. Explant used Media composition
1 Inflorescence node Inflorescence internode Axillary bud with inflorescence stalk ½ MS(control)
2 Inflorescence node Inflorescence internode Axillary bud with inflorescence stalk ½ MS+ 2 mg/L BAP+ 0.5mg/L NAA
3 Inflorescence node Inflorescence internode Axillary bud with inflorescence stalk ½ MS+ 2.5 mg/L BAP+ 0.5mg/L NAA
4 Inflorescence node Inflorescence internode Axillary bud with inflorescence stalk ½ MS+ 2.5 mg/L BAP+ 1mg/L NAA
5 Inflorescence node Inflorescence internode Axillary bud with inflorescence stalk ½ MS+ 3 mg/L BAP+ 0.5 mg/L NAA

Table 2: Different explants of Phalaenopsis and basal media with different hormonal combinations.

Observations recorded

  • percentage propagation of each explant after three week
  • presence of phenolic
  • explant response
  • days required for explant response in culture media

Experiment -1

Materials: Explants showing maximum percentage survival at culture initiation stage with minimum contamination as well as fastest response in culture media selected from experiment ii was used for further studies.

Design: CRD

Treatments: 5

Replication: 5

Experimental method: The explant selected from experiment II which showed the fastest response in half strength Murashige and Skoog media with different combination of BAP and NAA was used for the standardization of tissue culture technique in Phalaenopsis genotype. Those explants showed positive responses was sub cultured in the same media and maintained for the formation of protocorm like bodies (PLBs). The cultures were incubated under a 16-hour photoperiod. Subculture was performed every two week. Developed PLBs were then transferred into half-strength MS medium without any plant growth regulator. The pH of the media was adjusted to 5.6-5.8. These cultures were then incubated under a 16-hour photoperiod. Mean number of PLBs were calculated after tenth week of inoculation.

Observations recorded: Comparison of percentage propagation of explant in selected basal media with different combinations of BAP and NAA

  • Days for emergence of PLBs in different media combination
  • Days for emergence of plantlets from protector like bodies
  • Number of plantlets generated per explant

Results

The outcomes of the present study are reported in the following sections. The most important studies that were undertaken are listed below.

  • Identification of best explant from selected genotype for propagation
  • Standardization of tissue culture media for selected Phalaenopsis

Experiment-2

Inflorescence stalk, intermodal segment of inflorescence stalk and inflorescence stalk with axillary bud were used for in vitro culture. Flowering mother plants of Phalaenopsis Chian Xen Diamond * Tinny Honey selected based on inflorescence characters kept at the College of Agriculture, Vellayani, were used as explant sources.

Standardization of surface sterilization technique

Table 3 shows observations on the percentage of explant survival one week after inoculation. Explants were subjected to pretreatments prior to surface sterilization. Under running tap water, the explants were carefully cleaned. They were then immersed in a 0.5 percent bavistin solution containing two drops of pill and 0.5% bactericide for 40 minutes before being thoroughly rinsed with distilled water. Surface sterilization treatments with mercuric chloride (0.1%) and alcohol (70%) were applied to the inflorescence nodal, intermodal segments of inflorescence node, and axillary bud with inflorescence stalk for varying time durations (5 min, 8 min, 10 min, and 12 min).

On half strength MS media with different combination of BAP and NAA, four treatments were evaluated to examine the effect of surface sterilization treatments on microbial contamination and percentage explant survival (Table 3). Surface sterilization treatments had different impacts on different explants of P8. The treatment TS3 (0.1 percent mercuric chloride for 10 minutes+70% alcohol for 10 minutes) was determined to be the most effective for surface sterilization, with an explant survival rate of 80 percent (inflorescence stalk and intermodal segment of inflorescence stalk) and 60 per cent (axillary bud with inflorescence) (Figures 3-10).

Treatment number Treatments Duration (min.) Percentage survival at culture initiation stage
Inflore scence stalk Internodal segment of inflorescen ce stalk Axillary bud with inflorescenc e stalk
ST1 0.1% HgCl2 5 20 20 0
70% alcohol 5
ST2 0.1% HgCl2 8 40 40 20
70% alcohol 8
ST3 0.1% HgCl2 10 80 80 60
70% alcohol 10
ST4 0.1% HgCl2 12 60 60 40
70% alcohol 12

Table 3: Standardization of surface sterilization treatments for inflorescence stalk.

Effect of different hormonal combinations on various explants

For the study, explants were inoculated in ½ MS with various combinations of growth regulators BA (2 mgl-1-3 mgl-1) and NAA (0.5 mgl-1-1 mgl-1) (Figure 1). Response of each explant along with percentage establishment in culture and days required for response were recorded in the (Table 4).

Sl. No. Media composition Explant used Explant response Percentage of culture establishment Days required for sprouting
1 ½ MS (control) Inflorescence stalk with axillary bud No Change Nil Nil
Inflorescence stalk No Change Nil Nil
Inter nodal segment of Inflorescence stalk No Change Nil Nil
2 ½ MS+2 mg/L BAP+0.5mg/L NAA Inflorescence stalk with axillary bud No Change Nil Nil
Inflorescence stalk Sprouting 66 34
Inter nodal segment of Inflorescence stalk No Change Nil Nil

Table 4: Response of various explants to combinations of growth regulators.

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Figure 1: Phalaenopsis genotype selected for further studies-Phalaenopsis Chian Xen Mammon * Tinny Honey.

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Figure 2: Stage of explants used for inoculation.

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Figure 3: Inoculation of explants on culture medium; (A) Infloescence stalk; (B) Internodal segment of inflorescence stalk; (C) Axillary bud with inflorescence stalk.

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Figure 4: Response of inflorescence stalk to combination of growth regulators. (a) Bulging of inflorescence stalk 22 days after inoculation in ½ MS+ 2.5 mg/L BAP+1 mg/L NAA ;(b) Bulging of inflorescence stalk 16 days after inoculation in ½ MS+3 mg/L BAP+0.5 mg/L NAA.

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Figure 5: Response of inflorescence stalk to combination of growth regulators. (a) Sprouting of inflorescence stalk 34 days after inoculation in ½ MS+2 mg/L BAP+0.5mg/L NAA ; (b) Sprouting of inflorescence stalk 25 days after inoculation in ½ MS+2.5 mg/L BAP+ 0.5 mg/L NAA.

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Figure 6: Regeneration of shoots and roots from inflorescence stalk in ½ MS+2 mg/L BAP+ 0.5 mg/L NAA. (a) Shoot initiation seven weeks after inoculation; (b) Shoot elongation 12 weeks after inoculation; (c) In vitro rooting 18 weeks after inoculation.

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Figure 7: Regeneration of shoots and roots from inflorescence stalk in ½ MS+2.5 mg/L BAP+0.5 mg/L NAA. (a) Shoot initiation three weeks after inoculation; (b) Shoot elongation seven weeks after inoculation; (c) In vitro rooting 13 weeks after inoculation.

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Figure 8: PLBs formation from inflorescence stalk. (a) 14 weeks after inoculation in ½ MS +3mg/L BAP+0.5 mg/L NAA; (b) 16 weeks after inoculation in ½ MS+2.5 mg/L BAP+1 mg/L NAA.

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Figure 9: PLBs subcultured on basal medium without.

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Figure 10: Plantlets regenerated from PLBs after 12 week of.

Among the explants studied, only the inflorescence stalk responded by bulging and sprouting. In culture media, the inflorescence stalk with axillary bud and the intermodal section of the inflorescence stalk remained intact. Within 16 days of inoculation, bulging of the inflorescence stalk was observed with 50 per cent propagation in ½ MS with 3 mg/l BAP and 0.5 mg/l NAA. Within 25 days of inoculation, sprouting from the node of a mature inflorescence stalk was observed in with 2.5 mg/l BA and 0.5 mg/l NAA with 83 per cent propagation. As a result, the inflorescence stalk was used as an explant in further research (Figures 3 and 4).

Experiment 2

Inflorescence stalk was inoculated in half strength Murashige and Skoog (½ MS) medium with different concentrations of BAP and NAA for Protocorm Like Bodies (PLB) formation.

Design: CRD, Treatments: 5, Replication: 5, Treatments

T1-½ MS (control)

T2-½ MS+2 mg/L BAP+0.5mg/L NAA T3-½ MS+2.5 mg/L BAP+0.5mg/L NAA T4-½ MS+ 2.5 mg/L BAP+ 1 mg/L NAA T5-½ MS+3 mg/L BAP+0.5 mg/L NAA

Morph genic responses of inflorescence nodal explant cultured in media containing different combinations of growth regulators were recorded (Table 5). Within 14 weeks of inoculation, protector-like bodies (PLBs) were distinguished from the inflorescence stem in ½ MS with 3 mg/L BAP and 0.5 mg/L NAA (Plate 13). Average number of PLBs that arose from inflorescence stalk was found to be maximum in ½ MS+3 mg/L BAP+ 0.5 mg/L NAA (Table 6). Within three weeks of inoculation, ½ MS with 2.5 mg/L BAP and 0.5 mg/L NAA displayed inflorescence stalk shoot initiation response (Plate 12). From the same medium, shoot elongation needed only seven weeks while root regeneration taken thirteen weeks. ½ MS with 2 mg/L BAP and 0.5 mg/L NAA displayed inflorescence stalk shoot initiation response within seven weeks after inoculation (Plate 11).

Media Explant response Percentage propagation of explant Days for emergence of shoots/PLBs
T1 Nil Nil Nil
T2 Shoots initiation 66 49
T3 Shoots initiation 83 21
T4 PLB formation 22 112
T5 PLB formation 50 98

Table 5: Morph genic responses of inflorescence nodal explant cultured in media containing different combinations of growth regulators.

Media Number of PLBs per explant
½ MS+2.5 mg/L BAP+1mg/L NAA (T4) 6
½ MS+3mg/L BAP+0.5mg/L NAA (T5) 9

Table 6: Average number of PLBs that arose from inflorescence stalk.

Regeneration of plantlets

PLBs were transferred to basal medium without plant growth hormones (Plate 14). After 12 weeks of being placed into media without plant growth regulators, all PLBs germinated into plantlets (Plate 15). When a Protector-like Body (PLB) is grown in basal medium without growth regulators, it retains its ability to develop new leaves, roots, or even shoots since it has leaf and root primordial. Around 9 plantlets were regenerated per inflorescence stalk (Table 7).

Media Days for emergence of plantlets from protector like bodies Number of plantlets regenerated per inflorescence stalk
½ MS w/o PGR 84 9

Table 7: Regeneration of plantlets from flower stalk node.

Discussion

Floriculture has emerged as a massive entrepreneurial opportunity for small and marginal farmers, as well as a means of earning foreign currency. Cut flowers, pot plants, cut foliages, seeds, bulbs; tubers, rooted cuttings, and dried flowers or leaves are all products of the floriculture industry. Floriculture has grown in importance as the demand for flowers has increased, making it one of the most important commercial trades in Indian agriculture. Rose, carnation, chrysanthemum, gerbera, gladiolus, orchids, anthodium, tulip, and lily are the most important floricultural crops in the international cut flower trade. Orchids account for a significant portion of the global floriculture trade, both as cut flowers and as potted plants, and are estimated to account for approximately 10% of the international fresh cut flower trade.

Tropical and subtropical countries, many of which have automated and effectively managed big greenhouses, have produced the majority of the world's orchids. Phalaenopsis is the most widely traded potted orchid, with outstanding breeding and micro propagation technological achievements in countries such as Belgium, the Netherlands, Taiwan, and Thailand. The ease of controlled floral introduction for scheduled and year round production is another reason for its popularity. Cut orchids, such as Indicium, Vanda, Cymbidium, and Dendrobium are widely used as cut flowers around the world.

Dendrobium and Phalaenopsis plants were the major orchid genera for exports. Thailand is the world's leading producer and exporter of tropical orchids. Orchid exports generate approximately 2.5 billion baht in revenue for Thailand each year. Taiwan's orchid kingdom expanded its global leadership by adding Australia and Brazil as export markets. Orchids from the Taiwan are exported to 36 countries in North America, Northern Europe, and South Africa. Thailand is the largest exporter of orchid cut flowers to India, accounting for 80.67 percent of total imports, followed by the Netherlands (15.54 percent), New Zealand (2.29 percent), and China (1.5%). Singapore's imports of fresh orchids from India were only US$ 1379.3, accounting for

0.02 percent of the country's total imported products in 2007. As a result, considering the country's proximity and India's East Policy, there are numerous opportunities to increase India's exports to Singapore.

Due to the severe coronavirus pandemic and global economic disruption since the beginning of 2020, orchid marketing has shifted to a greater share of digitalization or E-commerce, coexisting with traditional flower stores and supermarkets. Non-traditional technologies, such as biotech-oriented efficient breeding, aid in the development of more orchid products or novel new orchid hybrids, which may open up new avenues for orchid production in the future.

Standardization of surface sterilization treatment for explants

The biggest obstacle encountered during the establishment of tissue culture explants was contamination induced by bacteria and fungi. Because the mother plants had been exposed to field conditions for a long period, microbes harbored the plant tissues, resulting in systemic infection during the culture of explants taken from them. During the early phases of culture, a high rate of bacterial and fungal contamination was detected in the current study. Various surface sterilization methods were used in order to get contamination-free culture.

When surface sterilization was performed using 0.1 percent mercuric chloride in combination with 70% alcohol for 10 minutes, the percentage survival of inflorescence stalks and intermodal segment of inflorescence stalk on half strength MS medium was determined to be the highest (80%). The sterilization treatment with

0.1 per cent mercuric chloride and 70 per cent alcohol (ST3) was shown to be very successful regardless of the explants. In comparison to other surface sterilization techniques, sterilized explants had a higher proportion of survival in the culture medium. Most of the strategies discovered for in vitro propagation of Phalaenopsis, according to and, involve propagation via seedling or culturing the dormant buds found at the basal section of the inflorescence. Axillary bud with inflorescence stalk showed 60 per cent survival in culture media after surface sterilization with TS3 treatment.

Response of different explants on culture medium: Only the inflorescence stem responded by bulging and sprouting when explants were inoculated in ½ MS with various combinations of growth regulators BA (2-3 mgl-1) and NAA (0.5-1 mg l-1). The axillary bud and the intermodal region of the inflorescence stalk remained intact in culture conditions. In ½ MS with 3 mg/l BAP and 0.5 mg/l NAA, bulging of the inflorescence stem was seen 16 days after inoculation, with 50% propagation. In ½ MS with 2.5 mg/l BA and 0.5 mg/l NAA, sprouting from the node of a mature inflorescence stalk was observed with 83 percent propagation within 25 days after inoculation. As a result, the stalk of the inflorescence was employed as an explant in the further studies. Explants were utilized in the culture of orchid species in a variety of ways. Four mass propagation protocols have been established. PLB regeneration from inflorescence node, shoot tips, root tips, and stem segments through direct embryogenesis was determined to be one of the finest techniques in Phalaenopsis, according to Chen et al. (2000) and Zhang and Fang (2005).

Standardization of tissue culture protocol for selected phalaenopsis genotype

Protocorm-Like Bodies (PLBs) were differentiated from the inflorescence stalk in ½ MS with 3 mg/L BAP and 0.5 mg/L NAA within 14 weeks of inoculation. According to Stacey D. Novak et al. (2014), hormone-depleted medium or decreased levels of auxin in combination with cytokinins generally create PLBs from callus. Arditti and Ernst (1993), reported that auxins, particularly NAA, alter the process of regeneration in monopodial epiphytic species, acting synergistically on the production of PLBs. ½ MS with 2.5 mg/L BAP and 0.5 mg/L NAA showed shoot initiation response from inflorescence stalk within three weeks of inoculation. Shoot elongation was observed within seven weeks and root regeneration within 13 weeks from same medium. Shoot development from nodes of a stem or stem-like tissue, such as rhizomes or inflorescence stalks, is a common in vitro response to exogenously given cytokine and auxin in many orchid species. In addition, some orchid tissue culture studies found that axing alone or in combination with cytokines increased overall shoot growth. The influence of auxin on new root production has been widely investigated in model plant systems, according to report that several techniques of propagation for Phalaenopsis have been developed through in vitro culture of various parts including shoot tips, flower stalk nodes, buds, root tips, and rhizome segments with varying responses. Although other portions of the plant can be utilized as initial explants for growing PLBs, foliar explants are more convenient to obtain and do not require the mother plant to be sacrificed. According to, unlike the utilization of inflorescence components, foliar explants are not limited in availability. Suggested that, in order to produce more PLBs, half strength MS was utilized, which produced an average of 9 PLBs per explant, compared to

PLBs per explant for VW: The formation of Protector Like Bodies (PLBs), according to, is a significant strategy for micro propagating orchids. All PLBs germinated into plantlets 12 weeks after being transferred into basal media without plant growth regulators. Exogenous Auxins (NAA and IAA) and Cytokinin (BAP) can induce cell proliferation and differentiation to create somatic embryos or PLBs, in studies conducted by. PLBs can germinate into plantlets when grown in media without plant growth regulators, according to studies by. Around 9 plantlets were regenerated per inflorescence stalk nodal segment. A continuous sub culturing of PLBs can generate more number of plantlets.

References

Author Info

S Parvathy*
 
Department of Agriculrural Sciences, Savitribai Phule Pune University, Pune, India
 

Citation: Parvathy S (2022) Standardization of Tissue Culture Techniques in Phalaenopsis Orchids. J Plant Biochem Physiol. 10:289

Received: 11-Mar-2022, Manuscript No. JPBP-22-16244; Editor assigned: 14-Mar-2022, Pre QC No. JPBP-22-16244; Reviewed: 28-Mar-2022, QC No. JPBP-22-16244; Revised: 11-May-2022, Manuscript No. JPBP-22-16244; Published: 18-May-2022 , DOI: DOI: 10.35248/2329-9029.22.10.289.

Copyright: © 2022 Parvathy S. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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