INTRODUCTION
Research rationale
As we approach the sixth great extinction, the urgency to protect biodiversity has intensified, largely due to human activities and population growth The degradation of biodiversity poses a significant threat, with over 12.5% of plant species at risk of extinction (Walter & Gillett, 1998) To combat this crisis, it is essential to implement diverse conservation strategies aimed at preserving and protecting rare species These strategies may include facilitating the migration of species to buffered environments that enhance their adaptability to changing conditions (Swarts & Dixon).
Orchids, belonging to the Orchidaceae family, are the largest group of flowering plants with nearly 25,000 species However, their populations in the wild are experiencing significant declines, with a high proportion of threatened genera and species This alarming trend highlights the vulnerability of orchids, making conservation efforts crucial for their survival.
& Dixon, 2009) They are exposed to the extrinsic threads from the over collecting and habitat change from human beings, as well as intrinsic (Swarts & Dixon,
Orchids face significant threats not only from external factors but also from their own vulnerabilities, particularly in relation to their symbiotic relationships with mycorrhizal fungi and their characteristics as epiphytes and lithophytes While mycorrhizal fungi generally support orchid health, climate change can disrupt this balance, putting orchids at risk (Dearnaley, 2007) Additionally, the ancient traits of epiphytes and lithophytes may hinder their survival as global warming forces plants to migrate to higher altitudes, creating challenges for orchids in adapting to these changes (Swarts & Dixon).
Anoectochilus setaceus Lindl., a member of the Orchidaceae family, is recognized for its significant horticultural, medicinal, and culinary applications Its high economic value and numerous health benefits contribute to its popularity in various industries.
A.setaceus Lindl in Vietnam is overexploited in the wild, leading to exhaustion (Bân, 2005) In 2007, in the Decree No 32/2006 / CP and the Vietnam Red Book,
A.setaceus Lindl were classified into group IA (a, c, d) a group of endangered forest plants (EN), and a group of plants banned from commercial exploitation and trade Therefore, it is very necessary to research the propagation method and cultivating tincture in an artificial environment (in vitro), contributing to the conservation of this precious medicinal herb.
Research’s objectives
The objective of the study was to examine the best condition for
Anoectochilus setaceus Lindl stems can be effectively propagated using in vitro techniques While previous studies have indicated that orchid seeds are preferable for propagation due to their germination rates (Dutra et al., 2008; Godo et al., 2010; Park et al., 2000; Zeng et al., 2012), the seasonal nature of stem and top growth makes them a more viable option for improving productivity and restoring biodiversity.
This study evaluated the optimal medium formula for A setaceus Lindl by analyzing shoot regeneration rates and lengths Building on previous research from the Biotech laboratory at Thai Nguyen University of Agriculture and Forestry (TUAF), the impact of varying concentrations of growth regulators (BAP and Kinetin) on shoot regeneration rates and the quality of regenerated shoots was assessed Following regeneration, the shoots were transferred to different media with varying organic compound compositions to facilitate shoot multiplication The findings aim to identify the most effective culture medium and explants for in vitro conservation and propagation of A setaceus.
Anoectochilus setaceus Lindl can be identified.
Significance of the study
In vitro refers to experiments conducted outside of a living organism, aiming to preserve the organism while providing essential nutrients for tissue culture in a lab setting This method offers a controlled environment for vulnerable species to adapt before reintroduction into their natural habitats By utilizing in vitro techniques for the propagation of A setaceus Lindl., this study contributes to the conservation of this valuable orchid and supports biodiversity preservation.
Scope and Limitations
The experiment faced several limitations that could affect the results Unequal distribution of agar during medium preparation resulted in some media being semi-fluid instead of semi-solid, which may have caused explants to fall due to insufficient support.
LITERATURE REVIEW
Orchid conservation status
Anthropogenic factors, such as illegal overcollection and land clearance, are driving wild orchids toward rarity (Cribb et al., 2003; Koopowitz, 2001) This unsustainable exploitation significantly reduces orchid diversity (IUCN, 1999) Additionally, ecosystem disruption from hunting can destabilize the food chain, increasing grazing pressure on orchids and heightening their vulnerability to damage (Swarts, 2007) Human impacts, including rapid industrialization and urbanization, exacerbate threats like disease, pests, intense fires, salinization, and desertification, all of which challenge orchid habitats and conservation efforts (Sahagian, 2000) Habitat changes pose significant risks, as disturbed ecosystems are likely to see a decline in orchid abundance, particularly those reliant on mycorrhizal associations (Brundrett, 2006; Cribb et al., 2003) Furthermore, climate change is making epiphytic and lithophytic orchids more vulnerable due to shifts in their habitat, while specialized pollinators and seed dispersal agents face potential extirpation, further increasing extinction risks for orchids (Dixon).
In vitro orchid conservation requires strategies that are well-informed about the growing environment to ensure the long-term sustainability of both orchids and their ecosystems Conservationists should focus on assisting with the migration of orchids both inside and outside their native ranges to promote effective preservation efforts.
2.1.1 Anoectochilus setaceus Lindl Conservation status
Anoectochilus setaceus Lindl is a member of the Orchidaceae family, which encompasses approximately 30-40 species found primarily in tropical regions These species are distributed from India to the mountainous areas of Southeast Asia and Japan (Acharya et al., 2009).
Anoectochilus setaceus Lindl usually grows under the canopy of primeval forests, evergreen dense forests, tropical forests, on limestone slopes, at an altitude of 800-
Anoectochilus setaceus Lindl., a herbaceous plant found at elevations of 1600 m, features elongated, succulent rhizomes and 2-6 leaves Its smooth, hairless pneumatophores and rhizomes can appear white-green or reddish-brown The plant produces self-clustered flowers at the stem's top, with a flower axis measuring 5-20 cm and often covered in reddish-brown hair, bearing 4-10 flowers (Sherif et al., 2012) Historically, China has utilized tinctures from this herb (Bulpitt, 2005) Research indicates that Anoectochilus setaceus contains various beneficial chemical compounds, including alkaloids, flavonoids, glycosides, 4-hydroxycinnamic acid, β-sitosterol, and kinsenoside (Bajracharya et al., 2003; Shiau et al., 2002; Sukamto, 2011) In traditional Taiwanese medicine, both fresh and dried tinctures are used to address ailments such as chest and abdominal pain, diabetes, nephritis, fever, high blood pressure, impotence, and various organ disorders Additionally, the herb exhibits properties like anti-viral, anti-inflammatory, liver protection, and potential in treating liver diseases, tumors, cardiovascular issues, osteoporosis, fatigue, aging, and immunosuppression (Fay & Chase, 2009) Kinsenoside, a compound extracted from Anoectochilus setaceus, has shown effective anti-hypertensive effects and plays a role in cardiovascular function (Huang et al., 2014).
The natural distribution of Anoectochilus setaceus Lindl in Vietnam spans several regions, including Lào Cai (Sapa), Hà Giang (Quản Bạ), Yên Bái, Vĩnh Phúc (Tam Đảo), Hà Tây (Mỹ Đức: Chùa Hương), Quảng Trị (Đồng Chè), Kontum (Đắc Tô: Đắc Uy), and Gia Lai (Kbang: Kon Hà Nừng) The density of this plant varies significantly, ranging from critically low levels of 20 plants per hectare in Tam Dao National Park to as high as 250 plants per hectare, as recorded in Xuan Son National Park (Phe et al., 2010).
The natural Anoectochilus setaceus Lindl has been overharvested for its medicinal and ornamental properties, commanding a price of $3,200 per kg for dried specimens and $300-$320 per kg for fresh plants This orchid can be propagated through tissue culture using its stems, seeds, and vegetative parts, with countries like China, Taiwan, and Japan leading in production and export While orchids naturally rely on insect pollination due to their unique hermaphroditic flower structure, artificial pollination is often employed to ensure high cross-pollination rates and the creation of desired hybrids This method is cost-effective and yields healthy, disease-free plants, although orchid seeds are scarce and have a low germination rate, resulting in a lengthy flowering period for quality blooms.
When a plant has 3-5 robust leaves and abundant roots, it can be easily propagated by separating and growing the sturdy plant independently This classic propagation method is cost-effective and widely practiced, drawing on the experience and customs of workers However, it faces challenges such as slow growth (yielding only 2-4 new plants per year), low seed quality, potential degeneration of flowering plants over time, and an increased risk of viral diseases, which can ultimately diminish flower quality, as noted by Nguyen Xuan Linh (1998).
Therefore, it is a need to generate another method with ultimate advantages and overcomes the obstacles.
In vitro
In vitro, a Latin term meaning "inside the glass," refers to studies conducted outside of a living organism This laboratory method is commonly utilized in the examination of bacteria and cultured tissues, where an artificial environment is created to support the growth and analysis of these biological samples.
In vitro techniques are extensively utilized in orchid conservation, providing valuable insights into the physiological, genetic, and molecular characteristics of these plants while effectively shortening the prolonged juvenile phase.
In vitro conditions can significantly accelerate the flowering period of Oncidium varicosum, which was observed to bloom after just 8 to 9 months, compared to the 3 years required for flowering in a natural environment.
The flowering of orchids seems to be the most desirable view for anyone, which can take up to 13 years in a natural condition to observe (Hossain et al.,
In vitro techniques offer significant advantages in manipulating and accelerating plant processes, such as flowering and fruit production, reducing timelines from years to just months (Hossain et al., 2013) By providing a controlled environment for plant tissue culture, researchers can effectively observe and study these elusive and seasonal processes, enhancing their understanding and application in agriculture (Bhadra & Hossain, 2003; Chia et al., 1999; Ziv & Naor, 2006).
The first practical method for in vitro propagation was established by Knudson in 1922 through the successful asymptotic germination of orchid seeds He developed an innovative technique for propagating plants under axenic conditions, which has since become a foundational element of plant tissue culture and modern plant biotechnology Subsequent studies, such as those by Kishor et al (2008) and Kishor & Sharma, have further explored the germination of immature orchid seeds or embryos in vitro.
Wetmore (1946) and other researchers demonstrated that various parts of wild grape plants can be cultured under optimal conditions Lon and Ball (1946) further revealed that the ability to form callus is enhanced when culturing plant parts like leaves, stems, and flowers While nutrient requirements vary for different plant parts, common needs include carbon sources, such as sugars, and macro-elements like nitrogen, phosphorus, potassium, and calcium, along with micro-elements like magnesium, iron, manganese, cobalt, and zinc Additionally, essential substances such as vitamins (B1, B6, B3) and growth regulators are necessary To support the growth and development of cultured organs, regular transfers to fresh media are crucial For tissue culture, low-nitrogen organic compounds, including amino acids, sugars, and inositol, must also be incorporated along with the standard nutritional ingredients.
In the case of tissue culture, growth regulators play a more important role because the isolated tissues do not have the ability to synthesize these substances
Although plant cell and tissue culture media are very diverse, they all include some basic ingredients are as follows: (1) Macro and micro mineral salts,
Vitamins, carbon sources such as certain sugars, growth regulators, and additional organic substances like coconut water, yeast extract, potato extract, and dried banana powder play crucial roles in the growth and differentiation of plants cultivated in vitro Additionally, substances that alter environmental conditions, such as agars, are essential for creating optimal growth conditions.
In static cultures, using liquid media can lead to tissue sinking and subsequent death due to oxygen deprivation To prevent this issue, agar, a starch derived from seaweed, is added to thicken the culture medium, allowing tissue to be cultured on its surface Typically, agar is utilized in concentrations ranging from 0.6% to 1%.
Cytokines, derivatives of adenine, are essential hormones that play a crucial role in cell division, apical dominance, and shoot differentiation in tissue culture The most commonly used cytokinins include 6-benzylaminopurine (BAP), 6-benzyladenin (BA), 6-γ-γ-dimethyl-aminopurine (2-iP), N-(2-furfurylamino)-1-H-purine-6-amine (kinetin), and 6-(4-hydroxy-3-methyl-trans-2-butanylamino) purine (zeatin) Among these, zeatin and 2-iP are naturally occurring cytokinins, while BA and kinetin are synthetic Typically, these compounds are prepared in dilute solutions of NaOH or HCl for use in various applications.
Coconut water, first utilized in tissue culture by Van Overbeek et al in the early 1940s, has since been recognized for its beneficial effects on plant cells by numerous researchers Rich in organic compounds, minerals, and growth promoters, coconut water has been effectively used to stimulate differentiation and rapid shoot multiplication across various plant species Typically sourced from selected fruit varieties, it is processed and preserved for fresh use or sold in bottled form by chemical companies The processing involves protein removal and membrane filtration for disinfection, followed by refrigeration While protein residues do not adversely affect tissue or cell culture growth, they can cause precipitation when stored in refrigerated conditions.
METHODOLOGY
Effects of basal culture medium on shoot regeneration
Stem explants were collected from the mother plant in the nursery and thoroughly washed to remove any unnecessary parts After soaking in dilute soapy water for 5 minutes, they were rinsed under running water to eliminate dirt and soap residue, followed by three rinses with sterile distilled water The samples were then sterilized in an incubator, shaken with 70% alcohol for 60 seconds, and rinsed three times with sterile distilled water Finally, the stem samples were cultured on a different medium for regeneration.
Medium formulas were based on 2 original medium formulas of (Murashige
& Skoog, 1962)- MS and (Chu, 1975)– N6 (Appendix A, B)
This study experimented with 4 medium formulas (pH= 5.8)
MS MS + 30g saccarose/ L, 5g agar / L ẵ MS ẵ MS + 30g saccarose/ L, 5g agar / L
Monitoring criteria: Rate of regeneration Tracking time: 2 weeks
The most sufficient formula (Formula S) whose explants perform the best was used for further experiments described in the following sections.
Effects of 6-Benzylaminopurine (BAP) and Kinetin on shoot regeneration
After 2 weeks of incubation in the boot medium (Formula S), sterile live specimens were transferred to a shoot regeneration medium, which was stimulating new shoots to form Suitable shoot regeneration medium was medium ẵ MS and growth regulators at different concentrations
3.2.1 Effect of BAP on shoot regeneration
Table 2: Effect of BAP concentration on shoot regeneration
3.2.2 Effects of Kinetin in shoot regeneration
Table 3 : Effect of Kinetin concentration on shoot regeneration
Monitoring criteria: Rate of regenerated samples
Commented [TT1]: Some descriptions needed Never let a content with a table only
3.3 Effects of some organic compounds (coconut water, potatoes, carrots) on multiplication of cultured samples
Medium ẵ MS, with the subsidy of the most suitable growth regulator concentration was used to culture sample in this experiment, with the further organic composition described below
Table 4: Composition of multiplication medium
5 100 ml/L Coconut water + 10g/L Potato + 10g/L Carrot
Monitoring criteria: Shoot multiplier rate Tracking time: 6 weeks.
RESULTS
Effects of culture medium formula on shoot regeneration
4.1.1 Effects of culture medium formula on shoot regeneration rate
Table 5: Shoot regeneration rate in culture medium formula
Figure 2: Shoot regeneration rate in culture medium formula
The shoot regeneration rate, measured as the number of shoots per sample, was monitored over a two-week period Results indicated that the MS medium yielded the highest regeneration rates, with averages of 1.67 shoots per sample in the first week and 2.4 shoots per sample in the second week, as detailed in Table 5.
Sh o o t regen er at io n r at e (sh o o ts/ ex p la n t)
Figure 2 with the slopest line, MS seemed to have the most significant change in regeneration rate throughout the weeks as it changed by 0.87 (from 1.26 in Week
1 to 2.13 in Week 2) Ranking in the third and the fourth position in the graph were ẵ N6 and N6
4.1.2 Effects of culture medium on average regenerated shoot length
Table 6: Average shoot length in culture medium formula
Medium No of tested explant
Figure 3: Change in length of newly regenerated shoots in culture mediums Table 6 showed the average length of shoot recorded in each week, from
Week 0 (initial length) till the end of tracking duration in Week 2 As the result,
Figure 3 subtracted the length in each record to the initial length, for the rise in length of shoots regenerated each week
Table 6 indicates that the ẵ MS formula achieved the longest shoot lengths during both tracking weeks, measuring 1.67 cm and 3.56 cm This demonstrates its effectiveness in promoting shoot elongation, as further illustrated in Figure 3, where ẵ MS recorded the highest shoot length While MS ranked second in regeneration rate among the formulas studied, N6 outperformed ẵ N6 in terms of shoot length increase over the weeks, as shown in Figure 3.
Figures 2 and 3 demonstrate that ẵ MS emerged as the most effective formula for shoot regeneration Consequently, ẵ MS was selected for the subsequent experiments in this study.
Effects of 6-Benzylaminopurine (BAP) and Kinetin in shoot regeneration stage
4.2.1 Effects of BAP on shoot regeneration stage
4.2.1.1 Effects of BAP on shoot regeneration rate
Table 7 Shoot regeneration rate of explants distributed in medium with different BAP concentrations
Medium Shoot regeneration in… ẵMS +
Figure 4 Shoot regeneration rate of explants distributed in medium with different BAP concentrations
Sh o o t regen er at io n r at e (sh o o ts/ ex p la n t)
The shoot regeneration rate exhibited an upward trend from Week 2 to Week 4, followed by a plateau from Week 4 to Week 6 for the control medium, or a gradual increase at a low rate until Week 6 Additionally, data from Table 7 and Figure 4 indicate that higher concentrations of BAP corresponded to an increased number of generated shoots.
4.2.1.2 Effects of BAP on average regenerated shoot length
Table 8 Average regenerated shoot length of explants distributed in medium with different BAP concentrations ẵMS +
Figure 5 Change in length newly regenerated shoot distributed in medium with different BAP concentrations
Table 8 showed the average length of shoot recorded in each week, from
Week 0 (initial length) till the end of tracking duration in Week 2 As the result,
Figure 5 subtracted the length in each record to the initial length, for the rise in length of shoots regenerated each week
According to Figure 5 , medium including 0.8 mg/L BAP acquired the longest shoots after a takeover in Week 4, followed by 0.2 mg/L BAP, 0.4 mg/L
BAP, and lastly 0 mg/L BAP
The optimal concentration of 0.8 mg/L BAP has been identified as the most effective for the in vitro propagation of Anoectochilus setaceus Lindl., due to its superior ability to promote shoot regeneration and elongation.
4.2.2 Effects of Kinetin shoot on regeneration stage
4.2.2.1 Effects of Kinetin on shoot regeneration rate
Table 9 Shoot regeneration rate distributed in medium with different Kinetin concentrations ẵMS +
The shoot regeneration rate was evaluated across various Kinetin concentrations, as shown in Table 9 The data highlights the changes in the number of shoots per explant over the monitoring period The optimal performance was observed at a Kinetin concentration of 0.8 mg/L, yielding an average of 2.27 shoots/explant in Week 2, 2.73 shoots/explant in Week 4, and 3.13 shoots/explant in Week 6.
Figure 6 illustrates two distinct trends observed over six weeks of tracking media with varying concentrations of Kinetin (1 mg/L, 0.8 mg/L, and 0.4 mg/L) The media with Kinetin exhibited a linear growth trend, while the other media showed a rapid increase in regeneration rates up to week 4, followed by a slight rise until week 6 Notably, all media containing Kinetin demonstrated higher regeneration rates compared to the control medium without Kinetin Among the Kinetin concentrations, 0.8 mg/L proved to be more effective than 1 mg/L, and a reduction in Kinetin concentration correspondingly decreased the number of shoots recorded per explant.
Sh o o t regen er at io n r at e (sh o o ts/ ex p la n t)
4.2.2.2 Effects of Kinetin on regenerated shoot length
Table 10 Average regenerated shoot length distributed in medium with different Kinetin concentrations ẵ MS +
Figure 7 Change in length of regenerated shoot distributed in medium with different Kinetin concentrations Table 10 showed the average length of shoot recorded in each week, from
Week 0 (initial length) till the end of tracking duration in Week 6 As the result,
Figure 7 subtracted the length in each record to the initial length, for the rise in length of shoots regenerated each week
The study demonstrated an increase in average shoot length across all tested formulas, as illustrated in Figure 7 Notably, while both 0 mg/L and 1 mg/L Kinetin started from the same baseline, their effects on shoot length diverged over time By Week 6, 1 mg/L Kinetin emerged as the optimal concentration for promoting shoot length extension.
Accounting for its excellent performance in regenerating more shoots and elongating shoots, 1 mg/L Kinetin became the best concentration of Kinetin recommended to use.
Effects of some organic compounds (coconut water, potatoes, mashed carrot)
4.3.1 Effects of some organic compounds (coconut water, potatoes, mashed carrot) on shoot multiplication rate
Table 11 Shoot multiplication rate based in organic compounds
No of shoots Shoots/explant No of shoots Shoots/explant No of shoots Shoots/explant
The monitoring of shoot regeneration rates in organic compounds reveals significant changes in the number of shoots per explant over time, as shown in Table 11 The optimal performance was achieved with a mixture of 100 ml/L coconut water, 10 g/L potato, and 10 g/L carrot, resulting in an average of 3.07 shoots/explant at Week 2, 4.47 shoots/explant at Week 4, and 5.47 shoots/explant at Week 6.
Figure 8 demonstrates the shoot multiplication rates across five studied mediums during the final phase of the research Overall, the combination of all components emerged as the most effective medium for shoot multiplication Following this, the medium containing 100 ml/L coconut water and 10 g/L potato showed a notable rate of shoots per sample The lower section of the graph illustrates the performance of the other mediums.
Sh o o t regen er at io n r at e (sh o o ts/ ex p la n t)
100ml/L Coconut water 15g/L Potato 15g/L Carrot
100 ml/L Coconut water + 10g/L Potato + 10g/L Carrot shoots in medium with individual organic composition (coconut or potato or carrot)
4.3.2 Effects of some organic compounds (coconut water, potatoes, mashed carrot) on multiplied shoot length
Table 12 Average regenerated shoot length based on organic compounds
100ml/L Coconut water 15g/L Potato 15g/L Carrot
100 ml/L Coconut water + 10g/L Potato + 10g/L Carrot
Figure 9 Change in length of multiplied shoot based on organic compounds
Table 12 showed the average length of shoot recorded in each week, from
Week 0 (initial length) till the end of tracking duration in Week 6 As the result,
Figure 9 subtracted the length in each record to the initial length, for the rise in length of shoots regenerated each week
The average length of newly multiplied shoots was analyzed, revealing that the medium comprising all studied compositions produced the longest shoots, measuring 4.81 mm A closer examination of individual organic compositions highlighted that the 15 g/L carrot was the only organic source that did not show positive growth, as the increase in shoot length from Week 4 to Week 6 was less significant than the growth observed from Week 2 to Week 4.
DISCUSSION AND CONCLUSION
Discussion
5.1.1 Effects of culture medium on shoot regeneration
The study concluded that MS is a more suitable basal formula compared to N6, whether at half or full strength It suggests that the performance of shoot possibilities is influenced by the salt composition of the medium.
The study by Kriswanto et al (2017) indicates that MS medium outperforms N6 in promoting shoot proliferation, with half-strength formulations yielding more shoots than full-strength counterparts This trend is consistent across both MS and N6 media Consequently, utilizing half the concentration of each component in the studied media is recommended, as it enhances cultivation performance while also conserving resources and providing economic advantages.
The study demonstrated that the ẵ MS formula excelled in both criteria for shoot regeneration, making it the most effective option Consequently, ẵ MS was selected for subsequent experiments and is highly recommended for the propagation of A setaceus.
5.1.2 Effects of BAP and Kinetin on shoot regeneration
5.1.2.1 Effects of BAP on shoot regeneration
This study investigated the effects of varying concentrations of BAP in the medium, ranging from 0 mg/L (control) to 0.8 mg/L The optimal concentration identified was 0.8 mg/L, as illustrated in Table 7 and Figure 4 The results indicated that increasing the amount of BAP in the cultivation medium led to improved outcomes across both evaluated criteria.
In Week 4, as illustrated in Figure 4, the rate of shoot regeneration began to change less rapidly, indicating the onset of the stationary phase According to Ding et al (2013), the initial four weeks represent a critical developmental stage in the in vitro cultivation of orchids, known as protocorm-like bodies formation During this stage, these protocorm-like bodies transition into the vegetative phase, where the production of shoots becomes less likely.
The results illustrated in Figures 4 and 5 clearly demonstrate that BAP significantly enhances the number of new shoots, as evidenced by the superior performance of media containing BAP at any concentration compared to the control medium.
Although 0.8 mg/L BAP contributed to the accomplishment of the longest shoot, as can be seen in Figure 5 , the gap between the highest, second highest or even third highest regeneration rate was not significant For that reason, the use of less BAP in cultivating medium does not seem to drop the productivity of shoot elongation However, with the purpose of improve the shoot regeneration in both criteria, it is still recommended to use 0.8 mg/L BAP for the best performance
5.1.2.2 Effects of Kinetin on shoot regeneration
Figure 6 illustrates that the regeneration rates of all media containing Kinetin surpassed those of the control medium without it, demonstrating Kinetin's effectiveness as a growth regulator for shoot regeneration Notably, the optimal concentration was found to be 0.8 mg/L, which yielded better results than 1 mg/L, indicating that the quantity of Kinetin does not directly correlate with the number of newly regenerated shoots Additionally, timing emerged as a significant factor influencing regeneration outcomes.
In a study examining the effects of Kinetin concentrations on shoot length, it was found that concentrations of 0.8 mg/L, 0.4 mg/L, and 0.2 mg/L were more effective than 1 mg/L in promoting shoot elongation from Week 2 to Week 3 Further research is recommended to explore the optimal timing for Kinetin application to maximize its effectiveness.
In the comparison of growth stimulators for in vitro conservation and propagation of A setaceus Lindl., BAP demonstrated superior efficiency over Kinetin, as illustrated in Figures 4, 5, 6, and 7.
5.1.3 Effects of organic compounds on shoot multiplication
This study demonstrated that the optimal combination of all tested compositions significantly enhanced the multiplication stage of A setaceus Lindl during in vitro conservation and propagation Specifically, the use of 100 ml/L coconut water combined with 10 g/L of nutrients yielded the best results.
Potato + 10g/L Carrot is highly recommended as an organic compound
5.1.4 Implementation of in vitro propagation in A.setaceus Lindl conservation and management
Future orchid conservation efforts should focus on artificial propagation to generate potential populations, as highlighted by Wraith et al (2020) The decline of A setaceus Lindl is expected to continue without practical implementation of conservation strategies Bridging the gap between theory and practice is essential, as Fay (2018) suggests that orchids should be conserved in sterilized conditions to restore species In vitro techniques must complement in situ conservation efforts for A setaceus Lindl Collaborative efforts between research institutes and conservationists are crucial for successful rehabilitation into the wild (Pedersen et al., 2018) Evaluating transplantation sites requires consideration of threats, land regions, conservation priorities, mycorrhizal associations, and pollinator availability (Wraith et al., 2020) Utilizing a spatial map with multi-criteria analysis (MCA) can help define potential distribution areas for the transplantation of A setaceus Lindl.
Protecting threatened orchids from illegal collection and logging is a critical priority for governments and policymakers, as highlighted by Wraith et al (2020) Effective biodiversity management is essential to minimize human impact on conservation efforts Implementing proper governance can help mitigate these effects, such as promoting ecotourism over traditional tourism, which can significantly reduce human interference with natural habitats (N D Swarts & Dixon, 2009) Additionally, it is crucial for local collectors to understand the exploitation risks and endangered status of A setaceus Lindl.
Therefore, raising education and awareness programs must be conducted This recommendation might also change a livelihood for people who used to depend on forestry resources, and orchid (Wraith et al., 2020)
To enhance conservation efforts for A setaceus Lindl., it is crucial to implement stricter policies and update its conservation priority profile, as the last revision of the Vietnam Red Book was in 2007 (Wraith et al., 2020) Given that Vietnam is one of the leading countries in orchid exploitation and trade, timely updates are essential to address the increasing threats to this species (Wraith et al., 2020).
Conclusion
The implementation of in vitro technique into the conservation of
A.setaceus Lindl promises to bring a high productivity of propagation, and a buffered site for challenge adaptation for rehabitatant The result of this study is only used for the conservation of A.setaceus Lindl., any commercial application from this study is not recommended The most suitable basic medium for conservation and propagation A.setaceus Lindl in vitro is ẵ MS Between studied growth regulators, BAP is highly recommended in use rather than Kinetin, thanks to its excellent performance with both tracking criteria, and to avoid the complicated use of Kinetin due to its dependent factor - timing When it comes to shoot multiplication in medium containing organic compounds, it is highly recommended to use 100 ml/L Coconut water + 10g/L Potato + 10g/L Carrot for the best record
In a nutshell, the most efficiency medium for shoot regeneration of
Anoectochilus setaceus Lindl., based on studied factors, is ẵ MS+ 0.8 mg/L BAP
To effectively recover Anoectochilus setaceus Lindl., a collaboration among conservationists, research institutions, and the government is essential Utilizing a mixture of 100 ml/L coconut water, 10 g/L potato, and 10 g/L carrot can support this initiative Efficient conservation of this valuable species requires management strategies that prioritize minimizing human impact on its natural habitat.
Raising awareness about the critical conservation status is essential to transforming the livelihoods of individuals who have historically relied on forest resources.
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