J Plant Biotechnol (2023) 50:108-114
Published online June 5, 2023
https://doi.org/10.5010/JPB.2023.50.014.108
© The Korean Society of Plant Biotechnology
Correspondence to : e-mail: nhhoang@hcmus.edu.vn
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
In vitro conservation is one of the most effective strategies for rare plant protection, especially for orchid species. To maximize the success rates of in vitro explant establishment (stage I) in conservation programs, the application of tissue culture additives such as Plant Preservative Mixture™ (PPM™) should be emphasized. In this study, we used Dendrobium thyrsiflorum Rchb.f. (1875) seeds and seedlings as a model for the evaluation of PPM™’s phytotoxicity in the meristematic tissues of epiphytic orchids. PPM™ had no observable inhibitory effect on protocorm, shoot, or root development when it was supplemented at 0.1%. PPM™ supplementation caused adverse effects on D. thyrsiflorum explants at concentrations > 0.2%. At high concentrations, young in vitro seedlings showed damage, especially at the root tissue level. Based on this model, supplementation of 0.1-0.2% PPM™ to culture media was successfully implemented to establish in vitro cultures of other rare orchid species in our conservation program.
Keywords protocorm, micropropagation, agriculture, production, Nervilia
Anthropogenic climate change and over-exploitation are significant causes of species extinction, including rare members of Orchidaceae (Wraith and Pickering 2018). In a Southeast Asian country, such as Vietnam, there are 68 orchid species listed on the IUCN Red List of Threatened Species Vietnam (2007). Among those, 22
To date, in vitro shoot propagation or asymbiotic seed germination is considered a reliable method for the conservation of orchids (Hoang et al. 2016; Pujasatria et al. 2020; Santos-Díaz et al. 2022). Especially the number of explants that are available for in vitro conservation projects can be seasonal or very limited in quantity (Sarasan 2010). During field surveys, specimens of endangered species may take days to locate and approach; severe damage and microbial contamination may prevent conservation efforts (Arab et al. 2014; Hoang et al. 2016; Izarra et al. 2020). Hence, implementing low-cost and innovative solutions to improve explant survival could facilitate conservation projects in under-development countries (Agrawal et al. 2010).
The Plant Preservative Mixture™ has antimicrobial activity that can eliminate a wide range of microbial organisms and prevent biofilm formation (Compton and Koch 2001). Its main component is a thermostable isothiazolone compound, includes 5 - chloro - 2 - methyl - 3(2H) - isothiazolone (MCI) and 2 - methyl - 3(2H) - isothiazolone (MI) (Patent No. 5,750,402). Due to its thermostability, the compound can be added directly to the culture medium before autoclave (Guri 1998). PPM™ is commonly used as a biocide in plant tissue culture (Faizy et al. 2017; Givnish et al. 2016; Leão et al. 2020; Rihan et al. 2012; Romadanova et al. 2022). We believe the full potential of PPM™ is not limited to commercial micropropagation, but it can be a powerful solution for in vitro conservation. Unfortunately, no literature could adequately address the value of PPM™ for rare plant protection.
In this research, we focused on the potential use of PPM™ for in vitro orchid conservation. We evaluated the effect of PPM™ on the germination, growth, and development of
The
The
Three-month old seedlings
To evaluate the potential use of PPM™ for culture establishment of other rare orchids in our conservation program, we used in vitro explants of
In this research, a one-factor simple experiment design was used. Replicates’ positions in the growth chamber were randomized using Microsoft Excel®. Chi-squared analysis was used for qualitative data such as seed germination rates. Statistical analysis was performed using R software (version 4.2.2).
Most tropical countries with the highest biodiversity also have low-income populations. Plant conservation and reintroduction research are often underestimated and poorly funded (Heywood 2017). To establish a sustainable yet high-impact plant conservation program in this context, the working procedures must be simple, low-cost, and effective (Ribaudo 2017). This study highlighted the use of commercial products for rare explants in general. Simple and effective in vitro explant establishment results are the foundation for more successful in vitro conservation and restoration projects in low-income countries.
The active ingredients in PPM™ are mainly isothiazolones, including methylchloroisothiazolinone and methylisothiazolinone (US patent US5750402A). These biofilm inhibitor compounds could be harmless to plant tissue at low levels and therefore were used as preservatives or germicidal (Guri et al. 1998). Our data showed no inhibitory effect on
Table 1 Seed germination rates and developmental stages of
Treatment | No. of seeds | Germination rate (%) | Developmental stage with damage (10 weeks) | |
---|---|---|---|---|
6 weeks | 10 weeks | |||
0.0% PPM™ | 317 | 88.64 | 88.33 | - |
0.1% PPM™ | 260 | 90.38 | 88.85 | - |
0.2% PPM™ | 383 | 84.33 | 82.33 | - |
0.4% PPM™ | 341 | 78.01* | 66.86** | IV |
0.8% PPM™ | 274 | 59.85** | 45.26** | III |
1.6% PPM™ | 367 | 28.88** | 6.54** | II |
The asterisks * and ** indicate significant chi-square differences between treatments with PPM™ and control (0.0% PPM™), with significance levels set at 0.05 and 0.001, respectively.
At the high level of PPM™ supplementation (above 0.2%),
Seeds of Orchidaceae members have morphological dormancy (Baskin and Baskin 2001). The underdeveloped embryos with dozens of cells required either endomycorrhizal fungus or exogenous sugar supplementation for their further germination stages (Rasmussen 1992). Seed maturation is merged into embryo growth and protocorm development (Fang et al. 2021). After a successful germination initiation, marked by water imbibition, the protocorms develop further into seedlings with true leaflets (Hoang et al. 2016). Normal embryo survival and protocorm development of
Three-month old
Table 2 Response of 3-month-old
Treatment | Percentage of seedlings with damage symptoms (%) | |||||
---|---|---|---|---|---|---|
Total | Leaf | Root | ||||
10 days | 20 days | 10 days | 20 days | 10 days | 20 days | |
0.0% PPM™ | 0.00 | 0.00 | NA | NA | NA | NA |
0.1% PPM™ | 0.00 | 0.00 | NA | NA | NA | NA |
0.2% PPM™ | 6.66 | 20.00* | 0.00 | 0.00* | 100.00 | 100.00 |
0.4% PPM™ | 50.00** | 56.67** | 0.00** | 11.76** | 100.00 | 100.00 |
0.8% PPM™ | 93.33** | 96.67** | 78.57* | 96.55 | 100.00 | 100.00 |
1.6% PPM™ | 100.00** | 100.00** | 86.67 | 100.00 | 100.00 | 100.00 |
The asterisks * and ** indicate significant chi-square differences between treatments with PPM™ and control (0.0% PPM™), with significance levels set at 0.05 and 0.001, respectively. NA: not available.
As mentioned above, seedlings showed damaging signs at different levels. PPM™ damage symptoms were commonly observed on roots but rarely on leaves except for very high concentrations, e.g., 0.8 and 1.6% (Fig. 2). The supplementation of PPM™ at 0.1% and 0.2% could not affect the growth, but from 0.4% to 1.6% inhibited growth and decreased the seedlings’ height and biomass after 20 days (Table 3). Growth inhibition when PPM™ is supplemented at levels above 0.2% is also observed on a few other species, such as Walnut (
Table 3 Growth of
Treatment | Height (mm) | Fresh weight (mg) | Dried weight (mg) | ||||||
---|---|---|---|---|---|---|---|---|---|
0 DAT | 10 DAT | 20 DAT | 0 DAT | 10 DAT | 20 DAT | 0 DAT | 10 DAT | 20 DAT | |
0.0% PPM™ | 8.75 | 9.04a | 9.63ab | 6.52 | 6.79 | 8.03 | 0.80 | 0.90 | 1.10 |
0.1% PPM™ | 10.26 | 10.93a | 11.57b | 11.34 | 12.31 | 14.50 | 1.20 | 1.70 | 1.90 |
0.2% PPM™ | 10.73 | 10.99a | 11.51ab | 14.59 | 16.04 | 17.21 | 1.40 | 1.50 | 1.70 |
0.4% PPM™ | 9.39 | 9.53a | 9.90ab | 9.98 | 9.73 | 9.62 | 0.90 | 0.80 | 0.90 |
0.8% PPM™ | 9.97 | 10.10a | 10.05a | 8.52 | 8.32 | 8.19 | 1.00 | 0.90 | 0.80 |
1.6% PPM™ | 9.31 | 9.48a | 9.29a | 9.33 | 7.44 | 6.60 | 0.90 | 0.70 | 0.60 |
Each treatment had 10 replicates, with 30 seedlings in total (3 subreplicates). The means of the seedling height, fresh weight, and dried weight were calculated using data from 10 replicates with 3 seedlings in each tube. Mean height values with different letters are significantly different at α= 0.05 according to one-way ANOVA and Tukey post-hoc test. Fresh weight and dried weight data were pooled. DAT: day after transplant.
In fact, by testing sensitive materials such as orchid embryos and seedlings, media supplemented with low dosage (0.1% PPM™) is considerably safer to use for rare explant establishment. During conservation programs, quality and amount of field-collected plant material are usually low (Sarasan 2010). The result could be poor in vitro establishment efficiency and a high-chance of losing valuable explants. This data is significant for in vitro conservation of rare plant species. We successfully used different versions of basal media supplemented with 0.1% PPM™ for in vitro culture establishment of rare species in our conservation program, including
In summary, we used
This research is funded by Vietnam National University, Ho Chi Minh City (VNU-HCM) under grant number C2020-18-02.
J Plant Biotechnol 2023; 50(1): 108-114
Published online June 5, 2023 https://doi.org/10.5010/JPB.2023.50.014.108
Copyright © The Korean Society of Plant Biotechnology.
Tran Trung Chanh ・Nguyen Tan Huy ・Nguyen Thu Ha ・Khanh Le ・Nguyen Huu Hoang
Research Center for High-Tech Application in Agriculture (RCHAA), University of Science, Vietnam National University Ho Chi Minh City, 227 Nguyen Van Cu, Ho Chi Minh city, Vietnam
Cisbay Global Inc., 6389 San Ignacio Avenue, San Jose, California, United States of America
Correspondence to:e-mail: nhhoang@hcmus.edu.vn
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
In vitro conservation is one of the most effective strategies for rare plant protection, especially for orchid species. To maximize the success rates of in vitro explant establishment (stage I) in conservation programs, the application of tissue culture additives such as Plant Preservative Mixture™ (PPM™) should be emphasized. In this study, we used Dendrobium thyrsiflorum Rchb.f. (1875) seeds and seedlings as a model for the evaluation of PPM™’s phytotoxicity in the meristematic tissues of epiphytic orchids. PPM™ had no observable inhibitory effect on protocorm, shoot, or root development when it was supplemented at 0.1%. PPM™ supplementation caused adverse effects on D. thyrsiflorum explants at concentrations > 0.2%. At high concentrations, young in vitro seedlings showed damage, especially at the root tissue level. Based on this model, supplementation of 0.1-0.2% PPM™ to culture media was successfully implemented to establish in vitro cultures of other rare orchid species in our conservation program.
Keywords: protocorm, micropropagation, agriculture, production, Nervilia
Anthropogenic climate change and over-exploitation are significant causes of species extinction, including rare members of Orchidaceae (Wraith and Pickering 2018). In a Southeast Asian country, such as Vietnam, there are 68 orchid species listed on the IUCN Red List of Threatened Species Vietnam (2007). Among those, 22
To date, in vitro shoot propagation or asymbiotic seed germination is considered a reliable method for the conservation of orchids (Hoang et al. 2016; Pujasatria et al. 2020; Santos-Díaz et al. 2022). Especially the number of explants that are available for in vitro conservation projects can be seasonal or very limited in quantity (Sarasan 2010). During field surveys, specimens of endangered species may take days to locate and approach; severe damage and microbial contamination may prevent conservation efforts (Arab et al. 2014; Hoang et al. 2016; Izarra et al. 2020). Hence, implementing low-cost and innovative solutions to improve explant survival could facilitate conservation projects in under-development countries (Agrawal et al. 2010).
The Plant Preservative Mixture™ has antimicrobial activity that can eliminate a wide range of microbial organisms and prevent biofilm formation (Compton and Koch 2001). Its main component is a thermostable isothiazolone compound, includes 5 - chloro - 2 - methyl - 3(2H) - isothiazolone (MCI) and 2 - methyl - 3(2H) - isothiazolone (MI) (Patent No. 5,750,402). Due to its thermostability, the compound can be added directly to the culture medium before autoclave (Guri 1998). PPM™ is commonly used as a biocide in plant tissue culture (Faizy et al. 2017; Givnish et al. 2016; Leão et al. 2020; Rihan et al. 2012; Romadanova et al. 2022). We believe the full potential of PPM™ is not limited to commercial micropropagation, but it can be a powerful solution for in vitro conservation. Unfortunately, no literature could adequately address the value of PPM™ for rare plant protection.
In this research, we focused on the potential use of PPM™ for in vitro orchid conservation. We evaluated the effect of PPM™ on the germination, growth, and development of
The
The
Three-month old seedlings
To evaluate the potential use of PPM™ for culture establishment of other rare orchids in our conservation program, we used in vitro explants of
In this research, a one-factor simple experiment design was used. Replicates’ positions in the growth chamber were randomized using Microsoft Excel®. Chi-squared analysis was used for qualitative data such as seed germination rates. Statistical analysis was performed using R software (version 4.2.2).
Most tropical countries with the highest biodiversity also have low-income populations. Plant conservation and reintroduction research are often underestimated and poorly funded (Heywood 2017). To establish a sustainable yet high-impact plant conservation program in this context, the working procedures must be simple, low-cost, and effective (Ribaudo 2017). This study highlighted the use of commercial products for rare explants in general. Simple and effective in vitro explant establishment results are the foundation for more successful in vitro conservation and restoration projects in low-income countries.
The active ingredients in PPM™ are mainly isothiazolones, including methylchloroisothiazolinone and methylisothiazolinone (US patent US5750402A). These biofilm inhibitor compounds could be harmless to plant tissue at low levels and therefore were used as preservatives or germicidal (Guri et al. 1998). Our data showed no inhibitory effect on
Table 1 . Seed germination rates and developmental stages of
Treatment | No. of seeds | Germination rate (%) | Developmental stage with damage (10 weeks) | |
---|---|---|---|---|
6 weeks | 10 weeks | |||
0.0% PPM™ | 317 | 88.64 | 88.33 | - |
0.1% PPM™ | 260 | 90.38 | 88.85 | - |
0.2% PPM™ | 383 | 84.33 | 82.33 | - |
0.4% PPM™ | 341 | 78.01* | 66.86** | IV |
0.8% PPM™ | 274 | 59.85** | 45.26** | III |
1.6% PPM™ | 367 | 28.88** | 6.54** | II |
The asterisks * and ** indicate significant chi-square differences between treatments with PPM™ and control (0.0% PPM™), with significance levels set at 0.05 and 0.001, respectively..
At the high level of PPM™ supplementation (above 0.2%),
Seeds of Orchidaceae members have morphological dormancy (Baskin and Baskin 2001). The underdeveloped embryos with dozens of cells required either endomycorrhizal fungus or exogenous sugar supplementation for their further germination stages (Rasmussen 1992). Seed maturation is merged into embryo growth and protocorm development (Fang et al. 2021). After a successful germination initiation, marked by water imbibition, the protocorms develop further into seedlings with true leaflets (Hoang et al. 2016). Normal embryo survival and protocorm development of
Three-month old
Table 2 . Response of 3-month-old
Treatment | Percentage of seedlings with damage symptoms (%) | |||||
---|---|---|---|---|---|---|
Total | Leaf | Root | ||||
10 days | 20 days | 10 days | 20 days | 10 days | 20 days | |
0.0% PPM™ | 0.00 | 0.00 | NA | NA | NA | NA |
0.1% PPM™ | 0.00 | 0.00 | NA | NA | NA | NA |
0.2% PPM™ | 6.66 | 20.00* | 0.00 | 0.00* | 100.00 | 100.00 |
0.4% PPM™ | 50.00** | 56.67** | 0.00** | 11.76** | 100.00 | 100.00 |
0.8% PPM™ | 93.33** | 96.67** | 78.57* | 96.55 | 100.00 | 100.00 |
1.6% PPM™ | 100.00** | 100.00** | 86.67 | 100.00 | 100.00 | 100.00 |
The asterisks * and ** indicate significant chi-square differences between treatments with PPM™ and control (0.0% PPM™), with significance levels set at 0.05 and 0.001, respectively. NA: not available..
As mentioned above, seedlings showed damaging signs at different levels. PPM™ damage symptoms were commonly observed on roots but rarely on leaves except for very high concentrations, e.g., 0.8 and 1.6% (Fig. 2). The supplementation of PPM™ at 0.1% and 0.2% could not affect the growth, but from 0.4% to 1.6% inhibited growth and decreased the seedlings’ height and biomass after 20 days (Table 3). Growth inhibition when PPM™ is supplemented at levels above 0.2% is also observed on a few other species, such as Walnut (
Table 3 . Growth of
Treatment | Height (mm) | Fresh weight (mg) | Dried weight (mg) | ||||||
---|---|---|---|---|---|---|---|---|---|
0 DAT | 10 DAT | 20 DAT | 0 DAT | 10 DAT | 20 DAT | 0 DAT | 10 DAT | 20 DAT | |
0.0% PPM™ | 8.75 | 9.04a | 9.63ab | 6.52 | 6.79 | 8.03 | 0.80 | 0.90 | 1.10 |
0.1% PPM™ | 10.26 | 10.93a | 11.57b | 11.34 | 12.31 | 14.50 | 1.20 | 1.70 | 1.90 |
0.2% PPM™ | 10.73 | 10.99a | 11.51ab | 14.59 | 16.04 | 17.21 | 1.40 | 1.50 | 1.70 |
0.4% PPM™ | 9.39 | 9.53a | 9.90ab | 9.98 | 9.73 | 9.62 | 0.90 | 0.80 | 0.90 |
0.8% PPM™ | 9.97 | 10.10a | 10.05a | 8.52 | 8.32 | 8.19 | 1.00 | 0.90 | 0.80 |
1.6% PPM™ | 9.31 | 9.48a | 9.29a | 9.33 | 7.44 | 6.60 | 0.90 | 0.70 | 0.60 |
Each treatment had 10 replicates, with 30 seedlings in total (3 subreplicates). The means of the seedling height, fresh weight, and dried weight were calculated using data from 10 replicates with 3 seedlings in each tube. Mean height values with different letters are significantly different at α= 0.05 according to one-way ANOVA and Tukey post-hoc test. Fresh weight and dried weight data were pooled. DAT: day after transplant..
In fact, by testing sensitive materials such as orchid embryos and seedlings, media supplemented with low dosage (0.1% PPM™) is considerably safer to use for rare explant establishment. During conservation programs, quality and amount of field-collected plant material are usually low (Sarasan 2010). The result could be poor in vitro establishment efficiency and a high-chance of losing valuable explants. This data is significant for in vitro conservation of rare plant species. We successfully used different versions of basal media supplemented with 0.1% PPM™ for in vitro culture establishment of rare species in our conservation program, including
In summary, we used
This research is funded by Vietnam National University, Ho Chi Minh City (VNU-HCM) under grant number C2020-18-02.
Table 1 . Seed germination rates and developmental stages of
Treatment | No. of seeds | Germination rate (%) | Developmental stage with damage (10 weeks) | |
---|---|---|---|---|
6 weeks | 10 weeks | |||
0.0% PPM™ | 317 | 88.64 | 88.33 | - |
0.1% PPM™ | 260 | 90.38 | 88.85 | - |
0.2% PPM™ | 383 | 84.33 | 82.33 | - |
0.4% PPM™ | 341 | 78.01* | 66.86** | IV |
0.8% PPM™ | 274 | 59.85** | 45.26** | III |
1.6% PPM™ | 367 | 28.88** | 6.54** | II |
The asterisks * and ** indicate significant chi-square differences between treatments with PPM™ and control (0.0% PPM™), with significance levels set at 0.05 and 0.001, respectively..
Table 2 . Response of 3-month-old
Treatment | Percentage of seedlings with damage symptoms (%) | |||||
---|---|---|---|---|---|---|
Total | Leaf | Root | ||||
10 days | 20 days | 10 days | 20 days | 10 days | 20 days | |
0.0% PPM™ | 0.00 | 0.00 | NA | NA | NA | NA |
0.1% PPM™ | 0.00 | 0.00 | NA | NA | NA | NA |
0.2% PPM™ | 6.66 | 20.00* | 0.00 | 0.00* | 100.00 | 100.00 |
0.4% PPM™ | 50.00** | 56.67** | 0.00** | 11.76** | 100.00 | 100.00 |
0.8% PPM™ | 93.33** | 96.67** | 78.57* | 96.55 | 100.00 | 100.00 |
1.6% PPM™ | 100.00** | 100.00** | 86.67 | 100.00 | 100.00 | 100.00 |
The asterisks * and ** indicate significant chi-square differences between treatments with PPM™ and control (0.0% PPM™), with significance levels set at 0.05 and 0.001, respectively. NA: not available..
Table 3 . Growth of
Treatment | Height (mm) | Fresh weight (mg) | Dried weight (mg) | ||||||
---|---|---|---|---|---|---|---|---|---|
0 DAT | 10 DAT | 20 DAT | 0 DAT | 10 DAT | 20 DAT | 0 DAT | 10 DAT | 20 DAT | |
0.0% PPM™ | 8.75 | 9.04a | 9.63ab | 6.52 | 6.79 | 8.03 | 0.80 | 0.90 | 1.10 |
0.1% PPM™ | 10.26 | 10.93a | 11.57b | 11.34 | 12.31 | 14.50 | 1.20 | 1.70 | 1.90 |
0.2% PPM™ | 10.73 | 10.99a | 11.51ab | 14.59 | 16.04 | 17.21 | 1.40 | 1.50 | 1.70 |
0.4% PPM™ | 9.39 | 9.53a | 9.90ab | 9.98 | 9.73 | 9.62 | 0.90 | 0.80 | 0.90 |
0.8% PPM™ | 9.97 | 10.10a | 10.05a | 8.52 | 8.32 | 8.19 | 1.00 | 0.90 | 0.80 |
1.6% PPM™ | 9.31 | 9.48a | 9.29a | 9.33 | 7.44 | 6.60 | 0.90 | 0.70 | 0.60 |
Each treatment had 10 replicates, with 30 seedlings in total (3 subreplicates). The means of the seedling height, fresh weight, and dried weight were calculated using data from 10 replicates with 3 seedlings in each tube. Mean height values with different letters are significantly different at α= 0.05 according to one-way ANOVA and Tukey post-hoc test. Fresh weight and dried weight data were pooled. DAT: day after transplant..
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