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 Dendrobium species are endangered (Ban 2007). The demand for rare orchid specimens can sometimes become extreme. For example, Dendrobium anosmum var alba was traded for thousands of US dollars at its peak, ‘fueled’ both orchid smugglers and local collectors. Over-collecting, thus, threatened many tropical orchid species, while reintroduction efforts are still a not common conservation strategy in underdeveloped countries (Seaton et al. 2013).

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 Dendrobium thyrsiflorum Rchb.f. seeds and seedlings. As a model, Dendrobium thyrsiflorum Rchb.f. is affected and threatened in its natural habitat due to its ornamental value and over-exploitation (Zhu et al. 2019). We successfully applied these results in the culture establishment and conservation of other rare orchids, such as Dendrobium officinale and Nervilia spp. To our knowledge, this study is the first one to promote and emphasize the unique value of commercially available formulations, such as PPM™, for in vitro rare plant conservation.

The Dendrobium thyrsiflorum Rchb.f. seeds and seedlings were used as model explants to evaluate the effectiveness of PPM™ on Dendrobium germination and in vitro development. Ripen unopened capsules were collected from Cat Tien National Park (Dong Nai, Vietnam). Capsules were covered in plastic bags, then transferred to the laboratory for experimenting. The sterilization process was performed in a bio-safety cabinet. The capsules were surface sterilized in 5% NaClO solution for 15 minutes, followed by flaming twice with 96% ethanol. After surface sterilization, the capsule was slit, and seeds were collected. Seeds were dried in 10 mL scintillation jars using silica beads for 24 hours. After that, the vials are sealed and stored in a refrigerator at 4°C (Shao et al. 2020; Zi et al. 2014).

Effects of PPM™ concentrations on Dendrobium thyrsiflorum Rchb.f. in vitro seed germination

The D. thyrsiflorum Rchb.f. seed vials were transferred from the refrigerator to room temperature before use. Seeds were weighed in sterilized containers. The orchid seed sterilization solution contained 5% NaClO, 96% C₂H₅OH, and sterilized distilled water with 1:1:18 ratio, respectively (Hoang et al. 2016). The seed sterilization solution was added to the seed containing jar and shaken for one minute. The seeds were washed three times with sterilized distilled water. After that, the seeds were suspended with 2 mL of sterilized distilled water. 50 µL of suspension solution was transferred on sterilized filter paper (1 cm × 2 cm), and seed numbers were counted. We transferred the filter paper to culture tubes containing 10 mL ½ MS (Murashige and Skoog 1962) supplemented with 30 g/L sucrose, 7 g/L agar, and 0.0, 0.1, 0.2, 0.4, 0.8, and 1.6% PPM™, respectively. PPM™ was added into the medium before pH adjusting to 5.8 and autoclaving at 121°C/1 atm. Each culture tube was considered a replicate. Each treatment had totally of 6 replicates, and each replicate contained 40-50 seeds as sub-replicates (260-383 seeds per treatment). The chamber temperature was 25 ± 2°C, 40 - 50%, humidity, and illuminated with 30 µMm^-2s^-1 growth light LED. During the 10-week experiment, seed germination and seedling development stages are categorized using the description from Hoang et al. (2016).

Effects of PPM™ concentrations on D. thyrsiflorum Rchb.f. in vitro seedlings growth

Three-month old seedlings D. thyrsiflorum Rchb.f. (approximately 1 cm height) on ½ MS medium supplemented with 30 g/L sucrose, 7 g/L agar culture were used as material. The seedlings’ weight and height were measured before transferring to culture tubes containing 10 mL solidified ½ MS culture medium supplemented with 0.0, 0.1, 0.2, 0.4, 0.8, and 1.6% PPM™, respectively. The experiment had six treatments, each with 10 culture tubes (n=10), and each tube had three seedlings (30 sub-replicates per treatment). Every 10 days, seedling fresh weight and dried weights were collected. Growth changes (height, fresh, and dried increases) were measured by deducing parameters between each growth interval. The culture chamber condition was described above.

Application of low PPM™ concentration on other orchids’ explant establishment

To evaluate the potential use of PPM™ for culture establishment of other rare orchids in our conservation program, we used in vitro explants of Dendrobium officinale, Nervilia fordii, Nervilia plicata of MS media supplemented with 0.1% PPM™. The explants were sterilized and transferred to MS media supplemented with 0.1% PPM™. General explant growth and development were observed.

Statistical analysis and data analysis

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 Dendrobium thyrsiflorum embryo explants at low levels, such as 0.1- 0.2% (Table 1). After 10 weeks of in vitro culture, the germination rate of Dendrobium thyrsiflorum seeds was not affected at the 0.1-0.2% PPM™ concentrations. No statistical differences were detected between the control and low-level treatments. No unusual organogenesis was observed, and protocorms grew normally on this media for months after experimenting (Table 1 & Fig. 1). Arditti and Yam also documented this low dosage for orchid seed (Arditti and Yam 2017) or melon explants (Compton and Koch 2001).

Table 1 . Seed germination rates and developmental stages of D. thyrsiflorum after 6 and 10 weeks of culture on MS (Murashige & Skoog) medium supplemented with different PPM™ concentrations.

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..

Figure 1. Seed germination of D. thyrsiflorum after 10 weeks of culture at different PPM™ concentrations. “V” refers to developmental stage V without damage at PPM™ concentrations of 0.0%, 0.1%, and 0.2%. “IV*” (at 0.4% PPM™), “III*” (at 0.8% PPM™), and “II*” (at 1.6% PPM™) refer to the damage that appeared in developmental stages IV, III, and II, respectively. Scale bar = 200 μm

At the high level of PPM™ supplementation (above 0.2%), Dendrobium thyrsiflorum protocorm explants were either damaged or starting to show inhibitory effects (Table 1). Stages of protocorm development were delayed as a sign of growth inhibition (Fig. 1). Browning and necrotic tissue were detected on protocorms. Growth inhibition by PPM™ supplementation was also documented by Paul et al. in Arabidopsis thaliana (Paul et al. 2001) and Niedz and Bausher in Citrus sinensis (Niedz and Bausher 2002). Besides growth inhibition, PPM™ can also negatively affect organogenesis in sensitive crops, such as petunia (Compton and Koch 2001) and chrysanthemum (George and Tripepi 2001).

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 D. thyrsiflorum Rchb.f. on medium supplemented with low PPM™ concentration (Fig. 1) indicate that the recipe could also be used safely for seedlings explants, which commonly have higher tolerance.

Three-month old Dendrobium thyrsiflorum Rchb.f. seedlings developed normally on MS media supplemented with 0.1% PPM™ during the experiment and months later. However, signs of damage were observed in the seedlings on media supplemented with higher PPM™ concentration (0.2% to 1.6%), particularly at root zones (Fig. 2). In seedlings with damage, root tissue demonstrated higher sensitivity to PPM™ than leaf tissues; there are no significant - differences among the percent of leaves and roots damaged soon after 10 days on media supplemented with 1.6% PPM™ or 20 days on media supplemented with 0.8% PPM™ (Table 2).

Table 2 . Response of 3-month-old D. thyrsiflorum seedlings after 10 and 20 days of culture on MS (Murashige & Skoog) medium supplemented with different PPM™ concentrations (30 replicates for each treatment).

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..

Figure 2. Adaptation of D. thyrsiflorum seedlings after 20 days of culture at different PPM™ concentrations. The 3-month-old seedlings showed normal development after culture at 0.0% and 0.1% PPM™ (N). However, at 0.2% and 0.4% PPM™, signs of damage were observed in roots (RD). At higher PPM™ concentrations (0.8% and 1.6%), signs of damage were also observed in leaves (LD). Scale bar = 5 mm

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 (Juglans regia L.) (Kushnarenko et al. 2022), American birch, chrysanthemum, and rhododendron (George and Tripepi 2001).

Table 3 . Growth of D. thyrsiflorum after 0, 10, and 20 days of culture at different PPM™ concentrations.

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 Dendrobium officinales (Critically Endangered-IUCN Red List), Dendrobium anosmum, Nervilia plicata, or Pyrenaria jonquieriana (unpublished data).

In summary, we used Dendrobium thyrsiflorum Rchb. f. as a model explant to illustrate the effectiveness of PPM™ supplementation to culture media at low concentrations (e.g., 0.1%). Similar to many other Ag-formulation, PPM™ usage is commonly seen in the commercial sector. We discussed PPM™’s potential in Stage I - explant establishment for rare explants. In the extinction era, we firmly believe that enhancement solutions, such as PPM™ and many other innovative agriculture solutions, should be discussed and explicitly implemented for plant conservation.

This research is funded by Vietnam National University, Ho Chi Minh City (VNU-HCM) under grant number C2020-18-02.