Conservation Horticulture: In Vitro Micropropagation and Acclimatization of Selected Florida Native Orchids

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Andrew MullinDepartment of Earth and Environment, Florida International University, 11200 SW 8th Street, Miami, FL 33199

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Bárbara Nogueira Souza CostaDepartment of Earth and Environment, Institute of Environment, Florida International University, 11200 SW 8th Street, Miami, FL 33199

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Jason DowningFairchild Tropical Botanic Garden, 10901 Old Cutler Road, Coral Gables, FL 33156

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Amir Ali KhoddamzadehDepartment of Earth and Environment, Institute of Environment, Florida International University, 11200 SW 8th Street, Miami, FL 33199; and Fairchild Tropical Botanic Garden, 10901 Old Cutler Road, Coral Gables, FL 33156

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Florida is home to 106 native orchid species, of which 77 are listed as endangered or threatened by the State of Florida. The Institute for Regional Conservation (IRC) has classified 62 of these species as either critically imperiled, imperiled, or rare in South Florida. Because of lack of endosperm, orchid germination rates are very low in nature, as they depend on an obligate relationship with mycorrhizal fungi for nutrients. Most orchid seeds can be germinated in vitro without the need for specific mycorrhizal fungi. This study aims are to establish a fast and efficient protocol for in vitro seed germination using different nutrient media and plant growth regulator (PGR) combinations, and to optimize seedling acclimatization protocols using different greenhouse media. To determine germination preferences, three different in vitro seed germination media were tested supplemented with PGRs, including 1) Murashige & Skoog (MS) (control), 2) MS supplemented with 1.5 mg/L 6-benzylaminopurine (BAP), and 3) MS supplemented with 1 mg/L BAP and 0.5 mg/L 1-Naphthaleneacetic acid (NAA) on Cyrtopodium punctatum, a state-listed endangered, IRC critically imperiled epiphytic orchid. There was no significant difference amongst the treatments after 2 and 6 weeks of seed sowing culture. To understand post-culture survivorship, two epiphytic and one terrestrial native species (Trichocentrum undulatum, Encyclia tampensis, and Oncidium ensatum) were chosen for the acclimatization study with two commercially available potting substrates (coir, sphagnum). To measure seedling growth rates, phenotypic measurements [leaf number, leaf length, root length, plantlet height, light intensity, pH, and electrical conductivity (EC)] and Soil Plant Analysis Development (SPAD) and Normalized Difference Vegetation Index (NDVI) values were recorded monthly for five months. All media/PGR combinations resulted in an initial high percentage of stage I growth but inhibited Protocorm-like bodies (PLBs) development, suggesting more research is necessary to determine later improvements or detriments to MS basal media with BAP and NAA. Comparing acclimatization media with the three species of orchid chosen for this experiment, neither O. ensatum nor E. tampensis showed a marked preference for sphagnum moss or coir. However, T. undulatum did perform better with coir compared with sphagnum. This research will help botanical gardens and commercial plant tissue culture laboratories to have a better understanding on selection of PGR combinations for in vitro cell culture and acclimatization media on increasing the viability and plant health and decreasing the mortality of endangered plants.

Abstract

Florida is home to 106 native orchid species, of which 77 are listed as endangered or threatened by the State of Florida. The Institute for Regional Conservation (IRC) has classified 62 of these species as either critically imperiled, imperiled, or rare in South Florida. Because of lack of endosperm, orchid germination rates are very low in nature, as they depend on an obligate relationship with mycorrhizal fungi for nutrients. Most orchid seeds can be germinated in vitro without the need for specific mycorrhizal fungi. This study aims are to establish a fast and efficient protocol for in vitro seed germination using different nutrient media and plant growth regulator (PGR) combinations, and to optimize seedling acclimatization protocols using different greenhouse media. To determine germination preferences, three different in vitro seed germination media were tested supplemented with PGRs, including 1) Murashige & Skoog (MS) (control), 2) MS supplemented with 1.5 mg/L 6-benzylaminopurine (BAP), and 3) MS supplemented with 1 mg/L BAP and 0.5 mg/L 1-Naphthaleneacetic acid (NAA) on Cyrtopodium punctatum, a state-listed endangered, IRC critically imperiled epiphytic orchid. There was no significant difference amongst the treatments after 2 and 6 weeks of seed sowing culture. To understand post-culture survivorship, two epiphytic and one terrestrial native species (Trichocentrum undulatum, Encyclia tampensis, and Oncidium ensatum) were chosen for the acclimatization study with two commercially available potting substrates (coir, sphagnum). To measure seedling growth rates, phenotypic measurements [leaf number, leaf length, root length, plantlet height, light intensity, pH, and electrical conductivity (EC)] and Soil Plant Analysis Development (SPAD) and Normalized Difference Vegetation Index (NDVI) values were recorded monthly for five months. All media/PGR combinations resulted in an initial high percentage of stage I growth but inhibited Protocorm-like bodies (PLBs) development, suggesting more research is necessary to determine later improvements or detriments to MS basal media with BAP and NAA. Comparing acclimatization media with the three species of orchid chosen for this experiment, neither O. ensatum nor E. tampensis showed a marked preference for sphagnum moss or coir. However, T. undulatum did perform better with coir compared with sphagnum. This research will help botanical gardens and commercial plant tissue culture laboratories to have a better understanding on selection of PGR combinations for in vitro cell culture and acclimatization media on increasing the viability and plant health and decreasing the mortality of endangered plants.

Orchidaceae is one of the largest and most diverse flowering plant families in the world with more than 25,000 different documented species (Chugh et al., 2009). Florida is home to 106 native orchid species, of which 77 are listed as endangered or threatened by the State of Florida (Anderson, 2010). The IRC has classified 62 of these species as either critically imperiled, imperiled, or rare in South Florida (Gann et al., 2001). The rarity of these species is due to poaching and habitat destruction (Gann et al., 2001; Junk et al., 2006; Luer, 1972).

The seeds of orchids are pin-sized and dust-like, and lack endosperm (Pant et al., 2011). This is an evolutionary tactic that allows the seeds to be dispersed great distances by wind (Barthlott et al., 2014); however, because of the lack of endosperm, they depend completely on a mycorrhizal fungus for all of its nutritional requirements during the germination phase (Rasmussen, 1995). As a result of this obligate relationship, orchids have very low germination success rates in the wild (Dearnaley, 2007). Orchid seeds are of simple design; however, the seedcoat is very tough and can be difficult to penetrate, which poses problems for germination as well as viability testing (Van Waes and Debergh, 1986). The viability of orchid seed also varies from the time of pollination to maturity, as time progresses, the seedcoat becomes more and more impenetrable, leading to a lessened germination percentage (De Stefano et al., 2022; Dowling and Jusaitis, 2012). Upon germination, orchid seeds have six different stages of growth: seed imbibition, seed enlargement, protocorm formation with shoot apex and rhizoids, protocorm with developing leaves and rhizoids, seedling formation, and seedling development with evident roots and two or more leaves (Srivastava et al., 2015).

PGRs, such as auxins and cytokinins, are often added to orchid micropropagation media to help germination success and overall plant vigor. The three PGRs commonly used during in vitro micropropagation are auxins, cytokinins, and gibberellins (Arditti and Ernst, 1993, Khoddamzadeh et al., 2011). One of the most widely used auxins is NAA. Auxins aid in the process of cell growth and expansion (Vendrame and Khoddamzadeh, 2016), whereas another important PGR widely used is BAP, which is responsible for cell division. There have been many studies showing the importance of growth regulators in the in vitro micropropagation of orchids, such as Bautista-Aguilar et al. (2021) in Vanilla planifolia, Do et al. (2019) in Paphiopedilum hybrids, Huang and Fang (2021) in Cymbidium goeringii, Mi et al. (2021) in Bletillastriata, Chookoh et al. (2019) in Tolumnia, and De Stefano et al. (2022) in Epidendrum nocturnum. However, a fast protocol has yet to be developed for the species used in this project.

Because of the high seedling mortality during the introduction and reintroduction stages, acclimatization, which is the process of transferring plants from an aseptic artificial environment to the outside world, is a vital final step of in vitro micropropagation. In a study from Dutra et al. (2009), coconut husk was used effectively during the acclimatization stage in a native Floridian epiphytic orchid C. punctatum, demonstrating that 35-week-old seedlings potted in coconut husk had a 90% survival rate. In another study on a terrestrial threatened Florida orchid, Bletia purpurea, plants were watered once weekly under 50% shadecloth with the application of N20–P20–K20 fertilizer weekly, yielded positive results 15 weeks later, with a 98% survival rate (Dutra et al., 2008). In another study on a terrestrial old-world orchid, Paphiopedilum armeniacum, plantlets were acclimatized comparing four different media of 1) Chilean sphagnum moss, sieved peat, and Zhijing stone; 2) mixed medium composed of Zhijing stone for orchids, sieved peat, and shattered fir bark; 3) mixed medium with commercial sand for orchids, sieved peat, and shattered fir bark; and last, 4) a mixed medium of Zhijing stone for orchids, coconut bran, and shattered fir bark. Survival and growth percentages were measured 90 d after acclimatization, and optimal results were observed with highest plantlet survival percentage (85.3%) using Zhijing stone for orchids under a water curtain every 1 to 2 d (Zhang et al., 2015), the least effective media was the orchid sand mix, with a survival rate of 74.3%. All four media demonstrated that ample drainage was important with this frequent watering schedule. This study aimed to establish a fast and efficient protocol for in vitro seed germination using different nutrient media and PGR combinations, and to optimize seedling acclimatization protocols using different greenhouse media.

Materials and Methods

In vitro seed germination.

To determine in vitro germination preferences, three different combinations of seed germination media and PGRs were tested on a state-listed endangered epiphytic orchid C. punctatum (Fig. 1), including 1) MS medium (Murashige and Skoog, 1962), 2) MS + 1.5 mg/L BAP, 3) MS + 1 mg/L BAP + 0.5 mg/L NAA, comparing the beginning stages of germination. C. punctatum is a critically imperiled epiphytic native orchid that inhabits remote in coastal dry forests and cypress domes of Everglades National Park and Big Cypress National Preserve of southern Florida with very low population due to anthropogenic factors (Fig. 2). Dry seeds of C. punctatum collected from a naturally occurring individual at Fairchild Tropical Botanic Garden (FTBG) 11 months prior were sterilized and agitated in a wash of 1% sodium hypochlorite for 5 min in a small container, then centrifuged to separate the seed from the bleach solution. After repeating these steps three times, a pipette was used to collect ≈50 to 125 seeds and were then inoculated into the three media treatments under a laminar flow hood and placed in the orchid laboratory at FTBG. The seeds were placed inside petri dishes with full-strength MS media, MS supplemented with 1.5 mg/L BAP, and MS supplemented with 1 mg/L BAP and 0.5 mg/L NAA. All media were supplemented with 20 g sucrose. The media were adjusted to pH 5.6 with 1 M KOH or HCl before autoclaving for 15 min at 121 °C. Plant growth regulators were added before autoclaving. Initial measurements were taken every 2 weeks, and stages of seed germination/growth were compared. Two weeks later, the initial readings were recorded. Stage 1, stage 2, and mortality seed counts were performed in each petri dish under microscope and noted.

Fig. 1.
Fig. 1.

Cyrtopodium punctatum in situ Everglades National Park. (Photo taken by Andrew Mullin.)

Citation: HortScience 57, 9; 10.21273/HORTSCI16672-22

Fig. 2.
Fig. 2.

Native range of Cyrtopodium punctatum (IRC, 2020).

Citation: HortScience 57, 9; 10.21273/HORTSCI16672-22

Acclimatization.

Three native species of endangered orchid were chosen for the acclimatization portion: T. undulatum (Fig. 3): State of Florida status: Endangered, IRC South Florida Status: Critically Imperiled (Fig. 4); O. ensatum (Fig. 5): Endangered, critically imperiled (Fig. 6); E. tampensis (Fig. 7): Commercially exploited (Fig. 8), secure. Young plantlets of O. ensatum, E. tampensis, and T. undulatum (50) were acquired from FTBG’s Million Orchid Project. Two acclimatization media, sphagnum moss (Better-Gro™) and coir (RHP), were chosen as a result of higher survivorship and overall plant health on previous screening study. Plantlets ranging from 9 to 12 months in 2.5-inch pots were taken from FTBG and brought to the Florida International University Biology Department greenhouse. To determine health and survivorship during acclimatization, leaf number, root length, leaf length, SPAD-502, and NDVI (GreenSeeker) values were evaluated. Root length was only measured after deflasking and at the last measurement (6 months old). A SPAD meter was used to measure chlorophyll content, whereas the NDVI meter was used to determine the difference between the transmittance of a red (650 nm) and infrared (IR) (940 nm) light, generating a numerical value to indicate the chlorophyll content (Yuan, 2016). The following formula describes how the NDVI index is calculated,
NDVI=(NIR-RED)(NIR+RED),
where NIR = reflection in the near-IR spectrum, and RED = reflection in the red range of the spectrum (Jiang et al., 2006). To obtain accurate measurements, three necessary readings were taken per plant ≈1.5 feet away. Root and leaf measurements were taken and averaged, and pH and EC values were obtained using a leachate of all plants in each treatment with average greenhouse temperature of 23 to 25 °C.
Fig. 3.
Fig. 3.

Trichocentrum undulatum in situ, Everglades National Park. (Photo taken by Andrew Mullin.)

Citation: HortScience 57, 9; 10.21273/HORTSCI16672-22

Fig. 4.
Fig. 4.

Map of Trichocentrum undulatum native range in Florida (IRC, 2020).

Citation: HortScience 57, 9; 10.21273/HORTSCI16672-22

Fig. 5.
Fig. 5.

Oncidium ensatum (Sauleda, 2014).

Citation: HortScience 57, 9; 10.21273/HORTSCI16672-22

Fig. 6.
Fig. 6.

Map of Oncidium ensatum native range in Florida (IRC, 2020).

Citation: HortScience 57, 9; 10.21273/HORTSCI16672-22

Fig. 7.
Fig. 7.

Encyclia tampensis in Big Cypress National Preserve. (Photo by Drew Mullin.)

Citation: HortScience 57, 9; 10.21273/HORTSCI16672-22

Fig. 8.
Fig. 8.

Map of Encyclia tampensis native range in Florida (IRC, 2020).

Citation: HortScience 57, 9; 10.21273/HORTSCI16672-22

Experimental design and statistical analysis.

The germination experiment was established in a completely randomized design (CRD), with three treatments and 10 replications per treatment for a total of 30 petri dishes. Acclimatization experiment was established in a CRD, with two treatments with six replications per treatment for a total of 12 plants per species. The data for the seed germination were submitted to two-way analysis of variance (ANOVA) followed by Fisher's least significant difference test, which were performed using GraphPad Prism version 9.3.1 for Windows (GraphPad Software, San Diego, CA, www.graphpad.com). The data for the acclimatization were submitted to ANOVA and the means compared by Tukey’s test (P ≤ 0.05) using the statistical program SISVAR (Ferreira, 2011).

Results

In vitro seed germination media in C. punctatum.

To determine the best in vitro seed germination media, three different media supplemented with PGRs were tested on C. punctatum. Media included 1) MS, 2) MS + 1.5 mg/L BAP, and 3) MS + 1 mg/L BAP + 0.5 mg/L NAA comparing germination stages. Data were not significantly different 2 weeks after inoculation between the germination media for the percentage of dead seeds, and stage 2 (testa ruptured/germinated) in C. punctatum (Fig. 9). But differences were observed among the in vitro seed germination media for stage 1 (swelling seeds, ungerminated). The MS + 1.5 mg/L BAP + 0.5 NAA exhibited the greatest swelling, stage 1 (96.85%), compared with MS media (94.29%) 2 weeks after culturing. All seeds demonstrated strong initial growth in all the germination media with imbibition occurring in almost every seed (Fig. 9).

Fig. 9.
Fig. 9.

In vitro Seed Germination of Cyrtopodium punctatum in MS (control), after 2 weeks and 6 weeks in culture. Media: 1) Murashige and Skoog (MS), 2) MS + 1.5 mg/L 6-benzylaminopurine (BAP), 3) MS + 1 mg/L BAP + 0.5 mg/L 1-Naphthaleneacetic acid (NAA) with 10 replications. (A) Stage 1: Swelling seeds, ungerminated. (B) Stage 2: testa ruptured, germinated. ns = not significant. *Significant by Fisher's least significant difference test (P ≤ 0.05).

Citation: HortScience 57, 9; 10.21273/HORTSCI16672-22

No significant differences between the treatments were observed 6 weeks after culturing for stage 1 and stage 2 (Fig. 9). However, significant differences were observed among the media for dead seeds 6 weeks after culturing. Results showed that MS + 1 mg/L BAP + 0.5 mg/L NAA exhibited the highest percentage (7.88%) of dead seeds, compared with MS (3.28%), and MS + 1.5 mg/L BAP (2.86%).

Figure 10A shows dry E. tampensis seed. All seeds had begun the first phase of growth, which was imbibition and swelling of the testa (Fig. 10B). Some seeds showed emergence of the testa out of the seedcoat and the beginning of stage 2, which was the germination stage (Fig. 10C).

Fig. 10.
Fig. 10.

Images of orchid seeds. (A) Scanning electron microscope image of dry Encyclia tampensis seed. (B) Seed stage 1, imbibition in Cyrtopodium punctatum. (C) Seed stage 2, germination/testa in C. punctatum ruptured 6 weeks after culturing. Arrow shows the embryo outside of the seedcoat.

Citation: HortScience 57, 9; 10.21273/HORTSCI16672-22

Acclimatization media in O. ensatum, T. undulatum, and E. tampensis.

Acclimatization is an important process to support successful plantlet transplantation from in vitro to ex vitro environments (Hazarika, 2003; Dohling et al., 2012; Sherif et al., 2018). This process allows in vitro grown plantlets to adapt to the natural environment, which normally has higher light intensity and lower humidity compared with in vitro conditions (Hazarika, 2003).

The O. ensatum plantlets in sphagnum exhibited 100% mortality, and 67% mortality in coir after 90 d, therefore the data related to 150 d after transplantation was not shown for this species in this project. No significant differences were observed among coir and sphagnum on leaf number (LN), leaf length (LL), SPAD values, and pH in O. ensatum plants (Table 1). Differences were observed (P ≤ 0.05) in root length, NDVI values, and EC between the two acclimatization media 30 days after transplantation (DAT) (Table 1). Root length was significantly (P ≤ 0.05) greater in coir (4.28 cm) than sphagnum (3.55 cm). NDVI values were significantly (P ≤ 0.05) greater in coir (0.51), compared with sphagnum (0.35) (Table 1). EC measurements were significantly (P ≤ 0.05) greater in sphagnum (531.50 µs) compared with coir (436.50 µs). SPAD values were greater in coir (14.55) compared with sphagnum (10.88) (Table 1).

Table 1.

Leaf number (LN), root length (RL), leaf length (LL), chlorophyll content - soil plant analytical development (SPAD), normalized difference vegetation index (NDVI), pH, and electrical conductivity (EC) of Oncidium ensatum plants under different types of media 30 d after transplantation (DAT).

Table 1.

Santos and Smozinski (2015) observed higher values for the number of leaves and vertical length of orchid plants (Epidendrum ibaguense) grown in coconut fiber when compared with plants grown in sphagnum. This was observed in this study regarding O. ensatum, LN was higher in sphagnum 30 DAT. However, it is important to emphasize that the coir media resulted in lower mortality rate than sphagnum media in O. ensatum, which means that the coir exhibited a higher survival rate in O. ensatum. In contrast, other research found different results, with the highest survival rate with 79%, in orchid plantlets of Gastrochilus matsuran grown in sphagnum media compared with 1) brick, stone, and wood chips; and 2) orchid stone, mountain stone, and wood chips (Kang et al., 2020).

No significant differences were observed between acclimatization media (sphagnum and coir) on LL, although LN was higher in coir (4.75 cm) compared with sphagnum (4.42 cm) in T. undulatum 30 DAT (Table 2). Significant differences were observed between acclimatization media, the SPAD values were higher using coir (24.81) compared with sphagnum (20.35) at 150 DAT, the LL was greater in sphagnum (6.63 cm) media comparing LL with coir (5.22 cm) at 30 DAT, and the EC was greater in coir (469.67 µs) compared with sphagnum (375.00 µs) at 150 DAT (Table 2).

Table 2.

Leaf number (LN), root length (RL), leaf length (LL), chlorophyll content - soil plant analytical development (SPAD), normalized difference vegetation index (NDVI), pH, and electrical conductivity (EC) of Trichocentrum undulatum plants under different types of media 30 and 150 d after transplantation (DAT).

Table 2.

Sousa et al. (2015) found no significant differences in the number of leaves and roots of Brassavola tuberculata for the substrates sphagnum and coconut fiber. The same was observed in this experiment, in which there was no significant difference between treatments for the number of leaves in T. undulatum. However, they found significant differences between treatments for survival rate and LL of B. tuberculata, where sphagnum provided the highest survivability (67.7%). Results were similar to this experiment for LL, because sphagnum provided the highest value for the LL of T. undulatum. In addition, regarding B. tuberculata, EC was greatest in coir compared with sphagnum. These findings support the results demonstrated in this study, in which coir provided a significantly (P ≤ 0.05) higher value of EC compared with sphagnum 150 DAT in (Table 3).

Table 3.

Leaf number (LN), root length (RL), leaf length (LL), chlorophyll content - soil plant analytical development (SPAD), normalized difference vegetation index (NDVI), pH, and electrical conductivity (EC) of Encyclia tampensis plants under different types of media 30 and 150 d after transplantation (DAT).

Table 3.

Root length was significantly (P ≤ 0.05) greater in coir (1.54 cm) compared with sphagnum (1.07 cm) 30 DAT (Table 3). NDVI values were significantly (P ≤ 0.05) greater in coir (48) than sphagnum (43) 150 DAT (Table 3). No significant differences were observed among coir and sphagnum on NL, LL, SPAD values, pH, or EC 30 and 150 DAT (Table 3). NDVI values were significantly (P ≤ 0.05) greater in coir (0.48), compared with sphagnum (0.43) 150 DAT (Table 3).

SPAD and NDVI devices both measure the chlorophyll content of green plants, providing a general synopsis of their health. This is an important tool concerning orchid conservation because it allows for an immediate measurement of the viability of the plant. The health index of each plant can inform the user of possible pests, disease, or improper watering and/or fertilizer practices, all resulting in lower chlorophyll content (Dunn et al., 2015; Freidenreich et al., 2019; Khoddamzadeh and Dunn, 2016). O. ensatum had higher SPAD and NDVI readings in coir 30 DAT, which resulted in a slower mortality rate, compared with sphagnum. T. undulatum, 150 DAT, exhibited significantly (P ≤ 0.05) higher readings for SPAD and NDVI in coir; however, sphagnum resulted in healthier plants. E. tampensis displayed higher SPAD and NDVI measurements for coir 150 DAT, which did result in healthier plants with longer and more leaves, compared with sphagnum.

Discussion

In general, seed germination of rare species of orchids is known to be lower and more complex compared with hybridized commercial varieties. Differences were observed among the in vitro seed germination media only for stage 1 (swelling seeds, ungerminated). The MS + 1.5 mg/L BAP + 0.5 NAA exhibited the greatest swelling, stage 1 (96.85%), compared with MS media (94.29%) 2 weeks after culturing (Fig. 9).

A very small variation in the concentration of PGRs and additives significantly affect the time period required and frequency of seed germination (Zeng et al., 2011). Studies have indicated that auxin influences protocorm development. In select orchid species, exogenous auxin application increased protocorm DNA content (Lim and Loh, 2003), diameters (Novak and Whitehouse, 2013), morphology (Hadley and Harvais, 1968), and numbers during germination (Miyoshi and Mii, 1995). Cytokinins generally promote the germination frequency, development, proliferation, and multiplication of protocorms, and development of seedlings, shoots, and plantlets (Hossain et al., 2010; Kalimuthu et al., 2007; Mahendran and Bai, 2009; Pathak et al., 2001; Utami et al., 2015). Consensus of researchers have indicated that a combination of auxin and cytokinins were most effective at promoting germination of orchid seed (Deb and Pongener, 2011; Godo et al., 2010; Hadley, 1970; Hadley and Harvais, 1968; Sharma and Tandon, 1986).

A study comparing in vitro seed germination media (Malmgren modified terrestrial orchid medium), modified Knudson C, and Phytotechnology (723) orchid seed sowing medium reported as low as 5% germination at 8 weeks in Calopogon tuberosus var. simpsonii (Kauth, 2005). In the same study, another terrestrial species Sacoila lanceolata var. lanceolata, comparing germination rates, found that fewer than 2% of seeds progressed to stage 2, regardless of media, and after 12 weeks, all seeds were dead. This might be explained by suggesting terrestrial orchids are more reliant on a mycobiont than epiphytic species, therefore symbiotic germination may be preferable. Different species of orchids may also have different germination preferences as to when seeds are collected relating to viability and seedcoat permeability (Arditti et al., 1985; Boland and Scott, 1991; Kauth, 2005). At FTBG, native species of orchid could take up to 6 months to germinate, and in the case of the Vanilla phaentha, 1 year is typical (unpublished data). In contrast to the native orchids, a commercially available Dendrobium orchid produced 100% germination in 45 d using Phytamax, compared with 93% germination in 49 d using MS media (Da Silva et al., 2015).

Seeds progressed readily into stage 1; however, rarely did seeds progress beyond stage 1 germination (swelling of embryo) during the culturing period of 2 weeks. Six weeks after culturing, stage 2 germination rates ranged from 0.95% to 2.15% across all treatments. Two PLBs were observed in MS + 1 mg/L BAP + 0.5 mg/L NAA after 14 weeks in culture. MS media resulted in higher percentages of stage 1 growth; however, other media, such as P723, resulted in lower initial germination rates, but higher survival rates and better overall seedling development (Dutra et al., 2009; Hoang et al., 2017; Johnson et al., 2007). Phytotech 723 is a diluted germination media containing half of the major inorganic salts, which has shown to be efficacious in the germination of terrestrial and epiphytic species (Phytotechnology Laboratories Catalog). A germination study comparing light and temperature conditions in the Florida native orchid, B. purpurea, found that average germination rates were best in a narrow temperature range of 19 to 29 °C under 100% darkness. The slowest germination rates were observed under cooler conditions, 11 to 22 °C (Johnson et al., 2011). This was similar to the culture room temperature (20 °C) used in this study for media maintenance and might be the reason for not having a high germination percentage during the culture period.

Studies comparing asymbiotic and symbiotic media often exhibit higher germination rates using symbiotic germination. In a study on Florida terrestrial orchid, Eulophiaalta, protocorm development was initiated faster using a symbiotic fungal isolate compared with five asymbiotic media (Johnson et al., 2007). In an attempt to speed germination rates, an external seedling heating pad was supplemented after 12 weeks, raising the ambient temperature above room temperature (20–25 °C) to 27 °C for 3 weeks. Similar to this research, the temperature increase resulted in the rapid formation of the two protocorms in MS + 1 mg/L BAP + 0.5 mg/L NAA.

For the acclimatization portion of this study, almost all of the O. ensatum plantlets died 90 DAT, with faster mortality in sphagnum compared with coir. LLs were significantly (P ≤ 0.05) greater using sphagnum (6.63 cm) in T. undulatum 30 DAT. SPAD and EC values were both significantly (P ≤ 0.05) greater in T. undulatum using coir 150 DAT. SPAD and NDVI devices both measure the chlorophyll content of plants, providing a general synopsis of their health. The health index of each plant can inform the user of possible pests, disease, or improper watering and/or fertilizer practices, all resulting in lower chlorophyll content (Dunn et al., 2015; Freidenreich et al., 2019; Khoddamzadeh and Dunn, 2016). Seedlings of O. ensatum had higher SPAD and NDVI readings in coir 30 DAT, which resulted in a slower mortality rate, compared with sphagnum. Seedlings of T. undulatum, 150 DAT, exhibited significantly (P ≤ 0.05) higher readings for SPAD and NDVI in coir; however, sphagnum resulted in healthier plants. Seedlings of E. tampensis displayed higher SPAD and NDVI measurements for coir 150 DAT, which did result in healthier plants with longer and more leaves, compared with sphagnum.

Seedlings of E. tampensis had significantly (P ≤ 0.05) longer leaf lengths using coir 30 DAT. The acclimatization results could be attributed to the different growing habits of these three orchid species, as well as their preferred habitat type. O. ensatum is a terrestrial species inhabiting hardwood hammocks in Everglades National Park. T. undulatum, is an epiphytic species with a very restricted distribution Florida, inhabiting only coastal prairies and dry tropical forests of Everglades National Park. In contrast, E. tampensis is a widespread epiphytic species that inhabits many different ecosystems, from prairies to hardwood hammocks. These differences may explain the substrate preferences demonstrated in this project. The two epiphytic species favored coir, because of the need for a dry-out period. Epiphytic species of orchids typically do not need organic material to provide moisture for their survival. Some studies also found no significant differences between treatments comparing growth characteristics. Sousa et al. (2015) found no significant differences on the number of leaves and roots in B. tuberculata using sphagnum and coconut fiber media. Similar findings were reported by Zandoná et al. (2014), where no significant differences were observed comparing shoot height, average length of roots, and number of roots for different types of substrates, such as sphagnum, carbonized rice husk, carbonized rice husk + sphagnum coconut fiber + carbonized rice husk in Arundina graminifolia. In a study on tomato plants, low EC resulted in sub-par fruit production (Ding et al., 2018). Overall, coir showed more promising results as an acclimatization media. This is likely because of the shorter duration of water retention compared with sphagnum, especially considering epiphytic and terrestrial orchids.

Conclusion

Although the data were not significantly different compared with in vitro seed germination media, MS + 1.5 mg/L BAP showed greater viability. This is very important especially in conservation horticulture and application of endangered orchids. In general, seed germination in native endangered orchids is known to be lower and more complex compared with hybridized commercial varieties. Although it remains unclear whether seed inhibition exhibited in this experiment is a result of evolutionary and genetic patterns (i.e., seed biology and/or inbreeding depression) or experimental conditions such as photoperiod, temperature, and mineral nutrition. Seedlings did initially benefit from the addition of a heat source, demonstrating the importance of the temperature parameter. More time also would be beneficial, because of the slow germination observed in this experiment. It is also indicated in the literature that symbiotic germination, although time consuming, may be more effective compared with asymbiotic germination. Among the acclimatization media, coir showed promising results in viability of the two epiphytic species, which might be because of the need for a dry-out period. The addition of other epiphytic and terrestrial species is necessary to understand substrate preferences and determine an efficient protocol for these endangered Florida orchids; future studies should include a genotyping, temperature, and light component.

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Contributor Notes

Thanks to Fairchild Tropical Botanic Garden’s Million Orchid Project for the lab and greenhouse spaces and materials and Conservation & Sustainable Horticulture Lab at Florida International University. Special thanks to Dr. Carl Lewis and Jay Arce. This research was partially funded by the U.S. Department of Agriculture National Institute of Food and Agriculture Hispanic-Serving Institutions Higher Education Grants Program (2016-38422-25549). In addition, thanks to Dr. Krish Jayachandran and Dr. Hong Liu for serving in thesis committee. This is contribution #1457 from the Institute of Environment at Florida International University.

A.A.K. is the corresponding author. E-mail: akhoddam@fiu.edu.

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    Fig. 1.

    Cyrtopodium punctatum in situ Everglades National Park. (Photo taken by Andrew Mullin.)

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    Fig. 2.

    Native range of Cyrtopodium punctatum (IRC, 2020).

  • View in gallery
    Fig. 3.

    Trichocentrum undulatum in situ, Everglades National Park. (Photo taken by Andrew Mullin.)

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    Fig. 4.

    Map of Trichocentrum undulatum native range in Florida (IRC, 2020).

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    Fig. 5.

    Oncidium ensatum (Sauleda, 2014).

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    Fig. 6.

    Map of Oncidium ensatum native range in Florida (IRC, 2020).

  • View in gallery
    Fig. 7.

    Encyclia tampensis in Big Cypress National Preserve. (Photo by Drew Mullin.)

  • View in gallery
    Fig. 8.

    Map of Encyclia tampensis native range in Florida (IRC, 2020).

  • View in gallery
    Fig. 9.

    In vitro Seed Germination of Cyrtopodium punctatum in MS (control), after 2 weeks and 6 weeks in culture. Media: 1) Murashige and Skoog (MS), 2) MS + 1.5 mg/L 6-benzylaminopurine (BAP), 3) MS + 1 mg/L BAP + 0.5 mg/L 1-Naphthaleneacetic acid (NAA) with 10 replications. (A) Stage 1: Swelling seeds, ungerminated. (B) Stage 2: testa ruptured, germinated. ns = not significant. *Significant by Fisher's least significant difference test (P ≤ 0.05).

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    Fig. 10.

    Images of orchid seeds. (A) Scanning electron microscope image of dry Encyclia tampensis seed. (B) Seed stage 1, imbibition in Cyrtopodium punctatum. (C) Seed stage 2, germination/testa in C. punctatum ruptured 6 weeks after culturing. Arrow shows the embryo outside of the seedcoat.

  • Anderson, PJ 2010 Native palms of Florida: Identification and regulation Florida Department of Agriculture and Consumer Services Division of Plant Industry

    • Search Google Scholar
    • Export Citation
  • Arditti, J. & Ernst, R. 1993 Micropropagation of orchids John Wiley and Sons New York

  • Arditti, J., Oliva, A. & Michaud, J.D. 1985 Practical germination of North American and related orchids-3-Calopogon tuberosus, Calypso bulbosa, Cypripedium species and hybrids, Piperia elegans var. elata, Piperiamaritima, Platantherahyberborea, and Platanthera saccata Am. Orchid Soc. Bull. 54 859 872

    • Search Google Scholar
    • Export Citation
  • Barthlott, W., Große-Veldmann, B. & Korotkova, N. 2014 Orchid seed diversity Englera 32 1 245

  • Bautista-Aguilar, J.R., Iglesias-Andreu, L.G., Martínez-Castillo, J., Ramírez-Mosqueda, M.A. & Ortiz-García, M.M. 2021 In-vitro conservation and genetic stability in Vanilla planifolia Jacks HortScience 56 12 1494 1498 https://doi.org/10.21273/HORTSCI16118-21

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Boland, T.J. & Scott, P.J. 1991 Ecological aspects of Aerthusa bulbosa, Calopogon tuberosus, and Pogonia Ophioglossoides (Orchidaceae) in eastern Newfoundland. I. flowering and fruiting patterns Rhodora 93 248 255

    • Search Google Scholar
    • Export Citation
  • Chookoh, N., Chiu, Y.T., Chang, C., Hu, W.H. & Dai, T.E. 2019 Micropropagation of Tolumnia orchids through induction of protocorm-like bodies from leaf segments HortScience 54 7 1230 1236, https://doi.org/10.21273/HORTSCI13747-18

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Chugh, S., Guha, S. & Rao, I.U. 2009 Micropropagation of orchids: A review on the potential of different explants Scientia Hort. 122 4 507 520 https://doi.org/10.1016/j.scienta.2009.07.016

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Da Silva, J.A.T., Cardoso, J.C., Dobránszki, J. & Zeng, S. 2015 Dendrobium micropropagation: A review Plant Cell Rep. 34 5 671 704 https://doi.org/10.1007/s00299-015-1754-4

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Dearnaley, J.D. 2007 Further advances in orchid mycorrhizal research Mycorrhiza 17 6 475 486

  • Deb, C.R. & Pongener, A. 2011 Asymbiotic seed germination and in-vitro seedling development of Cymbidium aloifolium (L.) Sw.: A multipurpose orchid Plant Biochem. Biotechnol. 20 1 90 95 https://doi.org/10.1007/s13562-010-0031-4

    • Crossref
    • Search Google Scholar
    • Export Citation
  • De Stefano, D., Souza Costa, B., Downing, J., Fallahi, E. & Khoddamzadeh, A.A. 2022 In-vitro micropropagation and acclimatization of Epidendrum nocturnum, an endangered native orchid using organic supplements Am. J. Plant Sci. 13 380 393 https://doi.org/10.4236/ajps.2022.133023

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Ding, X., Jiang, Y., Zhao, H., Guo, D., He, L., Liu, F., Zhou, Q., Nandwani, D., Hui, D. & Yu, J. 2018 Electrical conductivity of nutrient solution influenced photosynthesis, quality, and antioxidant enzyme activity of pakchoi (Brassica campestris L. ssp. Chinensis) in a hydroponic system PLoS One 13 8 e0202090 https://doi.org/10.1371/journal.pone.0202090

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Do, V.N.T., Hsu, S.T. & Lee, Y.I. 2019 Clonal propagation in-vitro of Paphiopedilum hybrids from adult plants HortScience 54 9 1565 1569 https://doi.org/10.21273/HORTSCI13791-18

    • Crossref
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