Asymbiotic germination and morphological studies of seeds of Atlantic Rainforest micro-orchids (Pleurothallidinae)

Franciele Marx Koene; Érika Amano; Eric de Camargo Smidt; Luciana Lopes Fortes Ribas

doi:10.1371/journal.pone.0243297

. 2020 Dec 14;15(12):e0243297. doi: 10.1371/journal.pone.0243297

Asymbiotic germination and morphological studies of seeds of Atlantic Rainforest micro-orchids (Pleurothallidinae)

Franciele Marx Koene ¹, Érika Amano ¹, Eric de Camargo Smidt ¹, Luciana Lopes Fortes Ribas ^1,^*

Editor: Jen-Tsung Chen²

PMCID: PMC7735581 PMID: 33315920

Abstract

The morphological and morphometric characters of seeds belonging to 11 species of the subtribe Pleurothallidinae using light and scanning electron microscopy were studied to understand the in vitro germination process. Qualitative data (color, shape, ornamentation) and quantitative ones were also evaluated in seeds and embryos (length, width, volume and air space percentage between the integument and the embryo). The viability of the seeds was evaluated by in vitro germination in woody plant medium (WPM), and by analysis of the developmental stages of protocorms until seedling formation (two to 24 weeks). Morphometric data showed variations within the genus Acianthera and between species of different genera. The best germination and protocorm formation responses occurred with Acianthera prolifera (92%) and Acianthera ochreata (86%), with the formation of seedlings after 12 and 16 weeks of sowing, respectively. The seeds and embryos of A. prolifera and A. ochreata were larger (length, width, and volume) with a structural polarity that may have facilitated their germination comparing to others studied species. Other characteristics of A. prolifera seeds that may have contributed to these results include the presence of a thin testa without ornamentation and a suspensor. The protocorms of Anathalis obovata, Dryadella liliputiana, and Octomeria gracillis developed slowly in the WPM, not reaching the seedling stage in 24 weeks of cultivation. This morphological and morphometric study contributes to the understanding of asymbiotic germination of some micro-orchid species.

Introduction

Orchids are suffering from an uncertain future through overexploitation, habitat loss due to human activities and the impact of climate change, and their survival is contingent on a variety of abiotic and biotic factors and their effect on orchid growth, development, and reproduction [1]. Their unusual physiology, seed structure, and germination pattern set them apart from other flowering plants. The seeds are dust-like, non-endospermic, and require a fungal stimulus for germination in nature [2, 3]. A unique characteristic of orchid seeds is that, rather than an endosperm, the air space surrounds a tiny globular embryo containing a small number of cells, which is protected by a membranous testa; the air-space volume in orchid seeds varies depending on the species [4, 5]. Seed morphological traits are thus related to biological and ecological processes like dormancy, germination, and seed dispersal [4].

Orchid propagation in nature is highly complex, involving specific mycorrhizal associations. In vitro methods, or plant tissue culture, can provide alternative approaches for both propagation and preservation, with asymbiotic germination representing an ideal system for studying the growth and development of seeds and seedlings [6, 7]. In vitro germination of orchids can help increase the effectiveness of conservation and breeding programs due to their high germination rates, which are commonly over 70% for epiphytic orchids, as opposed to under 5% in ex vitro conditions [8]. The culture media used for asymbiotic germination vary according to the species, and the most commonly used for the propagation of orchids are MS [9], VW [10] and KC [11, 12]. Koene et al. [13] recommended Woody Plant Medium (WPM) culture medium [14] for asymbiotic seed germination and plantlet development of A. prolifera when compared to MS, MS with half the salt concentration (MS/2) and KC. This culture medium was also better for germination and seedling development of other species of orchids, such as: Brasiliorchis picta (Hook.) R. B. Singer et al. [15] and Hadrolaelia grandis (Lindl.) Chiron & V. P. Castro [16].

The Orchidaceae is one of the largest and most diverse families of flowering plants in the world, with 25,000–28,000 species [2]. One of the subtribes of this family, Pleurothallidinae Lindl., belongs to Epidendroideae subfamily and Epidendreae tribe, consisting of approximately 5,100 species popularly known as micro-orchids [17]. This subtribe is found in the Neotropics, from Argentina to southern Mexico. Micro-orchids in the Atlantic Forest in Brazil, which is home to a large number of micro-orchid endemic species and is one of the most threatened extinction biomes on the planet, face extreme extinction pressures with a reduction in the original habitat of over 90% [18–20]. Most species of Pleurothallidinae have no great commercial appeal, due to the small dimensions of the plants and their flowers, as well as the difficulties of cultivation [18]. There are few studies of asymbiotic germination of micro-orchids, which have shown low germination rates and slow growth of seedlings [21, 22]. The best results were achieved by Koene et al. [13] with 79% of total germination of A. prolifera grown in WPM medium for 12 weeks. Studies on the morphology and morphometry of the seeds and their relationship with micro-orchid germination are also scarce.

Although the seeds of different orchid species have many similarities, there is significant variability in the size, shape, characteristics of the testa cells, the zones of adhesion, and sculptures constituted by the cellular wall and cuticular material [23]. Studies on seed morphology and morphometry have achieved substantial contributions to the taxonomy, phylogeny, and phytogeography of this group [24, 25]. Meanwhile, these studies can also enhance understanding of the in vitro germination process, which in turn can accelerate the production of seedlings for future reintroduction into their natural habitat, aiding in the conservation of the Pleurothallidinae. This study was carried out with 11 native species belonging to six genera of the Atlantic Forest (Fig 1) that have not been evaluated for the threat of extinction by the Flora of Brazil [26, 27], as follows: Acianthera aphthosa (Lindl.) Pridgeon & M. W. Chase, Acianthera hatschbachii (Schltr.) Chiron & van den Berg, Acianthera ochreata (Lindl.) Pridgeon & M.W. Chase, Acianthera prolifera (Herb. ex Lindl.) Pridgeon & M. W. Chase, Acianthera sonderiana (Rchb.f.) Pridgeon & M.W. Chase, Anathallis obovata (Lindl.) Pridgeon & M.W. Chase, Dryadella lilliputiana (Coan.) Luer, Dryadella zebrina (Porsch) Luer, Octomeria gracilis Lodd. ex Lindl., Pabstiella fusca (Lindl.) Chiron & Xim. Bols, and Stelis grandiflora Lindl. Acianthera aphthosa was the only species considered critically endangered (CR) in the Brazilian state of Espírito Santo by the Red List [28]. Five of these species are endemic to Brazil (A. ochreata, A. aphthosa, A. sonderiana, D. liliputiana, and O. gracilis) [26, 27]. Acianthera prolifera is rupicolous and A. ochreata is rupicolous and epiphytic, while all the other species in this study are epiphytes.

Fig 1 — A. *Acianthera aphtosa*, B. *Acianthera hatschbachii*, C. *Acianthera ochreata*, **D-E**. *Acianthera prolifera*, F. *Acianthera sonderiana*, G. *Anathallis obovata*, H. *Dryadella liliputiana*, I. *Dryadella zebrina*, J. *Octomeria gracilis*, K. *Pabstiella fusca*, L. *Stelis grandiflora*. Photos by Eric C. Smidt, except F by Luiz F. K. Varella.

This study aimed to analyze the morphological and morphometric characteristics of the seeds and relate them with the asymbiotic germination of species of the subtribe Pleurothallidinae. We demonstrate efficient methods of rapid seed germination and seedling development of some species and our results offer potential possibilities for reintroduction programs that can play a key role in reducing the extinction risk for species of micro-orchids.

Materials and methods

Seed materials

Mature capsules at dehiscence from manual cross pollination of 11 species of the Pleurothallidinae subtribe (Fig 2A) were collected (Fig 2B) from three to four plants of each species in a greenhouse of the Federal University of Paraná (UFPR), Curitiba, Paraná, Brazil. Table 1 shows the list of voucher specimens deposited at the Herbarium of the Botany Department, UFPR, and the seeds used for the morphological and morphometric studies for all species, and for asymbiotic germination.

Table 1. Species of the subtribe Pleurothallidinae used for morphological, morphometric, and in vitro germination analysis in this study.

Species	Voucher	In vitro germination
Acianthera aphthosa (Lindl.) Pridgeon & M. W. Chase	Koene, FM. 010	Yes
A. hatschbachii (Schltr.) Chiron & van den Berg	Oliveira, LRL. 019	No
A. ochreata (Lindl.) Pridgeon & M.W. Chase	Koene, FM. 005	Yes
A. prolifera (Herb. ex Lindl.) Pridgeon & M. W. Chase	Koene, FM. 001	Yes
A. sonderiana (Rchb.f.) Pridgeon & M.W. Chase	Koene FM. 009	No
Anathallis obovata (Lindl.) Pridgeon & M.W. Chase	Santos, MC. 020	Yes
Dryadella liliputiana (Coan.) Luer	Imig, DC. 381	Yes
D. zebrina (Porsch) Luer	Imig, DC. 405	No
Octomeria gracilis Lodd. ex Lindl.	Koene, FM. 014	Yes
Pabstiella fusca (Lindl.) Chiron & Xim. Bols	Koene, FM. 013	Yes
Stelis grandiflora Lindl.	Ignowski, H. 011	Yes

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Morphological and morphometric analysis of seeds

An average of 30 seeds per specimen was analyzed using a light (Fig 2C) and a scanning electron microscope (SEM). For SEM observations, the seeds were fixed with double-sided carbon tape and coated with gold. Qualitative data on the general seed morphology, including color, ornamentation, shape, micropillar opening, and the presence of a cuticular deposit, were analyzed. Qualitative and quantitative analyses were performed using a light microscope (Olympus BX41 with DC30 camera), and the following variables were measured for seeds and embryos: length (L), width (W), L/W ratio, volume, and percentage of air space between the testa and the embryo. The width and length were measured with a micrometer at the longest and widest axis of the seed. Seed volume was calculated as 2 [(W/2)² · (L/2) · (π/3)]. Embryo volume was calculated by using the formula 4/3 π · L/2 · (W/2)². The percentage of air space was calculated as [(seed volume—embryo volume) / seed volume] x 100. The terminology and methods adopted were those of: Arditti et al. [29, 30], Arditti and Ghani [24] and Barthlott et al. [25].

Seed viability

Tetrazolium test

Seeds (5 mg) were placed in a 1.5 mL Eppendorf microtube and pre-conditioned in a 10% sucrose solution at room temperature for 24 h. They were then immersed in a 1% tetrazolium solution for 24 h at 40°C in a water bath in the dark. The solution was drained from the tubes with a micropipette, and the seeds were washed twice with distilled water, following the methodology proposed by Hosomi et al. [31]. Red seeds were classified as viable and used for morphometric studies.

In vitro germination

Seeds were surface-sterilized by dipping into a 1% sodium hypochlorite (10–12% PA) solution (NaClO) (v / v) containing 0.1% Tween 20® (v / v) for 15 min while stirring. The seeds were then transferred to a funnel coated with sterile filter paper and washed six times with sterile distilled water. The seeds were dried on sterile filter paper and inoculated in Petri dishes (150 mm x 20 mm) containing 30 mL of the woody plant medium (WPM) [14]. The media were supplemented with 5.6 g L^-1 agar from HiMedia® (Mumbai, India) and 3% sucrose (w/v). The pH of the media was adjusted to 5.8 with 0.1 N NaOH or HCl before the addition of agar. Culture media were sterilized by autoclaving for 20 min at 121°C.

For the evaluation of seed germination about 500 seeds per Petri dish, with three Petri dishes per species, were inoculated. Five fields with 100 seeds per plate were marked and protocorm development was evaluated from two to 16 weeks based on the following stages: 1, seed with chlorophyllous embryo (Fig 2D); 2, testa ruptured/chlorophyllous protocorm (germination) (Fig 2E); 3, protocorm with apex and/or rhizoids (Fig 2F); 4, protocorm with one or two leaves (Fig 2G); 5, protocorm with two or more leaves and root (seedling) (Fig 2H). The germination rate was evaluated after four, eight, and 12 weeks of cultivation, and the average time (in days) to reach the stages was calculated for 24 weeks. The Petri dishes with seeds were maintained at 26±2°C/18±2°C (day/night), with a 16 h photoperiod under fluorescent lamps at a light intensity of 40 μmol m⁻² s⁻¹.

After seedling formation, the radicles can be cut, and explants cultured in a medium containing cytokinins to induce shoots (Fig 5C) or explants can be subcultured in flasks containing a medium supplemented with activated charcoal, where elongation and root development occur (Fig 5D). Finally, A. ochreata and A. prolifera plants were transplanted into sowing trays (3.5 cm²) containing commercial Forth® substrate, composed of a mixture of coconut fiber, Pinus bark, and charcoal with fine vermiculite Eucatex® (1:1) (v/v). The seedlings were acclimatized in a greenhouse at room temperature (25 ± 2°C day / 20 ± 2°C night), under a photoperiod of 12 h and a light intensity of 50 μmol·m⁻² ·s⁻¹

Statistical analysis

The experimental design was completely randomized. The data of the frequency (%) of the developmental protocorm stages were submitted to the Bartlett and the Shapiro-Wilk normality test and analysis of variance (ANOVA). The means were compared by the Tukey test at a level of significance of 5%. The statistical program used was PAST 3.3 software.

Results

Morphological and morphometric analysis of seeds

The seeds of the orchids studied exhibited diversity in their shape, size, volume, and seed coat (ornamentation) (Tables 2 and 3), as well as in their embryo morphometry (Table 4).

Table 2. Morphological seed characteristics of species belonging to the subtribe Pleurothallidinae.

Species	Color	Shape	Testa cell	Micropillar opening	Ornamentation
Acianthera aphthosa	Pale Yellow	Fusiform	Oblong	Yes	Papillae
A. ochreata	Brown	Ellipsoid	Oblong	Yes	Verrucosities
A. hatschbachii	Brown	Filiform	Oblong	Yes	Papillae
A. prolifera	Pale Yellow	Fusiform	Hexagonal	Yes	Absent
A. sonderiana	Brown	Clavate	Oblong	Yes	Verrucosities
Anathallis obovata	Pale yellow	Ellipsoid	Oblong	Yes	Absent
Dryadella liliputiana	Brown	Ellipsoid	Oblong	Yes	Verrucosities
D. zebrina	Brown	Ellipsoid	Oblong	No	Verrucosities
Pabstiella fusca	Brown	Clavate	Oblong	Yes	Papillae
Octomeria gracilis	Brown	Fusiform	Oblong	Yes	Papillae
Stellis grandiflora	Pale yellow	Clavate	Oblong	Yes	Absent

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Table 3. Morphometric data of seeds of species belonging to the subtribe Pleurothallidinae.

Species	Length	Width	Length/width Ratio	Testa cells^*	Volume
Species	(μm)	(μm)	Length/width Ratio	length- width	mm³ x 10⁻³
Acianthera aphthosa	513 ±24	115±21	1.075	9–12	1.401
A. hatschbachii	401 ±27	111±31	1.386	6–12	1.293
A. ochreata	473 ±24	139±18	1.768	10–12	2.392
A. prolifera	742±119	135±43	2.700	14–10	3.540
A. sonderiana	307±81	159± 18	1.120	3–6	2.032
Anathallis obovata	244±31	109±17	1.101	4–6	0.759
Dryadella liliputiana	265±42	146±20	1.125	2–5	1.479
D. zebrina	262±17	154±20	1.720	2–3	1.626
Octomeria gracilis	247±28	90±16	1.084	5–6	0.524
Pabstiella fusca	187±31	83±7	0.140	4–3	0.337
Stelis grandiflora	195±19	127±21	1.053	2–5	0.823

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The values represent the means and standard deviations.

*Testa cells: measurements on the largest length and width of the testa of the seed.

Table 4. Morphometric data of embryos of species belonging to the subtribe Pleurothallidinae.

Species	Length	Width	Volume	Air space	SV/EV^*
Species	(μm)	(μm)	(mm³ x 10⁻³)	(%)	SV/EV^*
Acianthera aphthosa	186±21	73± 14	0.5147	74.42	2.7220
A. hatschbachii	97±15	70± 15	0.2487	80.77	5.1990
A. ochreata	175±23	99±14	0.8976	62.48	2.6729
A. prolifera	216±47	80±9	0.7235	79.56	4.8929
A. sonderiana	84± 7	75±18	0.2473	87.83	8.2167
Anathallis obovata	76±17	69±6	0.1894	75.04	4.0074
Dryadella liliputiana	90±12	80±9	0.3014	79.61	4.9071
D. zebrina	84±7	79±7	0.2744	83.13	5.9256
Octomeria gracilis	77±7	71±5	0.2031	61.21	2.5800
Pabstiella fusca	69±8	64±7	0.1479	56.14	2.2785
Stelis grandiflora	79±5	75±7	0.2326	71.75	3.5382

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Values represent means and standard deviation.

* seed volume (SV) to embryo volume (EV) ratio

Pale yellow seeds were observed in A. prolifera, D. liliputianauana, and S. grandiflora, while the seeds of the other species studied were brown (Table 2).

The seeds had several shapes, including fusiform, filiform, ellipsoid, and clavate (Fig 3A–3K). Generally, cells were shorter at either pole, while the medial cells of the seed coat were elongated. The chalazal pole of the seeds was closed, and the micropillar end was open (Fig 3F), except for D. zebrina, which was also closed (Table 2). The testa cells observed were transparent, longitudinally oriented, and oblong (Fig 3L), except in A. prolifera, in which they were hexagonal (Fig 3M). The ornamentation pattern of the testa cells of some species included papillae or verrucosities (Fig 3N), though these were absent from A. prolifera, A. obovata, S. grandiflora and A. hatschbachii (Fig 3O) (Table 2). The number of testa cells ranged from 2 to 14 on the longest axis and 3 to 12 on the widest axis, with the highest number in A. prolifera and A. ochreata (Table 3).

The seeds showed high diversity in their size (length, width, and volume) (Table 3), as well as in the size and number of cells constituting the embryo (Table 4), despite their microscopic nature. The seed length ranged from 187±31 μm (P. fusca) to 742±119 μm (A. prolifera), while the width ranged from 83±7 μm (P. fusca) to 159± 18 μm for A. sonderiana (Table 3). A. prolifera and A. ochreata had length-to-width ratios of 2.700 and 1.767, respectively, while the L/W ratio in P. fusca was 0.140 (Table 3). A. prolifera had the largest seed volume (3.540 mm³ x 10−³) while the smallest was recorded in P. fusca (0.337 mm³ x 10−³) (Table 3).

The embryos were generally ellipsoidal and located at the center of the seed. Variation in length, width, and volume was also observed. The largest length was recorded for A. prolifera while A. ochreata had the largest width and volume, and P. fusca embryos had the smallest length, width, and volume (Table 4).

The largest percentage of air space was observed in A. sonderiana (87.83%), whereas the lowest percentage of air space was observed in O. gracilis (61.21%) and P. fusca (56.14%) (Table 4). The seed volume to embryo volume ratio was highest (8.2167) in A. sonderiana, followed by D. zebrina (5.9256), and A. hatschbachii (5.1990) and lowest in P. fusca (2.2785) (Table 4).

Anatomical analysis

Acianthera prolifera, A. obovata, O. gracilis, and P. fusca had a thin testa (0.01 μm), while the testa was thick in other species (0.03 μm). The cells of the apical pole of the embryos of A. aphthosa, A. ochreata, A. prolifera, and D. lilliputiana were smaller than those of the basal pole, while these cells were similar in size in the remaining species. Most of the species studied did not have a suspensor, which occurred only in A. prolifera and D. lilliputiana (Table 5). The presence of a cuticle surrounding the embryo was observed in A. aphthosa and P. fusca, though none of the species had a cuticle around the testa.

Table 5. Anatomical characteristics of the seed testa of the embryo cells, and presence or absence of suspensor and cuticle in species belonging to the subtribe Pleurothallidinae.

Species	Testa	Embryo cells	Suspensor	Cuticle
Species	Testa	Apical/basal pole	Suspensor	Cuticle
Acianthera aphthosa	Thick	Smaller/larger	Absence	Presence/embryo
A. ochreata	Thick	Smaller/larger	Absence	Absence
A. prolifera	Thin	Smaller/larger	Presence	Presence
A. sonderiana	Thick	Similar	Absence	Absence
Anathallis obovata	Thin	Similar	Absence	Absence
Dryadella liliputiana	Thick	Smaller/larger	Presence	Absence
D. zebrina	Thick	Smaller/larger	Absence	Absence
Octomeria gracillis	Thin	Similar	Absence	Absence
Pabstiella fusca	Thin	Similar	Absence	Presence/embryo
Stelis grandiflora	Thick	Similar	Absence	Absence

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In vitro germination

The seeds of all of the micro-orchid species studied exhibited chlorophyllous embryos after three to eight days of sowing (Fig 4A). The first asymbiotic germination responses (seeds with ruptured testa and chlorophyllous protocorm) occurred after seven days of sowing for A. prolifera and A. aphthosa, and between nine and 13 days for the other species (Table 6). In general, most of the seeds had a low germination rate up to eight weeks after sowing, except for A. prolifera (76%). By 12 weeks after sowing, the highest germination rates were obtained for A. prolifera, followed by A. ochreata and O. gracilis (92%, 86%, and 77%, respectively). Of the species studied, only A. prolifera, A. ochreata, A. aphthosa, and S. grandiflora reached the seedling stage, with the time to this stage varying between four and 12 weeks (Table 6, Fig 5A and 5B).

Table 6. Germination rate after four, eight, and 12 weeks in woody plant medium and the average time (days) for the protocorms development of the Pleurothallidinae species.

Species	Germination^* (%)			Developmental stages^** (days)
	4	8	12	I	II	III	IV	V
Acianthera aphthosa	7	38	41	4	7	14	60	121
A. ochreata	35	53	86	4	9	14	60	119
A. prolifera	39	76	92	3	7	10	35	90
Anathallis obovata	12	23	36	3	10	15
Dryadella lilliputiana	4	15	38	7	13	21
Octomeria gracilis	4	21	77	5	11	21
Pabstiella fusca	4	4		8	12
Stelis grandiflora	7	15	51	8	11	21	95	127

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* Evaluation after four, eight, and 12 weeks of sowing.

**Evaluation performed 16 weeks after sowing: I, seed with chlorophyllous embryo; II, ruptured testa (germination); III, protocorm with apex; IV, protocorm with one or two leaves; V, protocorm with leaves and root (seedling).

The seeds of A. obovata and D. liliputiana showed similar behavior in the WPM, with germination rates lower than 40%, and reached the stage of protocorm with apex without the development of leaves and radicle, which became visible by 15–20 days, though they did not progress to the other stages by the 24-week evaluation. Likewise, O. gracillis protocorms did not develop leaves, although the germination rate was high (77%) at 12 weeks (Table 6). The seeds of P. fusca had the worst germination response, with only 4% germination by eight weeks after sowing. Furthermore, the protocorms did not develop in the WPM, and they were necrotic.

The analysis of the rates obtained at each stage of protocorm development confirmed that A. prolifera and A. ochreata had the best germination response (Fig 4). After two weeks, A. prolifera had the highest proportion of seeds with chlorophyll embryos (55%), followed by A. ochreata (52%) and S. grandiflora (49%) (Fig 4A). During this period, the highest germination rates (ruptured testa with chlorophyll embryo) occurred in A. prolifera (44%) and A. ochreata (39%) and were significantly higher than the other species analyzed, in which germination rates were below 10%.

By four weeks after sowing, all species except P. fusca had protocorms with apex (Fig 4B). By six to eight weeks after sowing, A. prolifera had the highest rate of protocorms with apex (27%) (Fig 4C); it was also the only species to have protocorms with leaves (21%) (Fig 4D). Seedling development began at 12 weeks, with the largest number of seedlings found in A. prolifera (32%), followed by A. ochreata (20%). The protocorms of A. aphthosa and S. grandiflora grew slowly, with less than 10% forming seedlings, though a longer period of evaluation (Fig 4E) or subculture to the same medium may be necessary. The development of the protocorms of A. ochreata into seedlings was somewhat slower than that of A. prolifera. After elongation, A. ochreata (Fig 5E) and A. prolifera (Fig 5F) were then successfully acclimatized in a greenhouse with 80% survival after three months or 12 months.

Discussion

The species of the subtribe Pleurothallidinae showed considerable variation in the morphological characteristics of their seeds and embryos. The color of the seeds varied from pale yellow to brown. According to Swamy et al. [32], the microscopic size of orchid seeds makes it very difficult to visualize their color, but they are usually variations of yellow, brown, and white. Barthlott et al. [25] found that the most frequent color of seeds is brownish or dark brown, as observed in our study.

We observed seeds that were ellipsoid, clavate, fusiform, or filiform, shapes that have also been reported for other orchid species [30, 33–35]. Seed shape has an evolutionary significance, with fusiform seeds found in more primitive orchids and the various other forms in more evolved epidendroid orchids [36, 37]. Fusiform seeds in A. aphthosa, A. prolifera, and O. gracilis were also observed in this study.

The testa in the middle part of the seed tended to be oblong or rectangular with elongated cells in the longitudinal axis of the seed [25], though A. prolifera cells were hexagonal and A. hatschbachii cells were linear. The testa cells of A. obovata, A. prolifera, and S. grandiflora did not show ornamentation, while papillae or verrucosities were apparent in the seeds of the other species. The presence or absence of ornamentation is cited in the literature to delimit some genera of Orchidaceae [33]. This is not the case in our study since among the species of Acianthera, A. prolifera had no ornamentation, A. ochreata, and A. sonderiana had verrucosities, and A. aphthosa and A. hatschbachii had papillae. Thus, the presence of seed ornamentation did not affect the asymbiotic germination of micro-orchids.

Variations in seed size and embryos occurred in the genera studied. In our study, A. aphthosa and A. prolifera had medium-sized seeds (500–900μm) according to the classification of Barthlott et al. [25]. Meanwhile, A. hatschbachii, A. ochreata, A. sonderiana, A. obovata, D. zebrina, and O. gracilis had small (200–500 μm) seeds, while P. fusca and S. grandiflora had very small seeds (100–200 μm) [25]. The seeds and embryos of A. prolifera and A. ochreata were larger (length, width, L/W ratio, and volume) and had higher rates of asymbiotic germination. According to Arditti et al. [29], L/W ratios provide data on the relative degree of truncation. A. prolifera and A. ochreata had L/W ratios of 2.700 and 1.767, respectively, while P. fusca had an L/W ratio of 0.140. Seeds with an L/W ratio of under 6.0 are referred to as truncated seeds, while those with an L/W ratio of over 6.0 are referred to as elongated seeds [30]. Based on this classification, all seeds in our study are truncated. According to Arditti et al. [29], seed volume is a better measure of seed size in orchids, which is in line with the findings of our study. They also considered that the seed volume and seed size are directly proportional to each other.

The number of testa cells along the longitudinal axis of the seed was low (2–6) in D. liliputiana (2), D. zebrina (2), S. grandiflora (2), A. sonderiana (3), A. obovata (4), P. fusca (4), O. gracilis (5), and A. hatschbachii (6). On the other hand, there was a high number (9–14) of testa cells in A. aphthosa (9), A. ochreata (10), and A. prolifera (14). According to Barthlott et al. [25], the number of testa cells is coupled to cell division [38], a pattern that probably arises from slow or interrupted division in the integuments following fertilization. In other genera, where cell divisions continue, the seed coats are composed of numerous small cells. Seeds with only a few cells (five or fewer) along the longitudinal axis are especially common in Orchidaceae [25]. This feature is useful to characterize clades, usually at the subtribe level, with a high number of testa cells as the ancestral condition [25]. In addition, the species with a higher number of testa cells, A. ochreata and A. prolifera, showed a higher germination rate and plantlet formation in comparison with the other species analyzed.

As with the seeds, the embryos of A. prolifera and A. ochreata were also longer and wider, with greater volume. These species had higher germination rates after 12 weeks (92% and 86%, respectively). A similar result was reported by Tsutsumi et al. [39], who found that the larger embryos of Liparis fujisanensis F. Maek. ex Konta & S. Matsumoto were able to germinate more rapidly than smaller embryos. According to Yeung et al. [40], orchid embryos have fewer cells and are smaller than other flowering plant embryos. This can be a result of cells having a prolonged cell cycle time. The limited number of cells produced may also be due to the early cessation of mitotic activities.

Another characteristic that varied among the species of micro-orchids was the percentage of air space between the seed coat and the embryo. The highest percentage was observed in A. sonderiana (87.83%) and the lowest in O. gracilis (61.21%) and P. fusca (56.14%). Güler [41] also reported that the percentage of air space in certain species of Anacampis, Neotinea, and Orchis ranged from 56 to 80%. Similar results were obtained by Swamy et al. [32], who found air space percentages ranging from 86.29% in Cymbidium bicolor Lindl. to 47.39% in Coleogyne breviscapa Lindl. Seeds with higher percentages of air space are lighter and can be more buoyant, aiding in wind dispersal across large geographic areas [28, 42]. Similarly, the seed volume to embryo volume ratio (SV/EV) was highest in A. sonderiana and lowest in P. fusca. The SV/EV ratio varied significantly among the species of the genus Acianthera, ranging from 8.2167 in A. sonderiana to 2.6729 in A. ochreata. This characteristic did not have any effect on the germination responses of the studied species of micro-orchids.

In our study, a suspensor was only visible in the seeds of A. prolifera and D. liliputiana. Orchidaceae is comprised of species that may present or not a suspensor [40]. Suspensor plays an important role during embryonic development, facilitating the movement of nutrients from maternal tissues to the embryo [5]. This structure may have facilitated the higher germination rates of A. prolifera seeds since the germination response of D. lilliputiana was lower. Another difference was that the seed testa of D. lilliputiana was thicker, and the seeds smaller than those of A. prolifera.

Structural polarity can occur in orchid embryos of some species, with larger cells at the apex and smaller ones at the base of the embryo, while these poles are uniform in size in other species [5]. In this study, the size of the poles was different in five species. Of these, A. prolifera (92%) and A. ochreata (86%) had high germination responses. Germination rates were lower in other three species exhibiting polarity, A. aphthosa (38%) and D. liliputiana (38%). According to Yeung [5], the cells at the apical pole of the embryo will form a meristematic zone, and the basal cells are designated to house the symbiont upon seed germination. A similar result was observed in Phalaenopsis amabilis (L.) Blume, which also exhibited these differences at the poles of the embryo and germinated more easily [43]. Another study found no difference between the poles of Calypso bulbosa (L.) Oakes., which is considered a species of difficult germination [44]. The authors found the same results for P. fusca and S. grandiflora, which had lower germination rates and developed no seedlings during the observation period. Embryos of species with no differences in size at the cell poles require additional growth and development of the embryo itself. In nature, these requirements can be provided by mycorrhizae [40].

A cuticle was present around the embryo in A. aphthosa and P. fusca. According to Aybeke [42], cuticle formation can ensure the retention of moisture by the cells of the embryo and increase its viability, which the author observed in the embryo of Himantoglossum robertianum (Loisel.) P. Delforge.

The results of asymbiotic germination indicated the highest germination rates in A. prolifera (92%), A. ochreata (86%), and O. gracilis (77%) at 12 weeks after sowing in WPM. The first two species had larger seeds and embryos with structural polarity, and both had some embryos developing to the seedlings stage in 12 to 16 weeks of cultivation. Furthermore, the lack of an endosperm may result in the seed having a lower water holding capacity, resulting in a more rapid change in water content [40]. Meanwhile, seedlings did not develop in O. gracilis, despite the high germination rate, with no germinated protocorms exhibiting leaf development. Other species, such as D. liliputiana and A. obovata, had germination rates of 38% and 36%, respectively, at 12 weeks after sowing, with no protocorms forming seedlings by 24 weeks after sowing. In addition to the morphological and morphometric characteristics that varied widely among the species studied, WPM may not have provided ideal conditions for all species, or as it was observed in a previous study for A. prolifera, the medium MS/2 was better for initial germination and WPM for the formation of seedlings (79% germination after 12 weeks) [13]. According to Yeung et al. [40], initial media for germination may be adequate; however, for continual development, a more complex medium may be required. Alternatively, a subculture in the WPM could be necessary to stimulate the development of protocorms in other stages. The WPM has a low concentration of salts, less total nitrogen, and less ammonium than MS [45]. According to Suzuki et al. [46], the composition of the culture medium is essential for the success of the germination of orchid seeds, with results varying significantly from one species to another. Cultivating the seeds in a medium with an adequate nutritional composition helps to increase germination and, consequently, to produce large numbers of plants, which contributes to the conservation of endangered species [46].

The species with the worst germination response was P. fusca (4%), the protocorms of which were all necrotic by the fourth week after sowing in the WPM. This species had the smallest seeds and embryos, a cuticle covering the embryo and no structural polarity. For a majority of orchid seeds, even though the seed coat is thin, the addition of secondary walls, phenolic substances, and cuticular materials offer additional protection to the embryo within [40], and this may have hampered in vitro germination of P. fusca.

The culture medium used may not have been appropriate for this species, or disinfesting seeds with 1% sodium hypochlorite may have had a very strong effect since the seeds had a thin testa, i.e., this process may have caused damage to the embryos. Another factor is that some micro-orchids develop protocorms very slowly. For example, Anathallis adenochila (Loefgr.) F. Barros was found to take 12 months to develop seedlings (> 0.5 cm in height) [21]. In another species of the Pleurothallidinae subtribe, Restrepia brachypus Rchb.f., germination ranged from 7.96% in MS medium to 53.05% in Western (W) medium [22]. In addition, the protocorms of R. brachypus were subcultured for W medium and supplemented with banana pulp (60 g L^-1) for seedling development.

Another factor that may have influenced the low germination rates and slow protocorm development of some micro-orchids in our study may be the degree of seed maturity in asymbiotic germination. Yeung et al. [40] recommend that mature seeds of epiphytic orchids must be used for asymbiotic germination. The seeds of A. prolifera and A. ochreata could have been more mature than those of P. fusca when they germinated in vitro. In addition, an appropriate composition of the culture medium is assumed to be essential for the successful germination of immature orchids [47]. Factors such as degree of maturity of micro-orchid seeds need to be studied further for a better understanding of the germination of species that showed slow development and did not form seedlings in the WPM.

This study demonstrated considerable variation in the morphology, morphometry, and germination rates of the studied species. Germination and seedling formation of A. prolifera and A. ochreata occurred successfully in the WPM. Since germination was asynchronous, other formulations of the culture medium or supplements should be tested for their ability to accelerate the production of seedlings. Another approach would be to perform subcultures in the sowing stage so that there is no depletion of media components. Furthermore, evaluations should be carried out for more extended periods since some species grew very slowly, including A. obovata, D. liliputiana, and P. fusca.

A. ochreata and A. prolifera elongated and developed roots in WPM medium, supplemented with 1 g L^-1 activated charcoal (Fig 4D), as recommended by Koene et al. [13] in a previous study with A. prolifera. Finally, A. ochreata and A. prolifera achieved high survival rates [80%] after 12 months, when using commercial Forth® substrates, composed of a mixture of coconut fiber, Pinus bark, and charcoal with fine vermiculite Eucatex® (1:1). Koene et al. [13] had also successfully acclimatized A. prolifera (95% survival, after three months) using the same substrate and conditions of greenhouse. Therefore, our results offer potential possibilities for reintroduction programs in the future that can play a key role in reducing the threat of extinction for species of micro-orchids.

Conclusions

In this study, we demonstrate efficient methods of rapid germination and seedling development of different species of Atlantic Rainforest micro-orchids. Additional studies are needed to accelerate the propagation process for some of the species that germinated slowly or minimally in this study. However, the technique used may help in the massive and rapid propagation of certain species for reintroduction into degraded habitats.

Acknowledgments

The authors would like to thank CNPQ for the Master’s scholarship granted to Franciele Marx Koene.

Data Availability

All relevant data are within the manuscript.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0243297.r001

Decision Letter 0

Jen-Tsung Chen

18 Sep 2020

PONE-D-20-27111

Asymbiotic germination and morphological studies of seeds of Atlantic Rainforest micro-orchids (Pleurothallidinae)

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Reviewer #1: Comments: PONE-D-20-27111

In this manuscript, authors reported Asymbiotic germination and morphological studies of seeds of Atlantic Rainforest micro-orchids (Pleurothallidinae). After perusing the full manuscript, it was found that few similar papers have been published on this topic and it seems that this manuscript presents some applicable information to wide readers.

However, before a final decision, following issues should be addressed:

1. In Abstract: Add few starts lines about the background information of the research;

2. Add some most relevant keywords in the manuscript;

3. Revise the reference format as per journal author guidelines.

Reviewer #2: Dear Editor,

The paper describe 'Asymbiotic germination and morphological studies of seeds of Atlantic Rainforest micro-orchids (Pleurothallidinae)'. Orchid seeds are recalcitrant and the authors attempted to germinate it asymbiotic way.

The paper does not include novelty and including simple petri germination studies and some morphometric measurements.

The authors did not mention if the material terrestrial orchids or epiphytic orchids. They did not search whole literatures on orchids seed germination.

I am against to publication of this paper in Plos One.

Reviewer #3: PLOS ONE

Manuscript Number: PONE-D-20-27111

Title: A symbiotic germination and morphological studies of seeds of Atlantic Rainforest micro-orchids (Pleurothallidinae)

Article Type: Research Paper

Mona soliman

Review Report

The initial research articles in PLOS ONE describes the goals and the theories, methodologies and results of research and analysis of primary and unpublished studies. Original study papers can also include validation studies and disconfirmation of results that allow the exclusion and/or reproducibility of previously published findings by hypothetical assumptions. The manuscript entitled “A symbiotic germination and morphological studies of seeds of Atlantic Rainforest micro-orchids (Pleurothallidinae)” is interesting and mentioned to efficient methods of rapid propagation of different species of Atlantic Rainforest micro-orchids.

I recommend some minor changes before the paper is accepted in this periodical.

Abstract

Abstract is well written and informative

Introduction

The introduction is clearly and constructively written

Materials and Method

Line 123-132:

References are needed of all the above qualitative and quantitative methods

Line 141-149:

You must be subtitled to this paragraph to note the implications that the reader needs to understand……….thanks for your effort

Line 161-166:

In the results chapter, add the figures and tables to the correct place.

Results

These are nicely written

Discussion

The whole chapter deals with its purpose. The parameter studies are explained logically, but I think there should be a small reduction in discussions as far as possible

Comments for editor

Thank you for taking my manuscript review into consideration

I just need to check the paper again, as it is good, and only need minor corrections to be published and made available to readers.

Reviewer #4: All considerations were realized directly in the attached and revised version.

In addition:

About statystical analysis there are a mistake in comparisons of different species in Table 6. This comparison is similar to compare the natality of 'cats and lyons'. Also, comparisons between sizes of 'cats and lyons' is not biologically interesting. Also, a description about how many plants were used for measurements were not provided. In addition, the authors needs clarify if the time of evaluation was considered as treatment in the analysis. Tukey tests are not sound interesting in these cases, but only the standard deviation could be used for these analysis.

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Reviewer #4: Yes: Jean Carlos Cardoso

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PLoS One. 2020 Dec 14;15(12):e0243297. doi: 10.1371/journal.pone.0243297.r002

Author response to Decision Letter 0

3 Nov 2020

Responses to Reviewer 1

1. In Abstract: Add few starts lines about the background information of the research

Answer: Your request was accepted, see lines 25-28

2. Add some most relevant keywords in the manuscript

Answer: Relevant keywords have been added in blue

Keywords: Orchidaceae; micromorphology; morphometry, in vitro germination; woody plant medium; SEM.

3. Revise the reference format as per journal author guidelines.

Answer: The references format was reviewed and corrected as guidelines of Plos One

Responses to Reviewer 3

Materials and Method

1. Line 123-132:

References are needed of all the above qualitative and quantitative methods

Answer: The references were added, see lines 154-156

2. Line 141-149:

You must be subtitled to this paragraph to note the implications that the reader needs to understand……….thanks for your effort

Answer: Your request was accepted, see lines 159 and 166

3. Line 161-166:

In the results chapter, add the figures and tables to the correct place.

Answer: This has been corrected in the manuscript.

4. Discussion

The whole chapter deals with its purpose. The parameter studies are explained logically, but I think there should be a small reduction in discussions as far as possible

Answer: Your request was accepted.

5- About statystical analysis there are a mistake in comparisons of different species in Table 6. This comparison is similar to compare the natality of 'cats and lyons'. Also, comparisons between sizes of 'cats and lyons' is not biologically interesting. Also, a description about how many plants were used for measurements were not provided. In addition, the authors needs clarify if the time of evaluation was considered as treatment in the analysis. Tukey tests are not sound interesting in these cases, but only the standard deviation could be used for these analysis.

Answer: Statistical analysis was removed from the table 6.

Responses to Reviewer 4 (J. C. Cardoso)

Abstract

1. WPM medium is not a conventional medium for orchid germination. Authors have some reference support the use of this culture for this purpose? Because germination of orchids depends strong from the mineral composition.

References and the justification for using this medium were included in the manuscript.

Answer: WPM medium was previously tested for Acianthera prolifera and compared with other formulations (MS, MS/2 and KC) and showed better response, accelerating seedling development, so it was used to evaluate the germination of the other 10 micro-orchid species. It was also recommended for other species of orchids, such as: Brasiliorchis picta and Hadrolaelia grandis when compared to conventional medium such as: MS, MS/2, KC and VW.

The references are below and the justification for using the WPM medium were placed on the

lines 64-71 and 83-85 in the manuscript

Koene FM, Amano E, Ribas LLF. Asymbiotic seed germination and in vitro seedling development of Acianthera prolifera (Orchidaceae). South African Journal of Botany. 2019; 121:83-91.

Santos SA, Smidt EC, Padial AA, Ribas LLF. Asymbiotic seed germination and in vitro propagation of Brasiliorchis picta. African Journal of Biotechnology. 2016; 15:134–144.

Vudala SM, Ribas LLF. Seed storage and asymbiotic germination of Hadrolaelia grandis (Orchidaceae). South African Journal of Botany. 2017; 108:1–7.

2- What authors considering seedlings?

Answer: In our study is the stage that corresponds to protocorms with two or more leaves and roots (See Line 182). Some authors use plantlet for this stage. Seedlings are plants that develop from the embryo of a seed.

3- “The protocorms of Anathalis obovata, Dryadella liliputiana, and Octomeria gracillis developed slowly in the WPM, not reaching the seedling stage in 24 weeks of cultivation”. Is this a problem of culture media use or from the species?

Answer: These species were tested only in this medium. To be sure if it is a problem of the medium or species, studies are needed comparing various formulations of culture medium or a subculture in the WPM could be necessary. This was also discussed in the lines 460-467.

The two studies on seed germination of the subtribe Pleurothallidinae species, reported percentage of germination at most 50% testing various formulations of culture medium and slow development of protocorms, requiring a subculture for the formation of seedlings.

4- “This morphological and morphometric study contributes to the understanding of asymbiotic germination of some micro-orchid species” Please explain how?

Answer: The seeds and embryos of the species that germinated faster and with high percentages (A. prolifera and A. ochreata) presented some characteristics, such as: larger size and seed volume, structural polarity, higher number of testa cells and this may have contributed to this response. In our study the species with the lowest germination response was P. fusca (4%), this species had the smallest seeds and embryos, a cuticle covering the embryo and no structural polarity. We also observed that the percentage of air space between the seed coat and the embryo did not have effect on the germination responses of the studied species of micro-orchids. Further studies with other genera and species of the Subtribe Pleurothallidinae, and other culture media are needed to assess the influence of these morphological and morphometric characteristics, however, we achieved high germination percentages after six months of cultivation in WPM medium.

Introduction

5- Please the references follow numerical order, not alphabetic order. Please correct.

Answer: This has been corrected.

6- ... their high germination rates, which are commonly over 70%, as opposed to under 5% in ex vitro conditions. This is highly dependent on the species. Terrestrial orchids in general have many difficulties for germination using asymbiotic germination, compared with symbiotic.

Answer: It was clarified in the text that this statement is for epiphytic species at line 62. Discussion with species of terrestrial orchids was also excluded.

7- What the reason for choice eleven species and these is specifical? Occurrence in region?

Collection?

Answer: These species were previously pollinated and were selected for having mature capsules at the time of our study. Our goal was to compare the largest number of micro-orchid species, but we depended on the formation of capsules.

Material and methods

6- These species are uncommon! I recommended that the authors add a figure with the eleven species, if possible, at the reproductive stage.

Answer: A figure with the studied species was added as requested.

5- How was realized the pollination? Self-pollination? Crossing handling? Naturally?

If naturally, there are no possible hybridation?

Answer: Each species had three to four plants in a greenhouse, in which manual cross-pollination was performed.

6- How was the identification of species? Comparisons between the vouchers at herbarium?

Answer: The species were identified by comparisons between the vouchers at herbarium and with the help of the specialist in this Subtribe (Pleurothallidinae): Eric de Camargo Smidt (co-author) of this manuscript.

7- Recently legislation of Brazilian flora requires the registration of wild species in SISGEN system. Authors have these numbers for wild species collected in Brazil?

Answer: This registration is not required to studies with no accessions to the genes content or associated knowledge. Also, these plants came from outside Natural Protected Areas; they grow in periurban areas of Curitiba or purchased from reputable companies that produce and sell the species.

Results

8- 80% of survival for 3 months. For what species? In what environmental conditions? Please describe.

Answer: 80% of survival after three months for A. prolifera and A. ochreata. The environmental conditions in greenhouse were complemented. See lines 187-195 and 509-516.

Conclusions

Answer: They were corrected.

Figures 3 and 4

Very low quality figures

Answer: The figures were reviewed and prepared according to the guidelines of Plos One

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PLoS One. doi: 10.1371/journal.pone.0243297.r003

Decision Letter 1

Jen-Tsung Chen

19 Nov 2020

Asymbiotic germination and morphological studies of seeds of Atlantic Rainforest micro-orchids (Pleurothallidinae)

PONE-D-20-27111R1

Dear Dr. Ribas,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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PLoS One. doi: 10.1371/journal.pone.0243297.r004

Acceptance letter

Jen-Tsung Chen

4 Dec 2020

PONE-D-20-27111R1

Asymbiotic germination and morphological studies of seeds of Atlantic Rainforest micro-orchids (Pleurothallidinae)

Dear Dr. Ribas:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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All relevant data are within the manuscript.

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PERMALINK

Asymbiotic germination and morphological studies of seeds of Atlantic Rainforest micro-orchids (Pleurothallidinae)

Franciele Marx Koene

Érika Amano

Eric de Camargo Smidt

Luciana Lopes Fortes Ribas

Roles

Abstract

Introduction

Fig 1. Plants with reproductive structure of the studied species of the subtribe Pleurothallidinae.

Materials and methods

Seed materials

Fig 2. Acianthera ochreata.

Table 1. Species of the subtribe Pleurothallidinae used for morphological, morphometric, and in vitro germination analysis in this study.

Morphological and morphometric analysis of seeds

Seed viability

Tetrazolium test

In vitro germination

Fig 5. In vitro germination of micro-orchids.

Statistical analysis

Results

Morphological and morphometric analysis of seeds

Table 2. Morphological seed characteristics of species belonging to the subtribe Pleurothallidinae.

Table 3. Morphometric data of seeds of species belonging to the subtribe Pleurothallidinae.

Table 4. Morphometric data of embryos of species belonging to the subtribe Pleurothallidinae.

Fig 3.

Anatomical analysis

Table 5. Anatomical characteristics of the seed testa of the embryo cells, and presence or absence of suspensor and cuticle in species belonging to the subtribe Pleurothallidinae.

In vitro germination

Fig 4. Frequency (%) of the developmental protocorm stages of Pleurothallidinae species grown in vitro for two, four, six, eight, and 12 weeks in woody plant medium.

Table 6. Germination rate after four, eight, and 12 weeks in woody plant medium and the average time (days) for the protocorms development of the Pleurothallidinae species.

Discussion

Conclusions

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Jen-Tsung Chen

Roles

Author response to Decision Letter 0

Decision Letter 1

Jen-Tsung Chen

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Acceptance letter

Jen-Tsung Chen

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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