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

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.

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We studied the morphological and morphometric characters of seeds belonging 23 to 11 species of the subtribe Pleurothallidinae using light and scanning electron 24 microscopy to understand the in vitro germination process. The viability of the seeds 25 was evaluated by in vitro germination in woody plant medium (WPM), and by analysis 26 of the developmental stages of protocorms until seedling formation (two to 24 weeks). 27 Morphometric data showed variations within the genus Acianthera and between species 28 of different genera. The best germination and protocorm formation responses occurred 29 with Acianthera prolifera (92%) and Acianthera ochreata (86%), with the formation of 30 seedlings after 12 and 16 weeks of sowing, respectively. The seeds and embryos of 31 these species were larger (length, width, and volume) than the others, with a structural 32 polarity that may have facilitated their germination. Other characteristics of A. prolifera 33 seeds that may have contributed to these results include the presence of a thin testa Although the seeds of different orchid species have many similarities, there is 77 significant variability in the size, shape, characteristics of the testa cells, the zones of 78 adhesion, and sculptures constituted by the cellular wall and cuticular material [34]. 79 Studies on seed morphology and morphometry have achieved substantial contributions 80 to the taxonomy, phylogeny, and phytogeography of this group [1,7]. Meanwhile, these 81 studies can also enhance understanding of the in vitro germination process, which in 82 turn can accelerate the production of seedlings for future reintroduction into their 83 natural habitat, aiding in the conservation of the Pleurothallidinae. Our study was 84 carried out with 11 native species belonging to six genera of the Atlantic Forest that 85 have not been evaluated for the threat of extinction by the Flora of Brazil [8,9], as 86 follows: Acianthera aphthosa (Lindl.) Pridgeon  This study aimed to analyze the morphological and morphometric characteristics 99 of the seeds and relate them with the asymbiotic germination of species of the subtribe 100 Pleurothallidinae. We demonstrate efficient methods of rapid propagation of some 101 species and our results offer real possibilities for reintroduction programs that can play a 102 key role in reducing the extinction risk for species of micro-orchids. 103

Seed materials
105 Mature seeds of 11 species of the Pleurothallidinae subtribe ( Fig 1A) were 106 collected from naturally dehiscing capsules ( Fig 1B)  Morphological and morphometric analysis of seeds 120 An average of 30 seeds per specimen was analyzed using a light ( Fig 1C) and a 121 scanning electron microscope (SEM). For SEM observations, the seeds were adhered 122 with double-sided carbon tape and coated with gold. Qualitative data on the general 123 seed morphology, including color, ornamentation, shape, micropillar opening, and the 124 presence of a cuticular deposit, were analyzed. Quantitative analyses were performed 125 using a light microscope (Olympus BX41 with DC30 camera), and the following 126 variables were measured for seeds and embryos: length (L), width (W), L/W ratio, 127 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 129 volume was calculated as 2 [(W/2) 2 · (L/2) · (π/3)]. Embryo volume was calculated by 130 using the formula 4/3 π · L/2 · (W/2) 2 . The percentage of air space was calculated as 131 [(seed volume -embryo volume) / seed volume] x 100 [1,2]. About 500 seeds per Petri dish, with three Petri dishes per species, were 150 inoculated. Protocorm development was evaluated from two to 16 weeks based on the 151 following stages: 1, seed with chlorophyllous embryo (Fig 1D); 2, testa 152 ruptured/chlorophyllous protocorm (germination) (Fig 1E); 3, protocorm with apex 153 and/or rhizoids ( Fig 1F); 4, protocorm with one or two leaves; 5, protocorm with two or 154 more leaves and root (seedling) (Fig 1G). The germination rate was evaluated after four, 155 eight, and 12 weeks of cultivation, and the average time (in days) to reach the stages 156 was calculated for 24 weeks. The Petri dishes with seeds were maintained at 157 The seeds of the orchids studied exhibited diversity in their shape, size, volume, 177 and seed coat (ornamentation) (Tables 2 and 3), as well as in their embryo morphometry 178 (Table 4). 179 Pale yellow seeds were observed in A. prolifera, D. lilitipuana, and S. 180 grandiflora, while the seeds of the other species studied were brown (Table 2). 181 The seeds had several shapes, including fusiform, filiform, ellipsoid, and clavate 182 (Figs 2A-2K). Generally, cells were shorter at either pole, while the medial cells of the 183 seed coat were elongated. The chalazal pole of the seeds was closed, and the micropillar 184 end was open (Fig 2F), except for D. zebrina, which was also closed ( Table 2). The 185 testa cells observed were transparent, longitudinally oriented, and oblong (Fig 2L), 186 except in A. prolifera, in which they were hexagonal (Fig 2M). The ornamentation 187 pattern of the testa cells of some species included papillae or verrucosities (Fig 2N), 188 though these were absent from A. prolifera, A. obovata, S. grandiflora and A. 189 hatschbachii (Fig 2O) ( Table 2). The number of testa cells ranged from 2 to 14 on the 190 longest axis and 3 to 12 on the widest axis, with the highest number in A. prolifera and 191 A. ochreata (Table 3).  The seeds showed high diversity in their size (length, width, and volume) ( Table  208 3), as well as in the size and number of cells constituting the embryo (Table 4), despite 209 their microscopic nature. The seed length ranged from 187±31 µm (P. fusca) to 210 742±119 µm (A. prolifera), while the width ranged from 83±7 µm (P. fusca) to 159± 18 211 µm for A. sonderiana (Table 3). A. prolifera and A. ochreata had length-to-width ratios 212 of 2.700 and 1.767, respectively, while the L/W ratio in P. fusca was 0.140 (Table 3). A. 213 prolifera had the largest seed volume (3.540 mm 3 x 10 -3 ) while the smallest was 214 recorded in P. fusca (0.337 mm 3 x 10 -3 ) ( Table 3). 215 216  The embryos were generally ellipsoidal and located at the center of the seed. 224 Variation in length, width, and volume was also observed. The largest length was 225 recorded for A. prolifera while A. ochreata had the largest width and volume, and P. 226 fusca embryos had the smallest length, width, and volume (Table 4). 227 The largest percentage of air space was observed in A. sonderiana (87.83%), 228 whereas the lowest percentage of air space was observed in O. gracilis (61.21%) and P. 229 fusca (56.14%) (   In vitro germination

259
The seeds of all of the micro-orchid species studied exhibited chlorophyllous 260 embryos after three to eight days of sowing (Fig 3A). The first asymbiotic germination 261 responses (seeds with ruptured testa and chlorophyllous protocorm) occurred after 262 seven days of sowing for A. prolifera and A. aphthosa, and between nine and 13 days 263 for the other species (Table 6). In general, most of the seeds had a low germination rate 264 up to eight weeks after sowing, except for A. prolifera (76%). By 12 weeks after 265 sowing, the highest germination rates were obtained for A. prolifera, followed by A. The analysis of the rates obtained at each stage of protocorm development 293 confirmed that A. prolifera and A. ochreata had the best germination response (Fig 3). 294 After two weeks, A. prolifera had the highest proportion of seeds with chlorophyll 295 embryos (55%), followed by A. ochreata (52%) and S. grandiflora (49%) (Fig 3A). 296 During this period, the highest germination rates (ruptured testa with chlorophyll 297 embryo) occurred in A. prolifera (44%) and A. ochreata (39%) and were significantly 298 higher than the other species analyzed, in which germination rates were below 10%. 299 By four weeks after sowing, all species except P. fusca had protocorms with 300 apex (Fig 3B). By six to eight weeks after sowing, A. prolifera had the highest rate of 301 protocorms with apex (27%) (Fig 3C); it was also the only species to have protocorms 302 with leaves (21%) (Fig 3D). Seedling development began at 12 weeks, with the largest 303 number of seedlings found in A. prolifera (32%), followed by A. ochreata (20%). The 304 protocorms of A. aphthosa and S. grandiflora grew slowly, with less than 10% forming 305 seedlings, though a longer period of evaluation (Fig 3E) or subculture to the same 306 medium may be necessary. The development of the protocorms of A. ochreata into 307 seedlings was somewhat slower than that of A. prolifera. After seedling formation, the 308 radicles can be cut, and explants cultured in medium with cytokinins to induce shoots 309 ( Fig 4C) or explants can be subcultured for flasks with medium supplemented with 310 activated charcoal, where elongation and root development occur (Fig 4D). Finally, A. ochreata and A. prolifera plants were transplanted into sowing trays (3.5 cm 2 ) 312 containing commercial Forth® substrates, composed of a mixture of coconut fiber, 313 Pinus bark, and charcoal with fine vermiculite Eucatex® (1:1) (v/v). They were then 314 successfully acclimatized in a greenhouse with 80% survival after three months (Fig  315   4E) or 12 months (Fig 4F). of orchid seeds makes it very difficult to visualize their color, but they are usually 333 variations of yellow, brown, and white. Barthlott et al. [7] found that the most frequent 334 color of seeds is brownish or dark brown, as observed in our study. Color variation may also occur depending on the maturity of the seeds. A study found that mature seeds of 336 Angraecum spp. had brown testa that was less translucent, while the immature ones 337 were lighter and colorless [21]. 338 We observed seeds that were ellipsoid, clavate, fusiform, or filiform, shapes that 339 have also been reported for other orchid species [11,15,16,37]. Seed shape has an 340 evolutionary significance, with fusiform seeds found in more primitive orchids and the 341 various other forms in more evolved epidendroid orchids [12,40]. Fusiform seeds in A. prolifera and A. ochreata were larger (length, width, L/W ratio, and volume) and had 359 higher rates of asymbiotic germination. According to Arditti et al. [2], L/W ratios provide data on the relative degree of truncation. Acianthera prolifera and A. ochreata 361 had L/W ratios of 2.700 and 1.767, respectively, while P. fusca had an L/W ratio of 362 0.140. Seeds with an L/W ratio of under 6.0 are referred to as truncated seeds, while 363 those with an L/W ratio of over 6.0 are referred to as elongated seeds [3]. Based on this 364 classification, all seeds in our study are truncated. According to Arditti et al. [2], seed 365 volume is a better measure of seed size in orchids, which is in line with the findings of 366 our study. They also considered that the seed volume and seed size are directly 367 proportional to each other. 368 The number of testa cells along the longitudinal axis of the seed was low (2-6) 369 in D. liliputiana (2), D. zebrina (2), S. grandiflora (2), A. sonderiana (3), A. obovata 370 (4), P. fusca (4), O. gracilis (5), and A. hatschbachii (6). On the other hand, there was a 371 high number (9-14) of testa cells in A. aphthosa (9), A. ochreata (10), and A. prolifera 372 (14). According to Barthlott et al. [7], the number of testa cells is coupled to cell 373 division [28], a pattern that probably arises from slow or interrupted division in the 374 integuments following fertilization. In other genera, where cell divisions continue, the 375 seed coats are composed of numerous small cells. Seeds with only a few cells (five or 376 fewer) along the longitudinal axis are especially common in Orchidaceae [7].This 377 feature is useful to characterize clades, usually at the subtribe level, with a high number 378 of testa cells as the ancestral condition [7]. In addition, the species with a higher number 379 of testa cells, A. ochreata and A. prolifera, showed a higher germination rate and 380 plantlet formation in comparison with the other species analyzed. 381 As with the seeds, the embryos of A. prolifera and A. ochreata were also longer 382 and wider, with greater volume. These species had higher germination rates after 12 383 weeks (92% and 86%, respectively). A similar result was reported by Tsutsumi et al. 384 [38], who found that the larger embryos of Liparis fujisanensis F. Maek. ex Konta &