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.

Cryopreservation in liquid nitrogen of Brazilian orchid seeds Miltonia flavescens Lindl.

Miltonia flavescens is a species vulnerable to extinction, which justifies research on preservation methods. Cryopreservation in liquid nitrogen (LN) consists of maintaining biological material at a low-temperature (-196 °C). Thus, the aim of the experiment is to evaluate the influence of different cryoprotective solutions on cryopreservation in LN of the Brazilian orchid Miltonia flavescens seeds. The experiment was conducted in a completely randomized design (CRD), with eight treatments and six replications. The treatments were composed as follows: control; immersion in LN, no cryoprotectant adding; and immersion in LN, with the addition of cryoprotectants: sucrose 0,4 mol L-1; glycerol 2 mol L-1; protection by vitrification in solution (PVS)1; PVS2; PVS2 + 1% phloroglucinol, and PVS3. Except for the control treatment, which was kept in a freezer (10±2 °C), the others remained frozen for 15 days. After this period, the viability of the seeds was evaluated. These seeds were sown and, 30 days after germination, then the frequency of protocorm formation was verified. Before the cryopreservation, the seeds showed 75% viability and 9.5% water content. After cryopreservation, the seeds varied between 67 to 75% viability. However, treatment with glycerol 2 mol L-1 exhibited lower performance than the others (58%). The control treatment showed a higher percentage of protocorm formation (71%) followed by treatments PVS1 (63%), PVS2 (64%), and PVS2PHLO (66%). For the purpose of preserving Miltonia flavescens seeds in liquid nitrogen for a prolonged period, the treatments PVS1, PVS2, and PVS2PHLO proved to be viable and promising alternatives.

In Vitro Propagation of Gastrochilus matsuran (Makino) Schltr., an Endangered Epiphytic Orchid

Gastrochilus matsuran (Makino) Schltr. (Orchidaceae) populations are declining quickly because of overexploitation, climatic changes, and deforestation; therefore, mass-production protocols are required for this orchid. Natural propagation of this species is often hampered by meager seed germination and slow growth. Thus, our aim was to establish an effective protocol for the in vitro propagation of G. matsuran and reduce the risk of its extinction. We investigated the impacts of culture media, coconut water (CW), and plant hormones (gibberellic acid (GA3), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA), and thidiazuron (TDZ)) on asymbiotic germination, multiplication and conversion of protocorms, and plantlet development. Maximal seed germination (93.3%) was achieved on ½ MS medium without vitamins plus 5% CW, 1 µM NAA, and 1.5 µM GA3. Secondary protocorm formation was best achieved on ½ MS medium without vitamins plus 2 µM TDZ. The conversion of protocorms into seedlings was maximized by supplementation with 2 µM IBA or 1 µM NAA. Acclimatized plantlets that exhibited exuberant growth on sphagnum moss were reintroduced to tree trunks in a natural habitat, with a 67% survival rate. This in vitro propagation procedure would be helpful for the mass production and conservation of this rare epiphytic orchid.

In vitro Seed Germination, Protocorm Formation and Plantlet Regeneration in Aerides ringens Fisher

An attempt was made to in vitro seed germination and plantlet regeneration in Aerides ringens, a threatened, endemic and epiphytic orchid. Five basal media Knudson C (KC), Vacin and Went (VW), Burgeff’s (N3f), half strength MS, MS were examined for their effectiveness. Germination occurred in all the media. However, KC medium proved to be the best for seed germination (70.8%) and half strength MS was superior for the differentiation of protocorm like bodies (PLBs) into seedlings. The seeds were also cultured on various plant growth regulators (PGRs) to evaluate their effectiveness on seed germination and further differentiation. Among the PGRs tested, 0.57 μM IAA (IAA 89.3%), 4.64 μM Kn (88.6%) and 2.21 μM BAP (90.7%) and 0.57 μM IAA + 0.55 μM BAP (96.5%) were stimulated the germination and further differentiation of PLBs into seedlings. The concentrations of 2.21 μM BAP and combination of 0.57 μM IAA + 0.55 μM BAP stimulated the formation of MPLBs, which later differentiated into shoots and roots. Four potting substrates were evaluated for Ex vitro seedling survivability of which brick: charcoal: decaying litter: coconut husk (1 : 1 : 1 : 1) gave maximum survival rate (89.0%). Plant Tissue Cult. & Biotech. 29(1): 49-62, 2019 (June)

Germination and Protocorm Formation of Ophrys Sphegodes Mill. – In Vitro Protocol for a Rare Orchid Species

Summary Ophrys sphegodes Mill. is a wild orchid species which is threatened and protected due to its pollination biology, small seed and habitat destruction. The aim of this study was to establish asymbiotic germination protocol for the purpose of ex situ conservation. Two basal media Knudson C (KC) and Malmgren (MM), supplemented with organic additives (peptone (PE), L-glutamin (A)e, folic acid, casein hydrolysate (CA)) added separately and control media KC--C and MM--C were used in the present research. All the nutrition media contained 2% sucrose, 7% agar and 1% activated carbon, while their pH was adjusted to 5.8 ± 0.02 before autoclaving at 121 ° C for 20 minutes. The seeds were examined under two illumination conditions, 0/24 light/dark (L/D) and 16/8 L/D. The presented results indicate a huge influence of illumination and nutrition media on the seed germination and protocorm formation. The seed germination was overall significantly more successful in dark conditions (0/24 L/D) than with lighting (16/8 L/D). Protocorm, rhizoids and shoot formation were achieved only on the seeds cultured on MM medium, while the KC medium caused only swelling of the embryo. Organic additives had positive influence on the germination rate. According to the obtained results, the best germination rate and seedling development were achieved on MM-PE media, cultured in dark. The presented procedure accelerates the germination period and can provide a large number of plants in a relatively short period of time so it can be used for conservation programs and mass production protocol.

In vitro seed germination and seedling growth of Calanthe discolor Lindl

We investigated the effects of sodium hypochlorite (NaOCl) and culture medium on embryo swelling and germination of Calanthe discolor Lindl., and established a method for determining the swelling and protocorm formation of C. discolor seeds via in vitro examination of immature seeds. Treatment of immature seeds with NaOCl greatly enhanced the extent of embryo swelling and protocorm formation of immature zygote embryos compared to seeds without NaOCl treatment. The effects of the culture media were also evaluated with regard to embryo swelling and protocorm formation of in vitro cultured seeds with and without NaOCl treatment. Additionally, the effects of white fluorescent light and red and blue LED lights on seedling growth in in vitro culture were examined. The most suitable condition for seedling growth after 12 weeks of culture was the red LED light with POM medium. These results show effective asymbiotic germination and growth of C. discolor.

Evaluation of protocorm formation in meristematic regions of roots and leaves of Miltonia spectabilis moreliana (Orchidaceae).

The aim of this study was to evaluate the effect of 2.4-D and BAP on protocorm formation in meristematic regions of root and leaf of Miltonia spectabilis moreliana, and to test the effect of sucrose concentrations and pH levels on the growth of protocorms. Root and leaf segments of in vitro-germinated plants were cultured in MS/2 culture medium supplemented with 2.4-D and BAP, and kept in the dark for 210 days. The combination of 3 mg.L-1 2.4-D and 1 or 3 mg.L-1 BAP provided the best results. The concentrations of 15 and 30 g.L-1 sucrose, independently of the pH, were more efficient for the in vitro culture of this species, as they promoted an increase in the number and length of roots, height, and fresh weight of the plantlets. The pH was significant only in combination with 15 g.L-1 sucrose for the length of the largest root and fresh weight.

In vitro Seed Germination and Seedling Development of Esmeralda clarkei Rchb.f. (Orchidaceae)

Esmeralda clarkei Rchb.f. (Orchidaceae), an epiphytic orchid native to Nepal with high ornamental value grows from cool to intermediate climatic conditions with medium amount of light. In an attempt to preserve this important orchid by establishing an efficient in vitro regeneration protocol using seed culture was conducted. MS with BAP and NAA alone or in combination were used. In MS protocorms developed within 16 weeks followed by seedling development in 25 weeks of seed culture. Presence of BAP (0.5 mg/l) in MS improved seed germination by protocorm formation and seedling development in 17 and 26 weeks of seed culture, respectively. In 0.5 mg/l NAA supplemented media, development of protocorms was observed in 18 weeks and seedling growth was observed in 26 weeks of seed culture. Protocorm and seedling development were was delayed in other media. DOI: http://dx.doi.org/10.3329/ptcb.v22i2.14197 Plant Tissue Cult. & Biotech. 22(2): 107-111, 2012 (December)

Responses of seed germination and protocorm formation to light intensity and temperature in epiphytic and terrestrial Liparis (Orchidaceae)

Epiphytes begin their life history with seed germination on trees and live on the trees throughout the lives. In contrast, the seeds of terrestrial plants germinate on the ground. To clarify differences in early life history between epiphytes and terrestrial species, we compared the responses of in vitro asymbiotic seed germination and protocorm formation to various light intensities and temperatures between the epiphyte Liparis fujisanensis and two closely related terrestrial species, Liparis koreojaponica and Liparis kumokiri (Orchidaceae). Epiphytic germination was higher at low temperature and under high light intensity than that of the terrestrial species. Protocorms were formed only in the dark in the three species. Field measurements of temperature and photosynthetically active radiation in six natural habitats of the species from 2005 to 2006 showed that the habitats of the epiphyte were exposed to higher light intensities than those of the terrestrial species. The high germination capability of epiphyte seeds in wider ranges of light intensity and temperature may be beneficial to its seedling growth in the early growing season in exposed aboveground habitats.