In vitro development and acclimatization of Cyrtopodium aliciae L. Linden & Rolfe, an endemic species of the Chapada Diamantina

ABSTRACT: Orchids are valued as ornamental plants, bioindicators, and medicinal plants, which implies that some species may be over-collected. Some inhabit very fragile environments and are under threat by the misuse of habitats and anthropogenic impacts. The search for beautiful plants and flowers has increased the number of facilities for micropropagation either by seeding or by cloning plants using in vitro techniques. However, not all species have appropriate media for growth and development that would help in conservation efforts. Cyrtopodium aliciae is an endemic species of rupestrian grassland in Brazil t. It has appeal as an ornamental plant or for use in hybridisation programs dueo its small size and white brownish-purple dotted flowers. This study compared three different media, namely ½ concentration Murashige and Skoog (MS), Vacin and Wendt, and Knudson C, during plant growth and their effect on the acclimatization of Cyrtopodium aliciae. The number and length of shoots and roots, increase in mass, and survival in vitro and ex vitro were analyzed. The experiment was conducted as completely random with a factorial arrangement of treatments (3 × 3) with 10 repetitions per treatment containing 10 plants for the in vitro experiment and 3 repetitions of 10 plants for the ex vitro experiment. Cyrtopodium aliciae performed better in the ½ concentration MS medium with a higher increase in mass, plant development, and survival under both in vitro and ex vitro conditions.


INTRODUCTION
The genus Cyrtopodium genus is widespread across the Americas, and its highest diversity occurs in Brazil (FLORA DO BRASIL, 2020). Cyrtopodium plants can be large, such as the Cyrtopodium saintglerianum (FLORA DO BRASIL, 2020) or Cyrtopodium punctatum (DUTRA et al., 2009), or small, such as Cyrtopodium blanchetii (FLORA DO BRASIL, 2020), which all have long and racemose inflorescences and are underutilised as ornamental plants. However, these plants have great potential as they can hybridise with other genera, such as Galeandra, Cymbidium, Grammatophyllum, or Ansellia in subtribe Cyrtopodinae or with the sister subtribe Catasetinae (Catasetum, Cycnoches, Oliveira et al. Mormodes, and Clowesia). Many are used as medicinal plants ( ARAÚJO-LIMA et al., 2020) or as adhesives for many purposes. However, all these species face the risk of extinction, which is why their propagation is significant (DUTRA et al., 2009;SUZUKI et al., 2009). Cyrtopodium aliciae has an inflorescence crowded by white flowers with purplish-brown spots, bracts, sepals, floral segments with wavy margins, sympodial growth, and pseudobulbs. Moreover, fruit production occurs during the rainy season and ripening occurs during the dry season (SANTANA et al., 2016).
Orchids are valuable as ornamental or medicinal plants and are heavily collected for these reasons, thereby resulting in the transfer of wild germplasm to private collections (yAM & ARDITTI, 2009), which compromises the integrity of ecosystems. This environmental damage is difficult to repair and evaluate, especially with endangered species, because the scarcer the natural resource, the more difficult it is to return to its original state (RAMALHO & PIMENTA, 2010).
The commercialization of orchids has increased (HINSLEy et al., 2016(HINSLEy et al., , 2018 with the demand for other pot plants; the industry is an international business. Orchid cultivation is increasing from 6% to 9% annually in the global flower trade and can change the economic landscape of a country (SHARMA, 2019). In this context, Brazil needs to implement a quality model for the management of flowers and ornamental plant production (JUNQUEIRA & PEETZ, 2014). The growing importance of environmental preservation associated with the use of in vitro propagation techniques can minimize the effects of predatory collections of orchids, thereby allowing natural population maintenance and commercialization of propagated plants.
Orchid seeds consist of globular structures with few cells at maturity, very small undifferentiated embryos, and no reserve organs (LEE et al., 2008), and a lipid reserve. They do not have an appropriate enzyme system to convert lipid reserves into soluble sugars (LEE et al., 2006(LEE et al., , 2008MANNING & VAN, 1987;YAM & ARDITTI, 2009). They do not germinate properly; they develop into a structure called a protocorm, which later gives rise to roots and leaves (BASKIN & BASKIN, 2014). Under in vitro conditions, plant development occurs more quickly, thereby enabling the production of a greater quantity of more uniform seedlings in less time, in small places, and with a high phytosanitary attribute, minimising the extinction risk of these species (FARIA et al., 2006;MARTINI et al., 2001;SUZUKI & FERREIRA, 2007).
Although, much is known about germination and tissue culture, few attempts have been made with the genus Cyrtopodium (ARAÚJO, et al., 1999;DUTRA, et al., 2009;PEREIRA et al., 2015;PICOLOTTO et al., 2017;SOUSA et al., 2019) because it has been underutilised as an ornamental plant.
The change in the seedling development environment from in vitro to ex vitro triggers water stress, photosynthetic stress, changes in nutrient absorption, and phytosanitary problems (MORAES et al., 2009).
This study compared three different media during the in vitro and ex vitro micropropagation phases of Cyrtopodium aliciae for the production of this species given future multiplication, reintroduction in nature, and availability to the consumer market.
The seeds were disinfected with a sodium dichloroisocyanurate (5 g·L -1 ) solution for 10 min, washed three times with autoclaved distilled water, and dispersed in ½ concentration MS medium (MURASHIGE; SKOOG, 1962) containing 20 g·L -1 of sucrose as the carbon source. The pH was corrected to 5.6 and autoclaved at 121 °C and 1 atm. The medium was dispensed on Falcon dishes (60 mm × 15 mm), and left in a chamber with controlled temperature until use. After sowing, the plates were sealed with polyvinyl chloride (PVC) film and kept in a growth chamber at a temperature of 23 ± 2 °C and a photoperiod of 16 h of light.

Growth experiments
Thirty-day-old seedlings from the germination procedures were used in three different media (Table 1), namely ½ concentration MS medium (MURASHIGE & SKOOG, 1962), Knudson C (KC) medium (KNUDSON, 1946), and Vacin and Wendt (VW) medium (VACIN & WENT, 1949), with 20 g·L -1 of sucrose as the carbon source and 8 g·L -1 of agar in each medium. The pH of the media was adjusted to 5.6. The media were distributed in 50 mL aliquots each into 269 mL baby food jars and autoclaved at 121 °C and 105 kPa for 15 min. Ten seedlings were placed in each jar for a total of 300 plants per treatment. The jars were maintained in a growth chamber with an irradiance of 35 µmol photons·m -2 ·s -1 at 25 ± 1 °C and 16 h of light.
The in vitro development was evaluated every 90 d via weighing the fresh mass and counting the shoots and roots. The seedlings were weighed individually, the structures were counted, and the seedlings were replanted in the same type of fresh medium. The flasks were closed with two layers of PVC film.

Acclimatization
At the end of the in vitro phase, the seedlings were removed from the jars, washed carefully to remove any agar debris, and immersed in a solution consisting of 2 g·L -1 of methyl thiophanate (a fungicide agent) for 1 h (MACHADO NETO, 2019). The solution was then removed, and the seedlings were allowed to dry overnight.
Acclimatization was performed in a netted house with 80% shading (Polysombra, Polysack) and a plastic cover. The conditions outside the greenhouse included an average maximum temperature of 28.2 °C, an average minimum temperature of 16.2 °C, and average relative humidity of 53.6%. The seedlings were planted in community pots with sphagnum moss and pine bark (<5 mm; 1:1 v:v) used as substrates. The seedlings were watered daily (15 mm·m -2 ) and fertigated weekly using 0.3 g·m -2 of hydrosoluble fertilizer (20:20:20 analysis) supplemented with 0.1 g·m -2 of Ca(Cl 2 ) or MgSO 4 at two-week intervals. Seedling survival was evaluated after 90 and 180 d of in vitro cultivation according to the difference between the initial number and the final number of seedlings.

Survival and acclimatisation percentages
Survival and acclimatization percentages were calculated for in vitro and ex vitro conditions, respectively, as the percentage of the number of initial plants out of the final number of plants in each treatment.

Statistical analysis
The percentage of survival (%S) and acclimatization (%Ac) were transformed according to the equation . The data were expressed    as mean values. The experiment was conducted in a completely random design and analysed as a factorial arrangement (3 × 3) with three different media (½ MS, KC, and VW media) and three periods of evaluation (90, 180, and 270 d) with 10 repetitions each, 10 seedlings per repetition for the in vitro experiment, and 3 repetitions of 10 plants each for the acclimatization experiment. The data were subjected to analysis of variance, and when significant, the means were compared using Tukey's test (P ≤ 0.05) (FERREIRA, 2011).

RESULTS
No substantial growth was observed until 90 d of cultivation. There was a high growth of Cyrtopodium aliciae seedlings after 180 d of cultivation in the VW medium; however, after 270 d, the seedlings showing the greatest fresh weight were those growing in the ½ concentration MS medium (Figure 1). In the end, seedlings grown in both VW and KC media had an equivalent fresh weight but were twice as small as those grown in the ½ concentration MS medium.
The number of shoots obtained from different culture media are shown in figure 2A.
After 180 d of cultivation, there was greater growth of shoots in ½ concentration MS medium, whereas there was no difference between that in the VW and KC media. At the end of the experiment, more shoots were grown in the ½ concentration MS medium, the number did not differ in the VW medium. The KC medium showed fewer shoots. Figure 2B shows the roots growth data. After 180 d of cultivation, there was no difference in the number of roots in the VW and KC media; however, there was greater root development in the ½ concentration MS medium. After 270 d, plants that grew in the ½ concentration MS medium had twice the number of roots of seedlings that grew in the VM medium and had grown more than seedlings grown in the KC medium. Figure 3 shows the root and leaf lengths of Cyrtopodium aliciae after 270 d of cultivation. The root length did not differ among treatments; however, there was a greater improvement in leaf length in the KC and ½ concentration MS media.
The ½ concentration MS medium was superior for fresh or dry weight in Cyrtopodium aliciae (Figure 4). The other media did not differ and produced lighter plants.
Differences were observed in the final in vitro survival. The ½ concentration MS medium had the highest survival rate, followed by the KC and VW media, which had different survival rates ( Table 2). The ex vitro survival rate of Cyrtopodium aliciae differed in the three media analysed after 90 d (Table  2); plants derived from the ½ concentration MS medium had the highest survival rate, followed by the VW and KC media, which did not differ. After 180 d, plants derived from the ½ concentration MS medium had the highest survival rate, whereas those of plants derived from the VW and KC media were significantly different. When the media were compared among times in the same medium, no differences were observed in the ½ concentration MS medium-derived plants. However, a decrease in the survival rate of greater than 20% in the KC medium and 28% in the VW medium was observed. The leaf length was superior for the ½ concentration MS medium-derived plants, followed by that of the VW and KC media-derived plants, which were different. The shoot length was superior for the ½ concentration MS medium-derived plants, while there were no differences between that of the other media-derived plants.

DISCUSSION
The largest development of Cyrtopodium aliciae seedlings were achieved in the medium rich in nitrogen and potassium, namely ½ concentration MS medium (Table 1), for all variables. Cyrtopodium punctatum plants were cultivated efficiently in a commercial medium (Sigma P723) containing a quarter of the MS salts supplemented with organics (DUTRA et al., 2009); however, it showed high germination in the ½ concentration MS medium, which was similar to that in the Malmgren (MALMGREN, 1992)

and KC media.
Few studies have shown or compared the growth of Cyrtopodium species in synthetic media (DUTRA et al., 2009), compared growth regulators (RODRIGUES et al., 2015VOGEL & MACEDO, 2011), or compared symbiotic germination (GUIMARÃES et al., 2013;PICOLOTTO et al., 2017;SOUSA et al., 2019). However, looking at the tribe Cymbidieae, some studies have been conducted on Cymbidium (GOGOI et al., 2011;MAHENDRAN  Ciência Rural, v.53, n.5, 2023. Oliveira et al. et al., 2013;MOHANTY et al., 2012) and have compared different media and Catasetum (FERREIRA et al., 2018). The growth of shoots and roots and the accumulation of fresh mass were consistent throughout the cycle, except for the variables of leaf and root length. Cattleya seems to prefer MS medium over KC or other media (ABRÃO et al., 2014;JORGE et al., 2015;SUZUKI et al., 2009).
A study on Cattleya warneri grown for 180d on in vitro conditions showed that the MS medium provided significantly greater production of  fresh mass than the KC and VW treatments (JORGE et al., 2015). The author also suggested that potassium acts in osmotic control in plants and has a greater concentration compared with that in other media, such as KC and VW media, thereby resulting in a lower fresh mass owing to reduced amounts of potassium.
In contrast, KC medium proved to be more efficient in the initial development of protocorms after 120 d of sowing in vitro; however, after six months, the reduced concentration of potassium in the medium inhibited the accumulation of fresh mass in stems and roots. VW medium most effectively promoted the germination and growth of C. bicolor (SUZUKI et al., 2010). Shoot growth was higher in the MS medium but did not change in the VW medium. However, the KC medium showed lower shoot growth.
There was a reduction of more than 20% in the survival rate of Cyrtopodium aliciae at 180 d in the VW and KC media, whereas there was no change in that in the MS medium. The VW and KC media showed a reduction in shoot length (Table  2). Owing to the in vitro conditions, the remaining leaves reached senescence and/or did not survive the transplant, and each species had specificity and/ or environmental conditions for acclimatization. According to FRANCO et al. (2007), leaf abscission is related to a plant strategy that reduces respiratory demands and reduces the exposed area of unnecessary structures to reduce the effects produced by different types of stress in terms of air humidity and the substrate itself.

CONCLUSION
The production of Cyrtopodium aliciae can be easily achieved by in vitro culture in the ½ concentration MS medium, which provides the toughest seedlings that perform better during acclimatisation.

DECLARATION OF CONFLICT OF INTEREST
The authors declare no conflict of interest for this article. The founding sponsors had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, and in the decision to publish the results.   Upper case letters within periods and lower case letters for different means for the same species indicate a significant difference (P < 0.05).