In vitro germination, initial development and acclimatization of Cattleya nobilior Rchb. f. (Orchidaceae): an approach to curb the eventual endangerment of this exuberant, near-threatened Cerrado species Germinação in vitro, desenvolvimento inicial e aclimatização de Cattleya nobilior Rchb. f. (Orchidaceae): uma abordagem para evitar a eventual extinção dessa exuberante e quase ameaçada espécie de Cerrado

The objective of the present study was to evaluate the effects of different culture media on the in vitro germination and initial development of Cattleya nobilior, and to acclimatize this species using soybean straw as a substrate component, aiming the production of plants at large scale to reintroduce it in natural environments. Anatomical characteristics associated with development during acclimatization are also depicted. The influence of Murashige and Skoog, Knudson, and Vacin and Went culture media on the in vitro germination and protocorm development were assessed. Acclimatization was accomplished using different proportions of soybean straw (SS) and Bioplant (BP) as substrates. Anatomical studies were conducted on the leaves of plants during acclimatization. KC was the most suitable culture medium for both germination and initial development of C. nobilior. Regarding acclimatization, it is recommended that a substrate composed of 60% SR and 40% BP be initially used. Upon transfer to shade-house conditions, plants should be grown in a mix of 40% SR: and 60% BP. The leaf anatomical characteristics observed are typical of epiphytic orchids adapted to waterpoor environments, which indicates that SR did not negatively affect the development of the species. The in vitro propagation of C. nobilior as described herein is efficient for its multiplication for commercial and conservation purposes and SS can be used as an alternative component of the substrate for its acclimatization.


INTRODUCTION
In the Cerrado domain, the Orchidaceae family is considered the third most important in terms of representativeness (BARROS et al., 2015). In Brazil, 2,449 species According to the germination responses and respective basic nutritional needs of each species in the in vitro process, Stewart (1989) divides orchids into two large groups, the first group consisting of the species in which the seeds germinate in nutritionally simpler culture media such as KC and VW and a second group of the species that need nutritionally richer media, that is, with higher macronutrient content, such as MS.
Before being taken into the natural environment, plants produced in vitro require a gradual period of acclimatization so that they can survive ex vitro conditions. During In view of the above, the objective of this work was to evaluate the influence of different culture media on the in vitro germination and initial development of Cattleya nobilior (a near-threatened species) and develop a protocol to acclimatize this orchid species using soybean straw (Glycine max) as a substrate component. Morphological and anatomical characteristics associated with seedling development during acclimatization are also depicted.
Flowering occurs in August and September and, due to the beauty of its flowers (Fig. 1), the species experiences collection pressure by collectors and/or merchants (BIANCHETTI, 2007;RODRIGUEZ et al., 2009;BARROS et al., 2015), and may possibly be included in lists of endangered species if no action is taken to prevent it.
Even though it is not considered threatened with extinction, concern for the conservation status of this species is necessary, thereby making it impossible to frame it accurately and safely as threatened (BRASIL, 2008). At present, according to CNCFlora

Influence of culture media on in vitro germination and initial development
The following three culture media were tested: Knudson medium (1946) [KC], Vacin and Went (1949) [VW] and Murashige and Skoog (1962) [MS], at concentrations of 100 and 50% of their macronutrients (MS and ½MS, respectively). All media were supplemented with 0.4 mg.L -1 thiamine, 100 mg.L -1 of myo-inositol and 2% sucrose. The pH of each culture medium was adjusted to 5.8 ± 0.1 before the addition of 0.2% Phytagel. The media were sterilized in an autoclave at 121° C and 105 kPa for 20 min. Soon after sterilization, the seeds were inoculated on the media with the aid of a micropipette whose dosage was 250 μL of the aqueous suspension mentioned above, repetition was placed on a gridded slide to facilitate the counting of germinated seeds, which was performed under a Mikros stereoscopic microscope (São Paulo, Brazil).
Ninety days after the beginning of germination, the individuals contained in four replications, per treatment, were evaluated according to the number of organs formed.
Four stages of development were considered: stage 1 -protocorm (swollen green embryos); stage 2 -protocorm with one leaf; stage 3 -protocorm with two leaves and stage 4 -seedling (leaves and roots). Substantiated by the present evaluation, the growth index was calculated based on Spoerl (1948), that is, the percentage of individuals obtained at each stage of development, by repetition, was multiplied by the weights 1, 2, 3 and 4 according to the respective stages. The mean sum of all stages of development present was used to calculate the growth index of each repetition.

Acclimatization
The acclimatization process was carried out in two phases. In the first, 180 In the second phase, the plants (from the first phase) were transferred to individual plastic pots (7 cm high × 6 cm basal diameter) containing the same treatments described in the first phase except those containing only soybean straw.
These remained in the grow room for 10 days and were irrigated daily. Subsequently, they were transferred to a shade-house with 75% shading and received daily irrigation to the point of water saturation of the substrate. After 90 days, the same evaluations described in the first phase were performed.

Anatomical analyses
Anatomical analyses were performed on plant leaves at the end of the second acclimatization phase. These analyses were conducted at the Anatomy Laboratory in the  The results obtained for the germination of C. nobilior are presented in Table 1. and subsequent seedling growth, as will be discussed later.

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The germination percentage of orchids in asymbiotic cultures presents a percentage variation between 50 and 95%, and can reach up to 100%, as described by The results obtained for the initial development evaluated through the growth index are shown in Table 1. Figure

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Cattleya cultivation also reported that it is not recommended to use it as a single substrate.
The presence of straw in proportions between 20 and 80% added to Bioplant showed positive results for the species in the first acclimatization phase, presenting a survival percentage equal to or greater than 70%. This result is higher than that described by Dorneles and Trevelin (2011) in studies with a species of the same genus (C. intermedia) whose average survival percentage was 53% in Sphagnum substrate and

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conditions, which provides a reduction and mitigation of costs with commercial substrate, since soybean straw can be acquired at very low values or even at no cost, depending on the reuse or not of soybean bagasse by soybean producers.
In the second phase of acclimatization, there was a reduction in the percentage of survival of individuals in all treatments. Of these, the lowest rate observed was in the treatment that contained exclusively Bioplant probably because this substrate quickly loses moisture when exposed to the shade-house environment. Dorneles and Trevelin   Table 3 contains the results of the growth variables evaluated in both phases.
There were no significant differences among treatments for most of the variables analyzed in both phases. In relation to plant height, there were no significant differences among treatments in the first phase. In the second phase, a significant difference was

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Regarding the number of leaves, no significant differences were observed among treatments or losses of this organ during the two phases of acclimatization, that is, all plants remained with leaves of intense green color and apparently healthy, as can be observed in Figure 3c, corresponding to the second phase. Dorneles and Trevelin (2011) described that leaf permanence is probably associated with the specificities of each species and/or the physical and chemical conditions of the environment during the acclimatization process. As the atmospheric environment was quite uniform in the first phase of the present study (grow room with constant temperature and light intensity), the differences found are possibly a response to the substrate. In this sense, it is possible that in this phase the presence of soybean straw contributed to an average increase of 1 leaf at a proportion of 40%.
Concerning the variable of the number of roots, it was observed that in the first phase there were no significant differences among the treatments included in the interval between 0 and 60% soybean straw. However, the treatment containing 80% soybean straw caused a decrease in this variable when compared to the other treatments. In the second phase, the results showed no significant differences among treatments containing 20 to 80% soybean straw content. However, the treatment containing Bioplant as a single substrate resulted in a significant decrease in this variable. Regarding the length of the longest root, no significant differences were observed among most treatments in the first phase of acclimatization although in 80% soybean straw there was a decrease in this variable in relation to the other treatments.
In the second phase, only one difference was observed between the treatments containing 0 and 20% soybean straw, with a significant decrease in root length when Bioplant was used alone. However, the best results were observed in the interval between 20 and 60% soybean straw. Together, these results demonstrate that, in general, the different treatments did not strongly influence the formation and growth in extension of the roots.

Anatomical analyses
In cross-section (Fig.4), the leaves of C. nobilior commonly present a uniseriate epidermis and are hypostomatic. This characteristic was found in the leaf anatomy of other species of the same genus such as C. walkeriana, C. araguaiensis, and C. bicolor Around the atrium outside the ostiole, a pronounced cutinized thickening was observed that constitutes a stomatal crest which contributes to the formation of a suprastomatic chamber (Fig. 4d), similar to those observed by Zanega-Godoy and Costa (2003)  It is noted that on both sides of the leaf blade in cross-section, the external periclinal walls of epidermal cells are thick, almost always presenting convexity, especially along the margin. Also, the cuticle ( Fig. 4a and c) is thick along the margin and across the entire adaxial and abaxial surfaces homogeneously, however, in general the wall of the cells present in the adaxial surface is always thicker. Adjacent to the epidermis, a hypodermis was observed, formed by a layer of cells with different shapes from the rest of the parenchyma of the mesophyll. This layer was observed in the leaves of the plants of all acclimatization treatments. This result suggests that the proportional use of straw did not cause changes in C. nobilior. Vascular bundles (Fig. 4a) were observed in all leaves analyzed. This structure was also observed by Zanega- The mesophyll (Fig. 4b)

CONCLUSIONS
In summary, Knudson C medium was the most suitable for the seed germination and initial development of C. nobilior. With regard to the acclimatization process, it is recommended that the initial substrate be composed of 60% soybean straw and 40%