Optimal conditions for in vitro culture of Cattleya cernua, a small orchid native of Atlantic Forest and Cerrado

Márcio Hisayuki Sasamori Delio Endres Júnior Annette Droste About the authors

Abstract

Cattleya cernua is an epiphytic orchid native of the Atlantic Forest, Cerrado, Caatinga and Pampa. Aiming at the development of an in vitro conservation technology, plants were micropropagated through asymbiotic culture and the influence of different concentrations of sucrose (10, 30, 60 and 90 g L-1) and macronutrients (25, 50 and 100% MS) on survival and development was evaluated. Plant survival ranged between 47 and 100%. The interaction between macronutrients and sucrose influenced plant development. The aerial system of the plants was higher in 100% MS medium combined with 30 or 60 g L-1 of sucrose. The number of roots was higher with reduced macronutrients, combined with 30 or 60 g L-1 of sucrose. The length of the largest root was also higher when macronutrients were reduced but combined with 10 or 30 g L-1 of sucrose. The greatest mass was recorded when 30 g L-1 of sucrose was added to the three salt concentrations. Chlorophyll did not differ between plants grown with 30 or 90 g L-1 of sucrose. We recommend cultivating the plants in MS medium with 30 g L-1 of sucrose for better development of the aerial system. C. cernua can be asymbiotically micropropagated, facilitating ex vitro conservation strategies.

Key words:
carbon source; conservation; in vitro culture; nutrients; orchid

Resumo

Cattleya cernua é uma orquídea epífitica nativa da Floresta Atlântica, do Cerrado, da Caatinga e do Pampa. Visando ao desenvolvimento de uma ferramenta tecnológica in vitro para a conservação, plantas foram micropropagadas por meio da cultura assimbiótica, e a influência de diferentes concentrações de sacarose (10, 30, 60 e 90 g L-1) e macronutrientes (25, 50 e 100% MS) sobre a sobrevivência e o desenvolvimento das plantas foi avaliada. A sobrevivência das plantas variou entre 47 e 100%. A interação entre macronutrientes e sacarose influenciou o desenvolvimento das plantas. O sistema aéreo das plantas foi superior no meio 100% MS, combinado com 30 ou 60 g L-1 de sacarose. O número de raízes foi superior com macronutrientes reduzidos, combinados com 30 ou 60 g L-1 de sacarose. O comprimento da maior raiz também foi superior quando os macronutrientes foram reduzidos, mas combinados com 10 ou 30 g L-1 de sacarose. A maior massa foi registrada quando 30 g L-1 de sacarose foram adicionados às três concentrações de sais. A clorofila não diferiu entre plantas crescidas com 30 ou 90 g L-1 de sacarose. Nós recomendamos cultivar as plantas em meio MS com 30 g L-1 de sacarose para melhor desenvolvimento do sistema aéreo. C. cernua pode ser micropropagada assimbioticamente, facilitando estratégias de conservação ex vitro.

Palavras-chave:
fonte de carbono; conservação; cultura in vitro; nutrientes; orquídea

Introduction

Cattleya cernua (Lindl.) Van den Berg (basionym Sophronitis cernua (Lindl.) Lindl.) is a characteristic holoepiphytic orchid that reaches 15 cm in height and can occur both in the higher regions of trunks, in the primary and intermediate branches, and in areas more exposed to direct solar radiation (Cunha & Forzza 2007Cunha MFZ & Forzza RC (2007) Orchidaceae no Parque Natural Municipal da Prainha, RJ, Brasil. Acta Botanica Brasilica 21: 383-400.; Buzatto et al. 2010Buzatto CR, Ferreira PPA, Welker CAD, Seger GDS, Hertzog A & Singer RB (2010) The genus Cattleya Lindl. (Orchidaceae: Laeliinae) in Rio Grande do Sul state, Brazil. Revista Brasileira de Biociências 8: 388-398.; Schinini 2010Schinini A (2010) Orquídeas Nativas Del Paraguay. Rojasiana 9: 11-316.). During the reproductive period C. cernua presents a bright inflorescence of three to seven red-orange flowers (Buzatto et al. 2010Buzatto CR, Ferreira PPA, Welker CAD, Seger GDS, Hertzog A & Singer RB (2010) The genus Cattleya Lindl. (Orchidaceae: Laeliinae) in Rio Grande do Sul state, Brazil. Revista Brasileira de Biociências 8: 388-398.), giving the species high ornamental value (Moreira et al. 2014Moreira MM, Barberena FFVA & Lopes RC (2014) Orchidaceae of the Grumari restinga: floristic and similarity among restingas in Rio de Janeiro state, Brazil. Acta Botanica Brasilica 28: 321-326.). The tonality of the petals is the reason why the species is used to produce many interspecific and intergeneric hybrids (RHS 2016RHS (2016) Royal Horticultural Society. Available at <http://apps.rhs.org.uk/horticulturaldatabase/orchidregister/parentageresults.asp>. Access on 12 December 2016.
http://apps.rhs.org.uk/horticulturaldata...
), which has led to intense extractivism, resulting in reduced natural populations (Moreira et al. 2014Moreira MM, Barberena FFVA & Lopes RC (2014) Orchidaceae of the Grumari restinga: floristic and similarity among restingas in Rio de Janeiro state, Brazil. Acta Botanica Brasilica 28: 321-326.; Fig. 1a).

Figure 1
a. Cattleya cernua; b. plants 8 months after sowing; c. individualization of plants and start of experiment with different concentrations of macronutrients and sucrose; d. plants after 180 days growing in MS medium with 50% concentration of macronutrients and 30 g L-1 of sucrose. Bars = 10 mm.

The species is native to South America, occurring in Paraguay (Schinini 2010Schinini A (2010) Orquídeas Nativas Del Paraguay. Rojasiana 9: 11-316.), the northeast region of Argentina in Missiones (Johnson 2001Johnson AE (2001) Las Orquídeas del Parque Nacional Iguazú. LOLA, Buenos Aires. 282p.), and in Brazilian states in the South, Southeast, and Center-West regions as well as the state of Bahia, in the Atlantic Forest, Cerrado, Caatinga and Pampa biomes (van den Berg 2020van den Berg C (2020) Cattleya. In: Flora do Brasil 2020. Jardim Botânico do Rio de Janeiro. Available at <http://reflora.jbrj.gov.br/reflora/floradobrasil/FB582439>. Access on 10 April 2021.
http://reflora.jbrj.gov.br/reflora/flora...
). Although the species is widely distributed, information on occurrence records is scarce and sparse (Moreira et al. 2014Moreira MM, Barberena FFVA & Lopes RC (2014) Orchidaceae of the Grumari restinga: floristic and similarity among restingas in Rio de Janeiro state, Brazil. Acta Botanica Brasilica 28: 321-326.).

Biomes such as the Cerrado and Atlantic Forest are considered global biodiversity hotspots (Myers et al. 2000Myers N, Mittermeier RA, Mittermeier CG, Fonseca GA & Kent J (2000) Biodiversity Hotspots for conservation priorities. Nature 403: 853-858.) that have been highly degraded. Among the plant formations of the world similar to savannas, the Cerrado is recognized as one of the richest in biological diversity. On the other hand, in spite of its biological importance, the Cerrado has the smallest protected area among Brazilian biomes (MMA 2017MMA - Ministério do Meio Ambiente (2017) Biomas: cerrado. Available at <http://www.mma.gov.br/biomas/cerrado>. Access on 26 April 2017.
http://www.mma.gov.br/biomas/cerrado...
). Scientific information of Orchidaceae occurring in the Cerrado is scarce. The Atlantic Forest, another Brazilian biome with a high degree of biological diversity, is reduced to about 8% of its original area of approximately 1,315,460 km² (Fundação SOS Mata Atlântica 2019Fundação SOS Mata Atlântica (2019) Available at <http://www.sosma.org.br/nossa-causa/a-mata-atlantica/>. Access on 26 April 2019.
http://www.sosma.org.br/nossa-causa/a-ma...
). More than 20 thousand plant species are found in the Atlantic Forest, 40% of which are considered endemic (Fundação SOS Mata Atlântica 2019).

Due to intense environmental degradation of Brazilian biomes, in situ and ex situ conservation strategies for plants are extremely important for the conservation of species, especially epiphytic Orchidaceae, since several species of the family are listed as endangered (Martinelli & Moraes 2013Martinelli G & Moraes MA (2013) Livro vermelho da Flora do Brasil. Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rio de Janeiro. 1100p.). One of the ex situ conservation strategies is performed through in vitro propagation through sexually-produced seeds (Sasamori et al. 2015Sasamori MH, Endres Júnior D & Droste A (2015) Asymbiotic culture of Cattleya intermedia Graham (Orchidaceae): the influence of macronutrient salts and sucrose concentrations on survival and development of plantlets. Acta Botanica Brasílica 29: 292-298.), since in vitro germination enables the genetic variability of the plants to be maintained (Benson 1999Benson EE (1999) Plant Conservation Biotechnology. Taylor & Francis, London. 309p.; Pinto et al. 2010Pinto JRS, Freitas RMO & Praxedes SC (2010) Stimulation of in vitro development of Cattleya granulosa by sucrose. General and applied Plant Physiology 36: 183-188.). The asymbiotic culture results in a high seed germination rate (Pedrosode-Moraes et al. 2009Pedroso-de-Moraes C, Diogo JA, Pedro NP, Canabrava RI, Martini GA & Marteline MA (2009) Desenvolvimento in vitro de Cattleya loddigesii Lindl. (Orchidaceae) utilizando os fertilizantes comerciais. Revista Brasileira de Biociências 7: 67-69.; Pereira et al. 2015Pereira G, Albornoz V, Muñoz-Tapia L, Romero C & Atala C (2015) Asymbiotic germination of Bipinnula fimbriata (Orchidaceae) seeds in different culture media. Seed Science and Technology 43: 367-377.; Herrera et al. 2017Herrera H, Valadares R, Contreras D, Bashan Y & Arriagada C (2017) Mycorrhizal compatibility and symbiotic seed germination of orchids from the Coastal Range and Andes in south central Chile. Mycorrhiza 27: 175-188.) and rapid development of seedlings (Galdiano Júnior et al. 2013aGaldiano Júnior RF, Mantovani C, Faria RT & Lemos EGM (2013a) Concentração de sacarose no desenvolvimento in vitro e na aclimatação de Cattleya loddigesii Lindley. Semina: Ciências Agrárias 34: 583-592.), which are attributes not achieved by orchids in the natural environment. Moreover, it can be a highly efficient process for in vitro orchid propagation, since in laboratory conditions symbiotic seed germination requires previous isolation and culture of the fungi, and presents higher risk of contamination (Johnson et al. 2007Johnson TR, Stewart SL, Dutra D, Kane ME & Richardson L (2007) Asymbiotic and symbiotic seed germination of Eulophia alta (Orchidaceae) - preliminary evidence for the symbiotic culture advantage. Plant Cell, Tissue and Organ Culture 90: 313-323.; Abraham et al. 2012Abraham S, Augustine J & Thomas TD (2012) Asymbiotic seed germination and in vitro conservation of Coelogyne nervosa A. Rhich. and endemic orchid to Western Ghats. Physiology and Molecular Biology of Plants 18: 245-251.). After growth and acclimatization of the micropropagated orchid plants, individuals can be used in species conservation programs by reintroduction into natural habitats (Endres Júnior et al. 2015Endres Júnior D, Sasamori MH, Silveira T, Schmitt JL & Droste D (2015) Reintrodução de Cattleya intermedia Graham (Orchidaceae) em borda e interior de um fragmento de Floresta Estacional Semidecidual no sul do Brasil. Revista Brasileira de Biociências 13: 33-40.; Sasamori et al. 2015Sasamori MH, Endres Júnior D & Droste A (2015) Asymbiotic culture of Cattleya intermedia Graham (Orchidaceae): the influence of macronutrient salts and sucrose concentrations on survival and development of plantlets. Acta Botanica Brasílica 29: 292-298.), or be marketed as ornamental plants, thus alleviating pressure on natural populations (Mercier & Nievola 2003Mercier H & Nievola CC (2003) Obtenção de bromélias in vitro como estratégia de preservação. Vidalia 1: 57-62.).

In vitro propagation allows a high number of seedlings to be obtained from seeds, which contributes to the maintenance of the genetic characteristics and different physiological requirements of the plants (Pedroso-de-Moraes et al. 2009Pedroso-de-Moraes C, Diogo JA, Pedro NP, Canabrava RI, Martini GA & Marteline MA (2009) Desenvolvimento in vitro de Cattleya loddigesii Lindl. (Orchidaceae) utilizando os fertilizantes comerciais. Revista Brasileira de Biociências 7: 67-69.; Besson et al. 2010Besson JCF, Oliveira LK, Bonett LP & Stefanello S (2010) Source and concentration of carbohydrates on shoot growth and rooting of Miltonia flavescens Lindl. Revista Brasileira de Biociências 8: 9-13.; Pinto et al. 2010Pinto JRS, Freitas RMO & Praxedes SC (2010) Stimulation of in vitro development of Cattleya granulosa by sucrose. General and applied Plant Physiology 36: 183-188.). Although the technique of in vitro culture has been in use for years, studies prior to scale propagation are necessary for the establishment of a suitable culture medium and growing conditions, which will contribute to the ideal growth and development of the species in a short period of time (Endres Júnior et al. 2014Endres Júnior D, Sasamori MH & Droste A (2014) In vitro propagation of Anathallis adenochila (Loefgr.) F. Barros (Orchidaceae), a species endemic to southern and southeastern Brazil. Acta Botanica Brasilica 28: 489-494.; Sasamori et al. 2015Endres Júnior D, Sasamori MH, Silveira T, Schmitt JL & Droste D (2015) Reintrodução de Cattleya intermedia Graham (Orchidaceae) em borda e interior de um fragmento de Floresta Estacional Semidecidual no sul do Brasil. Revista Brasileira de Biociências 13: 33-40., 2016Sasamori MH, Endres Júnior D & Droste A (2016) Baixas concentrações de macronutrientes beneficiam a propagação in vitro de Vriesea incurvata (Bromeliaceae), uma espécie endêmica da Floresta Atlântica, Brasil. Rodriguésia 67: 1071-1081.). However, several studies have focused on the propagation of ornamental and commercial species, usually of artificial hybrid species (Faria et al. 2002Faria RT, Santiago DC, Saridakis DP, Albino UB & Araujo R (2002) Preservation of the Brazilian orchid Cattleya walkeriana Gardner using in vitro propagation. Crop Breeding and Applied Biothecnology 2: 489-492.; Pedroso-de-Moraes et al. 2012Pedroso-de-Moraes C, Souza-Leal T, Panosso AR & Souza MC (2012) Efeitos da escarificação química e da concentração de nitrogênio sobre a germinação e o desenvolvimento in vitro de Vanilla planifolia Jack ex Andr. (Orchidaceae: Vanilloideae). Acta Botanica Brasilica 26: 714-719.), and few are aimed at conservation of genetic diversity.

The abiotic conditions of in vitro culture need to be established for each orchid species in view of its peculiar physiological characteristics. The effect of different concentrations of macronutrients and sucrose on the in vitro development of Cattleya Lindl. species was assessed for C. violacea (Kunth) Rolfe (Galdiano Júnior et al. 2013bGaldiano Júnior RF, Mantovani C, Cassano AO & Lemos EGM (2013b) Desenvolvimento inicial e crescimento in vitro de Cattleya violacea (Kunth) Rolfe em diferentes concentrações de sacarose. Acta Amazônica 43: 127-134.) C. loddigesii Lindl. (Rezende et al. 2009Rezende JC, Ferreira EA, Pasqual M, Villa F & Santos FC (2009) Desenvolvimento in vitro de Cattleya loddigesii sp.: adição de reguladores de crescimento e sacarose. Agrarian 2: 99-114.) C. granulosa Lindl. (Pinto et al. 2010Pinto JRS, Freitas RMO & Praxedes SC (2010) Stimulation of in vitro development of Cattleya granulosa by sucrose. General and applied Plant Physiology 36: 183-188.) and C. intermedia Graham (Sasamori et al. 2015Sasamori MH, Endres Júnior D & Droste A (2015) Asymbiotic culture of Cattleya intermedia Graham (Orchidaceae): the influence of macronutrient salts and sucrose concentrations on survival and development of plantlets. Acta Botanica Brasílica 29: 292-298.), each species showing distinct responses in relation to morphometric variables. Sucrose, the main organic product added to culture medium, provides energy and carbon for the biosynthesis of structural and functional components (George et al. 2008aGeorge EF, Hall MA & De Klerk GJ (2008a) The components of plant tissue culture media II: organic additions, osmotic and pH effects, and support systems. In: George EF, Hall MA & De Klerk GJ (eds.) Plant propagation by tissue culture. 3rd ed. Springer, Dordrecht. Pp. 115-173.), since photosynthetically active radiation and CO2 concentration are insufficient for the optimal functioning of photosynthesis (George et al. 2008aGeorge EF, Hall MA & De Klerk GJ (2008a) The components of plant tissue culture media II: organic additions, osmotic and pH effects, and support systems. In: George EF, Hall MA & De Klerk GJ (eds.) Plant propagation by tissue culture. 3rd ed. Springer, Dordrecht. Pp. 115-173.; Xiao et al. 2011Xiao Y, Niu G & Kozai T (2011) Development and application of photoautotrophic micropropagation plant system. Plant Cell, Tissue and Organ Culture 105: 149-158.). Excess or deficiency of mineral nutrients in culture medium may be detrimental to the development of in vitro cultured seedlings (George et al. 2008bGeorge EF, Hall MA & De Klerk GJ (2008b) The components of plant tissue culture media I: macroand micro-nutrients. In: George EF, Hall MA & De Klerk GJ (eds.) Plant propagation by tissue culture. 3rd ed. Springer, Dordrecht. Pp. 65-113.), resulting in longer cultivation times for individuals and, consequently, increased costs, which can lead to greater sensitivity to external environmental stress during acclimatization and, therefore, to greater loss of micropropagated plants.

In order to obtain individuals for conservation purposes, the objective of the present study was to establish optimal conditions for asymbiotic in vitro propagation of C. cernua by evaluating the effect of different concentrations of macronutrients and sucrose on the survival and development of plants. It is expected that reduced mineral nutrients and increased sucrose concentration will contribute to increasing aerial and root systems of plants since the carbon source in the culture medium is essential for the in vitro growth phase of individuals (George et al. 2008aGeorge EF, Hall MA & De Klerk GJ (2008a) The components of plant tissue culture media II: organic additions, osmotic and pH effects, and support systems. In: George EF, Hall MA & De Klerk GJ (eds.) Plant propagation by tissue culture. 3rd ed. Springer, Dordrecht. Pp. 115-173.; Besson et al. 2010Besson JCF, Oliveira LK, Bonett LP & Stefanello S (2010) Source and concentration of carbohydrates on shoot growth and rooting of Miltonia flavescens Lindl. Revista Brasileira de Biociências 8: 9-13.). In addition, epiphytic plants are constantly exposed to the stress of nutritional deficiency in the natural environment (Benzing 2000Benzing DH (2000) Bromeliaceae: profile of an adaptive radiation. Cambridge University Press, Cambridge. 690p.) and may be adapted to a lesser requirement of mineral nutrients, and thus respond to lower mineral nutrients in in vitro culture medium.

Material and Methods

Mature capsules were collected from five plants of a Cattleya cernua population (one capsule per plant) in a forest fragment located in the municipality of Rolante, state of Rio Grande do Sul, Brazil, and taken to the laboratory. The capsules were washed in running water with commercial detergent, rinsed three times with sterilized distilled water and taken to a laminar flow chamber where they were disinfest for 30 seconds in 70% ethanol and then submerged in 2% sodium hypochlorite with Tween® 20 for 10 minutes. The capsules were then washed three times in sterile distilled water and opened with a scalpel to remove the seeds.

The seeds of all the capsules were pooled in a single sample and inoculated in vials (200 mL) containing 30 mL of MS medium (Murashige & Skoog 1962Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473-497.), supplemented with 30 g L-1 of sucrose and 10 g L-1 of activated charcoal, solidified with 6 g L-1 of agar (Kasvi). The medium was adjusted to pH 5.7 (Unemoto et al. 2007Unemoto LK, Faria RT, Vieira AOS & Dalio RJD (2007) Propagação in vitro de orquídeas brasileiras em meio de cultura simplificado. Revista Brasileira Agrociência 13: 267-269.) with 1N HCl by a pH meter (HI 2221, Hanna Instruments). The cultures remained in the growing room under controlled conditions with a photosynthetically active radiation (PAR) of 60 µmol m-2 s-1, a photoperiod of 12/12 h light/dark cycles and a temperature of 26 ± 1 °C.

After seed germination and protocorm development for eight months (Fig. 1b), the seedlings were transferred to vials (200 mL) containing 30 mL of the same medium used in the germination stage. Five seedlings were transferred to each vial, where they remained for additional 60 days, until attaining a height of about 1.1 ± 0.3 cm and roots of 3.0 ± 1.4 in length. The seedlings were then transferred to vials (200 mL; Fig. 1c) containing 30 mL of MS medium with the same concentrations of activated charcoal and agar and the same pH as the initial culture step. Combinations of three concentrations of the original macronutrient formula of MS (25, 50 and 100%; Tab. 1) and four concentrations of sucrose (10, 30, 60 and 90 g L-1) were evaluated. Fourteen replicates were performed for each combination of macronutrients and sucrose with five seedlings per vial for a total of 840 seedlings in 12 treatments.

Table 1
Composition of macronutrients of MS medium (Murashige & Skoog 1962Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473-497.) for the in vitro propagation of Cattleya cernua. 100MS = complete formulation of macronutrients; 50MS = reduction to 50% of macronutrients; 25MS = reduction to 25% of macronutrients).

After a period of 180 days under the same conditions of light and temperature as the initial culture step, plant survival was evaluated, using each vial as a replicate. The plants were removed from the vials and washed in running water. The following variables were evaluated for each plant: height of aerial part (HAP), number of leaves (NL), number of roots (NR), length of the longest root (LLR) and fresh mass (FM). The variables were measured using a caliper and a precision analytical balance. In addition to the morphological variables, levels of chlorophyll (a and b) and carotenoids in the leaves of plants from each treatment were also determined (Fig. 1d).

To determine the levels of photosynthetic pigments, leaf samples (distal half) were collected from propagated plants. Nine plants were selected for each treatment, which were divided into groups of three individuals, and leaf tissue samples were taken from each plant for a total of 20 mg for each group. For each 20 mg of leaf tissue, 1 mL of DMSO was added, in which the leaf tissue remained immersed for 24 hours in a water bath at 65 °C. Next, 100 uL triplicates of each 1 mL sample were removed and placed in each well of a 96-well cell culture dish for a total of nine samples per treatment. Readings were performed with a spectrophotometer (Spectramax M3®) at wavelengths of 665 nm, 649 nm and 480 nm. Concentrations were calculated according to the equations proposed by Wellburn (1994)Wellburn AR (1994) The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. Journal Plant Physiology 144: 307-313.: chlorophyll a (Chla) = 12.47A665 – 3.62A649; chlorophyll b (Chlb) = 25.06A649 – 6.5A665; and carotenoids (Car) = (1000A480 – 1.29 Chla – 53.78Chlb)/220.

Plant survival data were transformed into percentages. Data for height of the aerial part, leaf number, root number and fresh mass were natural log transformed (ln (x+1)). Data for length of largest root were square root transformed (root (x+1)). Plant survival in the treatments was compared by the Kruskal-Wallis test, followed by the Student-Newman-Keuls test at 5% probability. Data for morphological variables, as well as for leaf chlorophyll content, were compared by analysis of variance (two-way ANOVA), followed by the Bonferroni test at 5% probability. The analyses were performed using Biostat, version 5.3, and SPSS, version 20.0.

Results

In vitro propagation of Cattleya cernua with different concentrations of macronutrients and sucrose resulted in plant survival between 47 and 100% (Fig. 2). In general, the addition of sucrose combined with reduced macronutrients provided a survival percentage greater than 85%. On the other hand, treatments in which high concentrations of sucrose (60 and 90 g L-1) were combined with the complete concentration of macronutrients of MS medium (100% MS), proved detrimental to survival (Fig. 2).

Figure 2
Survival (mean ± standard error) of plants of Cattleya cernua propagated in vitro for 180 days at different concentrations of macronutrients and sucrose. 25MS and 50MS = 25% and 50% concentration of macronutrients, respectively; 100MS = complete concentration of macronutrients; 1, 3, 6 and 9% indicate concentrations of 10, 30, 60 and 90 g L-1 of sucrose, respectively. Different letters indicate significant differences between treatments according to Student-Newman-Keuls test at 5% probability. Kruskal-Wallis: H = 46.059; p < 0.001.

In general, the different concentrations of macronutrients and sucrose significantly influenced the growth of plants of C. cernua (Tab. 2). Increased macronutrient concentration in the culture medium at the different concentrations of sucrose (10, 30, 60 and 90 g L-1) generally contributed to a greater HAP. Thus, plants grown in treatments containing 100% of macronutrients had a significantly higher mean HAP (Tab. 2). Within each macronutrient concentration, the highest means for HAP were recorded for treatments containing 30 and 60 g L-1 of sucrose, while treatments containing 10 and 90 g L-1 of sucrose had significantly lower means for HAP (Tab. 2). Although there was a significant influence of sucrose and macronutrient concentration on HAP, this was not observed for the interaction between the concentration of both components (Tab. 2).

Table 2
Values (mean ± standard deviation) of height of the aerial part, number of leaves, number of roots, length of longest root and fresh mass of plants of Cattleya cernua micropropagated for 180 days in MS medium with different concentrations of macronutrients and sucrose.

The NL of micropropagated plants of C. cernua was influenced by macronutrient concentration, sucrose concentration and the interaction between these treatments (Tab. 2). The lowest means for

NL were observed in the presence of 60 and 90 g L-1 of sucrose when combined with the lowest concentration of MS salts (25MS). In addition, treatment with 30 g L-1 of sucrose also provided a significantly lower mean NL when combined with 25 and 50% of salts (Tab. 2). The different concentrations of sucrose, on the other hand, did not produce significant differences in NL in the treatments with 50 and 100% of macronutrients. When only 25% of the macronutrients of the MS medium were added, the lowest sucrose concentration (10 g L-1) was found to be beneficial for leaf production, with a significantly higher NL.

The root system of plants was also influenced by macronutrient concentration, sucrose concentration and the interaction between these treatments (Tab. 2). In general, the highest mean NR was observed when macronutrients were reduced in the culture medium, although a statistical difference was not observed in some treatments, such as 60 and 90 g L-1 of sucrose. When plants of C. cernua were cultivated in 25 and 100% of macronutrients, concentrations of between 30 and 90 g L-1 of sucrose were shown to be beneficial for root production. For the treatment with 50% of salts, the combination with 60 g L-1 of sucrose provided significantly higher mean NR. Reduction of macronutrients also provided the highest averages for root length of cultivated plants. The LLR was significantly greater for plants cultivated with 10 and 30 g L-1 of sucrose combined with 25 and 50% of macronutrients (Tab. 2). The sucrose concentration of 90 g L-1 was shown to be detrimental to root growth of C. cernua, as well as the total macronutrient concentration of MS medium.

In general, there was no influence of macronutrient concentration in the culture medium on plant FM, since there was no statistical difference between the concentrations of salts within each sucrose treatment. Sucrose concentration, on the other hand, influenced the fresh mass of plants, as did the interaction with mineral nutrient concentration of the medium. The highest mean values of FM were recorded for plants grown in the treatment with 30 g L-1 of sucrose. In addition, when half of the salt concentration was used, the FM of plants was higher when 10 and 60 g L-1 of sucrose were added. The mean FM of plants was also significantly higher in the treatment with the complete concentration of macronutrients combined with 30 and 60 g L-1 of sucrose (Tab. 2).

The levels of chlorophyll a, chlorophyll b and carotenoids of plants of C. cernua did not statistically differ between the different concentrations of macronutrient salts of the MS medium (25, 50 and 100%). The concentration of sucrose, on the other hand, influenced the levels of photosynthetic pigments, and the interaction between salts and sucrose was significant for chlorophyll a and carotenoids (Tab. 3). For chlorophyll a, the treatments with 30, 60 and 90 g L-1 of sucrose provided higher levels, except for individuals of the treatment with 50MS and 60 g L-1 of sucrose, which had a significantly lower mean. For chlorophyll b, plants of treatments with 30 and 90 g L-1 of sucrose had significantly higher averages (Tab. 3). The level of carotenoids was also significantly higher in the treatment with the highest concentration of sucrose (90 g L-1 of sucrose). Moreover, when the concentration of macronutrient salts of the MS medium was reduced to 25%, treatments with 30 and 60 g L-1 of sucrose also provided higher carotenoid levels in the cultivated plants (Tab. 3).

Table 3
Values (mean ± standard deviation) of chlorophyll (a and b) and carotenoid levels of plants of Cattleya cernua micropropagated for 180 days in MS medium with different concentrations of macronutrients and sucrose.

Discussion

High survival rates of Cattleya cernua were observed in most combinations of MS macronutrients and sucrose concentrations, with the exception of 100% macronutrients combinated with 6 or 9% sucrose. The use of 20 and 30 g L-1 of carbohydrate have been generally recommended for tissue culture, although studies have shown that different species respond better, or to lower concentrations of sucrose (Moreira et al. 2007Moreira BMT, Tomba EC & Zonetti PC (2007) Crescimento in vitro de plântulas de orquídea (Laelia purpurata lindl var venosa x Cattleya warneri T. Moore alba) sob diferentes concentrações de sacarose e frutose. Revista Saúde e Biologia 2: 16-21.; Pivetta et al. 2010Pivetta KFL, Martins TA, Galdiano Junior RF, Gimenes R, Faria RT & Takane RJ (2010) Crescimento in vitro de plântulas de Caularthron bicornutum em diferentes concentrações de sacarose. Ciência Rural 40: 1897-1902.; Galdiano Júnior et al. 2013aGaldiano Júnior RF, Mantovani C, Faria RT & Lemos EGM (2013a) Concentração de sacarose no desenvolvimento in vitro e na aclimatação de Cattleya loddigesii Lindley. Semina: Ciências Agrárias 34: 583-592.,bGaldiano Júnior RF, Mantovani C, Cassano AO & Lemos EGM (2013b) Desenvolvimento inicial e crescimento in vitro de Cattleya violacea (Kunth) Rolfe em diferentes concentrações de sacarose. Acta Amazônica 43: 127-134.; Martins et al. 2015Martins JPR, Pasqual M, Martins AD & Ribeira SF (2015) Effects of salts and sucrose concentrations on in vitro propagation of Billbergia zebrina (Herbert) Lindley (Bromeliaceae). Australian Journal of Crop Science 9: 85-91.; Koene et al. 2019Koene FM, Amano E & Ribas LLF (2019) Asymbiotic seed germination and in vitro seedling development of Acianthera prolifera (Orchidaceae). South African Journal of Botany 121: 83-91.), or to higher concentrations, between 40 and 60 g L-1 of the same carbohydrate (Rego-Oliveira et al. 2003Rego-Oliveira LV, Faria RT, Fonseca ICB & Saconato C (2003) Influência da fonte e concentração de carboidrato no crescimento vegetativo e enraizamento in vitro de Oncidium varicosum Lindl. (Orchidaceae). Ciências Agrárias 24: 265-272.; Besson et al. 2010Besson JCF, Oliveira LK, Bonett LP & Stefanello S (2010) Source and concentration of carbohydrates on shoot growth and rooting of Miltonia flavescens Lindl. Revista Brasileira de Biociências 8: 9-13.; Endres et al. 2014Endres Júnior D, Sasamori MH & Droste A (2014) In vitro propagation of Anathallis adenochila (Loefgr.) F. Barros (Orchidaceae), a species endemic to southern and southeastern Brazil. Acta Botanica Brasilica 28: 489-494.; Sasamori et al. 2015Endres Júnior D, Sasamori MH, Silveira T, Schmitt JL & Droste D (2015) Reintrodução de Cattleya intermedia Graham (Orchidaceae) em borda e interior de um fragmento de Floresta Estacional Semidecidual no sul do Brasil. Revista Brasileira de Biociências 13: 33-40.). This trend has also been observed for macronutrients, with C. cernua benefitting from lower concentrations, as do Anathallis adenochila (Loefgr.) F. Barros (Endres et al. 2014Endres Júnior D, Sasamori MH & Droste A (2014) In vitro propagation of Anathallis adenochila (Loefgr.) F. Barros (Orchidaceae), a species endemic to southern and southeastern Brazil. Acta Botanica Brasilica 28: 489-494.), Laelia anceps Lindl. (Ramírez-Mosqueda et al. 2019Ramírez-Mosqueda MA, Cruz-Cruz CA, Atlahua-Temoxtle J & Bello-Bello JJ (2019) In vitro conservation and regeneration of Laelia anceps Lindl. South African Journal of Botany 121: 219-223.) and Vriesea incurvata Gaudich. (Sasamori et al. 2016Sasamori MH, Endres Júnior D & Droste A (2016) Baixas concentrações de macronutrientes beneficiam a propagação in vitro de Vriesea incurvata (Bromeliaceae), uma espécie endêmica da Floresta Atlântica, Brasil. Rodriguésia 67: 1071-1081.), while other species like Miltonia flavescens (Lindl.) Lindl., Cattleya loddigesii Lindl. and C. intermedia Graham ex Hook., require higher concentrations of nutrients (Müller et al. 2007Müller TS, Dewes D, Karsten J, Schuelter AR & Stefanello S (2007) Crescimento in vitro e aclimatação de plântulas de Miltonia flavescens. Ciências Agrárias 29: 775-782.; Soares et al. 2009Soares JDR, Araújo AG, Pasqual M, Rodrigues FA & Assis FA (2009) Concentrações de sais do meio Knudson C e de ácido giberélico no crescimento in vitro de plântulas de orquídea. Ciência Rural 39: 772-777.; Sasamori et al. 2015Sasamori MH, Endres Júnior D & Droste A (2015) Asymbiotic culture of Cattleya intermedia Graham (Orchidaceae): the influence of macronutrient salts and sucrose concentrations on survival and development of plantlets. Acta Botanica Brasílica 29: 292-298.).

No evidence of deficiency and/or toxicity was found in the leaves of the propagated individuals of C. cernua both at low concentrations of macronutrients and at the total concentration of the MS medium. When high concentrations of macronutrients are added to the culture medium, such compounds may become toxic to plants, leading to irregular development or death of individuals (George et al. 2008bGeorge EF, Hall MA & De Klerk GJ (2008b) The components of plant tissue culture media I: macroand micro-nutrients. In: George EF, Hall MA & De Klerk GJ (eds.) Plant propagation by tissue culture. 3rd ed. Springer, Dordrecht. Pp. 65-113.). Likewise, low concentrations of nutrients in the culture medium can contribute to the formation of chlorosis and necrosis in leaves, since individuals experience deficiencies of nutrients that regulate metabolic processes (Marschner 2012Marschner H (2012) Mineral nutrition of highter plants. Academic Press, London. 672p.). Because it is considered one of the most nutrient-rich media, the use of MS may have contributed to the successful cultivation of C. cernua plants in the reduced nutrient treatments. Another important point to consider is the fact that the species possesses an epiphytic habit, thus allowing the micropropagated individuals to exhibit physiological adaptations to conditions of nutritional deficiency. For in vitro cultivation of Laelia anceps, the reduction of MS salts also did not harm its growth and development due to the low nutritional requirements since the species has an epiphytic habit (Ramírez-Mosqueda et al. 2019Ramírez-Mosqueda MA, Cruz-Cruz CA, Atlahua-Temoxtle J & Bello-Bello JJ (2019) In vitro conservation and regeneration of Laelia anceps Lindl. South African Journal of Botany 121: 219-223.). However, even if nutrient shortage allows the species to develop slowly, both in the natural environment and in in vitro culture, such a condition is not of interest in the propagation process. Plants of C. cernua grown in vitro had greater development of the aerial system in the medium with the complete concentration of macronutrients, which is thus a determinant factor for in vitro mass culture. This has been the objective of searches for a culture medium composition that contributes to the production of vigorous plants in a short period of time (Galdiano Júnior et al. 2013aGaldiano Júnior RF, Mantovani C, Faria RT & Lemos EGM (2013a) Concentração de sacarose no desenvolvimento in vitro e na aclimatação de Cattleya loddigesii Lindley. Semina: Ciências Agrárias 34: 583-592.,bGaldiano Júnior RF, Mantovani C, Cassano AO & Lemos EGM (2013b) Desenvolvimento inicial e crescimento in vitro de Cattleya violacea (Kunth) Rolfe em diferentes concentrações de sacarose. Acta Amazônica 43: 127-134.; Endres Júnior et al. 2015Endres Júnior D, Sasamori MH, Silveira T, Schmitt JL & Droste D (2015) Reintrodução de Cattleya intermedia Graham (Orchidaceae) em borda e interior de um fragmento de Floresta Estacional Semidecidual no sul do Brasil. Revista Brasileira de Biociências 13: 33-40.; Sasamori et al. 2015Sasamori MH, Endres Júnior D & Droste A (2015) Asymbiotic culture of Cattleya intermedia Graham (Orchidaceae): the influence of macronutrient salts and sucrose concentrations on survival and development of plantlets. Acta Botanica Brasílica 29: 292-298.).

The treatment with the highest concentration of sucrose (90 g L-1) was not beneficial for the development of plants, although increased sucrose in culture medium is essential as a carbon source. Carbohydrate added to the medium is used for the biosynthesis of structural and functional components for growth, and its ideal concentration depends on the requirements of each plant species (George et al. 2008aGeorge EF, Hall MA & De Klerk GJ (2008a) The components of plant tissue culture media II: organic additions, osmotic and pH effects, and support systems. In: George EF, Hall MA & De Klerk GJ (eds.) Plant propagation by tissue culture. 3rd ed. Springer, Dordrecht. Pp. 115-173.). The adequate supply of carbohydrate increases the reserves of starch and sucrose in the leaves of micropropagated plants, which then act as energy storage organs that will supply the acclimatization stage for the growth of new leaves adapted to the ex vitro environment, thus improving the performance of acclimatization (Capellades et al. 1991Capellades M, Lemeur R & Debergh P (1991) Effects of sucrose on starch accumulation and rate of photosynthesis in Rosa cultured in vitro. Plant Cell, Tissue and Organ Culture 25: 21-26.; Hazarika 2003Hazarika BN (2003) Acclimatization of tissue-cultured plants. Current Science 85: 1704-1712.; Fuentes et al. 2006Fuentes G, Talavera C, Desjardins Y & Santamaría JM (2006) Protocol to achieve photoautotrophic coconut plants cultured in vitro with improved performance ex vitro. In: Loyola-Vargas VM & Vázquez-Flota F (eds.) Plant Cell Culture Protocols. Humana Press Inc., Totowa. Pp. 131-144.).

During micropropagation, cultivated plants can undergo stress conditions, leading to the formation of morphological (hyperhydricity) and physiological changes (photosynthesis and gas exchange) (Ziv 1991Ziv M (1991) Quality of micropropagated plants -vitification. In vitro Cellular & Developmental Biology - Plant 27: 64-69.). When plants of C. cernua were removed from the vials, structures, such as leaves and roots, were broken during the handling of individuals, indicating the fragility of the plants grown in the treatment with 10 g L-1 of sucrose. Brittle organs, low cell wall strength and low chlorophyll content are characteristics of plants experiencing hyperhydricity (Franck et al. 2004Franck T, Kevers C, Gaspar T, Dommes J, Deby C, Greimers R, Serteyn D & Deby-Dupont G (2004) Hyperhydricity of Prunus avium L. shoots cultured on gelrite: a controlled stress response. Plant Physiology and Biochemistry 42: 519-527.; Kevers et al. 2004Kevers C, Franck T, Strasser RJ, Dommes J & Gaspar T (2004) Hyperhydricity of micropropagated shoots: at typically stress-induced change of physiological state. Plant Cell, Tissue and Organ Culture 77: 181-191.).

The morphological variables recorded for C. cernua corroborate the finding that macronutrient and sucrose concentrations are specific to the in vitro growth and development of various species, including orchids of the same genus. For the micropropagation of C. violacea, culture medium with 50% of macronutrient salts and between 20 and 30 g L-1 of sucrose provided greater values for aerial part height, number of leaves, fresh mass and the number and length of roots (Galdiano Júnior et al. 2013bGaldiano Júnior RF, Mantovani C, Cassano AO & Lemos EGM (2013b) Desenvolvimento inicial e crescimento in vitro de Cattleya violacea (Kunth) Rolfe em diferentes concentrações de sacarose. Acta Amazônica 43: 127-134.). In the cultivation of C. loddigesii, medium with 100% macronutrients and 60 g L-1 of sucrose stimulated the development of the root system; however, for the aerial system, the best treatments were with the addition of between 16 and 30 g L-1 associated with the growth regulator gibberellic acid (Rezende et al. 2009Rezende JC, Ferreira EA, Pasqual M, Villa F & Santos FC (2009) Desenvolvimento in vitro de Cattleya loddigesii sp.: adição de reguladores de crescimento e sacarose. Agrarian 2: 99-114.). When plants of C. granulosa were micropropagated in MS containing 45 g L-1 of sucrose, aerial part height, number of leaves and fresh root mass were higher than for plants micropropagated in the presence of 15 and 30 g L-1 (Pinto et al. 2010Pinto JRS, Freitas RMO & Praxedes SC (2010) Stimulation of in vitro development of Cattleya granulosa by sucrose. General and applied Plant Physiology 36: 183-188.). Plants of C. intermedia propagated in MS medium combined with 60 g L-1 of sucrose, as well as in medium with 50% macronutrients plus 45 or 60 g L-1 of sucrose, had the highest averages for both the aerial and root systems of individuals (Sasamori et al. 2015Sasamori MH, Endres Júnior D & Droste A (2015) Asymbiotic culture of Cattleya intermedia Graham (Orchidaceae): the influence of macronutrient salts and sucrose concentrations on survival and development of plantlets. Acta Botanica Brasílica 29: 292-298.).

As in other micropropagation studies (Araújo et al. 2006Araújo AG, Pasqual M, Pereira AR & Rocha HS (2006) In vitro growth of Laelia tenebrosa (Orchidaceae) in different concentrations of Knudson C salts and activated charcoal. Plant Cell Culture and Micropropagation 2: 61-67.; Tamaki et al. 2007Tamaki V, Mercier H & Nievola CC (2007) Cultivo in vitro de clones de Ananas comosus (L.) Merril cultivar ‘Smooth Cayenne’ em diferentes concentrações de macronutrientes. Hoehnea 34: 69-73.; Unemoto et al. 2007Unemoto LK, Faria RT, Vieira AOS & Dalio RJD (2007) Propagação in vitro de orquídeas brasileiras em meio de cultura simplificado. Revista Brasileira Agrociência 13: 267-269.; Martins et al. 2015Martins JPR, Pasqual M, Martins AD & Ribeira SF (2015) Effects of salts and sucrose concentrations on in vitro propagation of Billbergia zebrina (Herbert) Lindley (Bromeliaceae). Australian Journal of Crop Science 9: 85-91.; Sasamori et al. 2016Sasamori MH, Endres Júnior D & Droste A (2016) Baixas concentrações de macronutrientes beneficiam a propagação in vitro de Vriesea incurvata (Bromeliaceae), uma espécie endêmica da Floresta Atlântica, Brasil. Rodriguésia 67: 1071-1081.), the development of the root system recorded for C. cernua confirms that low concentrations of macronutrients may be beneficial to plants during in vitro cultivation. The use of media with reduced concentrations of macronutrients can stimulate root formation and growth (George et al. 2008aGeorge EF, Hall MA & De Klerk GJ (2008a) The components of plant tissue culture media II: organic additions, osmotic and pH effects, and support systems. In: George EF, Hall MA & De Klerk GJ (eds.) Plant propagation by tissue culture. 3rd ed. Springer, Dordrecht. Pp. 115-173.), which is important for contact with the substrate (Zhang et al. 2010Zhang Z, Song H, Liu Q, Rong X, Guan C, Peng J, Xie G & Zhang Y (2010) Studies on differences of nitrogen efficiency and root characteristics of oilseed rape (Brasica napus L.) cultivars in relation to nitrogen fertilization. Journal of Plant Nutrition 33: 1148-1459.). Tamaki et al. (2007)Tamaki V, Mercier H & Nievola CC (2007) Cultivo in vitro de clones de Ananas comosus (L.) Merril cultivar ‘Smooth Cayenne’ em diferentes concentrações de macronutrientes. Hoehnea 34: 69-73. suggested that low concentrations of nutrients in culture medium may induce the translocation of auxins, which are responsible for cell elongation, from leaves to roots (Marschner 2012Marschner H (2012) Mineral nutrition of highter plants. Academic Press, London. 672p.), thus contributing to the development of the root system and, consequently, to higher plant survival during ex vitro acclimatization (Besson et al. 2010Besson JCF, Oliveira LK, Bonett LP & Stefanello S (2010) Source and concentration of carbohydrates on shoot growth and rooting of Miltonia flavescens Lindl. Revista Brasileira de Biociências 8: 9-13.). On the other hand, very long branched roots are not recommended for in vitro culture because they make it difficult to wash and remove culture media adhered to the plants when they are removed from the vials. Short roots and/ or root primordia can contribute to the rooting of plants in acclimatization, since they are still in a stage of active growth (Woodhead & Bird 1998Woodhead JL & Bird KT (1998) Efficient rooting and acclimation of micropropagated Ruppia maritima Loisel. Journal of Marine Biotechnology 6: 152-156.; Costa et al. 2008Costa FHS, Pasqual M, Pereira JES, Rodrigues FA & Miyata LY (2008) Relação entre o tempo de enraizamento in vitro e o crescimento de plantas de bananeira na aclimatização. Revista Brasileira de Fruticultura 30: 31-37.).

In general, the morphological characteristics of orchid roots, including those of C. cernua, act directly on the biomass of propagated individuals through the incorporation of carbon (De Riek et al. 1997De Riek J, Piqueras A & Debergh PC (1997) Sucrose uptake and metabolism in a double layer system for micropropagation of Rosa multiflora. Plant Cell, Tissue and Organ Culture 47: 269-278.), which is obtained from the carbohydrate added to the medium. Sucrose added to media is the main source of carbon for the formation of structural skeletons and also serves to stimulate root growth and formation, with a concentration of 20 to 30 g L-1 of carbohydrate being recommended for rooting in vitro cultured plants (George et al. 2008aGeorge EF, Hall MA & De Klerk GJ (2008a) The components of plant tissue culture media II: organic additions, osmotic and pH effects, and support systems. In: George EF, Hall MA & De Klerk GJ (eds.) Plant propagation by tissue culture. 3rd ed. Springer, Dordrecht. Pp. 115-173.). Leaves and pseudobulbs of orchids have cells capable of storing large amounts of water and carbohydrates, while the roots possess a multiple epidermis (velamen) that acts as a “sponge” by storing moisture and nutrients (Benzing 1990Benzing DH (1990) Vascular epiphytes: general biology and related biota. Cambridge University Press, Cambridge. 354p.; Silva & Milaneze-Gutierre 2004Silva CI & Milaneze-Gutierre MA (2004) Caracterização morfo-anatômica dos órgãos vegetativos de Cattleya walkeriana Gardner (Orchidaceae). Acta Scientiarum, Biological Sciences 26: 91-100.). Due to these morphological characteristics of orchid roots and aerial system, the fresh mass of cultivated plants was not influenced by the concentration of macronutrients in the medium.

High concentrations of sucrose in the culture medium may also be harmful to plants by making it difficult to absorb water and nutrients (Paiva Neto & Otoni 2003Paiva-Neto VB & Otoni WC (2003) Carbon sources and their osmotic potential in plant tissue culture: does it matter? Science Horticulture 97: 193-202.; Fráguas et al. 2003Fráguas CB, Villa F, Souza AV, Pasqual M & Dutra LF (2003) In vitro growth of orchid seedlings obtained from hybridization between Cattleya labiata and Laelia itambana. Revista Ceres 50: 719-726.; Besson et al. 2010Besson JCF, Oliveira LK, Bonett LP & Stefanello S (2010) Source and concentration of carbohydrates on shoot growth and rooting of Miltonia flavescens Lindl. Revista Brasileira de Biociências 8: 9-13.; Koene et al. 2019Koene FM, Amano E & Ribas LLF (2019) Asymbiotic seed germination and in vitro seedling development of Acianthera prolifera (Orchidaceae). South African Journal of Botany 121: 83-91.). Indeed, when plants of the treatment with 90 g L-1 of sucrose were removed from their vials, their leaves were observed to be slightly dehydrated, indicating a possible alteration of the osmotic potential of the medium (Paiva Neto & Otoni 2003Paiva-Neto VB & Otoni WC (2003) Carbon sources and their osmotic potential in plant tissue culture: does it matter? Science Horticulture 97: 193-202.; George et al. 2008aGeorge EF, Hall MA & De Klerk GJ (2008a) The components of plant tissue culture media II: organic additions, osmotic and pH effects, and support systems. In: George EF, Hall MA & De Klerk GJ (eds.) Plant propagation by tissue culture. 3rd ed. Springer, Dordrecht. Pp. 115-173.). High concentrations of carbohydrates may also alter the structure of the photosynthetic apparatus, inhibiting chlorophyll synthesis and reducing the photosynthetic capacity of tissues (Silva 2004Silva JAT (2004) The effect of carbon source on in vitro organogenesis of chrysanthemum thin cell layers. Bragantia 63: 165-177.), which is quite low in the in vitro condition (Rolland et al. 2002Rolland F, Moore B & Sheen J (2002) Sugar sensing and signaling in plants. Plant Cell 14: S185-S205.). However, inhibition in the formation of chlorophyll a and b was not observed for plants of C. cernua in the treatments with higher concentrations of carbohydrate; in fact, there was significantly higher averages for the pigments in the treatments with high concentrations of sucrose. Reduced carbohydrate in the culture medium, on the other hand, can stimulate the plants to an autotrophic condition in vitro (Mosaleeyanon et al. 2004Mosaleeyanon K, Cha-um S & Kirdmanee C (2004) Enhanced growth and photosynthesis of rain tree (Samanea saman Merr.) plantlets in vitro under a CO2-enriched condition with decreased sucrose concentrations in the medium. Scientia Horticulturae 103: 51-63; Schmildt et al. 2014Schmildt O, Netto AT, Schmildt ER, Carvalho VS, Otoni C & Campostrini E (2014) Photosynthetic capacity, growth and water relations in ‘Golden’ papaya cultivated in vitro with modifications in light quality, sucrose concentration and ventilation. Theoretical and Experimental Plant Physiology 27: 7-18.), an event that was not observed for the plants of C. cernua cultivated at the concentration of 10 g L-1 of sucrose, which had significantly lower values for photosynthetic pigments.

The data obtained in the present study allowed the description of efficient in vitro culture of C. cernua. Plant growth was influenced by the interaction between macronutrient and sucrose concentrations, with the exception of height of the aerial part and chlorophyll b. Although reduced concentrations were beneficial for the root system, the treatment “100MS + 30 g L-1 of sucrose” had the best results for most of the evaluated variables, being the most suitable for in vitro culture of the species. The lower root length in this combination of macronutrients and sucrose could serve as an ally in the rooting process during plant acclimatization. The in vitro propagation of C. cernua can contribute to the conservation of this epiphytic species native to South America through indirect actions such as the marketing of plants cultivated in vitro, which would alleviate pressures on natural populations, and/or their use for environmental education. It can also contribute directly to conservation by providing individuals for use in reintroduction programs for this species.

Acknowledgements

The authors thank Universidade Feevale, for infrastructure and financial support; and the Brazilian Federal Agency, for the Support and Evaluation of Graduate Education (CAPES) for granting a doctoral scholarship to the first and second authors.

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Publication Dates

  • Publication in this collection
    09 July 2020
  • Date of issue
    2021

History

  • Received
    17 Oct 2019
  • Accepted
    22 May 2020
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