Impact of saline solution on growth and photosystem II during in vitro cultivation of Bromelia antiacantha (Bromeliaceae)

Rosiane Cipriano João Paulo Rodrigues Martins Luiz Carlos de Almeida Rodrigues Antelmo Ralph Falqueto Andreia Barcelos Passos Lima Gontijo About the authors

Abstract

In vitro cultivation is a technique with wide application for micropropagation. However, each species has specific mineral needs for this type of cultivation. The objective was to assess the impacts of the saline solution culture medium on the performance of the photosynthetic apparatus and growth of Bromelia antiacantha during in vitro cultivation, and thus to elucidate the mitigation of the nutritional imbalance that can interfere in the electron transport in the plants. Plants were cultivated in a salt concentration gradient of MS medium (0%, 25%, 50%, 75% or 100%). The growth traits and fluorescence a chlorophyll were analyzed. Intermediate concentrations of MS medium resulted in plants with a larger number of leaves and longer root length. The OJIP curves and results of the JIP test showed that the plants grown without MS salts presented less efficient photosystem II (PSII), as indicated by the performance index [Pi(total)]. In contrast, the intermediate concentrations (MS 25% and 50%) had a positive effect on the performance of the photosynthetic apparatus. The MS 25% medium can be used for in vitro cultivation of B. antiacantha, enabling the development of plants with suitable physiological qualities for planting in the field.

Key words:
bromeliad; chlorophyll a fluorescence; plant physiology; tissue culture

Resumo

Cultivo in vitro é uma técnica de grande aplicabilidade para micropropagação. Porém, cada espécie tem uma necessidade mineral mais adequada para o seu cultivo. O objetivo foi verificar os impactos da solução salina do meio de cultivo no desempenho do aparato fotossintético e crescimento de Bromelia antiacantha durante o cultivo in vitro, e dessa forma, elucidar a mitigação do desbalanço nutricional que poderia interferir na cadeia de transporte de elétrons nas plantas. Plântulas foram cultivadas em um gradiente de concentração do meio MS (0%, 25%, 50%, 75% ou 100%). Foram analisadas as variáveis de crescimento e a fluorescência da clorofila a. Concentrações intermediárias de sais do meio MS resultaram em plantas com maior número de folhas e comprimento de raiz. As curvas OJIP e a análise do teste JIP mostraram que as plantas cultivadas na ausência dos sais MS apresentaram menor eficiência do fotossistema II (FSII), como evidenciado por meio do índice de performance [Pi(total)]. Contrariamente, concentrações intermediárias (MS 25% e 50%) atuaram positivamente na performance do aparato fotossintético. O meio MS 25% pode ser utilizado para o cultivo in vitro de B. antiacantha, possibilitando o desenvolvimento de plantas com qualidades fisiológicas adequadas para o cultivo.

Palavras-chave:
bromélia; fluorescência da clorofila a; fisiologia vegetal; cultura de tecidos

Introduction

Ornamental bromeliads, native to the tropical and subtropical zones of Central and South America, are among the most commercially important ornamental plants in the world, occupying a valuable position in horticulture and the flower industry (Zhang et al. 2012Zhang F, Wang W, Ge Y, Shen X, Tian D, Liu J, Liu X, Yu X & Zhang Z (2012) Genetic relatedness among Aechmea species and hybrids inferred from AFLP markers and pedigree data. Scientia Horticulturae 139: 39-45.).

Among the most prevalent methods for large-scale propagation of horticultural species is tissue culture, mainly of ornamental species like orchids and bromeliads (Silva et al. 2017aSilva CS, Araújo LG, Sousa KCI, Silva DM, Sibov ST & Faria PR (2017a) In vitro germination and development of the Cerrado epiphytic orchid. Ornamental Horticulture 23: 96-100.; Lembrechts et al. 2017Lembrechts R, Ceusters N, De Proft M & Ceusters J (2017) Sugar and starch dynamics in the medium-root-leaf system indicate possibilities to optimize plant tissue culture. Scientia Horticulturae 224: 226-231.; Rosa et al. 2018Rosa WS, Martins JPR, Rodrigues ES, Rodrigues LCA, Gontijo ABPL & Falqueto AR (2018) Photosynthetic apparatus performance in function of the cytokinins used during the in vitro multiplication of Aechmea blanchetiana (Bromeliaceae). Plant Cell, Tissue and Organ Culture 133: 339-350.). The in vitro multiplication of bromeliads can be accomplished by direct or indirect organogenesis (Martins et al. 2014Martins JPR, Schimildt ER, Alexandre RS, Castro EM, Nani TF, Pires MF & Pasqual M (2014) Direct organogenesis and leaf-anatomy modifications in vitro of Neoregelia concentrica (Vellozo) L.B. Smith (Bromeliaceae). Pakistan Journal of Botany 46: 2179-2187.; Simão et al. 2016Simão MJ, Fonseca E, Garcia R, Mansur E & Pacheco G (2016) Effects of auxins and different culture systems on the adventitious root development ofPassiflora pohliiMast. and their ability to produce antioxidant compounds. Plant Cell, Tissue and Organ Culture 124: 419-430.). Studies of the growth and physiology of bromeliads in vitro also have been conducted (Martins et al. 2014Martins JPR, Schimildt ER, Alexandre RS, Castro EM, Nani TF, Pires MF & Pasqual M (2014) Direct organogenesis and leaf-anatomy modifications in vitro of Neoregelia concentrica (Vellozo) L.B. Smith (Bromeliaceae). Pakistan Journal of Botany 46: 2179-2187., 2016Martins JPR, Verdoodt V, Pasqual M & De Proft M (2016) Physiological responses by Billbergia zebrina (Bromeliaceae) when grown under controlled microenvironmental conditions. African Journal of Biotechnology 15: 1952-1961., 2018Martins JPR, Rodrigues LCA, Santos ER, Batista BG, Gontijo ABPL & Falqueto AR (2018) Anatomy and photosystem II activity of in vitro grown Aechmea blanchetiana as affected by 1-naphthaleneacetic acid. Biologia Plantarum 62: 211-221.; Simão et al. 2016Simão MJ, Fonseca E, Garcia R, Mansur E & Pacheco G (2016) Effects of auxins and different culture systems on the adventitious root development ofPassiflora pohliiMast. and their ability to produce antioxidant compounds. Plant Cell, Tissue and Organ Culture 124: 419-430.; Corredor-Prado et al. 2019Corredor-Prado JP, De Conti D, Roecker Júnior D, Cangahuala-Inocente GC, Guerra MP, Vesco LLD & Pescador R (2019) Proteomic identification of differentially altered proteins during regeneration from nodular cluster cultures in Vriesea reitzii (Bromeliaceae). Journal of Plant Growth Regulation 38: 586-599.). Martins et al. (2015)Martins JPR, Pascal 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. observed that the saline gradient of the culture medium developed by Murashige & Skoog (1962)Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473-497., better known as MS, can have a strong impact on the morphogenetic responses of bromeliads during in vitro cultivation.

The mineral nutrition is directly involved in plants’ metabolism, affecting their growth and development. This aspect needs to be observed in micropropagation programs, since plants need to absorb nutrients from the culture medium until they can be transplanted to ex vitro conditions (Akin et al. 2017Akin M, Eyduran E, Niedz RP & Reed BM (2017) Developing hazelnut tissue culture medium free of ion confounding. Plant Cell, Tissue and Organ Culture 130: 483-494.; Poothong et al. 2017Poothong S, Morré J, Maier CS & Reed BM (2017) Metabolic changes and improved growth in micropropagated red raspberry “Indian summer” are tied to improved mineral nutrition. In Vitro Cellular & Developmental Biology - Plant 53: 579-590.). The benefits of optimizing the nutrients in culture media are well documented for a wide range of species and applications (Hand & Reed 2014Hand C & Reed BM (2014) Minor nutrients are critical for the improved growth of Corylus avellana shoot cultures. Plant Cell, Tissue and Organ Culture 119: 427-439.; Tavares et al. 2015Tavares AR, Kanashiro S, Ribeiro RCS, Gonçalves AN & Jocys T (2015) Effect of phosphorus on in vitro growth and development of bromeliad Aechmea blanchetiana. Acta Horticulturae 1083: 241-248., 2017Tavares AR, Gonçalves AN, Ribeiro RCS, Jocys T & Kanashiro S (2017) Effect of nitrogen, phosphorus and calcium on in vitro growth and development of Aechmea blanchetiana. Acta Horticulturae 1155: 399-408.; Wada et al. 2015Wada S, Niedz RP & Reed BM (2015) Determining nitrate and ammonium requirements for optimal in vitro response of diverse pear species. In Vitro Cellular & Developmental Biology 51: 19-27.; Leljak-Levanic et al. 2016Leljak-Levanić D, Mrvková M, Turečková V, Pěnčík A, Rolčík J, Strnad M & Mihaljević S (2016) Hormonal and epigenetic regulation during embryogenic tissue habituation in Cucurbita pepo L. Plant Cell Reports 35: 77-89.; Akin et al. 2017Akin M, Eyduran E, Niedz RP & Reed BM (2017) Developing hazelnut tissue culture medium free of ion confounding. Plant Cell, Tissue and Organ Culture 130: 483-494.; Dias et al. 2017Dias GMG, Soares JDR, Ribeiro SF, Martins AD, Paqual M & Alves E (2017) Morphological and physiological characteristics in vitro anthurium plantlets exposed to silicon. Crop Breeding and Applied Biotechnology 17: 18-24.; Poothong et al. 2017Poothong S, Morré J, Maier CS & Reed BM (2017) Metabolic changes and improved growth in micropropagated red raspberry “Indian summer” are tied to improved mineral nutrition. In Vitro Cellular & Developmental Biology - Plant 53: 579-590.; Silva et al. 2017bSilva PPA, Kurita FMK & Tamaki V (2017b) In vitro propagation of Ananas comosus var. ananassoides (Baker) Coppens & F. Leal (Bromeliaceae). Científica 45: 313-320.; Assis et al. 2018Assis FA, Souza GA, Dias GMG, Assis GA, Rodrigues FA, Pasqual M, Costa BNS & Carvalho FJ (2018) Silicon and agar on in vitro development of cockscomb (Amaranthaceae). Brasília. Pesquisa agropecuária brasileira 53: 30-41.; Pérez-Alonso et al. 2018Pérez-Alonso N, Martín R, Capote A, Pérez A, Hernández-Díaz EK, Rojas L, Jiménez E, Quiala E, Angenon G, Garcia-Gonzales R & Chong-Pérez B (2018) Efficient direct shoot organogenesis, genetic stability and secondary metabolite production of micropropagated Digitalis purpurea L. Industrial Crops & Products 116: 259-266.). The success of micropropagation can depend on the nature and concentration of mineral nutrients in the medium, and this can vary and have different effects depending on the species, genotype or technique employed (Akin et al. 2017Akin M, Eyduran E, Niedz RP & Reed BM (2017) Developing hazelnut tissue culture medium free of ion confounding. Plant Cell, Tissue and Organ Culture 130: 483-494.; Poothong et al. 2017Poothong S, Morré J, Maier CS & Reed BM (2017) Metabolic changes and improved growth in micropropagated red raspberry “Indian summer” are tied to improved mineral nutrition. In Vitro Cellular & Developmental Biology - Plant 53: 579-590.). These differences have led to adaptations of the existing culture media and even development of new formulations (Greenway et al. 2012Greenway MB, Phillips IC, Lloyd MN, Hubstenberger JF & Phillips GC (2012) A nutrient medium for diverse applications and tissue growth of plant species in vitro. In Vitro Cellular & Developmental Biology - Plant 48: 403-410.; Poothong & Reed 2014Poothong S & Reed BM (2014) Modeling the effects of mineral nutrition for improving growth and development of micropropagated red raspberries. Scientia Horticulturae 165: 132-141.; Andrade & Tamaki 2016Andrade SV & Tamaki V (2016) In vitro growth of Nidularium Minutum Mez (Bromeliaceae) in different concentrations of nitrogen, phosphorus, potassium, and calcium. Journal of Plant Nutrition 39: 1634-1643.). Besides this, physiological disturbances of plants grown in vitro due to abiotic factors have often been reported (Magyar-Tábori et al. 2010Magyar-Tábori K, Dobránszki J, Bulley SM, Teixeira da Silva JÁ & Hudák I (2010) The role of cytokinins in hoot organogenesis in apple. Plant Cell, Tissue and Organ Culture 101: 251-267.; Martins et al. 2015Martins JPR, Pascal 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., 2016Martins JPR, Verdoodt V, Pasqual M & De Proft M (2016) Physiological responses by Billbergia zebrina (Bromeliaceae) when grown under controlled microenvironmental conditions. African Journal of Biotechnology 15: 1952-1961.).

Evaluations by the chlorophyll a fluorescence technique can enable verifying the physiological state of plants based on the detection of alterations in some components of photosystem II (PSII), components of the electron transport chain, and light-dependent photochemical reactions (Lotfi et al. 2018Lotfi R, Kalaji HM, Valizadeh GR, Khalilvand Behrozyar E, Hemati A, Gharavi-Kochebagh P & Ghassemi A (2018) Effects of humic acid on photosynthetic efficiency of rapeseed plants growing under different watering conditions. Photosynthetica 56: 962-970.). By means of the technique’s measurements, it is possible to detect changes in the bioenergy status of plants’ general photosynthetic apparatus (Borawska-Jarmułowicz et al. 2014Borawska-Jarmułowicz B, Mastalerczuk G, Kalaji MH, Carpentier R, Pietkiewicz S & Allakhverdiev SI (2014) Photosynthetic efficiency and survival of Dactylis glomerata and Lolium perenne following low temperature stress. Russian Journal of Plant Physiology 61: 281-288.), with numerous advantages. It is a very precise and nondestructive method that allows analyzing a large number of samples in a brief time interval (Zivcak et al. 2013Zivcak M, Brestic M, Balatova Z, Drevenakova P, Olsovska K, Kalaji HM, Yang X & Allakhverdiev SI (2013) Photosynthetic electron transport and specific photoprotective responses in wheat leaves under drought stress. Photosynthesis Research 117: 529-546.; Goltsev et al. 2016Goltsev VN, Kalaji HM, Paunov M, Bąba W, Horaczek T, Mojski J, Kociel H & Allakhverdiev SI (2016) Variable chlorophyll fluorescence and its use for assessing physiological condition of plant photosynthetic apparatus. Russian Journal of Plant Physiology 63: 869-893.). By applying the OJIP test, it is possible to obtain qualitative and quantitative visual information about the entire photosynthetic apparatus or about specific aspects of the PSII, intersystem and photosystem I (PSI). Besides this, the curves obtained from the OJIP test can be quantified by the JIP test, which supplies quantitative information about the productivity and efficiency of the photosynthetic apparatus (Kalaji et al. 2016Kalaji HM, Jajoo A, Oukarroum A, Brestic M, Zivcak M, Samborska IA, Cetner MD, Łukasik I, Goltsev V & Ladle RJ (2016) Chlorophyll a fluorescence as a tool to monitor physiological status of plants under abiotic stress conditions. Acta Physiologiae Plantarum 4: 1-11.; Rosa et al. 2018Rosa WS, Martins JPR, Rodrigues ES, Rodrigues LCA, Gontijo ABPL & Falqueto AR (2018) Photosynthetic apparatus performance in function of the cytokinins used during the in vitro multiplication of Aechmea blanchetiana (Bromeliaceae). Plant Cell, Tissue and Organ Culture 133: 339-350.).

Among the bromeliad species, Bromelia antiacantha Bertol. has strong potential for medicinal, nutritional, ornamental and industrial uses (Krumreich et al. 2015Krumreich FD, Corrêa APA, Silva SDS & Zambiazi RC (2015) Composição físico-química e de compostos bioativos em frutos de Bromelia antiacantha Bertol. Revista Brasileira de Fruticultura 37: 450-456.; Vallés & Cantera 2018Vallés D & Cantera AMB (2018) Antiacanthain A: new proteases isolated from Bromelia antiacantha Bertol. (Bromeliaceae). International Journal of Biological Macromolecules 113: 916-923.). In vitro propagation techniques have been widely studied and used for rapid multiplication of various economically important plant species, including other bromeliads such as Ananas comosus var. comosus (Scherer et al. 2015Scherer RF, de Freitas Fraga HP, Klabunde GF, Silva DA & Guerra MP (2015) Global DNA methylation levels during the development of nodule cluster cultures and assessment of genetic fidelity of in vitro-regenerated pineapple plants (Ananas comosus var. comosus). Journal of Plant Growth Regulation 34: 677-683.), Billbergia zebrina (Herb.) Lindl. (Martins et al. 2015Martins JPR, Pascal 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.), Vriesea cacuminis L.B. Sm. (Resende et al. 2016Resende CF, Ribeiro C, Mendes GC, Soares CQG, Braga VF, Cruz BP, Forzza RC & Peixoto PHP (2016) In vitro culture of Vriesea cacuminis L.B. Sm. (Bromeliaceae): an endemic species of Ibitipoca State Park, MG, Brazil. Iheringia, Série Botânica 71: 55-61.), Ananas comosus var. ananassoides (Baker) Coppens & F. Leal (Silva et al. 2017bSilva PPA, Kurita FMK & Tamaki V (2017b) In vitro propagation of Ananas comosus var. ananassoides (Baker) Coppens & F. Leal (Bromeliaceae). Científica 45: 313-320.), Vriesea reitzii Leme & Costa (Corredor-Prado et al. 2019Corredor-Prado JP, De Conti D, Roecker Júnior D, Cangahuala-Inocente GC, Guerra MP, Vesco LLD & Pescador R (2019) Proteomic identification of differentially altered proteins during regeneration from nodular cluster cultures in Vriesea reitzii (Bromeliaceae). Journal of Plant Growth Regulation 38: 586-599.), Aechmea ramosa Mart. ex Schult. f. (Faria et al. 2018Faria DV, Simão MJ, Cipriano R, Werner ET, Soares TCB, Aoyama EM & Gontijo ABPL (2018) In vitro morphogenesis and micropropagation of Aechmea ramosa var. ramosa Mart. ex Schult. f. (Bromeliaceae) from leaf explants. In Vitro Cellular & Developmental Biology - Plant 54: 530-536.), 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: 4., 2018Sasamori MH, Endres Júnior D & Droste A (2018) In vitro propagation of Vriesea incurvata: conservation of a bromeliad endemic to the Atlantic Forest. Iheringia, Série Botânica 73: 151-158.; Pulido-Rueda et al. 2018Pulido-Rueda EE, Milaneze-Gutierre MA & Negrelle R (2018) In vitro germination and growth of Vriesea incurvata Gaudich. (Bromeliaceae). Acta Agronómica 67: 140-145.), and Aechmea blanchetiana (Tavares et al. 2015Tavares AR, Kanashiro S, Ribeiro RCS, Gonçalves AN & Jocys T (2015) Effect of phosphorus on in vitro growth and development of bromeliad Aechmea blanchetiana. Acta Horticulturae 1083: 241-248., 2017Tavares AR, Gonçalves AN, Ribeiro RCS, Jocys T & Kanashiro S (2017) Effect of nitrogen, phosphorus and calcium on in vitro growth and development of Aechmea blanchetiana. Acta Horticulturae 1155: 399-408.; Martins et al. 2018Martins JPR, Rodrigues LCA, Santos ER, Batista BG, Gontijo ABPL & Falqueto AR (2018) Anatomy and photosystem II activity of in vitro grown Aechmea blanchetiana as affected by 1-naphthaleneacetic acid. Biologia Plantarum 62: 211-221., 2019Martins JPR, Rodrigues LCA, Silva TS, Santos ER, Falqueto AR & Gontijo ABPL (2019) Sources and concentrations of silicon modulate the physiological and anatomical responses of Aechmea blanchetiana (Bromeliaceae) during in vitro culture. Plant Cell, Tissue and Organ Culture 137: 397-410.; Rosa et al. 2018Rosa WS, Martins JPR, Rodrigues ES, Rodrigues LCA, Gontijo ABPL & Falqueto AR (2018) Photosynthetic apparatus performance in function of the cytokinins used during the in vitro multiplication of Aechmea blanchetiana (Bromeliaceae). Plant Cell, Tissue and Organ Culture 133: 339-350.), among others. In particular, Mercier & Yoshida (1998)Mercier H & Yoshida MK (1998) Bromelain activity in the leaf tissue of Bromelia antiacantha. Journal of the Bromeliad Society 48: 6-10. studied the activity of bromelain in the leaf tissues of B. antiacantha plants grown in vitro, but they did analyze the physiological quality of the plants.

Although complete MS medium (100%) is most often used for in vitro cultivation, for some species a dilution of macronutrients can produce better results (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: 4.). Dilutions MS medium salts have been used for in vitro cultivation of several bromeliad species, such as Ananas comosus var. ananassoides (Baker) Coppens & F. Leal (Silva et al. 2017bSilva PPA, Kurita FMK & Tamaki V (2017b) In vitro propagation of Ananas comosus var. ananassoides (Baker) Coppens & F. Leal (Bromeliaceae). Científica 45: 313-320.). The demand for mineral nutrients accompanies the specific life forms found within the group of bromeliads. In this context, knowing the effect of modifications of the traditional MS culture medium on the in vitro cultivation of B. antiacantha can support the development of a more suitable protocol for in vitro cultivation of this species. Therefore, the aim of this study was to observe the impacts of the saline solution of MS culture medium on the performance of the photosynthetic apparatus and growth of B. antiacantha during in vitro cultivation, and thus to elucidate the mitigation of nutritional imbalance, which can interfere in the electron transport chain of plants.

Material and Methods

Plant material

Bromelia antiacantha seeds were removed from ripe fruits collected from 15 matrix plants from areas in sandbank forest in the municipality of São Mateus, Espírito Santo state, Brazil. They were washed in tap water to remove the mucilage, dried on paper towels, placed in paper envelopes and stored at a temperature of 4 oC.

For performance of the experiment, a batch in which seeds from two or more fruits from each plant were mixed with seeds from other plants, to obtain a sample composite representative of the population diversity of B. antiacantha present in the collection areas.

In vitro cultivation

The seeds were disinfested in 70% ethanol for five minutes, followed by a 1% (v/v) sodium hypochlorite solution with three droplets of Tween 20 for five minutes. Then the seeds were washed three times in sterile distilled water. After disinfestation, the seeds were placed in flasks containing 50 ml of different concentrations (v/v) of MS medium salts (0%, 25%, 50%, 75% or 100%), obtained by serial dilution of the original composition proposed by Murashige & Skoog (1962)Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473-497.. All the media were supplemented with 30 g L-1 sucrose, solidified with 8 g L-1 agar, and the pH was adjusted to 5.8 before autoclaving at 120 ºC for 20 minutes. After inoculation, the plant material was kept for 90 days in a growth room with 16:8 hour photoperiod, under light intensity of 80 mmol.m-2.s-1 and temperature of 26 ± 1 ºC.

Plant growth

After cultivation for 90 days, the number of leaves, number of roots, aerial part length, root length and leaf area were recorded, in the last case using a leaf area meter (LI-COR L1-3100C). For analysis of the growth variables, 24 plants were collected at random and divided into six portions, thus composing six repetitions per treatment. For leaf area, five plants were used per treatment.

Chlorophyll a fluorescence analysis

The photosynthetic efficiency was analyzed between 8:00 and 10:00 a.m. by measurements of the chlorophyll a fluorescence using a Handy-PEA continuous excitation fluorometer (Hansatech, UK), according to the recommendations of Strasser et al. (2004)Strasser RJ, Tsimilli-Michael M & Srivastava A (2004) Analysis of the chlorophyll a fluorescence transient. In: Papageorgiou GC & Govindjee (eds.) Chlorophyll fluorescence: a signature of photosynthesis. Advances in photosynthesis and respiration series. Springer, Dordrecht. Pp. 321-362.. Before the readings, the leaves were adapted to the dark using leaf clips for 30 minutes, sufficient for complete oxidation of the photosynthetic system. Then a flash of light was emitted with saturation irradiance of 3,000 mmol photons m-2s-1 on the leaves, with duration of 1 second. The fluorescence intensity was measure at 50 ms, 100 ms, 300 ms, 2 ms, 30 ms and 1 s. Based on the OJIP fluorescence transient, the parameters were calculated as established by the JIP test. The interpretation and normalization of the parameters measured and calculated using this test were performed according to Strasser et al. (2004)Strasser RJ, Tsimilli-Michael M & Srivastava A (2004) Analysis of the chlorophyll a fluorescence transient. In: Papageorgiou GC & Govindjee (eds.) Chlorophyll fluorescence: a signature of photosynthesis. Advances in photosynthesis and respiration series. Springer, Dordrecht. Pp. 321-362. and Stirbet & Govindjee (2011)Stirbet A & Govindjee (2011) On the relation between the Kautsky effect (chlorophyll a fluorescence induction) and photosystem II: basics and applications of the OJIP fluorescence transient. Journal of Photochemistry and Photobiology B: Biology 104: 236-257.. The chlorophyll a fluorescence measurements were carried out with eight plants per treatment.

Statistical analysis

The experimental design was completely randomized and the data obtained were submitted to analysis of variance (ANOVA) and the means were compared by the Tukey test at 5% probability, using the SISVAR 5.4 software (Ferreira 2011Ferreira DF (2011) Sisvar: a computer statistical analysis system. Ciência e Agrotecnologia 35: 1039-1042.).

Results

In vitro growth

No differences were observed in relation to the control (MS 100%) in the treatments with different salt concentrations for number of roots. In MS 25% and MS 50% favored greater root length (Fig. 1). For all the variables related to growth of the aerial part, the treatment without addition of salts (MS 0%) presented the smallest values, mainly in relation to the plants grown in MS 50% medium.

Figure 1
Growth traits of Bromelia antiacantha plants grown in vitro for 90 days at different concentrations of MS medium salts. Means followed by the same letter, in each growth trait, do not different by the Tukey test at 5% significance.

Chlorophyll a fluorescence

The polyphasic OJIP curves of the plants presented typical polyphasic behavior, with increasing magnitude of the fluorescence signals from the basal level (called F0) to the maximum level (called Fm), with well-defined intermediate points J and I (Fig. 2a). In the O-J phase, there was a rise in the curves in all the treatments, a pattern that was most pronounced in the treatment with MS 0%. Starting at the J-I phase, the curve of MS 0% began to decrease.

Figure 2
a-b. Chlorophyll a fluorescence transients Bromelia antiacantha during in vitro cultivation in function of different concentrations of MS medium salts - a. fluorescence intensity; b. relative variable fluorescence [VOP = (Ft-F0) / (Fm-F0)] and kinetic differences of VOP [DVOP[treatment] - VOP[control]].

The relative variable fluorescence between points O and P (VOP) was greatest in plants grown in MS 0% (Fig. 2b). The kinetic difference curves of the transient fluorescence related to the control [DVOP(treatment) - VOP(control)] presented negative bands in phases O-J, J-I, and I-P in the treatments with MS 25% and MS 50%. Virtually no variations were observed of the parameter DVOP for the plants grown in MS 75% in relation to the control (MS 100%), while the MS 0% treatment was the only one that presented more pronounced positive bands.

The relative fluorescence between steps O (50 ms) and K (300 ms) [VOK = (Ft - F0) / (FK - F0)] was normalized and presented kinetic difference of DVOK [DVOK = VOK(treatment) - VOK(control)] also called L-band (Fig. 3a). The L-bands appeared at approximately 0.12 ms. The relative fluorescence between points O (50 ms) and J (2 ms) [VOJ = (Ft - F0) / (FJ - F0)] was normalized and is shown as kinetic difference [DVOJ = (VOJ (treatment) - VOJ (control)] (Fig. 3c), revealing K-band. Positive L- and K-bands were observed for the plants grown in MS 0%, which differed from the other treatments (Fig. 3b and 3d, respectively). On the other hand, negative L- and K-bands were observed in the other treatments, with the L-bands being most pronounced in the plants cultivated in MS 25% and MS 50%, which differed from the other treatments (Fig. 3d).

Figure 3
a-d. Chlorophyll a fluorescence transients Bromelia antiacantha during in vitro cultivation in function of different concentrations of MS medium salts - a. variable fluorescence between steps 0 and K [VOK = (Ft-F0) / (FK-F0)] and kinetic difference of VOK [DVOK = VOK(treatment) - VOK(control)]; b. means of VOK(0.12ms) followed by the same letter are not significantly different according to the Tukey test at 5%; c. variable fluorescence between steps O to J [VOJ = (Ft-F0) / (FJ-F0)] and kinetic difference of VOJ [DVOJ = (VOJ(treatment) - VOJ (control)]; d. means of VOJ(0.3ms) followed by the same letter are not significantly different according to the Tukey test at 5%.

The lowest values of F0 were observed for the plants grown in MS 100% and MS 75%, with differences only occurring in relation to MS 0%. The maximum photochemical efficiency values of PSII (jP0 = FV/FM = TR0/ABS) were lowest in MS 0% and differed from the other treatments (Fig. 4).

Figure 4
JIP test parameters as a function of MS concentrations obtained based on chlorophyll a fluorescence of Bromelia antiacantha. Means accompanied by an asterisk are significantly different by the Tukey test at 5% significance. F0 = initial fluorescence; φP0 = maximum quantum yield of primary photochemistry at (t = 0); ABS/RC = absorption flux per RC; TR0/RC = trapping flux (leading to QA reduction) per RC; ET0/RC = electron transport flux (further than QA-) per RC; DI0/RC = dissipated energy flux per RC (at t = 0); RC/CS = density of reaction center per cross section; Pi(TOTAL) = overall performance index, which measures the performance up until the final electron acceptors of PS I; SM/Tfmax = average fraction of open RC in the period of 0 to TFmax (time of maximum fluorescence production).

In general, the specific energy flux values per reaction center (RC) decreased with declining concentration of the MS medium, until the concentration of 25%, and the highest values were obtained in MS 0% (Fig. 4). For the energy transport flux per reaction center (ET0/RC) and the reaction center density per cross section (RC/CS), no differences were noted between the treatments. The photochemical performance index [Pi(TOTAL)] was lowest in MS 0%, and was significantly different from the other treatments. The SM/Tfmax (average fraction of open RCs in time period 0; Tfmax = time of maximum fluorescence production) did not differ between the different concentrations of salts in the MS medium.

Discussion

For Bromelia antiacantha, the concentrations of MS 25% and MS 50% favored longer root length, showing the advantage of using these dilutions to promote this growth trait. Dilution of the MS medium was observed to be advantageous for other species of bromeliads cultivated in vitro, such as Ananas comosus var. ananassoides (Baker) Coppens & F. Leal (Silva et al. 2017bSilva PPA, Kurita FMK & Tamaki V (2017b) In vitro propagation of Ananas comosus var. ananassoides (Baker) Coppens & F. Leal (Bromeliaceae). Científica 45: 313-320.). The dilution of the MS medium, and especially of the nitrogen compounds, increased the rooting and number of leaves in Vriesea incurvata cultivated in vitro (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: 4.). Smaller concentrations of nitrogen (15 mM) in the MS medium were suggested to optimize multiplication of A. comosus var. ananassoides (Silva et al. 2017bSilva PPA, Kurita FMK & Tamaki V (2017b) In vitro propagation of Ananas comosus var. ananassoides (Baker) Coppens & F. Leal (Bromeliaceae). Científica 45: 313-320.).

The observation of the difference of the relative variable fluorescence between F0 and FM (DVOP) revealed a large variation of fluorescence in the MS 0% medium, indicating that the plants submitted to this treatment suffered damage to the intersystem electron carriers, thus requiring a minimum concentration of salts in the MS medium. Nutrients such as Cu (micronutrient), Mg and Ca (macronutrients) are essential to plants as components of the photosynthetic apparatus and of enzymes that act during photosynthesis (Poothong et al. 2017Poothong S, Morré J, Maier CS & Reed BM (2017) Metabolic changes and improved growth in micropropagated red raspberry “Indian summer” are tied to improved mineral nutrition. In Vitro Cellular & Developmental Biology - Plant 53: 579-590.; Kwano et al. 2017Kwano BH, Moreira A, Moraes LAC & Nogueira MA (2017) Magnesium-manganese interaction in soybean cultivars with diferente nutritional requirements. Journal of Plant Nutrition 40: 372-381.). The absence of these nutrients limits the electron transfer process (Purohit 2018Purohit S (2018) Increased morphogenetic competence in Cuminum cyminum L. mediated through micronutrient manipulation. Journal of Medicinal Plants Studies 6: 141-144.), as observed in the results of DVOP during the in vitro cultivation of B. antiacantha.

Point J is directly involved in the constant changes in the transfer of electrons from quinone A (QA) to quinone B (QB) (Mehta et al. 2010Mehta P, Allakhverdiev SI & Jajoo A (2010) Characterization of photosystem II heterogeneity in response to high salt stress in wheat leaves (Triticum aestivum). Photosynthesis Research 105: 249-255.). This inflection in point J observed in the curve of MS 0% represents a double reduction of the pheophytin electron carriers, QA and QB (Chen et al. 2014Chen S, Strasser RJ & Qiang S (2014) In vivo assessment of effect of phytotoxin tenuazonic acid on PSII reaction center. Plant Physiology and Biochemistry 84: 10-21.), which possibly indicates damage caused by the absence of basic nutrients for this process. For point I, no substantial alterations were observed. This can be attributed to the distinct dissipative pathways that lead to complete closing of the PSII reaction center at point I, in which the alterations are related to events that occur before reduction of the pool of plastoquinones (PQ) (Chen et al. 2014Chen S, Strasser RJ & Qiang S (2014) In vivo assessment of effect of phytotoxin tenuazonic acid on PSII reaction center. Plant Physiology and Biochemistry 84: 10-21.).

Positive amplitudes of L- and K-bands are an indicator of a reduction of the plastoquinone pool in plants cultivated in MS 0%. These bands can be considered potential markers of disturbances before the appearance of visible signs of response to stress (Meng et al. 2016Meng LL, Song JF, Wen J, Zhang J & Wei JH (2016) Effects of drought stress on fluorescence characteristics of photosystem II in leaves of Plectranthus scutellarioides. Photosynthetica 54: 414-421.). The appearance of L-band is an indicator of energy connectivity or grouping of units of PSII, which presents a positive deviation when the connectivity is low (Yusuf et al. 2010Yusuf MA, Kumar D, Rajwanshi R, Strasser RJ, Tsimilli-Michael M, Govindje E & Sarin NB (2010) Overexpression of γ-tocopherol methyl transferase gene in transgenic Brassica juncea plants alleviates abiotic stress: physiological and chlorophyll a fluorescence measurements. Biochimica et Biophysica Acta - Bioenergetics 1797: 1428-1438.). The positive L-band in plants grown in MS 0% indicates some level of disturbance in the membranes of the thylakoid membranes, reducing the connectivity between the reaction centers (RCs) of PSII (Rosa et al. 2018Rosa WS, Martins JPR, Rodrigues ES, Rodrigues LCA, Gontijo ABPL & Falqueto AR (2018) Photosynthetic apparatus performance in function of the cytokinins used during the in vitro multiplication of Aechmea blanchetiana (Bromeliaceae). Plant Cell, Tissue and Organ Culture 133: 339-350.). Therefore, the absence of nutrients can impair the stability of the subunits of PSII, causing disturbances in the energy connectivity (Chen & Cheng 2010Chen LS & Cheng L (2010) The acceptor side of photosystem II is damaged more severely than the donor side of photosystem II in “Honeycrisp” apple leaves with zonal chlorosis. Acta Physiologiae Plantarum 32: 253-261.). A positive L-band can appear in plants suffering from nutritional deficiency, which suggests that the photosynthetic system increases the dissipation to improve the use of excitation energy. On the other hand, the treatment involving MS 25% presented negative L-bands, demonstrating better use of the excitation energy and improved connectivity between the reaction centers, and consequently better stability of the system.

Likewise, the presence of the K-band also indicates disturbances in PSII, which are related to the imbalance between the donation of electrons from the oxygen evolution complex (OEC) and the QA- electron acceptors (Kalaji et al. 2016Kalaji HM, Jajoo A, Oukarroum A, Brestic M, Zivcak M, Samborska IA, Cetner MD, Łukasik I, Goltsev V & Ladle RJ (2016) Chlorophyll a fluorescence as a tool to monitor physiological status of plants under abiotic stress conditions. Acta Physiologiae Plantarum 4: 1-11.). These are linked to dissociation of the OEC, which uses manganese (Mn) as an essential cofactor in the oxidation of water. The presence of a positive K-band reflects the inactivation of the OEC and/or increase in the size of the functional antenna of PSII (Yusuf et al. 2010Yusuf MA, Kumar D, Rajwanshi R, Strasser RJ, Tsimilli-Michael M, Govindje E & Sarin NB (2010) Overexpression of γ-tocopherol methyl transferase gene in transgenic Brassica juncea plants alleviates abiotic stress: physiological and chlorophyll a fluorescence measurements. Biochimica et Biophysica Acta - Bioenergetics 1797: 1428-1438.). The larger the amplitude of this band, the greater will be the inactivation of the OEC (Adamski et al. 2011Adamski JM, Peters JA, Danieloski R & Bacarin MA (2011) Excess iron-induced changes in the photosynthetic characteristics of sweet potato. Journal of Plant Physiology 168: 2056-2062.). Therefore, the appearance of a positive K-band in the plants grown in MS 0% can be related to low manganese content in the leaves, the result of the absence of mineral salts in the MS medium. However, the presence of negative K-bands with greater amplitude in the plants grown in MS 25% and MS 50% suggests the maintenance of activity of the OEC. This indicates better balance between the electron acceptor and donor sides of PSII.

The increase of F0 in the plants grown in MS 0% is related to the reduced energy capture rate by PSII and can be attributed to the smaller number of active reaction centers, in turn caused by the smaller energy transfer from the light-harvesting complexes of PSII (LHCII) to the reaction centers. That effect was probably caused by the limitation of nutrients essential for the functioning of the photosynthetic apparatus. This behavior is a consequence of the dissociation of the LHCII from the nucleus of PSII or the inactivation of the oxygen evolution complex (Mathur et al. 2011Mathur S, Mehta P, Jajoo A & Bharti S (2011) Analysis of elevated temperature induced inhibition of Photosystem II using chl a fluorescence induction kinetics. Plant Biology 13: 1-6.; Ghotbi-Ravandi et al. 2014Ghotbi-Ravandi AA, Shahbazi M, Shariati M & Mulo P (2014) Effects of Mild and Severe Drought Stress on Photosynthetic Efficiency in Tolerant and Susceptible Barley (Hordeum vulgare L.) Genotypes. Journal of Agronomy and Crop Science 403-415.). The results obtained for K-band and L-band suggest that the increased fluorescence intensity observed in F0 can be attributed both to the inactivation of the OEC and the reduction of the energy connectivity between the subunits of PSII. This increase of F0 was documented as one of the most direct signs of photoinhibition in plants (Lotfi et al. 2018Lotfi R, Kalaji HM, Valizadeh GR, Khalilvand Behrozyar E, Hemati A, Gharavi-Kochebagh P & Ghassemi A (2018) Effects of humic acid on photosynthetic efficiency of rapeseed plants growing under different watering conditions. Photosynthetica 56: 962-970.).

Besides this, the increase of F0 together with the decrease of FM causes a lower value of jP0 in MS 0%. The reduction of jP0 caused by stress or limitation of nutrients in the MS medium is considered to be a marker of damages in PSII (Martins et al. 2015Martins JPR, Pascal 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.; Falqueto et al. 2017Falqueto AR, Silva Júnior RA, Gomes MTG, Martins JPR, Silva DM & PartellI FL (2017) Effects of drought stress on chlorophyll a fluorescence in two rubber tree clones. Scientia Horticulturae 224: 238-243.). However, the photodamage observed can be considered a positive adaptation caused by the regulation of the photochemical and photoprotective mechanisms, such as dissipation of non-photochemical energy, state transition, cyclic electron flow around PSI or also the water oxidation cycle (Miyake 2010Miyake C (2010) Alternative electron flows (water-water cycle and cyclic electron flow around PSI) in photosynthesis: molecular mechanisms and physiological functions. Plant & Cell Physiology 51: 1951-63.; Rochaix 2011Rochaix JD (2011) Regulation of photosynthetic electron transport. Biochimica et Biophysica Acta 1807: 375-83.; Keren & Krieger-Liszkay 2011Keren N & Krieger-Liszkay A (2011) Photoinhibition: molecular mechanisms and physiological significance. Physiologia plantarum 142: 1-5.; Raven 2011Raven JA (2011) The cost of photoinhibition. Physiologia Plantarum 142: 87-104.; Duffy et al. 2013Duffy CDP, Valkunas L & Ruban AV (2013) Light-harvesting processes in the dynamic photosynthetic antenna. Physical Chemistry Chemical Physics 15: 18752-18770.). These mechanisms prevent the super-reduction of the electron transport chain of photosynthesis and diminish the potential for damages caused by oxidative stress (Carvalho 2008Carvalho MHC (2008) Drought stress and reactive oxygen species. Plant Signal Behav 3: 156-165.; Nishiyama et al. 2011Nishiyama Y, Allakhverdiev SI & Murata N (2011) Protein synthesis is the primary target of reactive oxygen species in the photoinhibition of photosystem II. Physiologia Plantarum 142: 35-46.; Kalaji et al. 2011Kalaji HM, Bosa K, Kościelniak J & Żuk-Gołaszewska K (2011) Effects of salt stress on photosystem II efficiency and CO2 assimilation of two Syrian barley landraces. Environmental and Experimental Botany 73: 64-72.; Campos et al. 2014Campos H, Trejo C, Peña-Valdivia CB, Garcia-Nava R, Conde-Martinez FV & Cruz-Ortega MR (2014) Photosynthetic acclimation to drought stress in Agave salmiana Otto ex Salm-Dyck seedlings is largely dependent on thermal dissipation and enhanced electron flux to photosystem I. Photosynthesis Research 122: 23-39.; Ali et al. 2018Ali S, Xua Y, Jia Q, Ma X, Ahmad I, Adnan M, Gerard R, Ren X, Zhang P, Cai T, Zhang J & Jia Z (2018) Interactive effects of plastic film mulching with supplemental irrigation on winter wheat photosynthesis, chlorophyll fluorescence and yield under simulated precipitation conditions. Agricultural Water Management 207: 1-14.).

The increase in the absorption flux (ABS/RC) and trapping flux of energy per reaction centers (TR0/RC) complemented by the increased flux of energy dissipated per active reaction center (DI0/RC) suggests existence of a mechanism to protect the plants, as also observed in Solanum nigrum L. during acclimation (Swain et al. 2010Swain SS, Tripathy T, Mohapatra PK & Chand PK (2010) Photosynthetic and transpiration responses of in vitro-regenerated Solanum nigrum L. plants to ex vitro adaptation. In Vitro Cellular & Developmental Biology - Plant 46: 134-141.). The energy transport flux represents the rate of reoxidation of QA- and its increase indicates that the plant is using light energy combined with a lower energy dissipation rate (DI0/RC) (Lotfi et al. 2018Lotfi R, Kalaji HM, Valizadeh GR, Khalilvand Behrozyar E, Hemati A, Gharavi-Kochebagh P & Ghassemi A (2018) Effects of humic acid on photosynthetic efficiency of rapeseed plants growing under different watering conditions. Photosynthetica 56: 962-970.). The lower values of DI0/RC observed in the treatments with MS 25%, MS 50%, MS 75% and MS 100% indicate these were the treatments when greater light energy use occurred. Another study demonstrated that the light reactions and electron transport during photosynthesis can be altered when plants face shortage of nutrients (Wagner et al. 2016Wagner H, Jakob T, Lavaud J & Wilhelm C (2016) Photosystem II cycle activity and alternative electron transport in the diatom Phaeodactylum tricornutum under dynamic light conditions and nitrogen limitation. Photosynthesis Research 128: 151-161.). This can explain the need for nutrients for better functioning of the photosynthesis process.

According to the values of DI0/RC observed, the energy from the increases of ABS/RC and TR0/RC did not contribute to the energy transport flux (ET0/RC), and instead was dissipated in the form of heat or emission of fluorescence (DI0/RC), as also observed by Araújo & Deminicis (2009)Araújo SAC & Deminicis BB (2009) Fotoinibição da fotossíntese. Porto Alegre. Revista Brasileira de Biociências 7: 463-472. and Falqueto et al. (2017)Falqueto AR, Silva Júnior RA, Gomes MTG, Martins JPR, Silva DM & PartellI FL (2017) Effects of drought stress on chlorophyll a fluorescence in two rubber tree clones. Scientia Horticulturae 224: 238-243.. The increase of DI0/RC observed in the plants grown in MS 0% medium, which differed from the other treatments, suggests activation of an energy dissipation mechanism in response to reduced assimilation of CO2 (Falqueto et al. 2017Falqueto AR, Silva Júnior RA, Gomes MTG, Martins JPR, Silva DM & PartellI FL (2017) Effects of drought stress on chlorophyll a fluorescence in two rubber tree clones. Scientia Horticulturae 224: 238-243.). The efficiency with which absorbed light energy is used also is modulated during deprivation of nutrients (Wagner et al. 2016Wagner H, Jakob T, Lavaud J & Wilhelm C (2016) Photosystem II cycle activity and alternative electron transport in the diatom Phaeodactylum tricornutum under dynamic light conditions and nitrogen limitation. Photosynthesis Research 128: 151-161.).

The total performance index [Pi(TOTAL)] is a plant vitality indicator and has also been considered a sensitive parameter to detect stress in plants (Yusuf et al. 2010Yusuf MA, Kumar D, Rajwanshi R, Strasser RJ, Tsimilli-Michael M, Govindje E & Sarin NB (2010) Overexpression of γ-tocopherol methyl transferase gene in transgenic Brassica juncea plants alleviates abiotic stress: physiological and chlorophyll a fluorescence measurements. Biochimica et Biophysica Acta - Bioenergetics 1797: 1428-1438.; Gururani et al. 2017Gururani MA, Venkatesh J, Ghosh R, Strasser RJ, Ponpandian LN & Bae H (2017) Chlorophyll-a fluorescence evaluation of PEGinduced osmotic stress on PSII activity in Arabidopsis plants expressing SIP1. Plant Biosystems - Taylor & Francis Online 16: 11:42.; Lotfi et al. 2018Lotfi R, Kalaji HM, Valizadeh GR, Khalilvand Behrozyar E, Hemati A, Gharavi-Kochebagh P & Ghassemi A (2018) Effects of humic acid on photosynthetic efficiency of rapeseed plants growing under different watering conditions. Photosynthetica 56: 962-970.), because its calculation includes parameters related to conservation of energy from photons absorbed by PSII (ABS), trapping of excitation energy (TR), conversion of excitation energy for the transport of electrons to the intersystem (ET) and reduction of the final acceptors of PSI (RE) (Redillas et al. 2011Redillas MCFR, Strasser RJ, Jeong JS, Kim YS & Kim JK (2011) The use of JIP test to evaluate drought-tolerance of transgenic rice overexpressing OsNAC10. Plant Biotechnology Reports 5: 169-175.). This parameter responds not only to losses in the activity of PSII, but also to damages related to PSI (Xiang et al. 2013Xiang M, Chen S, Wang L, Donga Z, Huanga J, Zhanga Y & Strasser RJ (2013) Effect of vulculic acid produced by Nimbya alternantherae on the photosynthetic apparatus of Alternanthera philoxeroides. Plant Physiology and Biochemistry 65: 81-88.). Therefore, the lower value of Pi(TOTAL) found in the plants cultivated in MS 0% medium can cause loss of the structure and function of PSI, inhibiting the donation of electrons and reduction of the final acceptors of PSI, consequently contributing to the reductions of Pi(TOTAL).

Conclusion

  1. Use of the MS 0% medium impaired the photosynthetic apparatus, while MS 25% produced the best results compared to the other concentrations of salts tested.

  2. The MS 25% medium can be used for in vitro cultivation of B. antiacantha, enabling the development of plants with suitable physiological qualities for ex vitro cultivation.

Acknowledgements

The authors would like to acknowledge the scholarship awarded by CAPES (Coordination for the Improvement of Higher Education Personnel).

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Publication Dates

  • Publication in this collection
    08 Mar 2021
  • Date of issue
    2021

History

  • Received
    08 July 2019
  • Accepted
    09 Mar 2020
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