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View Full Text-PDF Int. J. Curr. Res. Biosci. Plant Biol. (2021) 8(4), 1-10 International Journal of Current Research in Biosciences and Plant Biology Volume 8 ● Number 04 (April-2021) ● ISSN: 2349-8080 (Online) Journal homepage: www.ijcrbp.com Original Research Article doi: https://doi.org/10.20546/ijcrbp.2021.804.001 The in vitro banana-leaf-fragment technique preserves the photosynthetic apparatus Edilia De la Rosa-Manzano 1†, Manuel Jesús Cach-Pérez 2†, Cecilia Mónica Rodríguez-García 3,*, Leticia Peraza-Echeverría3, José Luis Andrade4, Blondy Beatriz Canto-Canché 3, Rosa Grijalva-Arango3 1Instituto de Ecología Aplicada, Universidad Autónoma de Tamaulipas. Av. División del Golfo No. 356, Col. Libertad. C.P. 87019, Ciudad Victoria, Tamaulipas, México. 2CONACyT - Departamento de Agricultura, Sociedad y Ambiente, El Colegio de la Frontera Sur. Carretera a Reforma Km. 15.5 S/N, Ranchería Guineo 2a. Sección, 86280, Villahermosa, Centro, Tabasco, México. 3Unidad de Biotecnología, Centro de Investigación Científica de Yucatán A.C. Calle 43 No. 130, Col. Chuburná de Hidalgo, 97200 Mérida, Yucatán, México. 4Unidad de Recursos Naturales, Centro de Investigación Científica de Yucatán A.C. *Corresponding author; e-mail: [email protected]; [email protected] Article Info Abstract Keywords: In vitro studies of plant-pathogen interactions using leaf-fragments remains controversial compared to Benzimidazole those studies under field conditions. The leaf-fragments technique, which predominantly uses Electron transport rate benzimidazole in the culture medium to retard senescence, has been reported as reliable, fast, and Grand Nain banana inexpensive for analysis of aggressiveness and resistance in the Mycosphaerella fijiensis-Musa spp. genotype interaction. However, no data have been published verifying whether in vitro banana leaf fragments Photosystem II maintain photosynthetic activity, which is a requirement for studying this interaction. In this study, Transpiration rate maximum quantum efficiency of photosystem II, electron transport rate, transpiration, carbon dioxide exchange, light saturation point, and stomatal density were evaluated in in vitro leaf fragments of the Grand Nain banana genotype. Furthermore, the same parameters were also attained for leaves from plants in the field (during two seasons) and greenhouse conditions. The photosynthetic yield was constant during the experiment in leaf fragments with benzimidazole, and the photosynthetic rates on day 30 were similar throughout the whole experiment. This study supports that the banana-leaf- fragment technique as such protects the photosynthetic apparatus and then is suitable for studies on interactions such as that of M. fijiensis-Musa acuminata. Received: 18 January 2021 Revised: 25 March 2021 Accepted: 29 March 2021 Published Online: 6 April 2021 † Equally contributed to this work. E. de la Rosa-Manzano et al. (2021) / The in vitro banana-leaf-fragment technique preserves the photosynthetic apparatus 1 Int. J. Curr. Res. Biosci. Plant Biol. (2021) 8(4), 1-10 Introduction studies. For this purpose, an efficient, artificial M. fijiensis inoculation protocol was developed using leaf Bananas and plantains are important products fragments from mature banana plants grown under worldwide, providing food and economic security in optimal physiological conditions (Abadie et al., 2008), many countries of Latin America, the Caribbean, but no physiological parameters have been measured in Africa, and Asia. Developed countries in Europe, North these fragments. Other authors are not convinced of the America, and other regions are the primary consumers, effectiveness of using leaf fragments in quantitative importing 93 % of the 20.2 million tonnes of banana, studies. For instance, when using banana leaf fragments excluding plantain, exported globally in 2019 (FAO to detect M. fijiensis and quantify its biomass, Arzanlou 2020). Because both banana and plantain crops are et al. (2007) observed significant data variation due to affected by fungal diseases, successful cultivar breeding artificial inoculation and deemed the results as depends on study of the plant-pathogen interactions. In unreliable. this regard, the banana-leaf-fragment technique was developed to evaluate banana genotype resistance to the Environmental physiology in banana genotypes under aggressiveness of Mycosphaerella fijiensis Morelet (El field conditions for transpiration and CO2 exchange Hadrami, 2000), a fungal pathogen responsible for black evaluation has been thoroughly reviewed (Turner et al., Sigatoka disease. The development of this reliable, fast, 2007). However, no reports exist on photosynthetic cheap, and accurate method has been vital to studying parameters in banana leaf fragments under in vitro this interaction. Many studies have used the detached conditions. The primary question is if under these leaf technique, since Yarwood described it in 1946, conditions the leaf fragments are photosynthetically using exogenous agents to prevent tissue senescence active and thus a reliable model to study the M. fijiensis- (Osborne, 1967 cited by Mahapatra et al., 2015). Musa spp. interaction. The goal of this study was to Because leaf senescence is primarily the breakdown of document how an edible banana genotype, such as chlorophyll, the colour change of the leaf indicates the Grand Nain, behaves photosynthetically in in vitro (via physiological and biochemical changes associated with leaf fragments) and to compare the same physiological senescence (Woo et al., 2019). The main advantages of parameters for plants in greenhouse and field the detached leaf technique are to save space and host conditions. Physiological measurements were maximum material, the low-cost of pathogen inoculum under quantum efficiency of photosystem II (Fv/Fm), electron study, the effortless and accuracy of the observations, transport rate (ETR), transpiration, carbon dioxide and the possibility of maintaining the most prominent uptake, light saturation point, and stomatal density. features and biochemical functions of plant leaves, including respiration, photosynthesis, and protein synthesis (Kamicoga, 2001). Materials and methods The use of benzimidazole (N, N’-methylphenyl-o- Plant material phenylenediamine) has been one of the more successful treatments for tissue culture of wheat, rice, and millet The most important worldwide commercial Cavendish (Person et al., 1957; Singh and Mishra, 1975; banana, the black Sigatoka-susceptible Grand Nain Mahapatra et al., 2015), as well as for banana leaf cultivar (Musa acuminata Colla, Cavendish subgroup, fragments maintained in vitro (El Hadrami, 2000). AAA), was selected to characterise the photosynthetic Addition of benzimidazole in the culture medium parameters in three conditions: in vitro leaf fragments, retards senescence of detached leaves and avoids leaves of plants growing in the greenhouse, and leaves respiratory rates increase. In vitro assays for resistance of plants growing in the field. against M. fijiensis are effective using banana leaf fragments or detached leaves for rapid, massive The plantlets obtained by organogenesis consisted of screening of Musa spp. in breeding programs for black three phases: induction, multiplication, and rooting (Ma leaf streak resistance, before carrying out final and Shii, 1972). These were removed from the flasks, confirmation trials under field conditions (Twizeyimana washed with tap water to eliminate agar medium, their et al., 2007). Quantifying fungal damage by real-time roots detached, transported to the greenhouse for polymerase chain reaction is important in plant transplanting on trays containing a mixture of soil and resistance/susceptibility and pathogen aggressiveness sunshine® (1:2, w/w), and covered with a dome of E. de la Rosa-Manzano et al. (2021) / The in vitro banana-leaf-fragment technique preserves the photosynthetic apparatus 2 Int. J. Curr. Res. Biosci. Plant Biol. (2021) 8(4), 1-10 translucent plastic during 7 days to allow 100 % of (n=5). Under in vitro conditions, parameters were survival. Plantlets watered with Hoagland medium measured at 1, 5, 15, and 30 days after they were placed (Hoagland and Arnon, 1950) weekly, were transplanted on culture medium (n=10). to polybags with the same soil mixture as above after 60 days. The leaf fragments were cut from the plants with a Maximum quantum efficiency of photosystem II (Fv/Fm) height of 75 cm (approximately in 4 months) and the and electron transport rate (ETR) were obtained using a first two open leaves had a size of 30 cm long, and 16 portable pulse-amplitude-modulated photosynthesis cm width. yield analyser (MiniPAM, Walz, Germany). The fibre- optic was kept at a constant distance of about 20 mm In vitro conditions: Material for analysis consisted of leaf and an angle of approximately 60° to the upper surface fragments (25 cm2). Only the first and second healthy of the leaf or leaf fragment. Measurements in the field green leaves, previously disinfected with commercial and greenhouse were taken every 3 h during the day bleach and rinsed three times with purified water, were (from 06:00 to 18:00 h), and those in in vitro conditions used to obtain the fragments. They were placed in Petri were taken twice, in the dark (06:00 h) and light (10:00 dishes containing agar [0.5% (w/v) Merck, Damstadt, h). The Fv/Fm was obtained as (Fm ‒ Fo)/Fm and PSII -1 Germany] without and with 200 mg L benzimidazole efficiency (Yield) as (Fm´ ‒ Ft)/Fm´, where Fm is (Fluka, Steinheim, Germany), on the base
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