Salinity-Induced Alterations in the Growth , Membranes and Osmolytes of Musa spp . cv . Caipira ( AAA ) During Micropropagation

The aim of this study was to determine the concentration and exposure time to NaCl suitable for the micropropagation of banana, through the analysis of growth traits. Banana propagules were inoculated in MS medium with different concentrations of NaCl (0; 50; 75 and 100 mM) for 120 days (multiplication and rooting, 60 days each), with monthly subcultures. These propagules were measured for plant height, number of leaves, sprouting rate, average number of formed propagules, rooting rate, root length and survival rate. After 30 days, NaCl reduced sprouting rate at multiplication; the number of leaves, rooting rate and root length in rooting; and the height and propagules number in both phases. After 60 days, the NaCl affected the sprouting rate and propagules number in the multiplication; length of root in rooting; and the height and number of leaves in both phases. After 120 days, the reduction in the survival rate was proportional to the increase of NaCl in the medium. Thus, it is concluded that NaCl reduces most of the growth traits and the treatments with 75 and 100 mM NaCl affected multiplication and in vitro rooting more intensely.


Introduction
Brazil is the third largest fruit producer in the world, with banana cultivation having highest ranking with an average annual production of 6.7 million tons (IBGE, 2017).Banana cultivation is widely exploited in the irrigated perimeters of the Northeast of Brazil and has been expanding in arid and semi-arid regions where soil salinity is associated with low precipitation, high surface evaporation, brackish irrigation and poor cultivation practices.
Around 20% of total cultivated land and 33% of irrigated agricultural land is estimated to be affected by high salinity worldwide (Shrivastava & Kumar, 2015).Soil salinity is one of the main environmental factors that limit global agricultural production (Bessa, Lacerda, Amorim, Bezerra, & Lima, 2017), interfering with water supply and nutrients which mainly affects the development, growth and survival of plants (Cavalcante, Cordeiro, Nascimento, Cavalcante, & Dias, 2010).stabilization and avoids the degradation of proteins under stress conditions, playing an important role in the osmotic adjustment (Farooq, Wahid, Kobayashi, & Fujita, 2009).
Due to large declines in productivity in salinised areas, research has been done in the search for genotypes that are more resistant to salinity and, in the specific case of bananas, that improve characteristics to those that are already genetically resistant to pests, such as Caipira (AAA), which is resistant to yellow sigatoka, black sigatoka, rhizome-borer and panama disease (Ramos, M. Leonel, & S. Lenoel, 2009).
Especially in banana cultivation, the use of micropropagated seedlings is of great importance in production systems, mainly due to rapid multiplication which guarantees the physiological, genetic, phytosanitary and uniformity of the crop (Oliveira, Pereira, Nietsche, Soouza, & Costa, 2014).However, before performing in vitro selection, it is necessary to identify the degree of resistance of the species when subjected to the stress factors, which determines the doses of the selected agent to be added to the culture medium.
Studies carried out with in vitro grown diploid genotypes of banana showed that the presence of NaCl in the culture medium significantly reduced the number of leaves, height, pseudocaule diameter and leaf area, and also the fresh and dry phytomass of both shoots as well as the root portion (Silva et al., 2009).In addition, in bananas of the Nanicão cultivar (Musa spp.AAA), a concentration of 120 mM NaCl reduced the number and size of the buds and the regeneration rate of plants in vitro (Ulisses et al., 2000).With the Grand Naine cultivar, the increase in saline levels in in vitro cultivation significantly reduced leaf length, umber of leaves, fresh matter yield, shoot number and bud survival rate (Macêdo et al., 2005).Although the study was carried out on the banana tree, the studied cultivars and the doses of NaCl used were different from those used by Ulisses et al. (2000) and Silva et al. (2009), thus showing that the in vitro response is to cultivar-and dose-dependent.Therefore it is necessary to carry out preliminary studies to determine the dose of NaCl added to the culture medium.
Thus, the objective of this study was to determine a NaCl concentration, and a duration of exposure to salt, suitable for studies involving micropropagation of the banana cv.Caipira, with an emphasis on characterising the effects of salinity on growth, water status and osmotic adjustment indicators.

Method
Propagules of banana (Musa spp.cv.Caipira), from the germplasm of the Empresa de Pesquisa Agropecuária do Rio Grande do Norte (EMPARN-Jiqui), with an average age of two months, were obtained in vitro.The study was divided in two stages that comprise the micropropagation, one referred to as multiplication and the second as the rooting stage.
During the multiplication phase, two-month-old defoliated banana propagules measuring approximately 1.0 cm were inoculated in MS medium (Murashige & Skoog, 1962) supplemented with 200 mg L -1 of inositol, 30 g L -1 of sucrose, 4.5 mg L -1 of 6-Benzylaminopurine (BAP), and containing different concentrations of NaCl (0 mM for the control; 50, 75 and 100 mM).The medium was solidified with 2 g L -1 of Phytagel TM and the pH adjusted to 5.7.The flasks, containing 40 mL of the nutrient medium, were sterilised for 20 min at 1.5 atm and then the propagules were inoculated in the different saline treatments.Twenty-five propagules were distributed per treatment, and one seedlot per inoculum was inoculated, totaling 100 experimental units.The flasks containing the propagules were kept in a growth room at a temperature of 25±5 ºC, light intensity of 30 μmol m -2 s -1 and a 12/12 hours (light/dark) photoperiod for 60 days.
The propagules were subcultured every 30 days under aseptic laminar flow hood conditions and evaluated for height, leaf number, sprouting rate [(seedlings/inoculated seedlings) × 100] and the mean number of seedlings formed (total of propagules formed/number of propagules that formed new propagules).After 60 days in multiplication medium, the propagules were transferred to the rooting medium with half of the nutrients available in the MS solution and supplemented with 100 mg L -1 of inositol and 15 g L -1 of sucrose.In the middle of the rooting period, the same concentrations of NaCl (0, 50, 75 and 100 mM) were added and the medium was solidified with 2 g L -1 Phytagel TM (pH 5.7).The propagules remained for a further 60 days in the rooting medium under the same environmental conditions described above.After 30 days, new subcultures were performed, with growth analysis (height and number of leaves of the propagules, sprouting rate, average number of propagules formed, and rooting rate [(inoculated plant/root propagating plant) × 100]).At the end of the 120 days of the micropropagation stages, the survival rate of the initially inoculated propagules was calculated.
After 120 days of micropropagation, the banana propagules were divided into leaves and roots and both parts were evaluated for water status indicators, represented by relative water content (RWC) and humidity (H), in addition to the occurrence of electrolyte leakage (EL) and osmotic adjustment indicators, namely concentrations of total soluble sugars (TSS), total free amino acids (TFAA) and proline (PRO).

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