Determination of Saline Tolerance Under In Vitro Conditions of Lettuce (Lactuca Sativa L.) Var. Crespa Induced Polyploidy
Argüelles Curaca Alexis*Hernández Amasifuen Angel David, Cortez Lázaro Anthony Apolinario, Barreto Romero Andrea Alhely, Díaz Pillasca Hermila Belba,Silva Vergara Mishell Zaori
Department of Plant Biotechnology, José Faustino Sánchez Carrión National University, USA
Submission:August 12, 2019Published: October 29, 2019
*Corresponding author:Argüelles Curaca Alexis, Department of Plant Biotechnology, José Faustino Sánchez Carrión National University, Huacho, Peru, USA
How to cite this article:Argüelles Curaca Alexis, Hernández Amasifuen Angel David, Cortez Lázaro Anthony Apolinario, Barreto Romero Andrea Alhely,Díaz Pillasca Hermila Belba, Silva Vergara Mishell Zaori. Determination of Saline Tolerance Under In Vitro Conditions of Lettuce (Lactuca Sativa L.) Var.Crespa Induced Polyploidy.Agri Res& Tech: Open Access J. 2019; 22(4): 556228. DOI: 10.19280/ARTOAJ.2019.22.556228
Abstract
Lettuce (Lactuca sativa L.) is a large-scale crop worldwide, which provides very few calories, high percentage of water, vitamins, minerals and fiber. But this plant species is relatively sensitive to salinity. Induction to polyploidy alters the number of chromosomes and, consequently, the proportion of allelic genes that contribute to the appearance of characters. Considering the problem generated by salinity in vegetable crops, it is intended to determine tolerance to saline stress under in vitro conditions of lettuce var. Crespa induced polyploidy, Lettuces were introduced in vitro culture, and subsequently were induced to chromosomal duplication with colchicine. It was considered to measure saline stress at different concentrations of NaCl, kept in a culture chamber under controlled temperature and photoperiod conditions for 30 days. Through the karyotype study, it was possible to determine that colchicine achieved chromosomal duplication of lettuce explants, which were initially diploid (2n = 18), and it was determined that polyploidy-induced explants showed greater dry weight per explant and area foliar.
Keywords:Egyptian Mongoose; Herpestes ichneumon; Ecology; Occurrence; Zoological Gardens; Gaza Strip
Introduction
At present, salinity is the most important environmental factor that limits the establishment and productivity of plants [1] causes countless physiological, morphological and biochemical effects in plants, such as decreased photosynthesis and quantitative and qualitative changes in Protein synthesis due to changes in gene expression due to salinity, among others [2]. Another physiological response of plants to salinity occurs by decreasing stomatal conductance; In this way, perspiration is reduced avoiding physiological drought to maintain the turgidity of the cells. The reduction of stomatal conductance implies the closure of stomata and is related, among other factors (light, humidity, CO2, temperature and air currents), with the decrease in the potential of foliar water, even above intense light. Stomatal closure reduces the entry of CO2 by inhibiting photosynthesis, resulting in a reduction in photosynthesis synthesis. In general, the consequence is the decrease in biomass production, such as roots, leaves, stems and seeds, related to the leaf area and the length of plants [3]. Lettuce (Lactuca sativa L.) belongs to the Compositae family; It is a food that contributes very few calories, high percentage of water (90-95%), vitamins (folates, pro vitamin A or beta-carotene and appreciable amounts of vitamin C), Minerals (potassium and magnesium) and fiber. The plant is annual and autogamous, its root never exceeds 25cm. deep, is pivotal, has a short cylindrical and branched stem, the inflorescence is composed of yellow floral chapters arranged in clusters or corymbs. As for the weather, lettuce can develop at temperatures of 6˚C; With a lot of difficulty. That is why cool temperate climates, with average monthly temperatures between 13˚C and 18˚C, allow cultivation throughout the year, although high temperatures also limit their development considerably [4]. This plant species is relatively sensitive to salinity, but such salt tolerance often varies within the same species. The threshold values for lettuce species are in the range of 1.0 to 1.4 dS m-1, and the slope for the decrease in yield, from 6.2 to 8% per dS m-1 [5]. Polyploidy is a natural phenomenon that occurs more frequently in plants than in animals and is considered a fundamental mechanism in the evolution of new species. About 40% of species of floral plants and between 70 and 80% of the herbs are polyploids. Many plants that are currently grown and exploited for world food are also polyploids. In the case of polyploidy induction, it is a procedure that gives the plant breeder the opportunity to modify a plant by altering the number of chromosomes and, consequently, the proportion of allelic genes that contribute to the appearance of Abstract Lettuce (Lactuca sativa L.) is a large-scale crop worldwide, which provides very few calories, high percentage of water, vitamins, minerals and fiber. But this plant species is relatively sensitive to salinity. Induction to polyploidy alters the number of chromosomes and, consequently, the proportion of allelic genes that contribute to the appearance of characters. Considering the problem generated by salinity in vegetable crops, it is intended to determine tolerance to saline stress under in vitro conditions of lettuce var. Crespa induced polyploidy, Lettuces were introduced in vitro culture, and subsequently were induced to chromosomal duplication with colchicine. It was considered to measure saline stress at different concentrations of NaCl, kept in a culture chamber under controlled temperature and photoperiod conditions for 30 days. Through the karyotype study, it was possible to determine that colchicine achieved chromosomal duplication of lettuce explants, which were initially diploid (2n = 18), and it was determined that polyploidy-induced explants showed greater dry weight per explant and area foliar. Keywords: Egyptian Mongoose; Herpestes ichneumon; Ecology; Occurrence; Zoological Gardens; Gaza Strip characters [6- 8]. Cassava plants obtained by colchicine treatment showed differences in the size of stomata when compared to the control, according to the correlation between the chromosomal number and the size of stomata observed in other plants, a group of polyploid plants of cassava [9]; thus having precedents of an alternative of detection in plants induced to polyploidy.
The use of the technique of induction of polyploidy in the improvement of plants of agricultural interest, has had promising results in much of the research with this technique, such as Matos [10] who managed to improve the production of aloe in quantities of 3 to 6 times higher compared to diploid plants. Considering the problem generated by salinity in vegetable crops, it is intended to determine tolerance to saline stress under in vitro conditions of lettuce var. Crespa induced polyploidy.
Materials and Methods
The plant material consisted of quality lettuce seeds and crespa variety obtained from a seed store in the Province of Huaura, department of Lima. Subsequently, the seeds were transferred to the facilities of the Plant Biotechnology Laboratory of the E.A.P Biology with a mention in Biotechnology for their introduction in vitro. First, a seed pre-disinfection process was carried out for 5 minutes with detergent and kept stirring slightly; a variant of the protocol described by Castilla y González [11] was used. In which the disinfection was carried out inside the laminar flow chamber, where it was disinfected with 70% alcohol for 1 minute and rinsed with distilled water, then it was immersed in 1.5% sodium hypochlorite for 10 minutes, it was added also a drop of tween 20, and finally rinsed 3 times with sterile distilled water. Once the seeds were disinfected, the seeds were then planted in jars with MS culture medium [12], whose pH was regulated at 5.8 and stored in the incubation chamber at 22°C regulated by photoperiods of light. In total 30 bottles were planted. When the first roots of the germinated seedlings were observed, chromosome duplication was induced, for which the seedlings were transferred to liquid media with colchicine for 24 hours. Subsequently it was performed in chromosomal analysis to determine the confirmation of duplication. Confirmed chromosomal duplication the seedlings were transferred to MS culture media, added with different concentrations of NaCl (Table 1). The variables to be evaluated were the fresh weight per seedling (FWS) and the leaf area (LA) of the seedlings after 30 days of transfer to saline stress treatments. The data obtained were studied by analysis of variance (ANOVA) and comparison test of means (Tukey, P≤ 0.05).
Results
Among the variables analyzed for the physiological potential of physic seeds in relation to the conduction system and the coloration of the capus, a significant difference was found for the isolated tutor factor in relation to the seedling length variable (CP) (table 2). There was interaction of the tutor with the coloring of the caprubble for the variables of first count of germination (PCG), germination (G) and germination Speed Index (IVG). Considering the variation attributed to the tutor in isolation, it is observed that vertically tutored plants presented higher averages for seedling length compared to non-tutored ones (table 3). These results can be justified because the tutoring provides the plants of Cape gooseberry better insolation and greater ventilation in the canopy and greater interception of the solar radiation, reducing the relative humidity of the air and renewing the concentration of carbonic gas in Atmosphere close to the leaves, which potentializes the photosyntic efficiency[20]. Thus, the vegetative development is favored and consequently there is greater availability of photoassimilates that directed to the fruits, increase their size and favor the vigor of the seeds.
Discussion
The karyotype study before the treatment with colchicine indicated that the seedlings were diploid (2n = 18), this corresponds to the statement by Archila et al. [5], that in the lettuce the genetic and chromosomal stability is maintained in conditions in vivo and in vitro. Later, after applying colchicine to the explants, variation in the chromosomal structures was obtained, obtaining results like Molero and Matos [13], who induced Aloe vera L. polyploidy through various colchicine treatments in order to induce tetraploidy in Ex vitro conditions from diploid plants. Both the leaf area and the fresh weight per explant expressed in Figures 2 & 3 respectively showed a decrease, since these parameters depend on cell expansion, which in turn are related to the massive cell division, but when found in a medium of osmotic difference it could affect the growth rate of the leaves [14]. In addition, the transport of sodium ions to dividing cells can affect cellular and physiological processes, as there is an increase in the concentration of sodium in the growing tissue, especially in species that have salinity sensitivity as it is the lettuce [15]. The study in the search for greater tolerance to saline stress in lettuce allows each time to use new methods of genetic improvement, which may eventually result in high-quality plant material, exploit and take more advantage of its agri-food uses.
Conclusion
It was determined that the seedlings of lettuce var. Crespa induced chromosomal duplication showed greater tolerance to saline stress, because a greater foliar area and fresh weight were expressed in the explants compared to the explants that were not induced.
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