Use of Response Surface Methodology on Medium Components Optimization for Embryogenic Callus Induction in Sugarcane (Saccharum Sp.) Cv. Cp 72- 2086

Lourdes Janeth Germán-Báez Autonomous University of Sinaloa Faculty of Chemical Biological Sciences: Universidad Autonoma de Sinaloa Facultad de Ciencias Quimico Biologicas David Ulises Santos-Ballardo Universidad Politécnica de Sinaloa: Universidad Politecnica de Sinaloa Mayra Esparza-Araiza Potosi Institute of Scienti c and Technological Research: Instituto Potosino de Investigacion Cienti ca y Tecnologica AC Eber Enrique Orozco-Guillen Universidad Politecnica de Sinaloa Junio Flores-Castellanos Universidad Autonoma de Sinaloa Sergio Medina-Godoy Instituto Politécnico Nacional Centro Interdisciplinario de Investigación para el Desarrollo Integral: Instituto Politecnico Nacional Centro Interdisciplinario de Investigacion para el Desarrollo Integral Sergio Hernández-Verdugo Universidad Autonoma de Sinaloa Angel Valdez-Ortiz (  avaldez@uas.edu.mx ) Autonomous University of Sinaloa: Universidad Autonoma de Sinaloa https://orcid.org/0000-00028064-197X


Introduction
Sugarcane (Saccharum o cinarum) is a tropical, semitropical and subtropical perennial grass belonging to the family of the poaceae. It is cultured in over 120 countries, primarily for its ability to store high used to develop and improve protocols for in vitro propagation of some sugarcane varieties, either through embryogenesis and/or organogenesis (Ramanand et al. 2006; Kumar and Sahoo 2009;Kavita et al. 2016;Salokhe 2021); nevertheless, previous reports indicate that some sugarcane genotypes are more prone to in vitro instability than others, possibly due to the interaction between genotype and culture medium. Thus, the establishment of a suitable culture medium is critical for in vitro propagation of sugarcane, especially when it is desirable that the protocol is based on somatic embryogenesis (Snyman et al. 2011;Maulidiya et al. 2020).
Induction of somatic embryogenesis has a great impact on the micropropagation of sugarcane, so it is widely used to generate somatic embryos with high regenerative frequency, allowing for obtaining a massive production of in vitro seedlings in short times (Ikeuchi et  During the formulation of a certain culture medium, the combination possibilities between different doses of nutrients and compounds to be used can be immense; thus, one of the options to formulate e cient culture media is the application of experimental design tools such as the response surface methodology (RSM), which is a collection of computer-based experimental designs to develop and adjust mathematical models using statistical techniques, determining the factor or combination of factors required to establish the optimal conditions for obtaining the best results, minimizing the number of The aim of this study was the determination of optimum amounts of the auxins 2,4dichlorophenoxyacetic acid (2,4-D) and indole-3-butyric acid (IBA), for embryogenic callus induction of Saccharum sp. CP 72-2086 by using response surface approach.

Plant Materials
For the experiments, meristems from sugarcane plants (Saccharum sp.) of the most important cultivar in Mexico (cv. CP72-2086), with growth time between 6 to 7 months were used. Plants with no visible signs of pests or diseases were selected (Fig. 1A). Cuttings of 50 ± 1 cm were obtained; these segments were washed with distilled water and commercial detergent before being stored in refrigeration at 4°C for 24 h.
The outer leaves were removed under aseptically conditions, and then immersed in an antioxidant solution for 1 h (0.3 g L − 1 of ascorbic acid). The apical meristems were obtained and placed in 1% Benzal solution for 10 min with constant movement; then, they were disinfected by immersion in 10% (w/v) NaOCl solution for 20 min adding two drops of Tween 80 per 100 mL, and washed three times with sterile distilled water (Fig. 1B). Later, cuttings were summited to a hydrothermal treatment at 50-55°C for 8 min, and nally rinsed three times with sterile distilled water (Parreño, 2012). Finally, disc meristems of 2 mm thick and 1.5 cm in diameter were excised and placed in the different callus inducing media (CIM)

Callus growth
Embryogenic callus were induced from each sample consisting of a disc of apical meristem; each one was weighed and cultured in an assay tube containing a CIM correspondent to each treatment. Samples were kept in controlled conditions in the darkness at 25 ºC for 60 days. After this, the percentage of samples with embryogenic callus (% SEC) and the weight gain on callus produced (ΔCW) (Eq. 1) per treatment were evaluated.
Where W F is the nal weight and W 0 is the initial weight of the explant.

Experimental design
An experimental factorial design with two process variables was used to optimize the balance of the phytohormones 2,4-D and IBA in the culture media composition for the maximum %SEC and ΔCW in sugarcane. The experimental design was determined by introducing the conditions in the software Design Expert 7.0 from which thirteen treatments were obtained, eight corresponded to factorial combinations of the independent variables and ve to central point's corresponding to the replicates (Table 1). Twenty samples for each treatment were evaluated. Furthermore, a central composite factorial design was used to nd the relationship between the independent variables (2,4-D and IBA), and the response variables (%SEC and ΔCW), through a second-order polynomial. Literature data and preliminary experiments were taken into account to determine the different amounts and combinations of the phytohormones. The model below shows the relationship between two independent variables X 1 = 2,4-D, X 2 = IBA; and two response or dependent variables Y 1 = %SEC, Y 2 = ΔCW, through a second-order polynomial (Eq. 2) Where β 0 is the compensatory term; β i is the dependent term of input factor X i ; β ii is the quadratic effect of the input factor X i ; and β ij is the linear-linear interaction effect between the input factor X i and X j .
Regression analysis was applied to the values of the response variables obtained experimentally, the nonsigni cant terms (p > 0.1) were eliminated and a new polynomial (prediction model) was recalculated for each response variable (Khuri and Cornell 1987). From each prediction model, response and contour surface graphs were constructed to study the effect of the process variables on each of the response variables analyzed, and those which presented the greatest signi cant effect on the % SEC and ΔCW were selected.

Optimization analysis
To obtain the optimum conditions for callus induction (searching the maximization of %SEC and the highest ΔCW), the Derringer function or desirability (D) was used. This methodology consists in adjusting the response variables (dependent) at the same time; which is necessary to nd optimal comprises between the total numbers of responses. The global D value was analyzed based on individual desirabilities. Statistical analyses were performed operating the Design-Expert software (Version 7.0.0, Stat-Ease Inc., Minneapolis, USA).

Results And Discussion
Induction of embryogenic callus Embryogenic callus was triggered in all tested treatments, with %SEC ranging between 10 and 100%.
After 60 days of explant cultivation, the weight gain on callus produced varied from 0.698 to 1.510 g. Despite in all treatments there was induction of callus, the best result was obtained using a combination of 0.5 mg L − 1 2,4-D + 5.25 mg L − 1 IBA, reaching values of %SEC = 100.0 and ΔCW = 1.510 g ( Table 1).
The independent and dependent variables were analyzed; the regression representations were adjusted for %SEC and ΔCW obtaining the following uncoded models: On the other hand, desirability function-based method was applied for the optimization of embryogenic callus induction, considering the maximum response values for %SEC and ΔCW (Fig. 3). To optimize the concentration of auxins needed to induce embryogenic callus, the desirability value obtained was 0.945, which is close to the maximum possible global desirability (value = 1); being the best predicted response corresponding to 2,4-D (0.5 mg L − 1 ) and IBA (3.53 mg L − 1 ); this values correspond to independent variables associated with the maximum overall desirability, where the predicted values of the model were 100.0% of SEC and 1.4323 g of ΔCW (Table 2). Related to the induced callus type, through the optimization tests and even under optimized conditions, different types of callus were obtained; thus, uffy and soft non-embryogenic callus were obtained from 8.61 mg L − 1 2,4-D + 8.61 mg L − 1 IBA (Fig. 4A); compact non-friable callus were induced from 5.25 mg L − 1 2,4-D + 0.5 mg L − 1 IBA (Fig. 4B); and combinations of non-embryogenic and embryogenic callus induced over the same apical meristem were also obtained (Fig. 4C). Finally, under the optimized conditions (0.5 mg L − 1 2,4-D + 3.53 mg L − 1 IBA) embryogenic callus was mainly obtained (Fig. 4D-F). It has been reported that as the formulation of the medium is modi ed, different types of callus are induced, which are classi ed based on their macroscopic characteristics (Ikeuchi et al. 2013

Conclusions
In order to nd a particularly suitable combination for e cient embryogenic callus induction of sugarcane cv. CP 72-2086, in the present work the RSM was applied as a mathematical method. Once the optimized conditions for the embryogenic callus induction were determined, it was possible to achieve results very close to the predicted values obtained by the regression models, with high regression coe cients, R 2 = 0.84 for %SEC and R 2 = 0.88 for ΔCW. Based on these results, we can verify that the RSM can be applied with great precision in biological study models, using minimal amount of resources and reaching good e ciency; in the present work an effective protocol to induce embryogenic callus for massive propagation of Saccharum sp. cv. CP 72-2086 was obtained through the use of RSM.