Micropropagation of Physalis species with economic potential

Submitted on August 26 , 2020 and accepted on February 14t , 2021. 1 Programa de Pós-Graduação em Agronomia (PPGA), Universidade Estadual do Oeste do Paraná, Marechal Cândido Rondon, Paraná, Brazil. fvilla2003@hotmail.com; luciana.sabini@hotmail.com; daniel_eafi@yahoo.com.br; edvan_costa@outlook.com; rittergio@hotmail.com; tatiane_eberling@hotmail.com *Corresponding author: fvilla2003@hotmail.com Micropropagation of Physalis species with economic potential


INTRODUCTION
Physalis is considered an exotic fruit with great economic potential, due to its adaptation to different Brazilian edaphoclimatic conditions. Its presence in supermarkets and local markets shows a trend of consumption by the population. Because it has high added value, fruit production is an alternative for small products that constitute the highest income per production area (Negri et al., 2016). Due to the potential of scientific interest it has increased in order to improve the production of culture, these show that the conditions for exploration for internal consumption and export (Chaves et al, 2005).
Asexual propagation is the main form of physalis multiplication, since this fruit has a large amount of seeds and a high germination rate, however it provides plantlets with high genetic variability, resulting in plants with different growth, vigor, performance and production of fruits, characteristics that are not interesting for commercial orchards . Tissue culture proposes obtaining large-scale plants, phytosanitary quality and high genetic fidelity. Among the challenges of this technique is mastering the various stages such as establishment and multiplication in vitro. (Mascarenhas et al., 2019).
The success of in vitro multiplication depends on the composition of the culture medium, which consists of essential elements for the growth of plants such as minerals, vitamins and carbohydrate source. These substances act as an energy source, provide control of tissue growth and regulate development in vitro. One of the ways to control these responses is through the concentration of macro and micronutrients that make up the culture media. (Oliveira et al., 2013a).
There are researches carried out by authors such as Chaves et al. (2005), Mascarenhas et al. (2019) with the Rev. Ceres, Viçosa, v. 68, n.6, p. 521-529, nov/dec, 2021 main commercial species (P. peruviana), but data are missing with the other species that have economic potential and that, because they have different morphoanatomical characteristics , genotypes may respond differently to in vitro processes (Costa et al., 2015). These characteristics are complex and may indicate differences in nutritional needs, concentration of growth regulators, site of excision, type of tissue and genotype and consequently interfere in the multiplication rate in vitro cultivation (Mascarenhas et al., 2019).
Considering the complexity of the micropropagation steps that involve several factors, adjustments to the in vitro multiplication protocols that already exist for the different Physalis species are necessary, so this study aimed to evaluate protocols of asepsis, composition and concentration of culture media in the establishment and multiplication of Physalis species in vitro.

Experiment I
The Physalis seeds used in this experiment were from ripe fruits collected in the, Germplasm Bank kept in the West State University of Paraná (Unioeste), Campus Marechal Cândido Rondon (PR). The species (Physalis peruviana, P. ixocarpa e P. minima) were properly identified and are categorized at the Unioeste's Herbarium. (HUNOP, Campus Cascavel).
After the withdrawal of fruits' seeds and the identification according to the species, they were taken to the Biotechnology Laboratory of the Pontifical Catholic University of Paraná (PUCPR), Campus Toledo (PR).
When received in the laboratory, the seeds of the three species (Physalis peruviana, P. ixocarpa e P. minima) were immediately taken to the laminar flow chamber BIOSEG 09 model, washed with distilled water and displayed in the asepsis protocols by immersion in: I = solution Tween 20 by 5' + 70% alcohol (A70) by 30" + Sodium hypochlorite (NaClO) by 3', II = A70 by 30" + NaClO by 3', III = A70 by 3' + NaClO by 10'. After the procedures, the seeds were washed four times in distilled water and autoclaved After the sanitization, 5 seeds were allocated by glass bottle, with a total capacity of 300 mL volum, containing 25 mL of media culture MS (Murashige & Skoog, 1962), added to 30 g L -1 of sucrose, 6 g L -1 of agar (Himedia ® ) and pH = 5,8, adjusted before autoclaving. During 30 days, the fungal and bacterian contamination existent at every 4 days was evaluated.
The experimental outline used was completely randomized, in factorial scheme 3 x 3 [asepsis methods x Physalis peruviana, P. ixocarpa e P. minima]. Containing 5 repetitions, 1 bottle (500 ml capacity) for repetition and 5 seeds of a species by bottle.
The experimental outline used completely randomized, in factorial scheme 3 x 3 [culture media x physalis species], containing 5 repetitions, 1 bottle (500 ml capacity) for repetition and 5 explants of a species by bottle.

Experiment III
After the experiment II analysis, the culture media that presented proper characteristics for the establishment of the in vitro physalis species was defined. So, the experiment III was constituted by 4 concentrations of medium MS (0, 50, 75 e 100%) and two physalis species (P. peruviana e P. minima). The culture media were added of 30 g L -1 of sucrose 6 g L -1 of agar (Himedia ® ) and pH = 5.8, adjusted before autoclaving.
The experimental outline used in this experiment was completely randomized, in factorial scheme 4 x 2 [concentrations of culture media x physalis species]. Containing 5 repetitions, 1 bottle (500 ml capacity) for repetition and 5 explants of a species by bottle.

Experiment IV
In this experiment four concentrations of sucrose were evaluated, these being 0, 15, 30, 60 g L -1 , for two species of physalis (P. peruviana e P. minima). The concentration of 100% of culture media (MS) was used, plus 6 g L -1 of agar (Himedia ® ) and pH = 5.8, adjusted before autoclaving.
The experimental outline was done entirely by chance, in factorial scheme 4 x 2 [concentrations of sucrose x species of physalis], containing 5 repetitions, 1 bottle (500 ml capacity) for repetition and 5 explants of a species by bottle.
For all the experiments, the bottles were covered with aluminum paper and autoclaved at 121 ºC and 1.2 atm of pression, during 20'. After the autoclaving, they were sealed with plastic film of PVC, in order to avoid contaminations and kept in wooden shelves (45 x 30 cm), in a growth room, with photoperiod of 16 h light and temperature ± 24°C.
In the experiments II, III and IV were used explants of pre-established plants in vitro from seeds were used. The explants from the third pricking-out were excised with the help of a scalpel, in laminar flow chamber in petri-dishes. These contained 1.5 cm of lenght, two opposite axillary buds and a pair of leaves.
After 30 days of the building of the experiments (II,III and IV), the number of plantlets sprouts, leaves and roots were evaluated. With the aid of a ruler, the lenght of the bigger root (cm) and the total of the plant (cm) were evaluated and by weighing on an analytical balance the fresh and dry plantlets biomass (g) was also evaluated. To obtain the dry plantlets biomass, they were put in white paper bags properly identified and taken to the to the forced ventilation oven at 65°C for 48 h.
The data obtained in all experiments was tabbed and the test of normality Shapiro-Wilk was applied. Afterwards, they were submitted to the analysis of variance and regression analysis for quantitative data, at 5% of error probability, being changed into (Y+1.0) 0.5, whenever necessary. For the results analysis, the Sisvar was used (Ferreira, 2011).

Experiment I
In Table 1 germination data (%), fungal and bacterial contamination (%) and total contaminaion (%) in seeds of physalis species is presented, where the significant effect for the first factor and the statistical difference for the species Physalis peruviana e P. minima was verified.
Better results in germination (%) in vitro were observed for P. peruviana, when using protocols II and III. For P. minima seeds, the highest germination percentage occurred when using protocols I and III; Protocol II showed a lower germination rate, a fact that may be related to a lower amount of reagents and exposure time. For P. ixocarpa, there was no statistical difference for germination between the protocols used. for all species when protocol III is used, good results are observed, which makes it possible to standardize the procedure with good results for the germination parameter.
Studies carried out by Chaves et al. (2005) showed that the rate of germination of P. peruviana was reduced, when solutions with calcium hypochlorite in seed asepsis were used, probably due to the high fungic contamination. The diferences in the germination rate of seeds among the distinct works were caused by the combination of several substances of asepsis. When the NaClO for asepsis was used, Pinheiro et al. (2016) noticed reduction in the fungic contamination, increase in the germinative potential in Cedrela fissilis seeds.
The low rate of total contamination (%) and all efficiency of protocols for this factor can be explained by the sequence of products used in seeds' asepsis. Besides that, it is taken for granted that there is a combination between chlorine and the membrane protein of microorganisms which form toxic compounds leading to the inhibition of essential enzymes for survival (Machado & Fernandes, 2018). The 70% alcohol is pointed out as a disinfectant and according to Tomazzi et al. (2019) when it is present at this concentration it evaporates slowly potentializing its action of denaturation of proteins in microbial cells when in contact with microorganisms.

Experiment II
In Table 2, it is observed that the number of regenerated seedlings, when compared to the culture media in Physalis peruviana there was no statistical difference and in P. ixocarpa there was a greater number of regenerated when in MS medium. In P. minima, there was the lowest number of seedlings regenerated when in medium Knudson. When the sprouts are analysed, for P.minima there was no difference between the WPM and MS medium. This variable had the same behavior for P. peruviana e P. ixocarpa, being the MS medium, the one that provided a greater number of sprouts for the species.
The MS medium is more concentrated in nitrogen in relation to other studied mediums, fact that influences in the synthesis of endogenous cytokinins, resulting in a number of sprous by species nodal segment as verified in the presente work (Jesus et al., 2010). When the number of leaves is analysed for the evaluated species, the MS medium was considered proper when compared to WPM e Knudson culture media. Oliveira et al. (2013a) obtained an average number of Physalis angulata in vitro leaves, similar to the one found in the present work.
A greater length of seedlings was verified in MS medium for the three species studied. Araujo et al. (2016) state that in addition to being rich in nitrogen, the MS medium also contains calcium four times more concentration than the other media, this is a structural component, responsible for the connection between pectins and groups of lipid acids, in addition, it acts as a signal, stimulating the production of auxin and cytokinin, endogenous hormones responsible for growth (Kerbauy, 2012).
Besides that, in works with fruit species in vitro, such as blackberry, pineapple and casserole, authors observed a good development having a greater number of leaves.sprouts and lenght of these plantlets, when propagated in a MS cultivation medium (Leitzke et al., 2010;Oliveira-Cauduro et al., 2016;Araujo et al., 2016).
When the fresh biomass was evaluated, the behavior was similar to the MS medium that presented better results for the three species, when compared to Knudson and WPM. These aspects show that culture media favors the assimilation of nutrients, fact that explains the better performance of plants in MS medium.
The evaluations that did not present a significant interaction are presented in Table 3, being those, a number of roots and, dry seedling biomass. It is noticed that he number of roots was samller for P. ixocarpa, when compared to the other species and the same behavior occurs to dry shoot biomass.
The MS cultivation medium promoted a bigger quantity of roots, being superior to the Knudson e WPM, ones that did not differ statistically between them. The presence of the boron, which is in bigger quantity in the MS medium, promote synthesis of indolacetic acid (AIA) and acts translocation of natural auxin, promoting the rooting. The bigger dry biomass values were found in the MS medium that is more concentrated in salts and nutrients, being an important attribute of growth (Moschini et al., 2019).

Experiment III
The factor evaluated the number of regenerated plantlets that did not present interaction and because of that, it was separately evaluated in Table 4, where we can observe that P. minima had a greateer number of regenerated plantlets compared to P. peruviana.
The Figure 2A presents a number of sprouts of P. peruviana e P.minima in concentrations of the MS medium, we notice that both species had greater values of sprouts when in a medium at concentration of 100%, growing in a linear manner. In Figure 2B, we can notice that the seedling lenght for P. minima obtained a linear growth according to the increase of the medium concentration, reaching its maximum value of approximately 3.0 cm in 100% of culture media. In P. peruviana it was noticed that there was no significant difference in the growth according to the variation of the medium culture.
For the number of leaves (Figura 2C), it is possible to verify that the species increased the quantity according to the increase of the concentration of the culture medium. The species Physalis minima, had its greater number of leaves in 100% of culture media, with approximately 7 leaves and the P. peruviana obtained at the maximun 3 leaves.
The phosphorus presents in the culture medium acts differently in the leaf expansion. The incorporation of this nutrient depends on the genotype of each species. Costa et al. (2015) observed that there was a greater number of leaves in the MS medium, however without statistical differences in MS medium with 50% of nitrate, working with hybrid basil, indicating that other factors can interfere in these characteristics.
The length of the larger root ( Figure 2D) of P. peruviana did not obtain statistical difference when the concentration of the culture medium varied. However, the P. minima varied a little its length and had its greater value of approximately 1.5 cm at 100% of medium.

Species of Physalis
According to Lemes et al. (2016), the plantlets present the need of low concentrations of nitrogen in the formation of roots and concentration of high levels of salts can affect negatively the development of roots. Therefore, the rooting can be related to each species' characteristics response to the in vitro stimuli.
Regarding the number of roots, there was no species x concentration of the culture medium. Therefore, only the concentration of the culture medium x number of roots was evaluated ( Figure 2E). It can be seen that the number of roots tended to grow, due to the presence of boron and other micronutrients that promote the synthesis of AIA (indolacetic acid) and the translocation of natural auxin, enabling rooting (Pasqual, 2001).
For fresh and dry seedling biomass, it was not observed any statistical difference between the concentra-

Experiment IV
In figure 1 the results found when physalis species (P. peruviana e P. minima) were submitted to the concentration of sucrose are shown.
It can be seen that the number of regenerated seedlings ( Figure 1A), in P. peruviana, increased with increasing sucrose concentrations and peaked at 44 g L -1 with an average of 5,05 regenerated seedlings and after this concentration there was a fall in regeneration. For P. minimal the same trend was observed, this species reached its maximum point in 31 g L -1 of sucrose with an average of 4,9 plantlets.
When the number of sprouts was evaluated ( Figure  1B), P. peruviana increased up to the concentration of 40 g L -1 of sucrose, when it obtained the average value of approximately 1.5 sprouts. P. minima had its maximum Rev. Ceres, Viçosa, v. 68, n.6, p. 521-529, nov/dec, 2021 point at 15 g L -1 with 1.3 sprouts and decreased with the increase of sucrose concentration.
Studies carried out by Poothong et al. (2020) showed that the reduced sucrose in the concentrations of 1.5% and 3% in the medium MS, increased the number of sprouts for raspberry, however the length of sprouts was greater when sucrose was not added to the media culture.
The length of the seedling ( Figure 1C) presented the greater average of explant growth (approximately 10cm) when used 45 g L -1 of sucrose for P. peruviana. In P. minima the greater average growth was approximately 5cm at the concentration of 30 g L -1 of sucrose.
A concentration of 15 g L -1 favored a quantity of leaves of P. minima, that at this concentration, it reached the average value of approximately 5 leaves by explant. P. peruviana had the maximum value of leaves of 3.6 when used 60 g L -1 of sucrose The excess of sucrose in the culture media can inhibit the chlorophyll synthesis and reduce the photosynthetic capacity of crops, besides that, due to the lack of CO 2 in the cultivation in vitro, a process of photosynthesis does not normally occur, so the explant depends of a source of energy that possibilitates the normal activity of cells physiological functions promoting the development of explants (Ayub et al., 2019).
The number of roots and the length of the biggest root for P. minima were better at the concentration of 30 g L -1 of sucrose, obtaining the values of approximately 3.5 roots and 3.3 cm. When P. peruviana was evaluated, the greater amount of roots was obtained when 60 g L -1 of sucrose was used, with approximately 8 roots for each explant and the biggest root length with 30 g L -1 having in average 6 cm.
For the formation of roots, energy that can be from photosynthesis or from other source of sugar is necessary. The exogenous carbon in the culture medium influences in the differentiation and growth of tissues, induction and differentiation of tissues and organs induction and differentiation. According to Calvete et al. (2002), the sucrose stimulates the rhizogenesis in vitro and in its absence, there was no rooting in strawberry explants.
The fresh biomass had an increase up to 40 g L -1 for P. peruviana and 24 g L -1 for P. minima, after these values, there was a reduction in the seedling's weight. However, when the fresh biomass is evaluated, the greater value obtained for the species is at 60 g L -1 of sucrose, what indicates that at this concentration, less water accumulation and greater salts accumulation occured.
For most of the parameters analysed, P. minima had its development favored at low sucrose concentrations (between 15 and 30 g L -1 ) when compared to the values of P. peruviana (between 45 and 60 g L -1 ), what indicates that the species genotype presents distinct needs for its development.

CONCLUSIONS
The protocols II e III were adequated for the germination P. peruviana, and I and III for P. minima, the three protocols were efficient for the control of fungus and bacteria.
The sucrose concentrations close to 50 g L -1 favored the establishment of P. peruviana of approximately 20 g L -1 favored the establishment of P minima.
The culture medium MS is the more indicated one for the in vitro establishment of P. peruviana, P. minima and P. ixocarpa.
The cultivation medium at the concentration of 100% obtained better values in the parameters of in vitro development evaluated for the species P. peruviana e P. minima.

ACKNOWLEDGMENT
To CAPES for granting a scholarship. There are no conflicts of interest.