Effect of the plant growth stimulant zeatin on regeneration capacity of some Physalis species in vitro culture

The aim of the study was to find an efficient culture medium for regeneration of Physalis species in vitro to provide their further propagation ex vitro and obtain fructiferous plants from the regenerants. Physalis peruviana L., P. ixocarpa Broth. (cv. Likhtaryk), and P. pubescens L. (cv. Zarynka) were taken as plant material for the research. Plant introduction into culture and regenerant production were carried out in vitro; the rooting of mature plants and obtaining plants with ripe fruits took place in a greenhouse and in open ground (ex vitro). To obtain regenerants, we used Murashige and Skoog (MC30) medium supplemented with the growth stimulant zeatin (Zea) at a concentration of 0.5–3 mg/l. The growth stimulant 6-benzylaminopurine (BAP) was used to elongate the regenerant stalks, and the growth stimulator α-naphthylacetic acid (NAA) was used to initiate root formation. Plant regeneration frequency and the number of regenerants per explant served as indicators of the efficiency of various zeatin concentrations on the physalis regenerative capacity. The most effective media for the shoot regeneration from cotyledonous leaf explants were MC30 + 1 mg/l Zea and MC30 + 2 mg/l Zea. Regeneration frequency on these media was 46.15 % and 53.84 % for P. ixocarpa (cv. Likhtaryk), 38.46 % and 45 % for P. peruviana, and 27 % and 34 % for P. pubescens (cv. Zarynka) respectively. The emerged regenerants were separated from explants and transferred to MC30 medium supplemented with 1 mg/l of BAP + 0.1 mg/l of NAA for stalk growth and rooting. After a month of cultivation, juvenile plants were obtained. They were transferred to a greenhouse for adaptation, and later to open ground at the experimental plot. Three months after the regenerant emergence, we obtained fertile plants, which bloomed and bore fruit. The regenerants for domestic varieties of P. ixocarpa (cv. Likhtaryk) and P. pubescens (cv. Zarynka) were obtained for the first time. We established a direct relationship between the concentration of zeatin and both the frequency of plant regeneration and the number of regenerants per explant.


Introduction
Physalis peruviana L., P. ixocarpa Broth., and P. pubescens L. are cultivated mainly in tropical and subtropical countries. One of the main useful component of various Physalis species is betulin, which has antitumor properties.
In Ukraine, the above-mentioned species are grown in botanical gardens and private collections. Physalis is a promising plant for obtaining recombinant proteins for pharmaceutical use. Materials dedicated to the editing of the P. pruinosa genome by the CRISPR-Cas method have recently been published (Lemmon et al., 2018). Physalis can be used as a model object to study the functioning of heterologous genes in its tissues and organs. Currently, sufficient amount of works on obtaining callus tissue and regeneration of various Physalis species have been conducted. A study of regenerative capacity was performed by a group headed by Rao (Rao et al., 2004), which resulted in obtaining regenerants for P. pubescens. Initially, callus tissue was grown from leaves and internodes, and then, the regenerants on a medium MS30 + 2 mg/l BAP + 0.5 mg/l NAA and on MS30 + 2.5 mg/l BAP + 0.5 mg/l NAA were obtained from it. K. Ramar and V. Ayyadurai investigated Physalis maxima regeneration capacity . H. Sandhya and R. Srinath obtained regenerants from nodal segments of Physalis minima (Sandhya, Srinath, 2015). K. Ramar with a group of scientists investigated regeneration capacity of Physalis peruviana. They obtained positive results of nodal segments and internode regeneration on the medium MS30 + 1.5 mg/l BAP + 0.5 mg/l gibberellic acid (GA3) + 0.5 mg/l 2,4-D; MS30 + 2 mg/l BAP + 1 mg/l GA3 + 1 mg/l 2,4-D, and that of leaf explants on MS30 + 2.5 mg/l BAP + 1 mg/l GA3 + 0.5 mg/l 2.4-D; MS30 + 3 mg/l BAP + 1 mg/l GA3 + 1 mg/l 2,4-D . K. Bergier with colleagues obtained Physalis ixocorpa regenerants from the "hairy root's culture" on the medium MS30 + 5 μm Kin + 1 μm BAP (Bergier et al., 2012). O. Kumar with a group obtained regenerants of Physalis angulata from meristems (Kumar et al., 2015). K. Swartwood and J. Van Eck received regenerants of Physalis pruinosa from hypocotyls explants (Swartwood, Van Eck, 2019). N. Assad-García obtained regenerants from the cotyledons of the 12-day-old seedlings of Physalis ixocorpa cv. Rendidora on the MS30 medium + 1 μM NAA + 12.5 μM BAP (Assad-García et al., 1992). P. Singh and colleagues received regenerants from nodal segments of P. peruviana on the MS30 medium + 2.5 mg/l BAP + 0.05 mg/l indolylbutyric acid (IBA) (Singh et al., 2016). A group of researchers headed by Otroshy received regenerants of P. peruviana on the MS30 medium + 4 mg/l BAP; MS30 + 1 mg/l Kin + 3 mg/l BAP from leaf explants and on the MS30 medium +2 mg/l Kin + 2 mg/l BAP; MS30 + 4 mg/l BAP + 1 mg/l Kin + 0.5 mg/l indolylbutyric acid (IBA) from nodular explants (Otroshy et al., 2013). Yaroshko and Kuchuk obtained regenerants of Physalis peruviana (Yaroshko, Kuchuk, 2019). Several scientific groups worked with Physalis minima (Afroz et al., 2009;Gupta, 1986;Sheeba et al., 2015;Mungole et al., 2011;Patel et al., 1987). Despite the achievements on this approach, there is still no works on the regeneration of domestic varieties of physalis in vitro.
Our objective was to find an efficient culture medium for the P. peruviana, P. ixocarpa, and P. pubescens regeneration in vitro in order to future obtaining adult plants from the regenerants ex vitro.

Materials and methods
The following species were used as plant material for investigations: Physalis peruviana, Physalis ixocarpa (cv. Likhtaryk), and P. pubescens (cv. Zarynka). The originator of Likhtaryk and Zarynka varieties is M.M. Gryshko National Botanical Garden of the National Academy of Sciences of Ukraine. The source material was taken from the collection fund of the department of cultural flora of the abovementioned institution (Rakhmetov et al., 2015).
Data collection and Statistical analysis. The efficacy of the used concentrations of growth stimulants for obtaining regenerants of species and varieties of the genus Physalis was determined by the following indicators: the number of regenerants obtained per one explant and the percentage of regeneration (regeneration frequency).
The number of regenerants was defined as a number of new young plants emerged from one explant. The regeneration frequency was calculated as a proportion (%) of the number of regenerated explants out of the total number of explants at the beginning of the experiment. The higher the percentage of regeneration was and the more regenerants were obtained from one explant, the more effective the concentration of growth stimulants used is considered.
Ten explants were used in each variant of experiments that was conducted in three replications. The data were analyzed using the general procedure in the Software Package STATISTICA Version 12. Spearman's test and standard error were used for statistical processing of the obtained data; the procedure was described in detail in our previous work (Yaroshko, Kuchuk, 2019). In this work, the effect of different concentrations of growth regulator zeatin was compared with the appearance of different numbers of regenerants per one explant.

Results and discussion
After cultivation of the explants on the MS30 medium with different Zea concentrations regenerants were obtained (Fig. 1, 2). The most effective medium for regeneration from leaf cotyledons was MS30 supplemented with 2mg/l Zea (Fig. 2, Table 1). Quite promising results of shoot regeneration were Вплив стимулятора росту зеатину на регенераційну здатність рослин деяких видів роду … Effect of the plant growth stimulant zeatin on regeneration capacity of some Physalis species …
The regeneration of Physalis ixocarpa (cv. Likhtaryk) on the medium MS30 + 2mg/l Zea was successful, 53.84 %, while on the medium MS30 + 1mg/l Zea it declined to 46.15 % (Fig. 2). The regeneration of Physalis peruviana was lower, 45 % and 38.46 %, respectively (Fig. 2).   Our data are consistent with other studies on the Physalis regenerative capacity. The majority of works were conducted with the use of BAP and Kin growth regulators and an addition of a third component Kumar et al., 2015;Gupta, 1986). We used only one regulator, zeatin, and received positive results.
In our previous work, we obtained regenerants for P. peruviana on the media MS30+ 1 mg/l kinetin (Kin) + 3 mg/l BAP and MS30 + 2 mg/l Kin + 1 mg/l BAP (33.33 % of regeneration on both) (Yaroshko, Kuchuk, 2019). In the current work, we got higher percentages of regeneration of the same species on the media MS30 + 1mg/l Zea and MS30 + 2mg/l Zea (38.46 % and 45 %). Thus, we can state that the MS30 media with Zea are more effective for obtaining Physalis regeneration than that with Kin or BAP.
According to the works of other researchers, the highest frequency of P. peruviana regeneration was obtained on the media with addition of BAP (concentration 1-3 mg/l) or Kin (1 mg/l) Bergier et al., 2012;Gupta, 1986). A number of regenerated plants averaged to 11 or 13 per one explant on the media with BAP or Kin, respectively. In our current work, we have achieved similar results on the media with 1 mg/l Zea (11 pc.) and 2 mg/l Zea (14 pc.) In the world literature, there is one published work on the regeneration of Physalis pubescens (Rao et al., 2004) and two works on the agrobacterial transformation and regeneration of Physalis ixocarpa (Bergier et al., 2012;Assad-García et al., 1992). Unfortunately, the regeneration percentage obtained in both species is not indicated in these papers. Therefore, we cannot compare the results of our study with those of other research groups.
In the course of our investigation, we found out that Physalis ixocarpa (cv. Likhtaryk) has the highest regeneration capacity among the three species studied (53.84 %). Such a regeneration percentage is sufficient to carry out genetic transformation of experimental plants. Thus, in the further research on Agrobacterium-mediated genetic transformation of Physalis plants, we will use Physalis ixocarpa (cv. Likhtaryk) as the most promising candidate.

Conclusions
Оur experiments resulted in definition of the most efficient culture media for regeneration of Physalis peruviana, P. ixocarpa (cv. Likhtaryk), and P. pubescens (cv. Zarynka): a percentage of shoot regeneration from cotyledon leaves was the highest on MS30 + 2mg/l of Zea and MS30 + 1mg/l of Zea. Then, the obtained regenerants were grown on the medium MS30 with 1 mg/l of BAP and 0.1 mg/l of NAA for elongating and rooting and, in a month, we got adult plants. Three month after the emergence of regenerants, the mature plants started blooming and bearing fruits.