Stevia rebaudiana is one of the potential sweet herbs in the family Asteraceae and commonly known as sweet leaf and commercially cultivated in many countries for multiple purposes (Amin et al. 2017). Obesity and overweight are one of the major risk factors, which causes several diseases such as hypertension, hyperlipidemia, diabetes, surgical risks, pulmonary and renal problems, pregnancy complications and certain type of cancer. Therefore, steviosides is one of the best strategies to control obesity and overweight (Gupta et al. 2013).
Plant tissue culture plays a key role in propagation, secondary metabolites production, genetic manipulation and conservation of plants. However, the in-vitro cultures of plant cells showed a genetic variation and it is also a characteristic of regenerated cells upon exposure to multiple culture conditions (Braun 1959). PGRs such as 2-4-D, NAA, BAP and salt stress are the major elicitors for genetic variability in plants which cause chromosomal instability, high level of polyploidy and shows visible differences in morphology (Sun et al. 2013; Pasqual et al. 2014; Silva and Carvalho, 2014; Khan et al. 2014). The genetic variation in tissue cultured-derived tissues received a great importance and was proposed by Larkin and Scowcroft (1981). The culture conditions might be mutagenic which induce genetic variation in regenerated plants obtained from somatic embryos, calli and tissues of plants that show phenotypic and as well as genotypic variation (Orbović et al. 2008). Moreover, dehydration stress and salt stress indicate a high level of similarity with respect to genetical, molecular biochemical and physiological effects. Many of salt responsive genes are involved in the regulation and encodes proteins of other salt responsive gene. Mostly transaction factors142 and protein kinase 143, these are the salt response regulatory genes and exposure of Stevia to salt stress and mannitol cause genetic variation in cultures in vitro (Pandey and Chikara, 2015). Therefore, the overall objective of the current study was to investigate the effect of salt stress on genetic variation in calli and shoot cultures of Stevia rebaudiana. The current study will help in understanding the genes responsible for salt stress and the calli cultures can be manipulated for such genes that can minimize the salt induced-stress in Stevia as well as in other medicinal plants.
Callus induction and shoot formation was obtained using the hormonal combination of previous research (Ahmad at al. 2016). The Stevia plant were taken from PCSIR Laboratories Complex, Peshawar. Callogenesis was obtained from leaf explant inoculated on MS medium containing 2,4-D (2.0 mg/L) in combination with BAP (2.0 mg/L). The shoots were obtained by exposing the callus to fresh media augmented with BAP (2.0 mg/L) and GA3 (0.5 mg/L). All cultured conditions were provided according to the protocol of Ahmad at al. (2016).
The calli and shoot cultures of Stevia was exposed to salt stress (100 mM NaCl). The flasks were placed in growth room for 30 days under complete cultural conditions. After 30 days, the cultures exposed to salt stress were removed from the flasks and washed with sterile distilled water and kept in liquid nitrogen for further analysis. Gene responsible for salinity stress was identified from previously available literature. The sequences of the gene and mRNA was retrieved from NCBI using the GenBank accession numbers. The total sequences were uploaded in the SnapGene (Molecular Biology software) for finding of potential restriction enzymes site to be digested with. The targeted genes UGT76G1, UGT85C2, UGT74G1 was taken from NCBI with GenBankID (LC312448.1, AY345978.1, AY345982.1). All the three sequences were BLAST with a shot gun transcriptome assemble, the blast result of these three genes match with Helianthus annuus UDP-glycosyltransferase (GenBank ID; XM-022127757.1). The sequence of UGT76G1, UGT85C2, UGT74G1 and Helianthus annuus UDP-glycosyltransferase showed 100 % similarity (Supplementary figure 1a, 1b and 1c).
Specific Primers was designed manually for the salinity stress responsible gene for the amplification of the total gDNA (genomic DNA) pool. The primers sequence was analyzed by OligoAnalyzer 3.1 (https://eu.idtdna.com/calc/analyzer) of “Integrate DNA Technology”. Primers were synthesized commercially from macrogen, advance Bioscience international (Supplementary table 1).
Further, the DNA was also isolated from control plant leaves, callus and in vitro shoots by CTAB method. The quantity of gDNA was analyzed by gel electrophoresis. The DNA was diluted up to 50 ng/µl for amplification of UDP-glycosyltransferases. Genomic DNA was used for PCR based amplification of the three genes using gene specific primers. For the amplification of the PCR, Dream taq, PCR Master Mix, was used. The PCR was operated according the profile given in Supplementary table 2. Each set of samples was restricted with same enzyme for finding any difference in the size of fragment and restriction site. Three different enzymes including ScaI, NcoI and DraIII were used in the current study for finding any differences among the genes. For restriction of other enzymes same protocol was followed (Supplementary table 3). A 25 µL reaction mixture comprised of 12.5 µL Dream Taq buffer with 0.5 mM MgCl2 and 10 µL of distilled water, 0.5 µL each of forward and reverse primers was added with 2 µL of template DNA was added. The reaction was placed in 37 ºC incubator for four hours for complete digestion with restriction enzymes.
Selected software’s, Satistix 8.1 (USA) and OriginLab 8.5 (USA) was used for measuring mean values and standard deviation.
It was observed that the 100 mM salt and PGRs induced 73.3 % callogenesis as compared to control media (93 %). The same media produced mean shoot length of 9 cm and shoot number (14) as compared to control cultures (11 cm and 20) after 30 days of inoculation. Hence from these results it is observed that saline stress has an effect on gene manipulation during in vitro induction of callus and shoot regeneration of stevia.
The genes responsible for salinity stress was identified from the previously available literature and was found that UDP-glycosyltransferase gene was responsible for salinity stress in S. rebaudiana. The complete gene of length 1377 bp and its mRNA sequences 1586 and 1554 was retrieved from NCBI using their GenBank ID as (for gene cds LC312448.1, for mRNA as AY345978.1 and AY345982.1; supplementary figure 2a, 2b and 2c).
Total gDNA was successfully isolated from the cultured callus as well as from the callus induced shootings/leaves of S. rebaudiana. The quantity of total gDNA was confirmed by Gel Electrophoresis and NanoDrop. RNA was removed from gDNA by treating with RNAse mix. Again, the quality was checked by Gel electrophoresis and good quality DNA was diluted to 60 ng/µL. The PCR was operated according the PCR profile. The fragments lengths of actual size were amplified by PCR as shown in figure 3.
The PCR amplified fragments containing the primers dimers seen at the bottom of the gel image was subjected to cleanup by using Axygen PCR clean up kit. PCR cleanup was successfully done by following the manufacturer protocol. The products were again analyzed by Gel Electrophoresis to test the quality of the products as shown in the Figure 4.
In order to determine the variation caused by salt stress. These samples were subjected to restriction with same enzyme as shown in Supplementary table 3 to analyze the fragments length. The UDP glycosyltransferase of both shoots from leaf and callus was successfully restricted with ScaI and the desired restricted fragments of 944 bp and 573 was obtained the same fragments length was also obtained when restricted the said sequence with SnapGene software. UDP glycosyltransferase mRNA was restricted with NcoI and again the same fragments length of 909 bp and 677 bp from both leaf and callus samples were obtained like previously. The same fragments were obtained when the sequence was analyzed in silico with SnapGene. mRNA was also restricted with DraIII successfully and the results was strange. In the mother plants the restricted fragments were of 1063 bp and 320 bp, and the same length that was expected from the restriction with SnapGene, while in the callus which was inoculated in salt containing media the product does not restricted. And we found only single band in the gel. The same experiment was repeated thrice but the results remain the same. Hence, from these results we assume that there is some type mutation arise due to which the restriction site for DraIII enzymes have been damaged and the fragment cannot be restricted with this enzyme anymore as the figure 5 elaborates all the restriction results.
In this study, the callus and shoot cultures were grown under in vitro condition these calli and shoot cultures were exposed to 100 mM salt stress and then analyzed for the identification of three genes including gene cds LC312448.1, for mRNA as AY345978.1 and AY345982. The exposure of these cultures to salts stress significantly affect the expression of these genes. The current results are in agreement with the data reported by Pandey and Chikara (2015). They also observed that the addition of different salt concentrations effected plant growth parameters and gene expression level involved in plant development and biosynthesis of secondary cell products. In Steviol glycoside biosynthesis pathway the transcript expression profiling of genes showed an increase in expression genes; UGT85C2, UGT76G1 and UGT74G1 in the 50, 75 and 100 mM NaCl concentration compared with control. As compared to UGT74G1 (Sativoside) the expression ratio of UGT76G1 (Rebaudioside A) was significantly increased by 67 %. Due to this it was concluded that salinity stress increases the sweetness of the plant than normal plant. while in drought stress condition, all the three gene were down regulated (Pandey and Chikara, 2015).
In this study, salt stress inhibited the plant growth parameters which are an agreement with the reports of Taffouo et al. (2009) and Kapoor and Srivastava (2010). The salt negatively affected photosynthetic pigments, growth hormones and enzymatic activities which showed inhibitory effect on plant growth and development. Furthermore, the decrease in stomatal conductance and evapotranspiration were also affected in Stevia exposed to salt stress (Acosta-Motos et al. 2015). In the previous study, Stevia was considered as a moderate salinity tolerant plant (Cantabella et al. 2017). Further it is concluded that lower concentration of NaCl caused a decreased in Steviol glycoside contents. Ceunen and Geuns (2013) found that under salt stress condition these metabolites play an important role in osmoprotactant molecule. Cantabella et al. (2017) found a great contribution in Stevia plant under salt stress to osmatic adjustment. The previous results showed that expression of KAH were increased at each concentration (50, 75, 100 mM) of NaCl. The expression ratio of the three genes may also be repressed by the varying concentration of NaCl. Some significant increase in their respective glucosides were observed with the over expression of some glycosyltransferase (Pandey and Chikara, 2015). In conclusion, three genes (UGT76GI, UGT85GI, UGT76GI) responsible for UDP glycoside transferase were isolated. Only the callus sample digested with DraIII showed a variation from the mother plant. Others genes did not show any variation.