Effect of Cytokinins on Micropropagation of Moringa Oleifera Lam. and Genomic Stability Assessment Using Flow-Cytometry

Rohit Bharati Czech University of Life Sciences Prague: Ceska Zemedelska Univerzita v Praze Moses Okao Czech University of Life Sciences Prague: Ceska Zemedelska Univerzita v Praze Katerina Hamouzová Czech University of Life Sciences Prague: Ceska Zemedelska Univerzita v Praze Eloy Fernandez-Cusimamani (  eloy@ftz.czu.cz ) Czech University of Life Sciences Prague: Ceska Zemedelska Univerzita v Praze https://orcid.org/0000-0001-7835-9310

Moringa has been found to enhance milk production for breastfeeding mothers (Siddhuraju and Becker, 2003).
Conventionally, Moringa is propagated through epigeal seed germination which is normally between 60 and 98 percent for fresh seeds (Nouman et al. 2012). However, seeds having a relatively longer juvenile phase and being heterogeneous in nature are not preferred (Jun-jie et al., 2017). In addition, the viability of seeds tends to decline rapidly (Fotouo-M et al., 2015). This phenomenon has been improved through vegetative propagation as Moringa can be propagated by stem cuttings where branch cuttings approximately one meter long are rooted and established in moist soil Ridzuan et al., 2020). However, stem cutting often affects yield and reduces the growth of the mother plant and at times, causes the death of the mother plant . To overcome these problems, plant tissue culture emerges as a better alternative to produce healthy plantlets without harming the mother plant within a shorter frame of time (Jamsheed et al., 2013).
Recently, numerous attempts have been made to propagate the plant by tissue culture with substantial progress. The progress includes shoot proliferation using nodal segment or callus supplemented with 0.5- Despite these studies, an e cient and complete protocol for mass propagation of this plant is still limited.
One of the major setbacks of the tissue culture technique is the putative occurrence of genomic variability among regenerants also known as somaclonal variations (Rout et al., 2006;). Genomic alterations such as endo-polyploidy, ampli cation, or deletion of DNA sequence, polyteny are observed during somatic differentiation (Schoenfelder and Fox, 2015). These alterations could be triggered by various factors such as long exposure to PGR, age of the culture, adaptive stress of in-vitro system, etc (Delporte et al., 2013;Clarindo et al.,2008). Hence, the assessment of genomic aberrations is a crucial step to achieving successful micropropagation while preserving the desirable characters in the regenerants. To date, the related studies had employed a single or small set of cytokinins within a single experiment.
However, in this study we had used the major cytokinins (BAP, TDZ, 2iP, Kinetin, and Zeatin) within a single experimental setup, to avert any experimental variations. Hence, this study aims to delve into the effects of major cytokinins within a single experimental setup. Additionally, it also aims to develop an e cient and complete micropropagation protocol of M. oleifera using nodal segment as explant while maintaining genomic stability.

Surface sterilization of explants
Firstly, explants containing 3-4 nodes were washed in running distilled water for 10min. This was followed by immersing the explants in 70% ethanol (v/v) for 2 min, then 1% solution of sodium hypochlorite (v/v) (NaClO, commercial bleach-SAVO,) containing two drops of tween 20 for 10 min. Subsequently, the explants were rinsed in sterile distilled water thrice and a rotary shaker was used for all rinses and surface sterilization. Thereafter, the sterile explants were transferred to the laminar ow hood for initiation. The percentage of clean culture were recorded after 14 days of inoculation.

Preparation of culture medium
Murashige and Skoog's (1962) basal medium was prepared by adding appropriate quantities of major and minor nutrients, vitamins, sucrose, myo-inositol, and agar to distilled water. The mixture was constantly stirred using a magnetic stirrer and when uniformity was achieved, the pH of the medium was adjusted to 5.7 ± 1. With continuous stirring, approx. 25-30 ml prepared media was dispensed into 100 ml conical asks, supplemented with respective growth regulators. The conical asks containing media were then covered using aluminium foil and sealed with para lm. Finally, the media was sterilized in an autoclave (Nuve 0T-032 model) at 121 o C for 20min before cooling and solidifying into a gel for proper explant anchorage.

Plant Growth Regulators treatment
The treatments consisted of ve different shoot induction media containing BAP, TDZ, Kinetin, Zeatin and N6-(2-isopentyl)adenine (2iP) at three concentrations (0.1, 0.5 and 1.0 mg/L) along with MS basal medium as control (0.0mg/L of growth regulator). After ve subsequent transfers (sub-culturing), in-vitro derived shoots were then transferred to root induction media with varying concentrations of Indole-3-acetic acid (IAA) (0.0, 0.1, 0.5 and 1.0mg/L). Accordingly, each treatment comprised of a minimum of 20 sampling units replicated three times.

Culture condition
The cultures were incubated in growth rooms at 24/20±1 o C and a 16/8h (light/dark) photoperiod under white uorescent lamps providing a light intensity of 2500lx. The relative humidity of the rooms was maintained between 65-70 %.

Ex-Vitro Transfer
In-vitro rooted explants were carefully removed from the growth media, washed with distilled water to remove adhering medium, and transferred into plastic pots containing sterile soil substrate and perlite in a ratio of 3:1 (v/v). Acclimatization was achieved by housing the plants under transparent polythene covering for 7 days while maintaining a high humidity in the chamber. The plants with pots were removed from the chamber and are kept under ambient conditions.

Flow cytometry analysis
Small sections of leaf sample (1cm 2 approx.) were chopped with a sharp razor blade in a petri dish containing 1 ml of Otto I buffer (0.5% Tween 20, 0.1 M C 6 H 8 O 7 ). The resultant suspension was then ltered through a 50 μM nylon mesh. Subsequently, 1 ml of Otto II buffer containing 4′,6-diamidino-2phenylindole (DAPI) (2 μg/mL) and 15 mM β-mercaptoethanol was added to the ltered sample solutions. A minimum of 10000 nuclei was recorded using the Partec PAS ow cytometer. Flomax software package (Version 2.3) was used to generate and evaluate the histogram of DNA content. The donor mother plant served as an internal standard. Random propagated plants were selected and utilized to assess genomic size stability. A minimum of 20 measurements was taken. Additionally, combined measurements were taken by chopping leaf samples from mother and micro propagated plants together.

Data collection and statistical analysis
After inoculation on shoot proliferation media, culture was maintained for 30 days before recording data on shoot growth and 15 days for root growth on rooting media. Growth parameters examined included the percentage of shoot regeneration and their numbers, shoot length, root numbers and lengths. All the recorded data were analyzed by Analysis of variance (ANOVA), followed by post-hoc assessment using Duncan's multiple range test at 5% signi cance.

Results And Discussion
In uence of cytokinins on shoot number and length It is a well-known fact that cytokinins play a vital role in shoot organogenesis (Hill and Schaller, 2013). In this study, the response of M. oleifera nodal explants to different cytokinins was tested (Table S1). The mean number of shoots per explant increased exponentially with the increasing concentration of all cytokinins studied (Fig. 1B). The only exception to this trend was in the explants treated with TDZ, where increment in concentration produced no shoots at all, rather had a detrimental effect on the explant (Fig. 1B, 2C). The highest number of shoots was observed in media supplemented with 1.0 mg/L of BAP producing an average of 8.0±1.20 shoots per explant (Fig. 1B). In addition to BAP, kinetin at a higher concentration of 1 mg/L also performed signi cantly well when compared to control in inducing new shoots up to 4.6±2.53 per explant (Table S1). Further, the highest shoot length was achieved by kinetin at the concentration of 1 mg/L producing a maximum average shoot length of 4.0±1.65 cm followed by BAP at a concentration of 1 mg/L that generated a maximum average shoot length of 3.9±0.84 cm (Fig.  1A). Although, the difference was statistically not signi cant (Table S1). Another trend that followed in all treatments was callusing at the base of the explants regardless of the cytokinin used, excluding the control (basic MS) (Fig. 2). Kinetin at lower concentrations, zeatin, TDZ and 2ip either produced statistically similar or lower number and length of shoots compared to the control. Furthermore, the survival rate among all treatments ranged between 80-100% except TDZ, where the survival rate was merely 10% at lower concentration and no explant survived at higher concentrations (Table S1).
In our study, BAP was found to be most effective in inducing multiple shoots. The trend of increasing shoot number in M. oleifera with increasing BAP concentration has been reported in several studies found that the number of shoots increased with increasing levels of BAP up to 1 mg/L which concurs with the results of the current study. Although, further increase in the concentration up to 3mg/L resulted in a short and fewer number of shoots. In another study conducted by Saini et al., 2012 from nodal segments of M. oleifera grown on MS media containing 1 mg/l BAP produced up to 9.0 ± 1.0 shoots per explant. However, the value dropped to 2.6 ± 0.5 and 2.3 ± 0.58 shoots per explant when the BAP was combined with Naphthaleneacetic acid (NAA) at 0.5 mg/L and 1 mg/L respectively. To that effect, several authors assert that plant growth regulators play antagonistic and synergistic roles to each other (Danilova, 2020). This means that the presence of auxins could have restrained the shoot enhancement abilities of cytokinin in those studies. Overall, from current and previous studies, it appears that BAP alone at a concentration of 1 mg/L is optimum for multiple shoot induction in this plant species.
On the other hand, shoot length was effectively in uenced by kinetin and was better than that of BAP but the difference was not signi cant between them. Even though the length of the shoots was higher, kinetin failed to produce a higher number of shoots in comparison to BAP (Fig. 1B). Moreover, it did not perform well in any of the previous studies on M. oleifera micropropagation when compared to BAP (Marfori, 2010, Abdullah et al., 2019. Recently, TDZ was also employed to assess its effect on shoot regeneration in M. oleifera. Concurring to the current study, no shoots were generated rather produced a high amount of callusing (Ridzuan et  In addition, zeatin and 2ip were also tested for their ability to induce and proliferation shoots. Although zeatin at all concentrations was able to produce a similar number of shoots as the control, it did not affect the shoot length of the explant and remained constant between all concentrations. Similarly, 2iP at all concentrations produced a similar number of shoots as the control, but it affected shoot length poorly and produced shorter shoots among all the treatments studied except TDZ.

Effect of auxin IAA on root induction
Normally, auxins are known to increase overall rooting percentages, produce more roots of better quality, speed up the process of root initiation in addition to consistent rooting (Hartmann et al. 2002;Blythe et al. 2007). In the current study, three concentrations of IAA were tested for inducing roots. All the concentrations of IAA were successful in inducing roots with the increasing concentration (Fig. 1C, 1D). The average number of roots and root length of 4.9±1.43 cm and 5.3±3.06 cm respectively was achieved at the concentration of 1 mg/L of IAA. Lower concentrations either produced statistically similar or lower number and length of roots compared to the control. However, the best rooting parameters were exhibited when in-vitro grown shoots were cultured on basic MS media producing an average number of roots and root length of 4.9±0.94 cm and 6.0±1.62 cm respectively (Table S2). Although this result was not signi cantly different from IAA incorporation at 1.0mg/L, it indicates that the level of endogenous auxin in the transferred shoots of M. oleifera was su cient for optimal root induction.
Reports from similar studies have provided differing results. For instance, a study by Gupta et al., 2020, reported that IAA at a lower concentration of 0.1 mg/L was effective in inducing rooting than higher concentration. On the other hand, a higher concentration of 1.75 mg/L of IAA was found to be more effective in a study by Shokoohmand and Drew, 2013. Differing from these ndings, effective rooting was observed on the basic MS in the current study. A similar observation was also made by  who reported optimum rooting without using growth regulators in this plant species.
The disparity between these reports could be due to genetic differences as different genotypes respond otherwise under similar conditions (Hartmann et al. 2002). These variations may also result from the invitro grown shoots having differing levels of juvenility. According to Blythe et al. 2007, juvenile plants are more responsive to auxin application than older plants. This means that the physiological state of the explant is very crucial in determining the level of rooting success. Even though longer roots indicate better rooting success, it may not necessarily translate to overall propagation success. This is because longer roots require more delicate handling during transplanting prior to hardening. Hence there is a potential risk of the roots breaking or getting damaged.

Ex-Vitro transfer and acclimatization
After successful in-vitro growth, the plants were transferred to the greenhouse for primary and secondary hardening. Due to the use of the transparent polythene covering for 7 days, a survival rate of 100% was achieved (Fig. 3). This result was better than what was recorded by Ochieng (2020) 95% respectively in this plant species. It appears that the use of the polythene covering contributed to achieving a better success rate than previous studies. However, a similar method was also used by Saini et al., 2012 where plants were kept in soil under transparent polythene for 15 days achieving a success rate of 80%. Unlike the current study, the long duration of 15 days in the polythene chamber and the use of soil for hardening might be the reason behind this lower success rate.

Flow cytometry analysis
One of the shortcomings of tissue culture is the occurrence of somaclonal variation within in-vitro derived plantlets. Therefore, the use of ow cytometry analysis to control genomic size is desirable, especially for plants of therapeutic compounds as their presence, composition and concentration should remain unchanged after micropropagation. Hence, ow cytometry was deployed to determine the genomic stability of M. oleifera regenerants. The results suggest that no aberrant plants were produced from the nodal explants regardless of the stage of sampling. This means that plants at all the stages assessed, generated gnomically stable plants as exhibited by single peaks attained at the gain value of 288 among measured samples (Fig. 4). The combination of donor and the micropropagated plants also resulted in a similar single peak suggesting no variation among them (Fig. 4). To the best of our knowledge, this is the rst study to utilize owcytometry to assess the genomic stability in micropropagated Moringa oleifera regenerants. Moreover, none of the previous studies on micropropagation of this species utilized any form of genetic analysis to assess the genomic stability except Avila-Treviño et al., 2017. Nonetheless, owcytometry has been utilized in numerous other plant species like Juniperus phoenicea by Loureiro et al., 2007 where genomic size stability was assessed of the plantlets following in-vitro multiplication and all plants produced were of uniform genomic size and had similar DNA content.

Conclusion
The surface sterilization protocol deployed in this study was su cient to obtain contamination-free sterile culture without affecting the explants. Among ve cytokinins studied, BAP at 1mg/L induced optimum shoots and is comparable to previous studies. Basic MS without auxin was found to be effective for root induction. Moreover, a 100% survival rate was achieved after ex-vitro transfer of the plantlets owing to the use of polythene coving for 7 days. Additionally, the assessment of genomic size stability did not show any variation between the mother plant and in-vitro plantlets. Overall, a reliable protocol for mass propagation of M. oleifera was established and this can further contribute to the future conservation and breeding efforts in this plant species.

Declarations Data availability
The data supporting the ndings of the current study are available within the article.
In uence of various PGRs on A) Shoot length B) Shoot number C) root number D) root length of M. oleifera explants in comparison to control (Basic MS).

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.