High frequency plant regeneration of Chrysopogon zizanioides via organogenesis and somatic embryogenesis-an underutilized pharmaceutically valuable biofuel plant

Vetiver (Chrysopogon zizanioides) is an essential oil-producing plant that has tremendous application in cosmetics, perfumery, and herbal medicine. Natural sterility and indiscriminate harvests lead to the risk of extinction of plant species in natural habitats. Therefore, a protocol for regeneration systems via organogenesis and somatic embryogenesis using node, leaf, and root explants has been standardized. The highest shoot regeneration frequency (72.2%) through organogenesis was attained from node explants on MS (Murashige & Skoog) medium comprising 2.0 mg L -1 BAP (“6-benzylaminopurine”). Concurrently, leaf explants cultivated on MS medium augmented by 1.0 mg L -1 , 2, 4-D (“2, 4-dichlorophenoxyacetic acid”) formed the optimal frequency (75.35%) of white friable compact (WFC) callus. However, the root explant was less responsive for WFC callus induction. Organogenic WFC callus cultivated on MS medium fortied by kinetin (1.0 mg L -1 ) as well as BAP (1.0 mg L -1 ) revealed the highest shoot regeneration eciency (75.49%) with 48 shoots per callus. Adventitious shoots obtained from node and WFC callus of both leaf and root explants cultivated on MS medium increased by NAA (2.0 mg L -1 showed the optimal rooting of 76.97%. Concomitantly, an elevated frequency of somatic embryogenesis (52.50%) was recorded from leaf explants on MS medium using BAP (0.5 mg L -1 ) & 2, 4-D (1.0 mg L -1 ). Leaf explants were superior to node and root explants for somatic embryo initiation. The cotyledonary embryos were eciently germinated into complete plantlets on a hormone-free MS medium. The plantlets gathered from organogenesis & somatic embryo genesis was effectively acclimatized into phenomenally similar plants. This technique may be applicable for wide-range propagation, genetic engineering, and the formation of bioactive compounds.


Introduction
Vetiver (Vetiveria zizanioides) is synonymously called as (Chrysopogon zizanioides) comes to the Poaceae family and is broadly cultured in tropical as well as sub-tropical areas for the production of essential oil. In spite of its economic importance, it has certain environmental signi cance such as soil moisture conservation, reduction in soil erosion, and deduction of heavy metals (Sudhishri et al. 2008;Minh and Khoa 2009;Leauagvutiviroj et al. 2010). Naturally, sterile plant and has no rhizome or stolons as seed material for cultivation, but conventionally propagated through root segments or slips.
The secondary metabolites present in vetiver root extract have been extensively used as a major ingredient in perfumery and cosmetic industries (Bhuiyan et al. 2008; Bhushan et al. 2013). Further, a variety of potential bioactivities have been noti ed previously from its extract including antimicrobial, herbicidal, and pesticide properties (Mao 2004; Koul 2008; Adam 2008; Sangeetha and Stella 2012). The pharmacological properties present in extract possess curative properties such as anti-in ammatory, antiseptic, sedative, vulnery, cicatrisant, aphrodisiac, digestive, haematinic, carminative, stomachic, antiasthmatic, antispasmodic, antihelmetic, antigout, and diuretic (Bhushan et al. 2013). Further, the viscous oil extorted from the roots of the vetiver has authenticated the presence of aroma and several potential therapeutic properties (Massardo and Pripdeevech et al. 2006;Rotkittikhum et al. 2010). The phytochemical components obtained from oil extracts include, khositone, vetivone, ß-Humulene, vetiverol, khusimol, vetivene, terpenes, benzoic acid, khusimone, Tripene-4-ol, ß-Humulene, epizizianal, vetivenylvetivenate, isokhusimol, ß-vetivone, vetivazulene (Pareek and Kumar 2011). Furthermore, the massive biomass derived from the entire plant could be an amenable and alternate resource for biofuel manufacture (Sun et al. 2014). Although C. zizanioides has a su cient quantity of varied secondary metabolites which could be utilized a novel phytomedicine, its population in natural habitat has become indiscriminately depleting due to pollen sterility, uncontrolled grazing, reaping by therapeutic professionals for making of folk medicine, deforestation, urbanization, and also due to unfavorable environmental factors.
Conventionally, vetiver has been propagated through root cuttings which have certain constraints such as inherent low viability, unsynchronized growth form, and also sluggish growth rate. These factors will impede the export of vetiver biomass across several nations. Thus, the actual requirement of biomass for oil extraction and biofuel production could not cope up with mounting commercial demands.
Henceforth, it is essential to propagate and conserve endangered or extinct germplasm by adopting the latest biotechnological technique i.e., regeneration via plant tissue culture (Deo et al. 2010; Anis and Ahmad 2016). Environmentally independent and year-round generation of genetically similar and valuable populations through micropropagation technique is highly indispensable to meet out the ever-increasing demands on commercial and industrial-scale (Hussain et al. 2018).
Despite the fact that there are a few explants with its pre-existing meristem of vetiver has been used to accomplish micropropagationstudies, such as shoot segment (Code et al. 2012), adventitious bud and axillary bud (Zhenrang et al. 2006;Kanokporn and Chonnikarn 2016), and in orescence (Sangduen and Prasertsongskun 2009) and reported with low regeneration frequencies. However, callogenesis and somatic embryogenesis have not been reported in this species. Herein, we developed e cient reliable and reproducible regeneration protocols through organogenesis and somatic embryogenesis from node, root, and leaf sheath explants of C. zizanioides.

Plant Materials
The propagating material clumps of C. zizanioides have been brought from a village, Nochikkadu, Cuddalore District, Tamil Nadu. The clumps were grown in the potting blend (compost, soil, and sand) in the proportion of 1:1:1 and maintained under shade in the pot culture yard of "Department of Genetics and Plant Breeding, Annamalai University, Annamalai Nagar", India. Explants were collected from ex-vitro grown plants for optimization of regeneration methodology in C. zizanioides.

Explants preparation
Node, leaf sheath, and root explants were gathered from 30-day old plants and twenty minutes of washing is done with tap water and disinfected using 1% bavistin with continuous agitation. The materials are again rinsed with owing tap water to eliminate residual bavistin. Again, explants were dipped in 0.1 percent "mercuric chloride" (HgCl 2 ) for 2 to 3 minutes, followed by 4 to 5 times cleaning with sterile ltered water to eliminate the traces of sterilizing agents. Explants such as nodal segments (5mm), leaf (5mm), and root segment (5mm) were cut away from the source plants with the support of sterilized sharp scalpels & forceps. Auxiliary buds and leaves and were cut off from the nodes and internodes by using a sharp scalpel blade. Identically, the cut may be given at the proximal end of the leaf sheath of explants.
Culture media and conditions MS basal medium (Skoog and Murashige 1962) was added with different doses of PGRs ("Plant growth regulators") including kinetin, NAA:"α-naphthaleneacetic acid", TDZ:"Thidiazuron", 2, 4-D and BAP. The carbon source incorporated in the medium is sucrose (30 g L -1 ) and phytagel was employed in all media as a gelling agent at a 4.0 g L -1 rate. These thermostable PGRs were applied before autoclaving of media. The pH of the medium was varied to 5.8 with 1 N HCl or1 N NaOH. The autoclaving of the culture media was done in an autoclave using steam sterilization on a 1.5 kg/cm 2 pressure and 121degrees Celsius for fteen minutes.
Twenty milliliters of medium were poured into sterile culture tubes (25 x 150 mm) and sealed with cotton plugs.
Cool white uorescent tubes were used in the chamber of plant growth to maintain a photoperiod of 16/8 hour with "light intensity"of 50 µmol m -2 s -1 ("Panasonic, Japan"). The PGR, as well as chemicals, were imported from Hi-media, and glasswares were brought from "Borosil", India. for 6 weeks and subcultured at 2 weeks intervals. Callus initiation Frequency of individual treatment was noted after 8 weeks of culture. The callus developmental nature was categorized as WFC ("white friable compact"), WNF ("white non-friable"), and no callus initiation (N). Twenty explants were subjected to individual replication, three replications per experiment, and treatment was performed three times. Organogenic calli resulting from the above trials were subjected to media with various intensities of NAA from 0.5 to 1.0 mg L -1 blended with numerous intensities of BAP (0.5, 1.0, 2.0, 3.0, 5.0 & 6.0 mg L -1 ) respectively for initiation of adventitious shoots.
Subculturing of these cultures was performed once in 2 weeks and continued for 6 weeks with the same medium concentration for adventitious shoot initiation. The fraction response of shoot organogenesis was observed from callus and a mean "number of shoots/callus pieces" after six weeks. The plantlet's nature was categorized as qualitatively +: "abnormal stunted shoots", ++: "normal leaves shoots", -: no shoots.

Hardening and Rooting
Transferring of shoots developed from "the nodes, internode explants, and shoots obtained from leaf sheath and root-derived calli to rooting media consisting of either hormone-free half-strength MS or half-strength MS + NAA (0.5 mg L -1 ) or full-strength MS basal medium or half MS + NAA (1.0 mg L -1 ) or full MS + NAA (2.0 mg L -1 ) or full MS + NAA (2.5 mg L -1 ) for 2-4 weeks. After root initiation from this culture, subculturing of inoculants into the fresh medium after 2 weeks was done and kept until four weeks. After four weeks fully developed rooted shoots have been uprooted from the media and cleaned thoroughly thrice with" sterile water to eliminate the adhered material and transferred to plastic pots with the potting mixture for hardening. Then the plastic containers were coated with plastic bags and holes were created for gaseous exchange. These potted plants were kept for 2 weeks in the chamber of plant growth and then shifted on to the greenhouse condition. The polyethylene covers were gradually detached from well-established plantlets. The plant survival ratio in the greenhouse and at the eld was assessed. After four weeks, the proportion of rooting response, the mean number of roots/shoots and root duration have been noted for each treatment. For each replication, ten adventitious shoots were utilized with 3 replications per experiment, and treatment was performed three times. The lower cut end of the leaf sheath explants was placed in a resting position and embedded in the culture medium. Subculturing of inoculants was done once in two weeks and kept until 4 to 6 weeks at 23 ± 2ºC, RH of 60-65 % and "light intensity" of 30 µmol m -2 s -1 within cool white pipes of the uorescent with a photoperiod of 16/8 hours in a growing chamber (Panasonic, Japan). The rate of explants developing "somatic embryos" was noted after four to 6 weeks and an average number of somatic embryos/explants was found after six weeks of inoculation. The different phases of somatic embryos such as cotyledonary, torpedo, heart, and globular, derived from MS medium consisting of 2, 4-D (1.0 mg L -1 ) as well as BAP of (0.5 mg L -1 ) were subjected to either MS + NAA of (0.5, 1.0 2.0 & 2.5 mg L -1 ) or half or full strength for the transformation of "somatic embryos" into entire plantlets. Subculturing of cultures was done at the interval of two weeks and kept up to six weeks. The regeneration frequencies of somatic embryos were noted after four to six weeks. Twenty explants were used with three replications per experiment and treatment was replicated three times. The fully developed plantlets with a well-organized rooting system were detached from the culture tube, and cleaned with sterile water to detach adhering agar, and were moved to sterilized potting blend and maintained in a greenhouse for acclimatization. The plantlet's survival ability was examined in both greenhouse and in eld conditions.

Statistical analysis
The CRD ("Completely randomized design") was employed as an experimental design for all experiments conducted in this research. The data collected from these experiments were analyzed through ANOVA ("analysis of variance") with the aid of SPSS Version 10 (SPSS, Chicago, IL) software. The signi cance differences within the treatment means were compared with the aid of DMRT ("Duncan's Multiple Range test") at a 5 percent probability level. The resulted data were represented as mean ± SE ("Standard Error").

Induction of multiple shoots
Nodal segments from one-month-old seedlings (Fig. 1a) were subjected to MS medium augmented with numerous kinetin, TDZ, and BAP levels for proliferation and induction of adventitious shoots. Shoots appeared from explants after two weeks of culture (Fig. 1b-f). However, completely emerged shoots and shoot elongation was found after four weeks of culture. Here the direct organogenesis pathway from the nodal segments without production of callus stage and visible shoot buds was identi ed within 4 weeks. The nodal explants cultivated on MS media augmented using BAP (1.0 mg L − 1 ) recorded high frequency (72.20 %) of shoot initiation with the maximum shoots number (10.04) and with the greater shoot duration of 8.1 cm (Table 1; Fig. 1h).

Callus Induction And Shoot Organogenesis
Leaf and root explants were nurtured onto MS medium amended with several levels of 2, 4-D (0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 3.0 mg L − 1 ) for callus induction. It was detected after 2 weeks (Fig. 1i) and a well-developed callus was noted after 4 weeks (Fig. 1j). Callus induction frequency of both explants was raised with the rise in levels of 2, 4-D from 0.2 to 0.5 mg L − 1 for root and from (0.5 to 1.0 mg L − 1 ) for leaf explants. Further rise in concentration decreased the potential of callus formation (Table 2). After eight weeks of cultivation, the highest rate of callus formation (75.35%) was identi ed to be 2, 4-D (1.0 mg L − 1 ) in (Fig. 1k) from leaf explants on MS and 64.28% from root explants cultivated on MS augmented by 2, 4-D (0.5 mg L − 1 ) in (Fig. 1O). Thus, leaf explant shows a better callus induction response than that root explants. There was no callusing response in case of nodal explants in the similar media containing 2, 4-D. Calli obtained from root explant at 2, 4-D from 0.2 mg L − 1 to 0.5 mg L − 1 along with leaf explant at 2, 4-D from 1.0 mg L − 1 to 1.5 mg L − 1 were"observed to be compact, friable as well as white in nature, whereas the rest were noted to be non-friable, white and spongy in nature (Table 2). These embryogenic WFC callus cultures procured from root explants and leaf explants were moved onto MS medium comprising numerous levels and blends of NAA along with BAP (Table 3). Both leaf sheath and root-derived calli showed the highest shoot regeneration rate on MS medium augmented using NAA of (1.0 mg L − 1 ) and BAP of (1.0 mg L − 1 ). Callus obtained from leaf demonstrated an optimum shoot regeneration frequency (75.49%) with 40 shoots per callus after six weeks of culture ( Fig. 1l-n). Similarly, root-derived calli also recorded the maximum frequency of shoot generation (60.20%) with 25 shoots per callus (Fig. 1p -r).This observation indicated that indirect organogenesis pathway is achievable from leaf and root explants of C. zizanioides   (Table 4). Root initiation was detected at the base of in vitro shoots after 2 weeks of inoculation (Fig. 1s) and a well-developed root system was visible after 4 weeks ( Fig. 1t and u). Results indicated that maximum response of root initiation (76.97%) was found with full-strength MS amended by NAA (2.0 mg L − 1 ) with an average of 16 roots per plant with the root duration of 4.01 ± 0.2 cm. However, there is less response of root initiation in the half-strength hormone-free medium.

Regeneration Via Somatic Embryogenesis
Node, leaf, and root explants were surface sterilized and inoculated in the MS medium augmented with many mixtures and levels of 2, 4-D as well as BAP for initiation of somatic embryos ( Table 5). The proximal part of the nodal segments was placed vertically in the culture tube, whereas leaf and root surface were allowed to rmly contact in the medium. Initiation of somatic embryos from explants was observed from all the treatments of 2, 4-D & BAP blends, indicating the optimization of PGRs for somatic embryogenesis. However, the greatest rate of somatic embryogenesis was noti ed from leaf (52.50%), followed by root (40.80%) and node segments (32.88%) on MS medium expanded by 2, 4-D (1.0 mg L − 1 ) as well as BAP (0.5 mg L − 1 ) ( Table 5). Somatic embryos of all stages were noted after 6 weeks of incubation. Concomitantly, leaf explant formed a substantially highest number of somatic embryos (46.2), followed by root (38.2) and node explants (23.6). Heart shape and torpedo embryos from node (Fig. 2a), cotyledonary stages from leaf (Fig. 2b), and roots (Fig. 2c) were observed during the 4th week of the culture period. Entire somatic embryos of all phases were acquired and transported to a hormonefree MS medium for germination into complete plantlets. Germination and proliferation of cotyledonary stage embryos (Fig, 2 d and e) into complete plants with an e cient rooting system were observed after 6 weeks ( Fig. 2f). Thereafter, in vitro established somatic plantlets were moved to sterile pot blend & adjusted in the greenhouse (Fig. 2h).

Hardening Of In Vitro Raised Plantlets
The better survivability of regenerated plantlets was achieved through hardening. In this study, the plantlets regenerated via organogenesis as well as somatic embryogenesis was moved to plastic containers covered with sand, sterilized soil along with FYM mix in a 1:1:1 ratio for acclimatization. These hardened plantlets were coated with a plastic sheet and drenched with a half-strength MS medium twice a week. Acclimatized plants were cultured in the chamber of a plant growth for 15 days and subsequently transferred to a greenhouse and then on the eld condition. The survival rates of regenerated plants were evaluated in the greenhouse as well as in eld conditions. Findings indicated that organogenic plantlets survived 70 percent in the greenhouse and 60% at eld conditions, whereas, somatic embryogenesis derived plantlets survived 78% at greenhouse conditions and 65% at eld conditions (Table 6). The complete protocol involving regeneration by organogenesis as well as somatic embryogenesis of C. zizanioides was presented in Fig. 3.  Here, cytokinins such as kinetin, TDZ, and BAP were used to assess the shoot induction e cacy from nodal explants of C. zizanioides. Among the three cytokinins tested, BAP exhibited maximum performance for adventitious shoot regeneration than TDZ and kinetin.

Hardening of somatic plants
Success in micropropagation is based on the successful ex vitro establishment of a fully regenerated in vitro plant. Fully grown healthy plants from experimental tubes were carefully removed and transferred to the potting mixture for acclimatization. The plantlet's survival rate was judged in the greenhouse and subsequently in eld conditions. Results revealed that the plantlets derived from somatic embryogenesis survived at higher rate when compared to organogenic plantlets (

Conclusion
The demonstrated protocol of plant regeneration via organogenesis and somatic embryogenesis using node, root, and leaf explants of C. zizaniodes. Callogenesis from leaf and roots explants may facilitate the extraction of novel bioactive compounds. The present protocol might open the ways to scale up research on genetic engineering to synthesize novel drugs, germplasm conservation, and micropropagation at industrial level.  on MS with BAP of (1.0 mg L− 1) and NAA of (1.0 mg L− 1) after six weeks (p), Many shoots initiation and elongation after 8 weeks (q & r); (s -u) Rooting of shoots on MS using NAA(2.0 mg L− 1) after two weeks (s) & four weeks (t and u); (v) Somatic plants acclimatized in pot mixture. Scale bars (a = 1 cm, b -f = 1 mm, g -i = 0.5 cm, k -n = 1 cm, q & p= 1mm, q -u= 1 cm, v = 2 cm).

Figure 2
Somatic embryogenesis in C. zizanioides from explants cultivated on MS forti ed using 2, 4-D (1.0 mg L− 1) as well as BAP (0.5 mg L− 1). (a-c) Clusters of somatic embryos from the node (a), asynchronous cotyledonary embryos from leaf (b) and root (c) after six weeks; (d -f) Germination of cotyledonary staged embryo on MS medium (d & e) after two weeks, complete plants with shoots and roots after four weeks (f); (g) Somatic plants acclimatized in pot mixture before transferring to the soil. Scale bars (a, b, c, d and e = 1 mm, f = 0.5 cm, g = 1 cm).

Figure 3
Outline of morphogenic pathways involved in micropropagation of C. zizanioides from different explants