About four million people in rural regions have entirely depended upon medicinal plants as a primary source of health care (Petrovska 2012). Plant tissue culture can be an alternate methodology for germplasm conservation and mass production of quality bio-active compounds (Ahmed et al. 2019; Grigoriadou et al. 2019). Hence, the current study was set to regulate reliable regeneration protocols via organogenesis & “somatic embryogenesis” in C. zizanioides.
Regeneration through direct organogenesis
Some of the reports on in vitro regeneration of vetiver using explants like leaf crown (Leupin et al. 2000), leaves (Mucciarelli et al. 1993), young inflorescence (Sangduen et al. 2009), meristem tip culture (Chitra et al. 2014) and mesocotyl culture (George et al. 1999). In this direct shoot organogenesis involving nodal segments has been successfully established from in vivo grown seedlings of C. zizanioides. Several reports have authenticated the superior role of nodal explant over the other types to accomplish micropropagation in medicinal plants such as Mentha piperita (Sujana 2011), Withania somnifera (Fatima 2012), Agastache foeniculum (Moharami 2014), Teucrium scorodonia (Makowczyńska 2016), and Spermacoce hispida (Deepak et al. 2019). Earlier literature has proved the existence of positive correlations among explant types, the shoot proliferation rate, and influences of cytokinin (Sajid et al. 2011; Dantu and Bhojwani 2013; Lebedev et al. 2018).
Here, cytokinins such as kinetin, TDZ, and BAP were used to assess the shoot induction efficacy from nodal explants of C. zizanioides. Among the three cytokinins tested, BAP exhibited maximum performance for adventitious shoot regeneration than TDZ and kinetin.
Yang et al. (2013) also stated about the effect of BAP in MS medium on direct regeneration in vetiver. The effect of BAP in shoot organogenesis was previously noted in several medical plants like Canscora decussate (Loganathan 2016), Scutellaria alpine (Grzegorczyk 2016), Dracocephalum forrestii (Weremczuk 2018), and Gymnema sylvestre (Isahet al. 2019). Interestingly, we noticed maximum shoot proliferation (72.2%) from node explant cultured onto MS fortified with BAP @ 1 mg L-1. Similar trends of findings were also observed in Securidaca longipedunculata (Lijalem et al., 2020) and Trichosanthes dioica (Tiwari et al. 2010). The nodal segment is considered to be the best explant type when compared to other types for initiation of direct shoot regeneration and proliferation and also requires only a lesser concentration of cytokinin, according to the Algabri et al. survey (2019).
Callus culture offers a wide variety of applications in pharmacological research, rather than direct organogenesis (Efferth 2018). The efficacy of callus initiation is reliant on the medium composition and types of the explants (Romano and Gonçalves 2013; Nordine et al. 2014). Leaf and root parts from in vitro seedlings of C. zizanioides were used as explants for callogenesis. In this study, even inflorescence of vetiver also used for callus development (Somporn 2003)using synthetic 2, 4-D, and auxin. 2, 4-D performs a significant part in plant tissue culture methods for callogenesis, which has been already stated in V. zizanioides (Mucciarelli et al. 1993; yang et al. 2008) proliferation as well as somatic embryogenesis (Gao et al. 2011). Further, it has been said that 2, 4-D has a noteworthy effect on the biochemical and molecular processes of callus with stimulating certain gene expression, controlling the metabolism of endogenous IAA as well as regulating DNA methylation (Pan 2010). The maximum frequency of friable compact callus was found in MS medium fortified using 2, 4-D at 0.5 mg L-1& 1.0 mg L-1 from root and leaf explants, respectively. Similar studies on callogenesis using leaf and root explants were reported in several medicinally important herbs such as Trachyspermum ammi (Fazeli-nasab 2018), Vitex negundo (Chowdhury 2011), Piper betel (Junairiah 2019), Ledebouria revoluta (Haque 2018), Celastrus paniculatus (Moola et al. 2020) and Rhinacanthus nasutus (Reshi et al. 2018), and Rosa hybrida (Liu et al. 2018). Further, callus is the most amenable and optimal tissue for the formation of novel secondary metabolites (Benjamin et al. 2019). It has been noticed that the leaf explants showed a better callusing response than root explants.
Regeneration from callus
The compact friable calli obtained from culturing of leaf and root explants were transferred onto shoot initiation medium augmented with varied levels and mixtures of BAP and NAA. It has been found that both the explants exhibited the highest frequency of shoot redevelopment on MS by NAA of 1.0 mg L-1 and BAP of 1.0 mg L-1 as stated in V. zizanioides (Sompornpailin et al. 2016; Sompornpailin 2009). Also the Zinc Oxide Nanoparticles showed some impact on calli derived regenerants in V. zizanioides (Khunchuay et al. 2017). Similar effects of auxin and cytokinin combinations for shoot initiation were well studied from several medicinal plant types like Rauvolfia serpentine (Singh et al. 2009), Celosia argentea (Bakar et al. 2014), Lallemantia iberica (Ozdemir et al. 2014), and Indigo feraviscosa (Rajabudeen 2016). Further, leaf-derived calli documented a better response that was produced from root explants on shoot regeneration over the calli.
Rooting of adventitious shoots
The rooting efficiency and survival rate of hardened plants at in vivo conditions are the factors for successful in vitro propagation (Reshi 2018). In vitro rooting hastened the adaptability of plants to ex vitro conditions (Krupa-Malkiewicz and Mglosiek 2016). Regardless of medium, auxin enhances the root initiation to in vitro regenerated adventitious shoots (Martins et al. 2013; Shekhawat and Manokari 2018). In the current work, full-strength MS medium amended with 2.0 mg L-1 NAA illustrated a better-rooting response than other levels. Concomitantly, the results of Widoretno et al. (2017) also corroborated the present results that NAA @ 1.0 mg L-1 noted better-rooting efficiency on C. zizanioides. 154 Concomitantly, the results of Widoretno et al. (2017) also corroborated the present results that NAA @ 1.0 mg L− 1 noted better-rooting efficiency on C. zizanioides. The NAA efficiency in the roots of in vitro regenerated shoots was well recognized in Rotula aquatica (Chithra 2004), Phyllanthus caroliniensis (Catapan et al. 2012), Andrographis paniculata (Hossain et al. 2016), Tinospora cordifolia (Mridulaet al. 2017).
Regeneration via somatic embryogenesis
Somatic embryos are the unicellular origin and can be maintained as an embryogenic culture for a long period (Tomiczak 2019). Here the somatic cells are differentiated into the whole plant by the embryogenesis process. In vitro embryogenesis is the direct approach where the somatic embryos are directly developed from explant tissues (Mazri and Meziani 2015). Many diverse and complex factors affect somatic embryogenesis (Deo et al. 2010). The concentration, type, and combination of regulators for plant growth are the main variables that influenced somatic embryogenesis as well as plant regeneration (De Almeida et al. 2012).
Here, node leaf, and root segments as explants for somatic embryogenesis have been accomplished. High-rate somatic embryogenesis was detected at BAP (1.0 mg L-1) and 2, 4-D (0.5 mg L-1) combinations from all explants. The mixtures of auxins with cytokinins were accounted for to induce considerable somatic embryogenesis in Fraxinus excelsior (Capuana et al. 2007),Eremochloa ophiuroides (Liu et al. 2008), Swertia chirayita (Chandra and Kumar 2014), and Lachenalia viridiflora (Kumar et al. 2016). The current research demonstrates that MS medium augmented with 2, 4-D and BAP is essential for friable embryogenic callus development in C. zizaniodes. The results of this research correspond to previous studies on somatic embryogenesis of Chrysanthemum indicum (Datta and Mandal 2005), “Brachiaria brizantha” (Cabral et al. 2011), Rosa hybrida (Bao et al. 2012), “Prosopis laevigata” (González-Buendíaet al. 2012), Vitis vinifera (Dai et al. 2015) and Curcuma amada (Raju et al. 2015).
The transformation of somatic embryos into mature plantlets is crucial for a successful in vitro plant regeneration system. The torpedo and cotyledonary embryos were harvested from 2, 4-D, and BAP treatments and were subjected to germinations. Results showed significant conversion and germinations of somatic embryos were noticed on half-strength MS medium. Published literature has confirmed the effectiveness of half-strength MS for “somatic embryo germination” in other types as well (Paul 2011; Raju 2013; Kumar, 2015; Khan et al. 2015). The simultaneous transformation of the shoot and root axis from the matured somatic embryos into plantlets is crucial in the somatic embryogenesis mode of regeneration (Pandey et al. 2013).
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 field conditions. Results revealed that the plantlets derived from somatic embryogenesis survived at higher rate when compared to organogenic plantlets (Table. 6). Regardless of the acclimatizing environment, similar observations on survival rate and adaptability were recorded from organogenesis and somatic embryogenesis derived plants (Bhagya et al. 2013; Deepak et al. 2019).