Regeneration of Hemidesmus indicus (L.) R. Br. using in vitro nodes: an alternative method for efficient multiplication of shoots

In vivo nodes of Hemidesmus indicus (L.) R. Br. induced healthy multiple shoots with branching in our earlier studies and thus in the present study, potency of in vitro nodes to regenerate shoots was evaluated. In vitro nodes were excised from eight-week-old shoots and placed in Murashige and Skoog’s (MS) medium fortified with sucrose (3%) and different concentrations of 6-benzyladenine (BA) and kinetin (Kn). After eight weeks, optimum of 5.42 ± 0.36 shoots with 100% response were regenerated in medium supplemented with BA (10 μM) and Kn (5 μM). These healthy shoots were placed in full, half and quarter strengths of liquid MS medium fortified with sucrose (1%) and α-naphthaleneacetic acid (NAA, 1-25 μM) for rooting. Among all the strengths of MS medium, full strength MS medium having 8 μM NAA formed maximum of 3.42 ± 0.55 roots (91.67% response) within four weeks. The protocol is in continuation with earlier study and it was confirmed that a single in vivo nodal explant can regenerate around 385 healthy elongated shoots within 4 months, which will help in mass-propagation of the species.


Introduction
Hemidesmus indicus (L.) R. Br. (Asclepiadaceae), commonly known as 'anantmool', is a slender, laticiferous, semi erect shrub widely used in Indian Systems of Medicine (Anonymous, 1997). The plant is prescribed for its properties to give longevity, rejuvenation and strengthening the bones and tissues and hence considered as 'Rasayana' drug (Puri, 2003). Traditionally the plant is used to cure scorpion sting and rat bite poisoning (Sharma et al., 1979) and also have uses to treat many ailments like blood diseases, diarrhoea, respiratory disorders, kidney and urinary disorders, skin diseases, syphilis and rheumatic fever (Nadkarni, 1989). The multipurpose medicinal attributes are because it has important secondary metabolites like lupeol, vanillin, rutin, 2-hydroxy-4-methoxybenzaldehyde and β-sitosterol (Chatterjee and Bhattacharya, 1955;Subramanian and Nair, 1968;Gupta et al., 1992;Nagarajan and Rao, 2003). National Medicinal Plants Board (NMPB) has reported H. indicus as one of the priority species because its annual requirement is 500-1000 MT (Ved and Goraya, 2007). To fulfil this demand, H. indicus is extensively collected from natural habitats and its vegetative propagation is slow, due to which it has become endangered (Sreekumar et al., 2000). Thus, since last two decades it is being imported from other countries like Myanmar, Mexico and Morocco (Planning commission, 2000).
Biotechnological tool like plant tissue culture is known to regenerate large number of plants in short time duration; and to achieve this leaf and nodal explants are mostly utilized. Nevertheless, regeneration through leaf explant is known to induce soma clonal variants and hence nodes are best suitable explants when the aim is to produce true-to-type plants (Pathak and Joshi, 2018). There are reports on H. indicus regeneration using nodal explants (Patnaik and Debata, 1996;Sreekumar et al., 2000;Misra et al., 2003;Saha et al., 2003;Shekhawat and Manokari, 2016;. However, the plant is a slow growing and sparingly branched in wild (Sreekumar et al., 2000) and hence use of nodal explant for plant regeneration may increase the threat on its population. George (1996) has depicted that the explants derived from shoot cultures have an early and greater capacity for regeneration due to juvenile nature as compared to in vivo plants. The utilization of in vitro nodes as an alternative to in vivo nodal explants for generating clonal plants has been reported in many endangered species (Baskaran and Jayabalan, 2005;Loganathan and Bai, 2014), whereas in H. indicus only few reports are documented (Sreekumar et al., 2000;Nagahatenna and Peiris, 2007). In previous study on in vivo nodal explants it was observed that the axillary buds of in vitro shoots had a tendency to form branches when grown in cytokinin fortified media .
Thus, the present study was focuses on the potency of in vitro nodes to form shoots through which clonal multiplication of H. indicus can be achieved and it will help in mass-propagation.

Materials and Methods
Plant material and culture medium for shoot regeneration Healthy twigs of H. indicus were collected from the Botanical Garden of The M.S. University of Baroda.
Nodal explants were excised, washed under running tap water for 1 h, surface sterilized and inoculated in earlier optimized media for shoot culture establishment i.e. MS (Murashige and Skoog, 1962) medium fortified with 6-benzyladenine (BA, 10 µM) and kinetin (Kn, 5 µM) . After eight weeks, these shoots were harvested from mother explant, all the leaves were trimmed off and in vitro nodes (2-3 cm long) were used as an explant for the present study. They were inoculated vertically into MS medium fortified with sucrose (3%) (SRL, Mumbai, India) and different concentrations of BA and Kn (5-20 µM) (SRL, Mumbai, India). pH of the medium was adjusted to 5.80 using NaOH/HCl (1 N) and agar (0.8%) (SRL, Mumbai, India) was used as the gelling agent. All the media were sterilized in autoclave at 121 ºC (15 psi) for 20 min.

Culture conditions
All the cultures were kept in culture room and maintained at 26 ± 2 ºC under 16/8 h (dark/light) photoperiod at 40 μmol/m 2 /s provided by cool-white fluorescent lights (Philips India Ltd., India).

Statistical analysis
The values are given as mean and standard error (SE) from twelve replicates. All the means were analysed using ANOVA (α = 0.05) and significant means were further analysed by Tukey's test using XLSTAT v2017.02.

Results and Discussion
Shoot regeneration In vitro nodes derived from eight-week-old shoot cultures were used as an explant for clonal multiplication in MS basal medium, which evoked a meagre response as it formed only 0.58 ± 0.22 shoots (41.67% response) (Table 1). Supplementing the medium with different concentration of BA (5-20 µM) slightly increased the culture response and the shoot number was increased to 1.83 ± 0.16 (100% response) when BA concentration reached to 10 µM. Further increase in concentrations to 15 and 20 µM failed to enhance the number of shoots. When BA was replaced with Kn in the medium it also resulted in a similar response as only 1.58 ± 0.28 shoots (83.33% response) were recorded in media containing 10 µM Kn (Table  1). Thus, both the cytokinins individually formed healthy axillary shoots, but they failed to induce multiples.
The response in terms of number of shoots was increased as the MS medium was augmented with both BA and Kn as it induced multiple shoots in most of the combinations. Eight weeks observations depicted that optimum 5.42 ± 0.36 shoots in 100% cultures were formed in medium supplemented with BA (10 µM) and Kn (5 µM) ( Table 1). In this combination the axillary bud beak was recorded within a week of inoculation ( Figure 1A). This bud elongated and formed shoot during second week and simultaneously bud break from the opposite node was also observed ( Figure 1B). These shoots started to elongate in third week and branching of shoots was occurred during fourth week ( Figure 1C). At the end of four weeks the shoots were sub-cultured which further proliferated and multiplied till the end of eight weeks ( Figure 1D). Thus, it was confirmed that in vitro buds, which were harvested after every eight weeks of culture period, retained the potency to form shoot cultures. Beneficial effect of cytokinins in axillary shoot regeneration is mainly attributed due to their involvement in processes like promotion of cell division, expansion, regulation of shoot formation and its multiplication (Mok and Mok, 2001). Synergistic combination of BA and Kn evoked optimum regeneration in the present study and the shoot number obtained in this protocol was higher as compared to previous report on same plant (Nagahatenna and Peiris, 2007). The combined effect of BA and Kn is also reported for maximum axillary bud multiplication in H. indicus (Shekhawat and Manokari, 2016). Similarly, in other medicinal plants like Eclipta alba (Baskaran and Jayabalan, 2005), Vanilla planifolia (Abebe et al., 2009), Pogostemon cablin (Swamy et al., 2010) and Enicostema axillare (Loganathan and Bai, 2014) BA with Kn induced optimum shoots from in vitro nodes. Moreover, the potency of in vitro nodes to regenerate shoots was less as compared to in vivo nodal explants of the same plant (Patnaik and Debata, 1996;Sreekumar et al., 2000;Misra et al., 2003;Saha et al., 2003), it is also in contradiction to the earlier report which states that the potency of in vivo and in vitro nodes is the same (Sreekumar et al., 2000). But this study is in continuation of previous work in which from single node around 11 shoots were formed within eight weeks, each shoot had an average 7 nodes . Thus approximately 77 in vitro nodes were obtained which were taken up for clonal multiplication of shoots and transferred to the media optimized in the present study. Total 5 shoots were regenerated with 100% response at the end of eight weeks which confirmed that around 77 × 5 = 385 shoots can be regenerated within 4 months from single in vivo nodal explant. Therefore, this method of using in vitro nodes for shoot regeneration is more efficient as compared to transferring of shoot clumps as reported for the same plant (Shekhawat and Manokari, 2016). The use of in vitro nodes will also resolve the problems reported for in vivo nodal explants of H. indicus such as leaf fall during culture multiplication, decrease in shoot number over successive passage, excessive callusing at the base of node and seasonal effect on regeneration frequency (Patnaik and Debata, 1996;Misra et al., 2003;Shekhawat and Manokari, 2016). In vitro rooting Elongated shoots were transferred to full, half and quarter strengths of liquid MS medium fortified with different NAA concentrations (1-25 μM). In all the strengths, basal MS medium failed to induce roots and further fortifying it with NAA resulted in induction of roots. In full strength MS medium, lower levels of NAA (1-4 μM) evoked poor response. The number of roots increased as the NAA level increased to 6 and 8 μM, and within four weeks optimum 3.42 ± 0.55 roots/shoot (91.67% response) were formed at 8 μM concentration in which 100% shoot survived ( Figure 1E). Further increase in concentration adversely affected shoot survival as well as rooting. It was noted that reducing the strength of MS medium to half and quarter couldn't evoke a better rooting response (Table 2). The MS media composition and auxins are two main factors which affects in vitro rooting (George and Sherrington, 1984). NAA has been reported to induce roots in Decalepis hamiltonii (Anitha and Pullaiah, 2002) and Stevia rebaudiana (Thiyagarajan and Venkatachalam, 2012). Similarly, in the present study rooting was achieved in full strength MS medium fortified with NAA (8 μM). It was also observed that increase in its concentration adversely affected the rooting response and this may be due to biosynthesis of ethylene by higher concentration of auxins that hinders root formation (Riov and Yang, 1989;Taiz and Zeiger, 2003). However, the number of roots formed in presence of NAA is less as compared to indole-3-butyric acid (IBA) fortified medium . Martin (2002) documented that superiority of IBA is due to its slow movement and better degradation as compared to NAA, which makes it more available in the medium and increases the rooting. There are reports which stated the superiority of IBA over NAA in H. indicus (Shekhawat and Manokari, 2016) and other plants like Tylophora indica (Thomas and Philip, 2005) and Rubia cordifolia (Khadke et al., 2013).

Conclusions
In vitro nodes of H. indicus have potency to regenerate multiple shoots within eight weeks. Hence present study along with earlier developed protocol can generate around 385 shoots from single in vivo nodal explant within span of four months. Utilization of in vitro nodes from shoot cultures will help in continuous multiplication of shoots without hampering the wild plants. This protocol is efficient for mass-propagation and conservation of this endangered species as well as extraction of valuable secondary metabolites.
Authors' Contributions AGJ and ARP: Designed and conceptualized the research. ARP: Performed the experimental work, analysed data and wrote the paper. AGJ: Supervised the work and revised the manuscript for important intellectual content. Both authors read and approved the final manuscript.