Momordica cymbalaria Fenzl. (Cucurbitaceae) is a climber and perpetual herb. It is also called as athalakkai. It climbs on the ground surface and supports by the help of tendrils. Momordica cymbalaria fruits resembled a small variety of Momordica charantia. The plant is available with fruits in various states of India such as Telangana, Madhya Pradesh, Karnataka, and Tamil Nadu states. During the rainy season, it is around the fences of farms . The plants die at the end of the season, but the underground tuberous roots were remained and emerge in the next season to maintain its perennial habits. This plant is not very popular because of its bitter taste and lack of understanding of its nutrient content .
Recent research has revealed that methanol extraction of M. cymbalaria has anticancer properties in aerial and underground parts compared to standard cyclophosphamide against ehrlich ascites carcinoma-induced cancer in rats . Fruit of M. cymbalaria consists of a high amount of fiber along with calcium, potassium, and C vitamin . According to the previous studies, fruit and root extracts of athalakkai were very useful in various treatments like diabetes, hypolipidemia [5, 6], diarrhea , and ulcer . For menstrual irregularities, antifertility, antiovulatory, abortifacient, cardioprotective properties, and hepatoprotective activity, roots of M. cymbalaria are used .
Rhizogenesis is the process of root formation in plants. The initiation of roots is one type of organogenesis . It is essential to have successful root growth, although root growth does not occur in the initial stage. After micropropagation, root formation is essential for the plant growth, although it does not occur in the initial stage.
The ability of the shoots to initiate root or plant to survive acclimatization on the concentration of cytokinins and auxins in the Murashige and Skoog (MS) medium is required.There are three stages such as induction, initiation, and elongation in the in vitro root development. The MS medium that contains auxins such as 2,4-D, α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), and indole-3-acetic acid (IAA) is a very suitable medium for the root and shoot proliferation. Cytokinins and auxins at high concentrations are favorable for shoot formation, but it restricts root formation. The MS medium without adding any plant growth regulators and less amount of auxin containing medium gives much rooting in many cucurbits . The combination of NAA and IBA is a very suitable combination than 2,4-D for the callus induction and direct rhizogenesis from leaf and stem explants of Heliotropium indicum . High proportion of rooting was recorded in Tricosanthes dioica by the combination of 0.5 mg/L of IBA and 2.0 mg/L of NAA . In Erythrina variegata, without any involvement of callus formation, high percentage of rooting was observed by using lower concentrations of NAA and 2,4-D .
The main objective of this experiment is the optimization of in vitro rooting and plant growth regulator conditions in different parts of M. cymbalaria. Our investigation of in vitro adventitious rhizogenesis from leaf and node explants of M. cymbalaria was not done until now. In this study, the protocol that is very useful for the extraction of bioactive compounds from the roots of medicinally valuable plants is also discussed.
2. MATERIALS AND METHODS
The tubular roots of M. cymbalaria Fenzl were collected in the rainy season, in the Jammikunta, Kamalapur Crop Farms, Warangal District, Telangana, India. The collected plants were maintained in the Departmental Greenhouse. Young, healthy plants were raised under in vivo condition and different explants like a leaf and nodes were washed in running tap water for half an hour and rinsed with labolene detergent for 3–4 times and again washed with running tap water. The rinsed explants were surface sterilized in 0.1% mercuric chloride for 3–5 minutes and rinsed with double sterilized distilled water for 3–4 times in the laminar airflow chamber and then left for air dry in a sterile environment.
|Table 1: Rhizogenesis from nodal explants of M. cymbalaria Fenzl.|
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The sterilized leaf and node explants were inoculated into the test tubes containing the MS medium with 0.8% agar-agar, 3% sucrose, and various concentrations of NAA, IBA, 6-Benzylaminopurine (BAP), and IAA at 0.5, 1.0, 1.5, 2.0, and 2.5 mg/L individually. The pH of the medium is regulated between 5.6 and 5.8 by the addition of 0.1 N NaOH or 0.1% HCl, then agar (0.8%) is added to the medium and heated for dissolving. The medium was sterilized in the autoclave at 121°C for 15 minutes below 15 psi. The culture tubes were maintained at temperature between 25°C ± 2°C for 16/8 hours with light and dark cycle. Fluorescent tubes (Philips, India) were used for regulating photoperiod.
Young healthy leaf and node explants selected from in vivo grown plants were inoculated on medium containing various concentrations of NAA, IAA, and IBA (0.5, 1.0, 1.5, 2.0, and 2.5 mg/L) (Tables 1 and 2). The NAA is a more suitable plant growth regulator for the induction of callus, stimulation of the cluster, and multiple roots than IBA and IAA. The percentage of induction of callus and proliferation of roots per explants was recorded after 4 weeks of culture. Rooted shoots should be carefully taken from the medium and thoroughly cleaned with distilled water. The obtained roots were stored in the shade for 15–30 days and then dried and preserved in a polyethylene cover for biological activity and phytochemical analyzes in future studies.
4. RESULTS AND DISCUSSION
When node and leaf explants were grown on MS basal media without hormones, no morphogenetic response was observed, whereas induction of callus was observed within one week of culturing on the MS medium enriched with various concentrations of auxins such as IBA, IAA, and NAA of 0.5, 1.0, 1.5, 2.0, and 2.5 mg/L individually (Tables 1 and 2, Fig. 1a, Fig 2a – c). The same callus was subcultured on the half-strength MS medium, as a result rhizogenesis was occurred (Fig. 1c – f, Fig. 2d – f).
|Table 2: Rhizogenesis from leaf explants of M. cymbalaria Fenzl.|
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Direct rhizogenesis also appeared in some node explants before callus induced completely on 0.5 mg/L of NAA alone with the half-strength MS medium (Fig. 1a). Different types of colors and textures of calli were produced in both node and leaf explants containing MS medium with various concentrations of NAA, IBA, and IAA (Tables 1 and 2). Green nodular callus formation has taken place in node explants on MS medium fortified with 0.5 mg/L of NAA alone (Fig. 1a). Brown and green colored callus was produced in leaf explants containing 0.5 and 1.0 mg/L of IBA, respectively (Fig. 2b and c).
The highest percentage of rhizogenesis was obtained from green compact callus on the half-strength MS medium containing 1.5 mg/L of NAA in node explants, but in the leaf explants, light green compact callus was produced a maximum percentage of roots containing a medium at 1.0 mg/L of IBA.
In this study, various concentrations of auxins, that is, 0.5, 1.0, 1.5, 2.0, and 2.5 mg/L were analyzed for their consequence on rhizogenesis. In node explants, a number of adventitious roots were initiated at 0.5, 1.0, and 1.5 mg/L of IAA, IBA, and NAA, respectively (Table 1), whereas in leaf explants, numerous root induction takes place at 1.0, 1.0, and 0.5 mg/L of NAA, IBA, and IAA, respectively (Table 2).
The percentage of rhizogenesis was decreased with an increase in the concentration of auxins NAA, IBA, and IAA alone on node and leaf explants. The same tendency is seen in all cultures, but in node explants, rhizogenesis capacity is somewhat different, which is 1.5 mg/L of NAA produced when compared with low concentration (Tables 1 and 2). Among the different concentrations of auxins, the highest mean number of root length was recorded at 1.5 mg/L of NAA alone in the node explant (Tables 1 and 2).
In general, auxins (IBA, NAA, and IAA) have been promoted a maximum percentage of rooting in plants. However, in this study, NAA is the most successful auxin for the induction of rooting in node explants. Similar findings were reported in tomato .
It was observed that both node and leaf explants do not have an equal potential to regenerate roots. Node explants have shown a higher percentage of rhizogenesis than leaf explants. Previous studies were also similar to our result, that is, various growth regulators influenced the induction of roots as well as their elongation [16, 17]. In Citrullus colocynthis, 2.0 mg/L of IAA and 1.5 mg/L of IBA were more suitable for the formation of cluster roots in stem explants and also 2.0 mg/L of 2,4-D, 1.5 mg/L of IBA, and 2.0 mg/ L of IAA are the best for the production of multiple root production in leaf explants . The discrepancy in rooting response may be a result of genotype or cultural conditions or other factors in plants.
On the other hand, Mahendranath et al.  reported that IBA has produced the maximum biomass when compared to IAA and also individually superior over IAA or NAA in the induction of rooting has been reported earlier in Psoralea corylifolia . A similar study was also observed in Withania somnifera , Morinda citrifolia , and Periploca sepium .
|Figure 1: Rhizogenesis from nodal explants of M. cymbalaria Fenzl. (a) Green nodular callus formation of node explants on the MS medium fortified with 0.5 mg/L of NAA. (b) Roots initiation from cut margins of node explant on the MS medium fortified with 0.5 mg/L of NAA. (c) Roots initiation from green compact callus on the MS medium fortified with 1.0 mg/L of NAA. (d) Elongation of roots after three weeks of culture on the half-strength MS medium fortified with 1.0 mg/L of NAA. (e) Formation of shoots and roots on the MS medium fortified with 1.5 mg/L of NAA. (f) Maximum number of roots after three weeks of culture on the half-strength MS medium fortified with 1.5 mg/L of NAA..|
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In this study, we have been observed that the IBA was found effective for the induction of maximum rooting in leaf explants. The influence of IBA on rhizogenesis has also been supported by Neto et al. .
Of the three auxins, NAA, IBA, and IAA, tested NAA is the most effective for the induction of roots in node explants (Table 1), whereas IBA is the most effective for rhizogenesis in leaf explants (Table 2).
|Figure 2: Rhizogenesis from leaf explants of M. cymbalaria Fenzl. (a) Callus initiation from cut margins of leaf explant on the MS medium fortified with 0.5 mg/L of IBA. (b) Light brown callus production on the MS medium fortified with 0.5 mg/L of IBA after 10–12 days of inoculation. (c) Induction of green callus on the MS medium fortified with 1.0 mg/L of IBA. (d) Initiation of roots from leaf callus cultures on 1.0 mg/L of IBA after four weeks of culture. (e) Formation of shoots and roots after subculture for two weeks on the MS medium fortified with 1.0 mg/L of IBA. (f) Maximum number of roots after three weeks of culture on the half-strength MS medium fortified with 1.0 mg/L of IBA.|
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The present study revealed that efficient rhizogenesis was achieved in M. cymbalaria Fenzl. Both leaf and nodal explants were responded for the rhizogenesis at different concentrations of auxins. Compared with leaf explants, nodal explants induced more number of roots (9.3 ± 0.61) by using NAA at 1.5 mg/L concentration. Depending on genotype and culture conditions, variation in rhizogenesis response may occur. In addition, this protocol is useful for the production of large amounts of bioactive compounds in certain medicinal plants. Therefore, the roots of M. cymbalaria contain a quercetin bioactive compound that is used in pharmacy for the design of anticancer. A unique characteristic of this study is the in vitro adventitious rooting and proliferation of leaf and node explants of M. cymbalaria, which have not been previously reported.
The financial assistance provided under the UGC-SAP-DRS-II program of Government of India for the Department of Biotechnology, Kakatiya University is gratefully acknowledged.
2. Nikam TD, Ghane SG, Nehul JN, Barmukh RB. Induction of morphogenic callus and multiple shoot regeneration in Momordica cymbalaria Fenzl. Indian J Biotechnol 2009; 8:442–7.
3. Chittapur R. Momordica cymbalaria nutritious underutilized vegetable taxonomy, nutritional, medicinal, propagation, hybridization and cytological aspects. Int J Argicul Sci Res 2015; 5(4):2250–7.
9. Raju K, Balaraman R, Firdous KMW, Vinoth Kumar M. Hepatoprotective effects of Momordica cymbalaria Fenzl against CCl4 induced hepatic injury in rats. Pharmacologyonline 2008; 1:365–74.
12. Bagadekar AN, Jayaraj M. Invitro rhizogenesis from leaf and stem callus of Heliotropium indicum L. Medicinal herb. Int J Plant Anim Environ Sci 2011; 1(2):1–5.
13. Kumar S, Singh M, Singh AK, Srivastava K, Banerjee MK. Invitro propagation of pointed guard (Trichosanthes dioica Roxb.) Indian Institute Veg Res 2003; 2(6):74–5.
14. Shasthree T, Imran MA, Mallaiah B. In vitro rooting from callus cultures derived from seedling explants of Erythrina variegata L. Current Trend Biotechnol Pharm 2009; 3(4):447–52.
16. Balvanyos I, Kursinszki L, Szoke E. The effect of plant growth regulators on biomass formation and lobeline production of Lobelia inflata L. hairy root cultures. Plant Growth Reg 2001; 34:339–45. CrossRef
17. Balestri E, Bertini S. Growth and development of Posidonia oceanica seedlings treated with plant growth regulators: Possible implications for meadow restoration. Aquat Bot 2003; 76:291–7. CrossRef
18. Ramakrishna D, Shasthree T. Adventitious rooting and proliferation from different explants of Citrullus colocynthis (L.) Schard an endangered medicinally important cucurbit. Asian J Biotechnol 2015; 7(2):88–95. CrossRef
19. Mahendranath G, Venugopalan A, Parimalan R, Giridhar P, Ravishankar GA. Annatto pigment production in root cultures of Achiote (Bixa orellana L.). Plant Cell Tiss Organ Cult 2011; 16:517–22. CrossRef
21. Sivanandhan G, Arun M, Mayavan S, Rajesh M, Mariashibu TS, Manickavasagam M, Selvaraj N, Ganapathi A. Chitosan enhances withanolides production in adventitious root cultures of Withania somnifera (L.) Dunal. Industrial Crops and Products 2012; 37:124–9. CrossRef
22. Baque MA, Lee EJ, Paek KY. Medium salt strength induced changes in growth, physiology and secondary metabolite content in adventitious roots of Morinda citrifolia: the role of antioxidant enzymes and phenylalanine ammonia lyase. Plant Cell Rep 2010; 29:685–94. CrossRef
23. Zhang J, Gao WY, Wan J, Li XL. Effects of sucrose concentration and exogenous hormones on growth and periplocin accumulation in adventitious roots of Periploca sepium Bunge. Acta Physiologiae Plantarum 2012; 34:1345–51. CrossRef
24. Neto VBP, Res LB, Finger FL, Barros RS, Carvalho CR, Otoni WC. Involvement of ethylene in the rooting of seedling shoot cultures of Bixa orellana L. In vitro Cell Dev Biol Plant 2009; 45:693–700. CrossRef