ABSTRACT
In vitro regeneration of plants is influenced to a great extent by genotype, the type of explant used, media composition and plant growth regulators. This research was devised to study the effect of explant source and different hormonal combinations on the in vitro regeneration of Heracleum candicans. Two different explants viz; leaf and petiole were used to investigate their morphogenic response on MS medium supplemented with different concentrations of auxins (2, 4- dichlorophenoxyacetic acid and indole 3-acetic acid) and cytokinins (6-benzyl amino purine and kinetin) either individually or in combinations. Petiole explants were most responsive for callus production as well as shoot regeneration. 2,4-D at 3 mg/L proved to be the best concentration for callus induction. For shoot regeneration, MS medium supplemented with BAP at 3 mg/l and IAA at 2 mg/l proved to be effective in production of 21±0.7 mean number of shoots in 90% of cultures. Regenerated shoots were separated and rooted on full strength MS basal medium producing 6.9 average numbers of roots per shoot in 90% of cultures. The present research yielded a suitable explant and optimum concentrations of different plant growth regulators for in vitro regeneration of this important medicinal herb.
Key words: Heracleum candicans, leaf, petiole, callus, shoot regeneration.
Heracleum candicans is a perennial herb endemic to the northwest Himalayas and is found growing in alpine zones of West Pakistan, Nepal, Bhutan, Afghanistan and India (Sharma and Wakhlu, 2001). Propagation occurs by means of seeds, however, the seed germination is poor. This poor germination coupled with unsustainable harvesting of plants from natural habitats for commercial utilization threatens the existence of this plant species (Kaul, 1989). Being the main source of xanthotoxin, it has an immense demand in pharmaceutical industries (BCIL, 1996). Xanthotoxin is widely used to treat leucoderma and to prepare suntan lotions (Kaul, 1989). The fruit is used as an aphrodisiac and nerve tonic (Satyavati and Gupta, 1987). Activity-guided isolation has also shown
heraclenin to be the anti-inflammatory principle present in H. candicans. Extracts of root and shoot showed antibacterial activity (Kaur et al., 2006). A lengthier vegetative growing phase coupled with low seed viability in this species (Butola and Badola, 2004, 2006) offer fewer chances to revive and shift into fresh habitats. Because of the high market price of its rootstock, it is one of the important medicinal plants exported from India (BCIL, 1996). Owing to its unsustainable harvesting in nature, this plant is categorized as endangered for northwest Himalayas (Anonymous, 1998; CAMP, 2003), and vulnerable for the Jammu & Kashmir State in India (Ved et al., 2003).
The instantaneous rising demand of herbal medicine is creating heavy pressure on some selected important medicinal plants in the wild due to overexploitation. Several of these medicinal plant species have slow growth rates, low population densities and narrow geographic ranges (Kala, 2009); therefore, they are more likely to undergo extinction (Anandanayaki, 2010). The vanishing of such dynamic and huge amounts of biodiversity presents one of the serious challenges for the world community to halt the devastation of plant diversity that is essential to meet the present and future needs of mankind. To regenerate organs from plant tissues, media components and hormone concentrations can be optimized. Tissue culture is not only a necessary enabling technology for transgenic plant production but is also used for in vitro propagation of valuable plants. Large-scale propagation is a prerequisite to meet the pharmaceutical needs, and also for effective conservation of endangered plant species. Tissue culture, from conservation point of view is important as it requires a small amount of propagules for mass multiplication providing huge quantity of raw material to herbal drug industry. Micropropagation of plant species has been found to be explant based. In case of Apiaceae family, different explants namely intermodal segments (Yoshimatsu and Shimomura, 1991), shoot tips (Wakhlu and Sharma, 1998; Rachetti and Biradar, 2016; Srilakshmi et al., 2016), single-node explants (Chandrika et al., 2015; Tuncer, 2017), hypocotyl explant (Thapa et al., 2015; Mandal and Sharma, 2016), leaf explant (Rao et al., 2015; Soorni and Kahrizi, 2015) and petiole explant (Askari-Khorasgani et al., 2013; Torabi et al., 2014; Sharma, 2015) have been used for in vitro regeneration of different plants. The present study is aimed at identifying the best type of explant as well as the most efficient growth regulator concentration and combinations for shoot formation and regeneration of H. candicans.
For standardization of in vitro regeneration protocols, two different explants viz; leaf and petiole were used. Leaf and petiole explants obtained from Kashmir University Botanical Garden (Voucher Specimen Number 2616-(Ref.No.F/Herbarium-Specimen COPT) KU/2017) were firstly washed with tap water in order to remove dust, dirt and other undesirable materials followed by washing with detergent solution (Labolene) containing 4 drops of Tween 20. This was followed by washing with tap water to eliminate the detergent. The explants were washed 2 to 3 times with double distilled water in a laminar air flow cabinet. Finally, the explants were sterilized with 2% sodium hypochlorite solution for different time durations. The surface disinfected explants were then washed 3 to 4 times with autoclaved double distilled water to remove the last traces of the sterilants. The disinfected plant material was then put into pre-autoclaved petri-dishes, cut into appropriate size and finally aseptically cultured on the medium. Each treatment involved about 10 to 30 explants and each experiment was repeated twice. Explants were inoculated onto MS basal medium (control) and MS medium fortified with different plant growth regulators both individually and in combinations. Throughout the experiments, culture room was kept entirely germ-free and explants were cultured under cool white light with 1500-3000 Lux light intensity. The temperature of the room was maintained between 22±4°C with 60-70% relative humidity and photoperiod of 16 h.
The cultures were regularly observed, changes in explant were recorded on weekly basis and the data was put into a suitable arrangement in tabulated form. The parameters recorded were induction of callus, texture of callus, shoot regeneration and root regeneration. Each experiment was repeated twice, data was analyzed by calculating Standard Error (SE) of various treatments and means were analyzed by analysis of variance (ANOVA).
During the present study, the comparative effect of two different explants viz. leaf and petiole and concentrations of auxins and cytokinins on the morphogenesis of H. candicans was analyzed. The results are summarized in Tables 1 to 4. The data revealed that there were significant differences in the type of explant used and the effect of different concentrations and combinations of 2,4-D, IAA, BAP and Kn.
Evaluation of the effect of explant type and hormonal concentration on callus induction
During the present investigation, callus induction from leaf and petiole explant was obtained on MS medium supplemented with auxins both individually and in combination with cytokinins. Among auxins, five different concentrations of 2,4-D were used for callus induction. Callus induction was observed in both the explants and callus induction percentage varied significantly among the two explants. The 2,4-D at 3 mg/L was the optimum or threshold concentration for callus induction in both the explants, however the number of days taken for callus induction were shortest in the case of petiole explants. In order to examine the effect of auxin-cytokinin combinations on callus induction, leaf and petiole explants were inoculated on MS medium supplemented with 2,4-D (3 mg/L) in combination with five different concentrations of Kn. The 2,4-D at 3 mg/L and Kn at 2 mg/L proved to be the optimal concentration for maximum callus induction, with 100% culture response occurring in the petiole explant. By increasing the concentration of Kn further, there occurs a continuing decrease in callus induction, because of the fact that increasing the ratio of cytokinin to auxin favors shoot regeneration.
Evaluation of the effect of explant type and hormonal concentration on shoot regeneration
During the present study, shoot regeneration was witnessed from both the explant types on MS medium supplemented with cytokinins individually and in combination with auxins. When MS medium was fortified, five different concentrations of BAP individually, BAP at 3 mg/L proved to be best medium for shoot regeneration with maximum number of shoots obtained from petiole explant in minimum number of days. Among five different auxin and cytokinin combinations used, BAP at 3 mg/L and IAA at 2 mg/L gave best results in both the explant types with maximum number of shoots regenerated from petiole derived calli. By increasing the auxin concentration from 2 to 5 mg/L in the medium, a significant decrease occurred in the frequency and number of shoots produced per explant with an increase in the amount of callus. For root formation, the in vitro raised shoots were subcultured on MS basal medium and MS medium containing IAA, NAA and IBA individually. In the present study, MS basal medium was effective for root regeneration. Among hormone supplemented medium, the most effective concentration at which maximum root induction was achieved was IAA at 2 mg/L.
The explant source is a vital factor for in vitro growth and development of plant species affecting callus production, shoot bud induction as well as regeneration and multiplication. During the present research, statistical analysis revealed that there were significant differences between the two explant types used. Petiole explant proved to be the most effective explant for both callus induction as well as shoot formation (Figure 1). Patra et al. (1998) developed a successful protocol for in vitro plant regeneration from callus derived from leaf explants in Centella asiatica (L.). Mohapatra et al. (2008) also performed in vitro studies in C. asiatica using leaf explant. The study revealed that frequency of multiple shoot regeneration was much higher on MS medium supplemented with combination of auxin and cytokinin than cytokinins used individually. Sharma and Wakhlu (2001) obtained similar results for adventitious shoot induction from petiole explants of H. candicans Wall. However, the researchers used 2,4-D rather than IAA used in the present study. Makunga et al. (2003) obtained direct shoot regeneration from petiole explant of Thapsia garganica on MS medium supplemented with 0.5 mg /L NAA and 1.5 mg/l BAP which is in contrast with our results. Sharma (2009) achieved callus initiation from petiole explants of H. candicans on MS medium supplemented with 0.5 mg/L 2,4-D and 0.5 mg/L BAP. In addition, the best combination of growth regulators for maximum callus growth was obtained upon subculture of callus to a medium supplemented with 1 mg/L 2,4-D and 0.25 mg/L Kn. Askari-Khorasgani et al. (2013) also used petiole explant for direct regeneration of Kelussia odoratissima. The maximum rate of shoot multiplication was achieved by inoculation of petiole explants on MS medium fortified with 2 mg/L BAP and 0.1 mg/L NAA. Sharma (2015) performed studies on callus induction from petiole explants in Ferula jaeschakeana in which explants were cultured on MS medium supplemented with 2,4-D for callus initiation. The best results of callus induction were obtained on medium fortified with 1 mg/L 2,4-D. The addition of Kn in combination with 2,4-D enhanced the callus formation from the explants.
The present study yielded a suitable explant and optimum concentrations of different plant growth regulators for in vitro regeneration of this important medicinal herb.
The authors have not declared any conflict of interests.
The authors appreciate all the researchers whose articles are quoted and incorporated in the references of the document.
REFERENCES
|
Anandanayaki S (2010). Comparative pharmacognostical studies on selected plants (pedalium murex roen ex. L. And martynia annua L.) A Thesis submitted to Tamil University.
|
|
|
|
Anonymous (1998). Conservation assessment and management plant workshop-briefing book. FRLHT, Bangalore, India.
|
|
|
|
|
Askari-Khorasgani O, Mortazaeinezhad F, Otroshy M, Golparvar AR, Moeini A (2013). Direct regeneration of an endangered medicinal plant Kelussia odoratissima. International Journal of Agriculture and Crop Sciences 5(17):1969-1974.
|
|
|
|
|
BCIL (1996). Sectoral study of Indian Medicinal Plants- status, perspective and strategy for growth Biotech Consortium India Ltd., New Delhi.
|
|
|
|
|
Butola JS, Badola HK (2004). Effect of pre-sowing treatment on seed germination and seedling vigour in Angelica glauca, a threatened medicinal herb. Current Science 87(6):796-798.
|
|
|
|
|
Butola JS, Badola HK (2006). Chemical treatments to improve seedling emergence, vigour and survival in Heracleum candicans Wall-(Apiaceae): a high value threatened medicinal and edible herb of Himalaya. Journal of Plant Biology 33(3):215-220.
|
|
|
|
|
CAMP (2003). CAMP Report: Conservation Assessment and Management Prioritisation for the Medicinal Plants of Jammu and Kashmir, Himachal Pradesh and Uttaranchal, Workshop, Shimla Himachal Pradesh. FRLHT, Bangalore, India 206.
|
|
|
|
|
Chandrika R, Shivakameshwari MN, TharaSaraswathi KJ (2015). Reduction of vitrification in in vitro shoot cultures of Eryngium foetidum L. - a potential aromatic and medicinal herb. Indian Journal of Plant Sciences 4(2):52-58.
|
|
|
|
|
Kala CP (2009). Ethnobotanical and Ecological Approaches for Conservation of Medicinal and Aromatic Plants. Acta Horticulturae 860(860):19-26
Crossref
|
|
|
|
|
Kaul MK (1989). Himalayan Heracleum Linn (Hogweed) - a review CSIR Jammu, India.
|
|
|
|
|
Kaur M, Thakur Y, Thakur M, Chand R (2006). Antimicrobial properties of Heracleum candicans Wall. Natural Product Radiance 5(1):25-28.
|
|
|
|
|
Makunga NP, Jager AK, Van Staden J (2003). Micropropagation of Thapsia garganica- a medicinal plant. Plant Cell Reports 21(10):967-973.
Crossref
|
|
|
|
|
Mandal J, Sharma P (2016). In vitro Micropropagation of Carum copticum L. Pharmaceutical Bioprocessing 4(3):047-051
|
|
|
|
|
Mohapatra H, Barik DP, Rath SP (2008). In vitro regeneration of medicinal plant Centella asiatica. Biologia Plantarum 52:339-342.
Crossref
|
|
|
|
|
Patra A, Rai B, Rout GR, Das P (1998). Successful plant regeneration from callus cultures of Centella asiatica (Linn.) Urban. Plant Growth Regulation 24(1):13-16.
Crossref
|
|
|
|
|
Rachetti BD, Biradar SR (2016). In vitro propagation of Centella asiatica L. by using coconut water and house hold sugar. Trends in Biotechnology Research 5(1):1-4.
|
|
|
|
|
Rao S, Usha K, Arjun (2015). Production of secondary metabolites from callus cultures of Centella asiatica (L.) Urban. Annals of Phytomedicine 4(1):74-78
|
|
|
|
|
Sharma RK (2009). Caulogenesis in Heracleum candicans Wall. International Journal of Plant Sciences 4(2):354-356.
|
|
|
|
|
Sharma RK (2015). Callus formation in Ferula jaeschkeana vatke. International Journal of Plant Sciences 10(1):98-101.
Crossref
|
|
|
|
|
Sharma RK, Wakhlu AK (2001). Adventitious shoot regeneration from petiole explants of Heracleum candicans wall. In Vitro Cellular and Developmental Biology-Plant 37(6):794-797.
Crossref
|
|
|
|
|
Soorni J, Kahrizi D (2015). Effect of Genotype, Explant Type and 2,4-D on Cell Dedifferentiation and Callus Induction in Cumin (Cuminum cyminum) Medicinal Plant. Journal of Applied Biotechnology Reports 2(3):265-270.
|
|
|
|
|
Srilakshmi A, Ugraiah A, Gayatri CM, Rajanna L (2016). Genetic Fidelity in Micropropagated Plantlets of Pimpinella tirupatiensis-an Endemic and Threatened Medicinal Plant Using RAPD and ISSR Markers. International Journal of Bioassays 5(6):4661-4666.
Crossref
|
|
|
|
|
Thapa A, Rathore SS, Sharma LK, Agrawal D, Saxena SN (2015). Plant regeneration in coriander (Coriandrum sativum L.) International Journal of Seed Spices 5(2):63-66.
|
|
|
|
|
Torabi S, Far MS, Omidi M, Gharakhanlou H, Saffar ZN (2014). A survey of the best growing conditions of in vitro culture in fennel different genotypes for mass production. International Journal of Biosciences 5(4):7-17.
Crossref
|
|
|
|
|
Tuncer B (2017). Investigation of the in vitro regeneration of some medical and aromatic wild plant species. Applied Ecology and Environmental Research 15(4):905-914.
Crossref
|
|
|
|
|
Ved DK, Kinhal GA, Ravikumar K (2003). CAMP Report: Conservation Assessment and Management Prioritisation for the Medicinal Plants of Jammu and Kashmir, Himachal Pradesh and Uttaranchal, Workshop, Shimla, Himachal Pradesh. FRLHT, Bangalore, India.
|
|
|
|
|
Wakhlu AK, Sharma RK (1998). Micropropagation of Heracleum candicans Wall: a rare medicinal herb. In Vitro Cellular and Developmental Biology-Plant 35(1):79-81.
Crossref
|
|
|
|
|
Yoshimatsu K, Shimomura K (1991). Efficient shoot formation on internodal segments and alkaloid formation in the regenerates of Cephaelis ipecacuanha A. Richard. Plant Cell Reports 9(10):567-570.
Crossref
|
|
