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Indo American Journal of Pharmaceutical Research, 2016

ISSN NO: 2231-6876

EFFECT OF EXPLANT SOURCES AND DIFFERENT CONCENTRATIONS OF PLANT GROWTH REGULATORS ON IN VITRO MICROPROPAGATION OF GLYCYRRHIZA GLABRA L. Yogesh Badkhane*, A.S. Yadav, A. Bajaj Molecular Biology and Seed Technology Laboratory, Govt. Motilal Vigyan Mahavidyalaya (MVM), Affiliated to Barkatullah University Bhopal – 462008 M.P. ARTICLE INFO Article history Received 18/06/2016 Available online 08/07/2016 Keywords PGR’S, Glycyrrhiza Glabra L., Micropropagation, Nodal Explants, Shoot Induction.

ABSTRACT In vitro explants response and regeneration capacity of Glycyrrhiza glabra L. was tested by using varying concentrations of BA, Kn, NAA, and IBA on MS media. Explant types exhibited a different response towards establishment and shoot proliferation with highest shoot number at different concentration of PGR’s. By contrast, the nodal expant has led to the highest percent establishment and remarkable enhancement in shoot proliferation. The percent explants establishment (66.67%) and shoot formation frequency was highest (3.67 shoots/explants) at the concentration of BA 2.0 mg/l. A good response was observed towards multiplication of shoot highest in 2.0 mg/l BA + 0.50 mg/l NAA which lead to the best rate of regeneration frequency (86.67%) and shoot formation (18.33 shoots/explants), whereas 1.0 mg/l BA + 0.25 mg/l NAA favored the highest shoot development (24.0 shoots/explants) with apical buds after 4th weeks of inoculation. The study on rooting capacity revealed that treatments 1.0 mg/l IAA proved to be superior with maximum 100% rooting, 16.0 roots/explant and 2.33 cm root length. Present investigation reports that the response of nodal explants found to be the best for explants establishment and hormonal concentrations respectively for regeneration of medicinally important plant of Glycyrrhiza glabra L.

Copy right © 2016 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Please cite this article in press as Yogesh Badkhane et al. Effect of explant sources and different concentrations of plant growth regulators on in vitro micropropagation of Glycyrrhiza glabra L .Indo American Journal of Pharmaceutical Research.2016:6(06).

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Corresponding author Yogesh Badkhane Research Scholar Dept. of Botany, Govt. Motilal Vigyan Mahavidyalaya, Bhopal, (M.P.) India. [email protected]

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INTRODUCTION Glycyrrhiza glabra L. belongs to the family Fabaceae, is a genus of perennial herb and under shrubs distributed in the subtropical and warm temperate regions of the world, especially in the Mediterranean countries and China. Glycyrrhiza glabra Linn. commonly known as liquorice and sweet wood in english, Jothi‐madh, Mulhatti in Hindi, Yashti‐madhuh, Madhuka in Sanskrit, Jashtimadhu, Jaishbomodhu in Bengali, Atimadhuranu, Yashtimadhukam in Telugu, Jethimadhu in Gujarati and Atimaduram in Tamil (Chopra et.al. 2002). Licorice extracts and its principle component, glycyrrhizin, have extensive use in foods, tobacco products, and snuff, and in traditional and herbal medicine. It is cultivated for its rhizomes (underground stems) that contain the compound glycyrrhizin, which is 50 times sweeter than sugar. It is cultivated in the Mediterranean basin of Africa, in southern Europe, and in India (The Indian pharmaceutical codex, 1953., African pharmacopoeia, 1985., Ghazanfar, 1994., Chin et al., 1992) widely cultivated in Punjab and sub Himalaya tracts (Dhuke et al., 2002), Baramulla, Srinagar, Jammu, Dehradun, Delhi and South India (Meena et. al., 2010). As per report given by task force, planning commission, New Delhi (2000) on conservation and sustainable use of medicinal plants Glycyrrhiza glabra L. has been listed as a medicinal plant in high demand require by Indian pharmaceutical industries. The demand of this plant as per the list was estimated 5000 tonnes per annum. Imports from Pakistan, Iran and Afghanistan are in huge demand which need to be supported by indigenous production. Currently no concerted effort has been made for the conservation and sustainable use of this plant species, regarding its careful cultivation and harvesting so as to replenish its exhausting supply in nature. Though the plant has immense medicinal value, and high demand it is gradually declining from the nature due to it’s over exploitation, lots of habitat environmental pollution and lack of indigenous production. That is why there is an urgent need of replenishment of the short supply and conservation of this exotic plant genetic resource. Generally Glycyrrhiza glabra L. is multiplied vegetatively by means of cuttings, but this process is slow in response and there is a constant need to maintain large number of mother plants stock (Sharma et al., 2010). From seeds plants are rather slow to grow (Brown, 1995) and the high seed cost is also extremely inhibitive in its production. Thus, large scale propagation through tissue culture technology is the only possible option to ensure the availability of quality planting material for large scale cultivation to meet the ever increasing pharmaceutical demand for this plant. As more uses of this plant are identified, the pressure on existing natural population would keep on increasing further and cause stress to the natural wild stands of this species unless cultivation of the species is carried out on large scale for its conservation and sustainable use. This can be ensured by the application of mass multiplication using biotechnological approaches. A lot of research work has been carried out in the field of its micro-propagation by using shoot tip, axillary buds and nodal explants in tissue culture. Thengane et al. (1998) have reported a stepwise protocol for the micro-propagation of Glycyrrhiza glabra L. on a simple minimal medium using shoot tip and nodal explants where very high multiplication rates with healthy root system were obtained. Sharma et al. (2010) reported rapid, efficient and high frequency regeneration of Glycyrrhiza glabra L., where using young leaf and shoot tips explants via callus mediated shoot Regeneration. Recently Yadav and Singh (2012) and Shrivastava et al. (2013) reported micropropagation studies on this species. These reports, however, were inadequate for large-scale propagation of this species. Therefore, in the present study an attempt was made to develop a protocol for in vitro propagation of Glycyrrhiza glabra L. with the aim to identify the most suitable explant source (i.e. shoot tip, young leaf and nodal segment) and the best concentration of growth regulators for successful in vitro propagation of Glycyrrhiza glabra L.

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Growth media: Leaf, nodal segments and shoot tip explants of Glycyrrhiza glabra L. were inoculated in MS medium with 5 different concentrations of BA (0.0, 0.5, 1.0, 2.0, 3.0 mg/l) for shoot initiation. Small shoots started appearing within 7-10 days of inoculation on three explants types- leaf, shoot tips and nodal segments. Sub-culturing was done frequently during the early stages of establishment to reduce the adverse effects of browning of tissues. The experiment was repeated 3 times to confirm the results of this experiment. The results of this experiment are tabulated in the Table No. 1 – 4 and graphically depicted in Fig. No. 1 - 4. On perusal of the data it is evident that the response of the treatment types differed based on the explant types. For shoot induction semi-solid MS (Murashige and Skoog, 1962) media supplemented with BA, KN, NAA, at varying concentrations were prepared. The new shoots induced from the in vitro cultures were further used as explants for multiple shoot regeneration. For in vitro rooting, individual shoots (3-5 cm) were cut from the proliferated shoot cultures and implanted on half strength MS with different concentrations of NAA, IAA and IBA. All media were supplemented with 30 g/l sucrose and gelled with 7 g/l agar. Prepared media were dispensed into 150 x15 mm culture tubes or 250 ml conical flasks according to experiment. The pH of the media was adjusted to 5.8 before autoclaving at 121 oC for 20 min. The cultures were incubated for 16 h photoperiod at 24 ± 2 OC under a fluorescent light.

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MATERIALS AND METHODS Procurement and preparation of explants: Healthy plant was collected from Prof. T.S. Murthy Science and Technology Station Mahaveer Colony, Obedulaganj dist. Raisen, and the plant will be planted in the Botanical garden of Govt. Motilal Vigyan Mahavidyalaya Bhopal, MP, India. Shoot tips, young leaf and nodal explants of Glycyrrhiza glabra L. were used for this experiment. The explants were washed thoroughly under running tap water, presoaked in liquid detergent for about 20 min and again washed with sterile distilled water for 5 min. They were then surface sterilized with 0.1% (w/v) mercuric chloride for 1 min, followed by five rinses with sterile distilled water inside a laminar air flow cabinet. The surface sterilized explants were sized to 1.0-1.5 cm in length and inoculated on the culture medium.

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Maintenance of in vitro cultures: Visual observation of culture was made every week. Data on shoot induction, proliferation and root induction were recorded after three weeks of inoculation and used for calculation. For each treatment 5 explants were used and all the treatments were repeated thrice. Hardening and acclimatization: The healthy rooted plantlets were taken out from the culture tubes, washed to make free from agar gel with running tap water and transplanted to plastic cups containing soil, sand and compost (1:1:1) for hardening. The plantlets were kept in a poly chamber for acclimation. Established plantlets were transplanted in open field under natural conditions and the survival rate was recorded. RESULTS Initiation and establishment of meristem culture The percent establishment and shoot formation varied in different explants at different levels of BA. Overall, the frequency of establishment in the case of nodal explant was highest (66.67%) with mean maximum 3.67±0.67 shoots/explants when cultured on MS medium with 2.0 mg/l BA. The lowest frequency of establishment (0%) with mean minimum shoot formation/explant of 0.0 was recorded in case of MS medium with both 0.5 mg/l BA and 1.0 mg/l BA in case of the leaf explant. Individually, percent establishment frequency ranged between 0% - 13.33% in leaf explants; between 26.67% - 53.33% in shoot tip explant and between 33.33% - 66.67% in case of nodal explants (Fig. 1, Table 1). The shoot formation/explant ranged between with 0.0 - 0.67±0.33 in leaf explant; between with 1.33±0.33 – 2.67±0.33 in shoot tip explant and 1.67±0.33 – 3.33±0.88 in nodal explant. In the case of leaf explants in all the treatment types tested only 13.33% leaves explants showing establishment with minimal shoot formation frequency 0.67±0.33 at concentration of BA 3.0 mg/l was recorded at the end of 4th week of culture (Table 1). The treatment with MS + BA (2.00 mg/l) was found significantly superior over other treatments types for establishment of primary culture (Plate 1). Frequency of establishment was maximum (66.67%) on nodal explants with 2.0 mg/l BA; with 3.67±0.67 mean shoot formation with shoots being robust and healthy as compared to other treatments (Fig. 1, Table 1).

Fig. 1 Effect of different BA concentrations on establishment of Glycyrrhiza glabra L. from Leaf, shoot tip and nodal explants.

Nodal explants Mean Mean explant number of Mean explant Treatments Establishment shoot/ Establishment Mean ± SE Explants Mean ± SE Mean ± SE MS + BA 0 0 0 0 MS + BA 0.5 0 0 1.67±0.33 MS + BA 1.00 0 0 3.00±0.58 MS + BA 2.00 0.33±0.58 0.67±0.67 3.33±0.88 MS + BA 3.00 0.67±0.33 0.670.33 2.67±0.88 CD at 5% 0.66 1.05 1.24 No. of treatments: 5; No. of Replicates: 3; No. of Explants / Replicate: 5. Data (±SE) were recorded after 4 weeks of culture.

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Mean number of shoot/ Explants Mean ± SE 0 3.0±0.58 2.33±0.33 3.67±0.67 3.00±1.00 1.33

Shoot Tip explants Mean Mean number of explants shoot/ Establishment Explants Mean ± SE Mean ± SE 0 0 1.33±0.33 2.00±0.58 2.33±0.33 2.67±0.33 2.67±0.33 2.00±0.58 2.33±0.67 1.33±0.33 1.99 1.94

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Leaf explants

Conc. of PGR mg/l

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Table: 1 Effect of different concentration of BA on the establishment and shoot formation of Glycyrrhiza glabra L. explants.

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Plate – 1.

(A)

(B)

(C)

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Multiplication of shoots The nodal and internodal explants derived from established in vitro cultures were re-inoculated on MS medium supplemented with different cytokinin + auxin combination for evaluation of their regeneration potential and shoot formation. Different concentrations of BA, Kn, NAA, and IBA (0.0, 0.5, 1.0, 2.0, 3.0 and 0.10, 0.0, 0.25, 0.50, 1.00 mg/l) were used in various combinations. As mentioned Table No. 2 and 3 the treatment combination MS + BA + NAA (0 + 0) (control) is not responsive and does not cause any callogenic or shoot multiplication induction. A treatment combination containing BA (2.0 mg/l) and NAA (0.50 mg/l) have shown the highest regeneration percentage (86.67%) of response. An enhancement in the induction of number of shoots/explant with mean maximum of 18.33±0.33 shoots/explant has been recorded. Mean shoot length recorded was 2.25±0.08 cm along with the emergence of large number of shoot buds at the base of the explants within four weeks of culture (Plate – 2 A & B). Whereas, the treatment combination of BA (0.5 mg/l) + NAA 0.10 mg/l) responded with poor regeneration (53.33%) with mean number of shoots per explants is 14.67±0.88 shoots/explant. Other combinations of treatments MS + BA + NAA (3.0 + 1.00) and MS + BA + NAA (1.0 + 0.25) have shown intermediate results without any specific pattern. The response of Kn + NAA combination was comparatively less potent as compared to BA+NAA treatment combination both in terms of number of shoots/explant as well as in regeneration capacity. As a result, lower numbers of shoots/explants were recorded on Kn + NAA supplemented medium. The effect of this combination was found to be highest in term of regeneration (53.33%) at 2.0 mg/l Kn + 0.50 mg/l NAA with mean shoots/explant of 5.0±0.58 and mean shoot length of 2.13±0.14 cm. Whereas, higher concentrations Kn (3.0 mg/l) + NAA (1.0 mg/l) showed minimal regeneration frequency (33.33%), regeneration of 3.67±0.33 shoots/explants and mean minimum shoot length of 2.14±0.18. Other combinations of treatments MS + Kn + NAA (1.0 + 0.25) and MS + Kn + NAA (0.5 + 0.10) have shown intermediate results (Fig. 2, Table 2). At last, the combination of Kn + IBA supplemented to MS media with varied concentrations were also tested for shoot proliferation and was comparatively found least effective of the three PGR combinations investigated. Both MS medium supplemented with BA + IBA (1.0 + 0.25) and BA + IBA (2.0 + 0.50) have exhibited the maximum regeneration of 46.67%, mean shoot formation of 06±0.58 shoots per explant with mean shoot length of 2.12±0.13 within four weeks of culture. Lower concentrations of Kn (0.5 mg/l) + IBA 0.10 mg/l) and higher concentration of responded poor regeneration (40%) with 2.21±0.18 mean shoots/explants and 2.21±0.18 mean shoot length was recorded. Both higher concentrations Kn (3.0 mg/l) + IBA 1.0 mg/l) and lower concentration MS + BA + NAA (0.5 + 0.10) combinations showed lower shoot proliferation, explant response, regeneration percentage and shoot length after two weeks of incubation and culture.

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A & C: Initiation and establishment of explants at 2.0 mg/l BA.

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Fig. 2 Effect of various cytokinin-auxin combinations on percentage regeneration of Glycyrrhiza glabra L. from in vitro derived explants of Glycyrrhiza glabra L. after 4 - weeks of incubation. Table: 2. Effect of various cytokinin-auxin combinations on direct adventitious shoot formation from in vitro derived explants of Glycyrrhiza glabra L. after 4 - weeks of incubation. Conc. Of PGR mg/l

Shoot length (cm) Mean ± SE 0 2.27±0.14 2.21±0.16 2.13±0.14 2.14±0.18 -

Kn + IBA Mean shoot formation Mean ± SE 0 4.00±0.58 5.00±0.58 6.00±0.58 4.67±0.88 1.88

Shoot length (cm) Mean ± SE 0 2.21±0.18 2.25±0.14 2.12±0.13 2.23±0.15 -

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The observation period of this experiment was further extended to six weeks. Shoot buds have shown excellent elongation when incubation was further extended to sixth week on the same medium. More specifically BA (2.0 mg/l) + NAA (0.50 mg/l) have induced outstanding enhancement both in number of shoots/explant 23.67±0.33 shoots per explant and the mean maximum shoot length of 2.36±0.07 cm. The regeneration percentage was maximum 93.33% at this concentration after six weeks of culture (Fig. 2, Table 3, Plate 2-C). Lower concentrations BA 0.5 mg/l + NAA 0.10 mg/l showed minimum 60% regeneration with 15.0±0.67 mean shoot/explants at 6 weeks of culture. Higher concentrations of BA (3.0 mg/l) + NAA (1.0 mg/l) responded with 86.67% percentage of regeneration with 16.67±0.67 mean shoots/explants. Lower concentrations of NAA (0.5 mg/l) + Kn (0.10 mg/l) responded with poor regeneration (46.67%) and produced a mean 10.67±0.88 shoots/explant and had mean shoot length of 2.34±0.12. Higher concentrations NAA (3.0 mg/l) + Kn 1.0 mg/l) showed 53.33% regeneration and proliferation by producing 12.33±1.45 shoot/explant was recorded after 6 th week of culture. The combination of Kn and IBA varied concentrations were also tested for shoot proliferation at the extended observation period. The combination of Kn (2.0 mg/l) with IBA (0.50 mg/l) showed 53.33% regeneration with mean shoot formation/explants was 7.00±0.58 and mean shoot length 2.29±0.12 was recorded after6 th weeks. Lower concentrations of Kn (0.5 mg/l) + IBA 0.10 mg/l) responded with comparatively poor regeneration (40%) with 5.33±0.67 mean shoots/explants. Higher concentrations Kn (3.0 mg/l) + IBA 1.0 mg/l) also showed minimal regeneration (40%) and mean shoot/explants 4.67±0.67 and 2.21±0.13 mean shoot length was recorded after 6 th weeks of tissue culturing (Fig. 2, Table 3).

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BA+NAA Kn + NAA Mean shoot Shoot Mean shoot formation length (cm) formation Mean ± SE Mean ± SE Mean ± SE 1 0 0 0 0 2 0.5 + 0.10 14.67±0.88 2.18±0.09 3.67±0.67 3 1.0 + 0.25 24.00±1.53 2.28±0.07 6.00±1.00 4 2.0 + 0.50 18.33±0.33 2.25±0.08 5.00±0.58 5 3.0 + 1.00 16.67±0.33 2.29±0.09 3.67±0.33 CD at 5% 2.57 1.94 No. of treatments: 5; No. of Replicates: 3; No. of Explants / Replicate: 5. Data (±SE) were recorded after 4 weeks of culture. S. No.

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Fig. 3 Effect of various cytokinin-auxin combinations on percentage regeneration from in vitro derived explants of Glycyrrhiza glabra L. after 6 - weeks of incubation. Table: 3. Effect of various cytokinin-auxin combinations on direct adventitious shoot formation from in vitro derived explants of Glycyrrhiza glabra L. after 6 - weeks of incubation. BA+NAA Kn + NAA Mean shoot Shoot Mean shoot formation length(cm) formation Mean ± SE Mean ± SE Mean ± SE 1 0 (Control) 0 0 0 2 0.5 + 0.10 15.00±0.67 2.31±0.11 10.67±0.88 3 1.0 + 0.25 24.00±1.53 2.35±0.07 7.00±1.53 4 2.0 + 0.50 23.67±0.33 2.36±0.07 10.33±1.86 5 3.0 + 1.00 17.67±0.67 2.34±0.08 12.33±1.45 CD at 5% 2.53 4.15 No. of treatments: 5; No. of Replicates: 3; No. of Explants / Replicate: 5 Data (±SE) were recorded after 6 weeks of culture Conc. Of PGR mg/l

Shoot length(cm) Mean ± SE 0 2.34±0.12 2.24±0.13 2.15±0.12 2.09±0.11 -

Kn + IBA Mean shoot formation Mean ± SE 0 5.33±0.67 8.67±1.20 7.00±0.58 4.67±0.67 2.30

Shoot length(cm) Mean ± SE 0 2.33±0.12 2.37±0.11 2.29±0.12 2.21±0.13 -

(A)

(B)

(A-B) Axillary shoot multiplication on BA + NAA (2.0 + 0.50 mg/l) after 4 th week of culture.

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Plate – 2.

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S. No.

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(C) (C) Axillary shoot multiplication on BA + NAA (2.0 + 0.50 mg/l) after 6 th week of culture.

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Fig. 4 Effect of different concentrations of various auxins showing percentage root induction in Glycyrrhizaglabra L. in MS half strength.

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Root Induction & Hardening Well grown shoots (3-4 cm) were subjected to various concentrations of auxin for studies on root formation in Glycyrrhiza glabra L. Within 10 days of incubation all the explants were involved in root formation. The root formation has varied with various treatment types of auxins or without auxins. For in vitro root induction half strength MS medium supplemented with different concentration (1.0, 2.0 and 3.0 mg/l) of NAA, IAA and IBA were used. The result of these treatments in terms of root induction and formation is depicted in Table 4 and Fig. 4) Overall perusal of the Table No. 4.56, 4.57 and Figure No. 4.44, 4.45, 4.46 reveals that explants inoculated in half strength MS medium without any growth hormone supplementation (control) failed to generate any root inducing response. At different auxin levels were used, where IAA showed high frequency 100% and have produced maximum root induction 16±0.58 mean number of roots/shoot with root length (2.33±0.33cm) at IAA (1.0 mg/l) proving ideal concentration for root induction (Plate 3 – A,B). The other two auxins tested for root induction viz. IBA and NAA gave positive but inferior results at all concentrations when compared to IBA containing medium. The response of IBA was also recorded considerable on root induction, inducing maximum root formation (5.67±0.33) with maximum root length (1.67±0.33cm) at (IBA 3.0 mg/l), while NAA in this concern, gave rise to root formation (3.67±0.33) with maximum root length (1.67±0.33cm) which is relatively low as compared to IAA and IBA. Further, increasing or decreasing the concentration of IBA did not improve the number or length of root but induce the adverse effect in the root formation. Rooted micro shoots of Glycyrrhiza glabra L. were taken out from culture tubes, and washed thoroughly under tap water to remove all the traces of culture medium and agar from the rooted microshoots. Microshoots with 3-4 cm long shoots and 10-15 roots were transferred to plastic bags containing sand, soil and vermicompost in a ratio of 1:1:1 and kept under controlled green house conditions for 15-days (Plate 3 - C). The plantlets so hardened for four weeks were subsequently transferred to open beds. Almost all the rooted microshoots (87%) established and survived after field transfer. Further, no morphological variation was observed among the in vitro raised plants of Glycyrrhiza glabra L. when compared with mother stock.

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Table 4. Effect of different concentrations of various auxins on root induction in Glycyrrhiza glabra L. in MS half strength. Growth Regulator Number of Conc. (mg l-1) % Rooting Roots/ plantlets Mean ± SE NAA IAA IBA Control (PGR free) 1.0 40.0 2.00±0.58 2.0 73.3 1.67±0.33 3.0 60.0 3.67±0.33 CD at 5% 1.39 1.0 100 16.0±0.58 2.0 93.3 13.3±0.88 3.0 86.67 11.0±1.00 CD at 5% 0.74 1.0 73.33 4.67±1.20 2.0 60.0 3.67±0.33 3.0 40.0 5.67±0.33 CD at 5% 1.66 No. of treatments: 5; No. of Replicates: 3; No. of Explants / Replicate: 5 Data (±SE) were recorded after 6 weeks of culture

Root length (cm) Mean ± SE 2.33±0.33 1.67±0.33 1.67±0.33 2.33±0.33 2.00±0.58 2.33±0.33 1.67±0.33 1.67±0.33 1.67±0.33 -

Field Survival (%) 33 47 53 87 80 67 53 53 40 -

Plate – 3

(C) (A) Thick white short stunted root formation on 1.0 mg/l IAA (B) Elongation of roots (C) Hardened Plants

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(B)

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(A)

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DISCUSSION Initiation and establishment of culture In the present investigation, different strategies were employed to achieve in vitro micropropagation of Glycyrrhiza glabra L. Three types of explants (leaves, shoot tips and nodal segments) from young plant were used for various studies. This studies deal with factors critical influencing axillary shoot proliferation (shoot initiation, shoot multiplication, in vitro rooting, and hardening). The three explant types i.e. leaf, shoot tip and nodal segments of Glycyrrhiza glabra L. were initially incubated on MS medium fortified with varying concentrations of BA (0.0, 0.5, 1.0, 2.0, 3.0 mg/l) only. The response in terms of shoot initiation and establishment has significantly varied both in different BA concentrations, as well as in different explant types (Plate 1). It is highly significant to report here that during the present investigation, when leaf explants were tested, with optimal concentrations of BA (3.0 mg/l) for shoot initiation (Table 4.1, Fig.4.1), only 13.33% leaf explants showed percent initiation and establishment. At this concentration mean explant response of 0.67±0.33/5 explant and mean number of 0.67±0.33/5 shoots/explant was recorded (Table 4.2). This is the first report where leaf explants of Glycyrrhiza glabra L. have shown direct regeneration and establishment at this concentration. It is worth mentioning that there is no previous report as per the detailed literature review undertaken on shoot development from leaf segments of Glycyrrhiza glabra L., hence this is a new finding of this investigation. Although it may be noted that many studies showing direct shoot organogenesis in other plant species i.e. Jatropha curcas (KhuranaKaul et al., 2010); Plectranthus neochilus (Motal et al., 2010); Launaea sarmentosa (Mahesh et al., 2012); Aquilaria malaccensis (Saikia & Shrivastava., 2015) etc. have been earlier reported. Shoot tip explant establishment also varied at different concentrations of PGR. Highest establishment response (53.33%) and maximum mean number of shoot formation of 2 ±0.58/5 explants was obtained in MS medium fortified with BAP 2.0 mg/l at three to four weeks of incubation (Table 4.1, 4.2). Each explant developed adventitious shoots that formed along the margins and numerous shoot buds at the base of explant. The treatment with MS + BAP (2.00 mg/l) was significantly superior to any other treatment for establishment of culture. Frequency of establishment was high with 2.0 mg/l BAP, on which shoots were active and healthy, compared to other treatments. This is in accordance with the earlier reported findings by Thengane et al., (1998), who obtained multiplication through shoot tip and nodal explantsin MS medium with BA (0.88-8.87 μM) was used. Direct shoot induction has been reported in several medicinal plant viz. Dendranthema morifolium L. (Waseem et al., 2011); Banana (Devendrakumar et al., 2013); Hybanthus enneaspermus (L.) Muell. (Sudharson et al., 2014). In the case of nodal explants also, varying concentration of BA showed variable frequency of percent establishment. The best concentration of BA (2.00 mg/l), has led to the highest percent establishment (66.67%) and remarkable enhancement in shoot proliferation, with maximum mean number of shoots (3.67±0.67)/5 explants (Table 4.1, 4.2). As for the lowest response (33.33%) is concerned, it was obtained in MS medium with BA 0.5 mg/l (Fig. 4.1, 4.2, 4.3). The stimulatory effect of BA on multiple shoot formation has been reported earlier in Glycyrrhiza glabra L. (Yadav and Singh, 2011) and in other medicinal plants like Aegle marmelos (Yadav and Singh, 2011) and Spilanthes acmella (Yadav and Singh, 2010).

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Root Induction & Hardening The regenerated shoots were excised and transferred on half strength MS medium supplemented with different concentration (1.0, 2.0 and 3.0 mg/l) of NAA, IAA and IBA were used. The present observation indicates that auxins were found to induce and enhance rooting from the basal cut ends of the shoots. Highest rate of rooting frequency (100%) was observed and have produced maximum root induction 16±0.58 mean number of roots/shoot with root length (2.33±0.33 cm) in the medium containing IAA (1.0 mg/l) proving ideal concentration for root induction. The result thus establishes that the auxin is very essential for in vitro root formation in Glycyrrhiza glabra L. Significant differences were observed among different auxin treatments used for rooting. Medium supplemented with IAA was found to be the best auxin for in vitro root regeneration with 100% rooting response in IAA (1.0 mg/l), followed by 73.33% in IBA (1.0 mg/l) and 40% in NAA (2.0 mg/l). The regenerated shoots failed to induce rooting on auxin-free medium (control) and subsequently led to drying of shoots, In contrary to the findings of present study, Kohjyouma et al., (1995) reported the highest frequency of root formation on MS medium supplemented with 0.05-0.10 mg/l NAA. Sawaengsak et al., (2011) reported rooting in half strength B5 salts supplemented with various concentration (0-5.0 mg/l) of IAA and IBA. The highest frequency of root formation was obtained on the medium supplemented with 5.0 mg/l of either IAA or IBA. However, whilst the addition of IBA in the rooting medium for explants was found more suitable for root induction than IAA containing medium in terms of the root length attained. Sarkar and Roy (2014) reported 100% rooting after one month of culture with root initiation (68%) in MS media supplemented with IAA (3.0 mg/l) in Glycyrrhiza

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Direct shoot regeneration The explants inoculated on cytokinin-auxin combination medium exhibited enhanced and earlier response as compared to single cytokinin containing media. However, encouraging results were obtained, when BA in combination with NAA at different concentration were investigated for multiple shoot induction. Yadav and Singh, (2012) reported the effectiveness of MS medium supplemented with BAP (2.00 mgl-1) + NAA (0.5 mgl-1), producing multiple shoots (3.0 ± 0.81) after 30 days of culture. The response of MS medium supplemented with BAP (2.00 mgl-1) + NAA (0.5 mgl-1) was found to be outstanding, while in the present study BA (1.0 mg/l) + NAA (0.25 mg/l) proved most efficient for axillary bud proliferation. Though the number shoots formed in the studies of Yadav and Singh (2012), were slightly higher (approximately 3.0 ± 0.81 shoots/explant) as compared to shoots formed. In the present study (24.0±1.53 shoots/explant) after the 4 weeks of culturing (Table 4.21, Fig. 4.21) and production of multiple shoots (28.0±1.53 shoots/explant) within 6-weeks of inoculation as reported in this study is highly efficient with high frequency shoot production (Table 4.24, Fig. 4.27).

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glabra L. In the present study 100% rooting was achieved on half strength MS medium with (IAA (1.0 mg/l) within 4 weeks of culturing from the regenerated shoots which is essential for mass production of this species. A large number of reports have been published recently that are in agreement with present findings where IAA has been found most effective auxin for in vitro rooting in other species viz; Citrus aurantifolia (Al-Khayri and Al-Bahrany, 2001); Vitis amurensis Rupr (Han et al., 2003); Codonopsis pilosula (Slupski et al., 2011); Calendula officinalis (Victorio et al., 2012); Jatropha curcas L. (Singh and Shetty, 2012); Mentha viridis L (Rahman et al., 2013); Piper longum L. (Padhan, 2015). CONCLUSIONS In conclusion we describe an effective and reproducible procedure of Glycyrrhiza glabra L. highlights the explants response on in vitro condition and development high frequency shoot multiplication by using various combination and concentration of Plant growth regulators. From our experimental data, it is evident that nodal explant showed highest percent of establishment at the concentration of BA 2.0 mg/l and regeneration frequency was highest in 2.0 mg/l BA + 0.50 mg/l NAA, whereas highest shoot development was observed at 1.0 mg/l BA + 0.25 mg/l NAA, while the maximum rooting was obtained on 1.0 mg/l IAA. Our aim is to describe an effective and reproducible procedure of Glycyrrhiza glabra L. micropropagation and a successful adaptation of plants to greenhouse condition. This producer can play an important role in genetic improvement studies and commercial purposes of this multipurpose medicinal plant. ABBREVIATIONS MS : Murashige and Skoog medium; PGR’s : Plant growth regulators; BA : 6-Benzyl adenine; Kn : Kinetin (6-furfuryl amino purine); NAA : α- naphthalene acetic acid; IAA : Indole-3-acetic acid; % : Percent/Percentage, CD : Critical Difference; SE : Standard Error; Govt. : Government

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REFERENCES 1. African pharmacopoeia, Vol. 1, 1st. ed. Lagos, Organization of African Unity, Scientific Technical & Research Commission, 1985, 131–134. 2. Al-Khayri J. M. and Al-Bahrany A. M., In vitro micropropagation of Citrus aurantifolia (lime) Current Science, 2001, Vol. 81, No. 9, 10. 3. Brown D., Encyclopaedia of Herbs and their Uses. Dorling Kindersley, London,1995, ISBN 0-7512-020-31. 4. Chopra R N, Nayar S L, Chopra I C. Glossary of Indian medicinal plants. NISCIR, CSIR, Delhi 2002. 5. Devendrakumar. D, Anbazhagan M. and Rajendran R., Effect of Benzyl Amino Purine (BAP) concentration on in vitro shoot proliferation of Banana (Musa spp.). International Journal of Research in Biotechnology and Biochemistry, 2013, 3(3): 31-32. 6. Dhuke J.A., Ducelllier J., Dhuke A.N. and Bogensehutz M.J., Handbook of medicinal herbs, New York, SRC Press, 2002, 2, 461. 2. 7. Ghazanfar SA, Handbook of Arabian medicinal plants. Boca Raton, FL, CRC Press, 1994, 110–111. 8. Han D. S., Niimi Y. and Wu J. Y., Micropropagation of Vitis amurensis Rupr.: An improved protocol. Vitis, 2003, 42 (3), 163– 164. 9. Khurana V. K.., Kachhwaha S., Kothari S. L., Direct shoot regeneration from leaf explants of Jatropha curcas in response to thidiazuron and high copper contents in the medium. Biologia Plantarum., 2010, Volume 54, Issue 2, pp 369-372. 10. Kohjyouma M., Kohda H., Tani N., Ashida K., Sugino M., Akihiko Y. and Horikoshi T., In vitro propagation from axillary buds of Glycyrrhiza glabra L. Plant Tissue Culture Letters, 1995, 12(2), 145-149. 11. Mahesh A., Thangadurai D., And Melchias G., Rapid in vitro plant regeneration from leaf explants of Launaea sarmentosa (Willd.) Sch. Bip. ex Kuntze. Biol. Res., 2012, vol. 45 no. 2. http://dx.doi.org/10.4067/S0716-97602012000200004. 12. Meena A. K., Singh A., Sharma K., Kumari S., Rao M.M. Physicochemical and Preliminary Phytochemical Studies on The Rhizomes of Glycyrrhiza Glabra Linn. International Journal of Pharmacy and Pharmaceutical Sciences, 2010, Vol. 2, Suppl 2. 13. MotaI M. S., BandeiraI J. de M., BragaI E. J. B., BianchiI V. J., PetersI J. A., In vitro shoot regeneration of boldo from leaf explants. Cienc. Rural, 2010, vol.40 no.10 http://dx.doi.org/10.1590/S0103-84782010005000173. 14. Murashige T. and Skoog F. C., A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant., 1962, 15, 473-479.

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ACKNOWLEDGEMENTS We are very thankful to the Principal Govt. Motilalal Vigyan Mahavidyalaya, Bhopal (M.P.) for providing us lab facility. Thanks are also due to Department of Higher Education, Govt. of M.P. I am deeply grateful to the University Grants Commission for awarding me Fellowship that gave me ample time to finish this research work.

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15. Padhan B., Regeneration of plantlets of Piper longum L. through in vitro culture from nodal segments, Journal of Applied Biology and Biotechnology, 2015, Vol. 3 (05), pp. 035-039. 16. Planning Commission, Report of the task force on conservation and sustainable use of medicinal plants. Planning Commission, New Delhi, 2010. 17. Rahman M. M., Ankhi U. R. and Biswas A., Micropropagation of Mentha viridis L.: An aromatic medicinal plant. Int. J. of Pharm. & Life Sci. (IJPLS), 2013, Vol. 4, Issue 9: 2926-2930. 18. Saikia M., and Shrivastava K., Direct shoot organogenesis from leaf explants of Aquilaria malaccensis Lam. Indian Journal of Research in Pharmacy and Biotechnology, 2015, 2320 – 3471. 19. Sarkar M. And Roy S. C., Rapid Scale Micropropagation of Glycyrrhiza glabra L. (Leguminoseae) A Valuable Medicinal Herb, IJSR, 2014, Vol. 3, Issue 3. 20. Sawaengsak W., Saisavoey T., Chuntaratin P., and Karnchanatat A., Micropropagation of the medicinal herb Glycyrrhiza glabra L., through shoot tip explant culture and glycyrrhizin detection. International Research Journal of Plant Science, 2011, Vol. 2(5) pp. 129-136. 21. Sharma A. K., Yadav A.S., Raghuwanshi D. K., Mir F. A., Lone S.A. and Rai A. K., An Improved Protocol for Rapid, Efficient And High Frequency Regeneration of Glycyrrhiza glabra L. Asian J. Exp. Sci., 2010, Vol. 24, No. 1 22. Shrivastava M., Purshottam D.K. Shrivastava A.K., Mishra P., In Vitro Conservation of Glycyrrhiza Glabra by Slow growth Culture. International Journal of Bio-Technology & Research (IJBTR), 2013, Vol. 3, Issue 1, 49-58. 23. Singh G. and Shetty S., Evaluation of in vitro rooting efficiency in the biodiesel plant, Jatropha curcas. Biotechnol. Bioinf. Bioeng., 2012, 2(1):591-596. 24. Słupski W., Tubek B., and Matkowski A., Micropropagation of Codonopsis pilosula (Franch.) Nannf by axillary shoot multiplication. Acta Biologica Cracoviensia Series Botanica, 2011, 53/2, pp 87–93, DOI: 10.2478/v10182-011-0031-2. 25. Sudharson S., Anbazhagan M., Balachandran B. and Arumugam K., Effect of BAP on in vitro propagation of Hybanthus enneaspermus (L.) Muell, an important medicinal plant. Int.J.Curr.Microbiol.App.Sci, 2014, 3(8) 397-402. 26. The Indian pharmaceutical codex, Vol. I. Indigenous drugs. New Delhi, Council of Scientific & Industrial Research, 1953, 112– 113. 27. Thengane S.R., Kulkarni D.K. and Krishnamurthy K.V., Micropropagation of licorice (Glycyrrhiza glabra L.) through shoot tip and nodal culture. In Vitro Cell. Dev. Biol – Plant, 1998, 34: 331-334. 28. Victório C.P., Lage C. L.S., Sato A., Tissue culture techniques in the proliferation of shoots and roots of Calendula officinalis1. Revista Ciência Agronômica, 2012, v. 43, n. 3, p. 539-545. 29. Waseem K., Jilani M. S., Jaskani M. J., Khan M. S., Kiran M. AND Khan U.G., Significance of Different Plant Growth Regulators on the regeneration of Chrysanthemum plantlets (Dendranthema morifolium L.) through shoot tip culture. Pak. J. Bot., 2011, 43(4): 1843-1848, 2011. 30. Yadav K, Singh N, In vitro propagation and biochemical analysis of field established wood apple (Aegle marmelos L.) Analele. Universităţii din Oradea – Fascicula Biologie., 2011, 18: 23-28. 31. Yadav K, Singh N., Factors influencing in vitro plant regeneration of Liquorice (Glycyrrhiza glabra L.) Iranian Journal of Biotechnology, 2012, Vol. 10, No. 3. 32. Yadav K. and Singh N., Micropropagation of Spilanthes acmella Murr. – An Important Medicinal Plant. Nature and Science, 2010;8(9).

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