Introduction

Glycyrrhiza glabra (family Leguminosae) is an important perennial cross-pollinated plant having diverse pharmacological effects including detoxication, antioxidant, antiulcer, anti-inflammatory, and antiviral properties [31]. The most important secondary metabolite isolated from roots and stolons of this plant is “Glycyrrhizin” which is used in the treatment of bronchial asthma, allergic, dermatitis, gastric ulcers, Adisson’s disease, and eczema [21].

Because of its diverse medicinal properties, it is widely used in the confectionary, food, and pharmaceutical industries and there is an urgent need of new approaches of producing G. glabra to meet the increasing demands [36]. The conventional method for propagation is through rhizomes, stolons, or other cuttings which is a commercial valuable part of the plant. The regeneration rate is also very slow and low [9, 29].

Although studies on in vitro culture of G. glabra have been developed with different purposes, such as for rapid production of high quality, disease-free and uniform planting material [4, 7, 21, 29, 31]. During the acclimatization stage of micropropagation, the in vitro-raised plants are subjected to environmental stress due to poor root, shoot, and cuticular development [32]. The incorporation of mycorrhization at this stage represents a biologic solution for reducing the weaning stress by solving the problems of transient transplantation, thus shortening the acclimatization period [27].

Considering the beneficial effect of arbuscular mycorrhizal fungi (AMF) on medicinal plants, more attention should be paid toward the combining of appropriate mycorrhizal fungi with in vitro micropropagation techniques [28].

To our knowledge, there is no information available about the role of AMF on micropropagated plantlets of G. glabra. Our work aims to examine the effect of AMF inoculation on survival and growth enhancement of in vitro raised plantlets of G. glabra that can be used for sustainable utilization and mass cultivation in the future.

Materials and Methods

Plant Material and Micropropagation Procedures

Healthy nodal segments (1.0–1.5 cm) excised from the plants growing in Herbal Garden of Botany Department, Kurukshetra University, Haryana, India, served as source of explants for micropropagation. Complete plantlets of G. glabra consisting of well-developed roots and leaves were obtained by direct regeneration from nodal explants on Murashige and Skoog [22] medium supplemented with BAP (2.0 mg l−1) following our modified protocol of Yadav and Singh [37].

Plant cultures were maintained in culture room, at a temperature of 25 ± 2 °C under photoperiod of 16 h having light intensity of 40 μmol m−2 s−1 provided by cool white fluorescent lamps with 60 % relative humidity.

After 75 days, when adequate rooted shoots were obtained, the in vitro raised plantlets (14 cm) after washing with fresh water to remove agar particles were transferred from the culture tubes to plastic cups (15 × 8 cm) holding 500 g of sterilized soil and sand (3:1) mixture. The cups were covered with transparent polythene bags (to maintain humidity) with holes (to provide aeration) and kept in culture room under the same light and temperature conditions provided during micropropagation.

Choice of AMF

Two dominant AMF, Glomus mosseae and Acaulospora laevis, were isolated from the rhizosphere soil of G. glabra by wet sieving and decanting technique given by Gerdemann and Nicolson [8].

Multiplication of AMF Cultures

The starter inoculum or pure culture of each selected dominant AM fungus was raised by “Funnel Technique” of Menge and Timmer [20].

AMF Inoculation Procedure

The acclimatized plantlets were transplanted into experimental earthen pots (30 × 30 cm) having sterilized sand:soil mixture (300:1,500 g). To each pot, 10 % inoculum consisting of AMF spores and fine roots of maize having mycelium/arbuscules/vesicles alone and in combination were added. The inoculum was put very close to rhizosphere of transplanted plantlets in the experiments and was transferred in glasshouse having constant temperature (28–30 °C) and relative humidity (65–75 %) for further studies. Light was provided by cool white fluorescent lamps (8,000 lux) under a 16-h photoperiod. The polyhouse also received sunlight. Plants were watered regularly and 50 ml per pot Hoagland nutrient solution (without KH2PO4) was given every 7 days.

The experiments were carried out in alone and combined inoculations. In the control, no inoculum was added.

Different growth and biochemical parameters were studied after 120 days of inoculation (DOI).

Growth and Morpho-Physiologic Parameters

Among various plant growth parameters, plant height (cm), number of leaves per plant, leaf area (cm2), fresh shoot weight (g), fresh root weight (g), dry shoot weight (g), and dry root weight (g) were observed. Plant dry weight was determined after drying the tissue in an oven at 80 °C for 48–72 h. Leaf area was measured using a leaf area meter (Leaf Area Meter 211; Systronics Ltd, Ahmadabad, India).

Biochemical Assays

Different biochemical parameters, viz., chlorophyll, total sugars, reducing sugars, and proteins, were estimated from the leaf tissues. The content of chlorophyll (a, b, and total) was measured according to Arnon [3], reducing and total sugars according to Hart and Fisher [10], and proteins by the method of Bradford [6].

Statistical Analyses

Each experiment consisted of 5 replicates per treatment and was repeated thrice. Data were analyzed for significance by one-way analysis of variance and the differences contrasted by a Duncan’s multiple range test (DMRT) at p ≤ 0.05. All statistical analyses were performed by means of the Statistical Package for Social Sciences (SPSS, version 11.5).

Results and Discussion

In vitro methods of propagation provide an alternate and effective means for rapid multiplication of species by the continuous production to meet the demand for commercial exploitation [15–17]. The beneficial effect of mycorrhizal inoculation has also been reported earlier in other micropropagated medicinal plant [11, 32, 33, 38, 39].

The survival rate and development of micropropagated plantlets of G. glabra have been improved by introducing effective mycorrhizal symbiosis with the plant. Table 1 shows the survival rate of G. glabra plants improved significantly when inoculated with different treatments of AMF at the beginning of the acclimatization stage. Inoculation with G. mosseae contributed to 100 % of plantlet survival as opposed to 66.6 % of survival in control (Table 1).

Table 1 Effect of AM fungi on the survival rate of micropropagated G. glabra after 120 days post-inoculation
Full size table

It is obvious that the integration of AMF was found to have a significant effect on the growth and development of micropropagated G. glabra plantlets. However, this improvement was considerably different with regard to the type of fungal strain used. In the present investigation, it was found that mycorrhizal inoculation increased the height of the plant in comparison to the control. The most effective results were observed in the dual combination of A. laevis + G. mosseae after 120 days of inoculation. Similar trend was observed in the case of leaves number per plant (Table 2). Significant increment in root length was observed with G. mosseae followed by dual combination of G. mosseae + A. laevis. The maximum leaf area was found in with the A. laevis treatment followed by dual combination of G. mosseae + A. laevis. A significant increment in leaf area registered with inoculated plant may be due to increased phosphorus uptake resulting in enhancement of biosynthesis processes, determining a faster growth and development, which leads to a greater leaf area [30].

Table 2 Effect of AMF on the various growth parameters of micropropagated G. glabra after 120 days of inoculation
Full size table

Biomass of all the inoculated plants of G. glabra increased significantly in terms of fresh and dry shoot weight after 120 days of inoculation. A significant increment in shoot biomass was recorded in dual combination of G. mosseae + A. laevis followed by G. mosseae (Table 2). AMF play a dominant role in increasing phosphorus solubilization and uptake of P, N, Ca, S, K, Mg, Mn, and Cl by plants [35]. It may be due to the reason that AMF hyphae growing through soil pore spaces can affect phosphate absorption beyond the depleted zone. The fungal hyphae transport phosphate over large distance into the root cortical cells [13, 24]. They also produce growth-promoting substances such as indole acetic acid-, cytokinins-, and gibberellin-like substances [12, 14, 25, 26].

The results of the present study clearly revealed the significant variations among different biochemical parameters treated with different AM fungus (Table 3). It can be stated that the leaf chlorophyll content recorded in the mycorrhizal plants was typically higher than non-treated control. Micropropagated plantlets inoculated with G. mosseae yielded the highest amount of total chlorophyll. In this study, increased chlorophyll “a” and chlorophyll “b” in plants is similar to the findings of Bavaresco and Fogher [5] on the effect of G. mosseae. Mycorrhizal-treated plants show a greater increase in the rate of photosynthesis than their controls which may be due to increase in the content of total chlorophyll [18]. Increased chlorophyll content after AMF inoculation may be due to the higher concentrations of Mg, Fe, and Cu in foliar tissues thereby influencing chlorophyll synthesis [19, 33].

Table 3 Effect of AMF on the various biochemical parameters (mg/100 mg fresh wt.) of micropropagated G. glabra after 120 days post-inoculation
Full size table

As compared to other biochemical traits, reducing sugar and total sugar contents showed a decrease was observed in different AMF inoculation than control (Table 3). It may be because the AMF always serves as a strong sink for sugars and utilize 10–20 % of net photosynthate in exchange for the transfer of nutrients to the host to lead a symbiotic life [1, 23].

Leaf protein showed a minor increase in G. mosseae inoculated plants followed by G. mosseae + A. laevis (Table 3). This is in accordance with Vazquez et al. [34] according to which the total soluble protein content was significantly higher in mycorrhizal than non-mycorrhizal plants Mycorrhizal formation is known to increase the expression of low molecular weight proteins as reported by Arines et al. [2] in Pisum sativum.

This technology may also be applied to improve the post-transplanting performance of many other multipurpose valuable plants for the sustainable cultivation.