Efficiency of liquid system for mass multiplication of Glycyrrhiza Glabra and evaluation of genetic fidelity of micropropagated plants.Introduction
The roots and rhizomes of the Glycyrrhiza glabra (L.) commonly known as "licorice" are the major source of triterpenoids saponin "glycyrrhizin". Besides, various other flavonoids, essential oils, alkaloids, polysaccharides and polyamines, fatty acids and amino acids are also known to be present in abundance in licorice (Nomura and Fukai, 1998). Existence of these chemical compounds in licorice is accountable for its high pharmaceutical value all over the world. The licorice extract is extensively used as a major ingredient of the drugs used for the treatment of gastric and duodenal ulcers. It is also used in various drugs as expectorant, anti tussive, demulcent and anti-inflammatory purposes (Hayashi et al., 1993). Owing to the sweetness of glycyrrhizin which is fifty times sweeter than sucrose the licorice extract is largely used as a flavoring agent and sweetener in confectionery industry. The conventional propagation of licorice is rather difficult, as poor flowering and poor seed viability along with very slow growth rate are the major limiting factors. Vegetative propagation using under ground stem runners is the only implemented method for its commercial cultivation. Because of these inherent problems of regeneration, cultivation of licorice has not been popularized from commerce point of view and a large amount of industrial requirement is not being fulfilled. Therefore, the establishment of a high through put in-vitro procedure of rapid clonal propagation would be highly enviable and of immense need. In our present study the efficiency of liquid culture for mass multiplication of G. glabra over conventional semi solid culture is analyzed. This communication explicit our empirical studies related with the standardization of culture conditions conducive for rapid clonal mass multiplication of G. glabra using liquid medium as well as the utility of a bench top stirred tank bioreactor for the clonal mass plant propagation. The micropropagated plants obtained from different culture conditions were finally transferred to soil.
The accomplishment of any micropropagation protocol largely depends upon the production of genetically akin plants (Rani and Raina 2000; 2003). The occurrence of concealed genetic effects arising via somaclonal variation in the regenerants can critically limit the broader utility of micropropagation system. Therefore, it is significant to establish the suitability of a micropropagation protocol developed with respect to the production of genetically akin plants and to make its ultimate use by entrepreneurs possible with out any ambiguity. Some such efforts have been made to assess the genetic fidelity of tissue culture derived plants of licorice during the present course of our study. For this purpose PCR amplification by using a set of MAP primers were carried out for randomly selected licorice plant grown in semi solid and liquid in vitro culture systems.
Materials and Methods
Plant Material and Establishment of Aseptic Cultures
Green and juvenile stem segments of 2-3 cm. length along with apical and lateral buds were used as explants. These explants were obtained from one-year-old Glycyrrhiza glabra plants grown in glass house conditions. The collected explants were washed under tap water for 2-3 h and then surface sterilized with 0.1% w/v Hg[Cl.sub.2] solution for 45 seconds. The surface sterilization was followed by thorough rinsing with sterile distilled water and subsequently these sterile nodal explants were aseptically transferred to the glass culture vessels (Borosil / Corning made test tubes of 25x150 mm and conical Erlenmeyer flasks of 100 ml capacity) containing growth medium. The growth medium consists of basal salts of MS medium (Murashige and Skoog, 1962) containing 3% w/v sucrose, 0.1% w/v myoinositol and agar (0.8% w/v). No growth hormones were added to this medium. All these aseptic cultures were maintained in culture room under controlled conditions (12:12 h light: dark regime, 3000-4000 lux intensity and 25[+ or -]2[degrees]C incubation temperature with 60% relative humidity).
Effects of Semi Solid and Liquid Medium on The Growth of Cultures
The apical and axillary shoots grown in aseptic stock cultures on MS basal medium were used for the experimental purposes. The stock cultures were maintained through regular sub-culturing of growing shoots on similar (MS basal) medium. By this, multiplication of propagules was achieved. For the comparative study, the explants obtained from regular sub-culturing of stock cultures were transferred to liquid and semi-solid MS basal medium supplemented with 1.0 mg[l.sup.-1] IAA. 30 ml of liquid medium was dispensed in 250 ml Erlenmeyer shake flasks and the cultures were incubated on a rotary shaker at 75 rpm under same culture condition as stated above.
Experimental Design and Statistical Analysis
The experiments related to growth responses of cultured licorice plants in two different media were performed in five replicates and repeated three times. The mean, standard error and one way ANOVA were calculated. No significant difference (at P=0.05) was observed in results.
Mass Multiplication of G. Glabra Plants in Bioreactor
On the basis of quality response of licorice plants in liquid medium, further experiments were stretched out and designed for mass multiplication using a bench top, stirred tank bioreactor (model Bio Flow-3000, M/s New Brunswick Scientific, USA) of 5 liter working capacity. This air-sparged top driven system had mechanical agitation through marine blade impeller. Liquid MS medium (2.5 liter) supplemented with 1.0 mg[l.sup.-1] IAA was dispensed in the culture vessel. During the experiments, an autoclavable nylon mesh (pore size 200[micro]m) was tightened just beneath the medium surface on the lower stainless steel semi-circular ring of the baffle assembly so that the explants do not submerge or sink to the bottom in the medium but remain floating over mesh support during the culture period. The influent air was passed in the medium through hydrophobic membrane filter (Whatman USA, 0.22[micro]) through air sparger. This complete unit was autoclaved at 120[degrees] C and 15 lbs. pressure for 15 min. in a vertical sterilizer.
Inoculum consisting of nodal explants (200 in number, 2-4 cm. in length with 2-4 nodes) was transferred from Erlenmeyer flasks to the sterilized reactor culture vessel under a clean air laminar hood provided with HEPA filter. All the probes were connected to the control module. Air sparger was connected to air compressor through silicon tubing fitted with 0.22[micro] Whatman filter paper. Culture conditions maintained during the experimentation were 12:12 light: dark regime, 25[+ or -]1[degrees] C temperature.
Hardening of in Vitro Raised Plants Under Glass House Conditions
Rooted plantlets growing under different in-vitro conditions i.e. agar supplemented semi-solid medium, in liquid culture medium in shake flasks and in bioreactor were gently washed under tap water to remove the traces of adherent culture medium and carefully transferred to the earthen pots containing sand: soil: farmyard manure (1:1:1 v/v).
Evaluation of Genetic Fidelity of Micropropagated Plants
Micropropagated plants of G. glabra cultured under different sets of experiments were randomly selected and subjected to RAPD. This procedure involved the isolation of high molecular weight DNA and its amplification through PCR. The DNA was extracted from the 18 randomly selected cultured plants along with control parent plant (growing in glass house) by the method described by Khanuja et al., 1999 and subjected to PCR amplifications using a set of 20 MAP primers (MAP 01 to MAP 20) procured from M/s Banglore, Gennie, India. The PCR reaction was carried out in a total volume of 25 [micro]l, with each reaction tube comprising of 25 ng of DNA, 0.3 units of Taq DNA polymerase, 100 [micro]l of each dNTPs, 1.5 mM Mg[Cl.sub.2] and 5 p mol of decanucleotide primers. Amplification reaction was carried out using the DNA engine thermal cycler (MJ Research, USA).
Results and Discussion
The present study was aimed to prove the efficiency of liquid culture system for rapid clonal multiplication of Glycyrrhiza glabra using shake flasks. Experiments were therefore performed to study the effect of liquid culture system on various growth parameters. MS basal medium supplemented with 1.0 mg[l.sup.-1] IAA and with or with out 0.8% w/v agar was used in the experiment and the growth response of the regenerated axillary shoots from the inoculated nodal explants (4 cm length with 2 nodes and considered as propagules) was recorded in both liquid and semi-solid culture medium. Cultures were incubated under similar culture conditions. The morphogenetic responses in terms of number of regenerated shoots / explant, number of nodes / shoot, length of inter nodal region, number of leaflets / shoot, size of leaflets, number of roots induced / explant, length of roots and total biomass / culture were recorded after 40 days of culture. Observations revealed that liquid medium supported improved growth responses of regenerated axillary as well as adventitious shoots as compared to the semi-solid medium (Table-1; Figure 1).
[FIGURE 1 OMITTED]
Though in both semi-solid and liquid culture conditions, the average number of nodes per shoot was almost equal (8 nodes per shoot). However, taking into account the number of axillary shoots regenerated per explant and the total number of nodes produced in all the axillary shoots (number of nodes per culture), a total of 44 nodal explants per culture could be obtained in liquid medium after 40 days of culture. Against this, only 15 nodes were obtained in the semi-solid medium, which served as control.
A significant difference in the length of regenerated axillary shoots was observed in liquid medium (13.68) as compared to semi-solid medium (10.70). The length of shoots ranged from 4.0 to 26.0 cms in cultures growing in liquid medium and 8.0 to 15 cms in cultures growing in semi solid medium. The size of internodal region in the regenerated shoots growing in liquid medium mainly contributed the elongation of axillary shoots. Concomitantly with axillary shoot growth, liquid culture also supported root induction and root growth in the regenerated shoots (Figure 1f). Roots generally appeared from the nodes at the base of axillary shoots in mother nodal explants. However, in due course of culture duration in liquid media, lower nodes of regenerated axillary / adventitious shoots also showed rhizogenesis though many nodes were always above the culture medium surface (figure 1f, indicated by arrow). This kind of morphogenetic response observed only with liquid system was further proved supportive during the establishment of micropropagated plants under soil (data not provided) as this kind of rhizogenesis appears to be more closely resemble with natural habit of Glycyrrhiza plants. Because of overall supportive effect, a total of 3.85 gm (F. wt.) biomass per culture was obtained after 40 days of culture duration compared to only 2.87 gm after same culture duration in semi-solid medium. Thus, in equal culture duration of 40 days, about 3 fold increase in propagable units (nodal segments) could be obtained. This will undoubtedly influence the efficiency of a micropropagation protocol, if such a procedure is adopted for commercial cultivation by any biotech industry. A mass of evidence have been accumulated from research reports that prove the competence of the use of shake culture technique for mass multiplication of plants (Ewelina Piatczak et al., 2005; Mehrotra et al., 2007; Maxwell et al., 2007). Further, several reports favors the shake culture techniques not only for mass multiplication of a number of plant species but also for decreasing the cost and time of the overall procedure (Kim et al., 2003; Teisson and Alvard et al., 1995; Tewary and Seibio Oka, 1999; Park et al., 2000; Kieffer et al., 2001; Firoozabady and Gutterson, 2003). Thus, the use of liquid culture system makes a perceptible progress towards the commercialization of the micropropagation procedure of a particular species (Gupta and Timmis, 2005; Mehrotra et al., 2007). In the light of above facts, the present study was carried out to elucidate the efficiency of liquid medium for licorice micropropagation by improving upon the existing protocol (Shah and Dalal, 1980; Kukreja, 1998). In agar based medium, the agar is the costliest ingredient as well as there are several studies that have been an evidence for the discouraging effects of agar on the growth of cultured tissue (Moreas-Cerdiera et al., 1995; Lucyszyn et al., 2007). Thus, the optimized liquid culture parameters for a species not only reduce the production cost but also improve the production efficiency. The potential of liquid cultures for enhanced bud regeneration from gentian is also well documented (Hosokawa et al., 1998). Better growth response in terms of shoot and root proliferation by the use of liquid medium has also been reported in various plants including ornamentals and trees (Chu et al. 1992; Adelberg, 2005). When shoot buds are cultured in liquid medium, growth responses varies with species and genera. Some plants grew fairly well in shake culture but others did not. The growth distinctiveness depends on the nature of the genera, species or cultivar. Hormones and mineral composition affect the growth of the tissues more positively in the liquid medium (Bhagyalaxmi 1995). This is because the tissues are completely submerged in the liquid medium, so that contact area of the cultures to the medium is wider in liquid than on the agar medium. The continuous shaking of cultures leads to the proper mixing of air as well as nutrients leading to their frequent availability to the growing tissue. Besides, continuous shaking also obstructs the apical dominance which ultimately leads to more adventitious branching (Wawrosch et al., 2005; Kongbangkerd and Wawrosch, 2003).
The study carried out at shake flask level had clearly revealed that liquid MS basal medium supplemented with 1.0 mg[l.sup.-1] IAA was the suitable medium for axillary shoot elongation as well as root induction in these shoots. Further, this culture medium significantly shortened the culture duration by efficiently producing complete plantlets only on one medium. Hence, the study was further expanded to the mass multiplication of this plant in bioreactor using the similar growth medium. Initially, sixty percent of inoculated explants exhibited axillary bud growth with in 20 days of incubation period. Lateral shoots elongated upto 6 cm in length. This response resulted in only a marginal increase (app. 1.4 fold over initial inoculum) after 20 days. During the culture period few explants remained in floating condition in the medium due to air current and agitation in the medium and many explants got submerged and settled down at the bottom of the culture vessel. Agitation through impeller in the medium forced the submerged explants to float and remained suspended in the medium. Some of these floating explants came into contact of marine impeller blades and got injured. Such injured explants also exhibited very poor response in terms of axillary shoot growth thereby resulting in low biomass yield. In the successive experiment the configuration of reactor vessel was modified. An Autoclavable nylon mesh was provided as a septum in the culture vessel just below the medium surface. This support prevented the sinking of explants to the bottom of the culture vessel and in addition provided a support for anchorage of roots originating from in vitro shoots. These shoots grew normally in upper section of culture vessel above the medium surface. This also avoided vitrification of shoots. The nylon septum divided the culture vessel into two halves. The impeller was placed in lower half of the vessel thus avoiding the direct contact of explants with impeller and prevented the tissue injury. 200 explants weighing 27.9 gm (F. wt.) were inoculated and incubated for 20 days. Growth response in terms of both axillary shoot initiation and elongation was observed in 80 % of inoculated explants in contrast to only 60 % response observed in previous experiment (Figure 2 a). The growing shoots were harvested and biomass yield of 295.95 g (F. wt.) was recorded after 20 days of culture (Figure 2 b). Thus, an increase of about 10.5 fold in biomass over initial inoculum was recorded. The major disadvantage of the conventional micropropagation technique is the difficulty in controlling the chemical and physical parameters of the culture vessel. Adopting bioreactors with liquid medium for micropropagation is important to overcome such limitations as well as due to the ease of scaling up (Preil, 1991) and resulting in low production costs. Generally bioreactors are used for large scale culture of somatic embryos but in recent past there is a collection of published reports in which bioreactors are reported to grow plants via organogenesis (Ziv, 2005 and references with in). The major limitation in the use of a bioreactor for plant propagation is the shear stress which leads to tissue injury and ultimate death of culture. To avoid this limitation bioreactors are designed and their configuration is modified on the basis of specific requirements of any particular plant species (Ingram and Mavituna, 2000; Curtis, 2005; Ziv, 2005). Only a few bioreactors are presently used for this purpose mainly due to the lack of systemic and factorial experiments on propagation using bioreactor which are needed to reveal the complex interaction between plant physiology and physical parameters of bioreactors.
In PCR amplification, among 20 primers 75% exhibited clear amplification. Remaining 25% comprising of 5 primers viz MAP 05, MAP 12, MAP 15, MAP 19 and MAP 20 did not showed any amplification. A total of 59 RAPD bands were produced out of which 40 bands were found to be monomorphic and 19 were polymorphic in nature (Table 2; Figure 3). Thus, 68% of monomorphism was observed in 18 randomly selected plants of G. glabra cultured under different experimental conditions along with parent plant. The similarity matrix was used to generate a graphic phenogram through unweighted pair-group method with arithmetic means (UPGMA) which reveals the similarity coefficient among all accessions ranged from 87% to 100% (figure 4). The most divergent pair having 87% similarity was accession 3 and 19 and 5 and 19 while rest of the in vitro raised plants exhibited a much narrower variability with similarity coefficients of 91% to 100%. Thus, showing that all sampled plants have a very narrow genetic base. The use of RAPD markers for the assessment of genetic fidelity among micropropagated plants are reported for various plant species (Rani and Raina 2000 and 2003; Gangopadhyay et al., 2004; Kawiak and ojkowska., 2004). In commercial industry, where micropropagation technology has been developed, their foremost concern is the maintenance of true-to-type nature of the micropropagated plants concerning explant source. This can be achieved by developing the micropropagation protocols based either upon axillary branching or somatic embryogenesis. These two methods are believed to give rise to genetically uniform and true-to-type plants, since the organized meristems do not undergo genetic changes that might arise during cell division or differentiation from callus cultures. However various physical and chemical factors during in vitro culture of plants can be extremely stress full on plant cells and tissue could be responsible for introducing some variations only superficially. The results in the present study in conjunction with earlier reports (Rani and Raina 2000; 2003; Modgil 2005) has convincingly accentuate upon the competence of in vitro micropropagation protocol using liquid culture system for mass multiplication of Glycyrrhiza glabra. However the use of bioreactors for the en mass multiplication of plants still require some more study and interpretation of intriguing aspects of various physical, chemical and biological parameters regarding growth and productivity.
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Authors (S.M. and A.K.K.) are thankful to Director, CIMAP, Lucknow for providing facilities to carry out the work.
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Shakti Mehrotra * (1), Bhartendu N. Mishra * (1) and Arun K. Kukreja (2)
* (1) Institute of Engineering & Technology Department of Biotechnology, Institute of Engineering & Technology, Lucknow, 226021, Uttar Pradesh India, E-mail: email@example.com
(2) Plant Tissue Culture Division, Central Institute of Medicinal & Aromatic Plants, (CIMAP); Lucknow, 226015, Uttar Pradesh, India, E-mail: firstname.lastname@example.org
* Corresponding Author
Table 1: Growth response of regenerated axillary shoots of G. glabra on semi-solid and liquid culture media. S. No. Growth Response of Response of parameters axillary shoots axillary on semi solid Shoots on liquid medium Medium 1. Inoculum F. 0.18 [+ or -] 0.0 0.18 [+ or -] 0.0 wt.(g) 2. Size of inoculum 4.0 [+ or -] 0.0 4.0 [+ or -] 0.0 (cms) 3. Number of shoots/ 2.0 [+ or -] 0.0 2.7 [+ or -] 0.4 explants 4. Length of shoots 10.7 [+ or -] 1.9 13.7 [+ or -] 0.89 (cms) (8-15) * (4-26) * 5. Number of nodes/shoot 8.0 [+ or -] 1.0 8.0 [+ or -] 1.02 6. Number of nodes/ culture 15.8 [+ or -] 2.3 44.0 [+ or -]5.5 7. Length of inter nodal segment (cms) 1.11 [+ or -] 0.32 4.1 [+ or -] 0.73 8. Leaf number/shoot 9.1 [+ or -] 1.3 10.4 [+ or -] 0.91 9. Leaf size (mm) 6.6 [+ or -] 1.7 9.1 [+ or -] 0.74 10. Number of roots/plant 7.2 [+ or -] 0.74 5.0 [+ or -] 0.89 11. Length of roots(cms) 8.5 [+ or -] 1.9 7.3 [+ or -]1.1 12. Total biomass / flask, F.wt.(g) 2.87 [+ or -] 0.26 3.85 [+ or -]0.21 Values given in parenthesis represent the range of shoot length Table 2: Monomorphic and polymorphic bands as observed with different primers Primers Total Monomorphism Polymorphism number Number of Percentage Number of Percentage of bands bands bands MAP 01 2 2 100.00 0 -- MAP 02 6 1 16.60 5 83.30 MAP 03 3 3 100.00 0 -- MAP 04 4 4 100.00 0 -- MAP 06 5 3 60.00 2 40.00 MAP 07 2 2 100.00 0 -- MAP 08 4 1 25.00 3 75.00 MAP 09 4 4 100.00 0 -- MAP 10 5 5 100.00 0 -- MAP 11 5 3 60.00 2 40.00 MAP 13 2 2 100.00 0 -- MAP 14 1 1 100.00 0 -- MAP 16 1 1 100.00 0 -- MAP 17 5 2 40.00 3 60.00 MAP 18 10 6 60.00 4 40.00