Abstract: Aloe (Aloe vera L., Family Liliaceae), with proven multiple medicinal values finds favour readily in Ayurevdic applications, is facing a serious threat to its population as well as biodiversity due to its popularly harvested aloe leaves by the local communities and herbal medicine vendors. To succeed in dealing with the specified problem along with the facilitation of the mass production of commercial level, constant supply of quality propagules can be possible through conservation of propagules in vitro. The present study is thus concerned with in vitro conservation of multiple shoot culture of aloe to achieve unbroken supply of propagules maintaining their genetic purity. Rhizomatous stem explants result multiple bud break in MS plus 0.25 mg L-1 of NAA and 1.5 mg L-1 of BAP. Separated shoot buds further result in shoot multiplication and proliferation on MS with 2.5 mg L-1 of BAP. In vitro generated multiple shoots were split into individual shoot and subcultured for further multiplication in a sustainable. Five subcultures were performed at an extended 5 month interval over a period of 25 months in the same medium. Plantlets regenerated after 1st subculture and plantlets from 5th subculture showed no significant difference in the phenotypic response. Genetic integrity of in vitro clones was tested using ISSR primers. All monomorphic bands in the ISSR assay, both for primary culture as well long term culture, ascertained their genetic integrity to a great extent.
INTRODUCTION
Aloe (Aloe vera L.) is a versatile plant of the family Liliaceae having wonderful healing and softening properties. It works immediately and effectively to heal and soothe skin injuries due to minor wounds and burns (Davis et al., 1994). It is now being marketed as a remedy for coughs, wounds, stomach and mouth ulcers, gastritis, diabetes, cancer, headaches, arthritis, immune-system deficiencies, and many other conditions (Leon, 2003). Presently it is used as an important ingredient in different cosmetic formularies (Ahmed et al., 2007). But, the uncontrolled collection and sale of aloe leaves result in destruction of the plants. Everybody, from the local communities to the herbal medicine vendors, popularly harvest aloe leaves, causing a serious problem to its population as well as biodiversity (Supe, 2007). Hence, it is utmost necessary to conserve this plant species for its sustainable use in future. With the aid of in vitro biotechnology, the additional conservation strategies are now being developed for several medicinal plants (Chaudhuri and Jha, 2004).
Long term conservation in vitro, is an important aspect of plant tissue culture. Elite germplasm of various rare and endangered species have been conserved in vitro (Martin and Pradeep, 2003; Tyagi et al., 2004; Islam et al., 2005; Gonzalez et al., 2006). There are several methods available for in vitro conservation of germplasm for virtually unlimited time span in limited space (Bhojwani and Razdan, 1996); as for example, cryopreservation, alginate encapsulation, use of in vitro minimum growth (Sarkar et al., 2005; Goncalves and Romano, 2007) and through extension of subculture period (Martin and Pradeep, 2003) but no such intervention reported yet in Aloe vera L. Moreover, establishment of aseptic in vitro culture and genetic stability of the individual species are the most important prerequisite to conserve the cultures for a long period of time. The present study is concerned with the conservation of aloe in vitro through a simple and efficient protocol maintaining the genetic integrity of the germplasm.
MATERIALS AND METHODS
Collection of explants and in vitro propagation: The whole experiment was carried out in the Green House and Plant Tissue Culture Laboratory of Biotechnology Department, Bidhan Chandra Krishi Viswavidyalaya, W.B., India during 2006-2009. Surface sterilized mature plants which are regenerated through suckers were grown under greenhouse condition. Forty five days old rhizomatous stems were collected, trimmed to 2 cm, surface sterilized and inoculated in artificial growth medium under laminar air flow. For establishment of explants, MS medium was supplemented with 0.25 mg L-1 of NAA and 1.5 mg L-1 of BAP. Induced buds were excised and inoculated on MS, fortified with 2.5 mg L-1 of BAP only, for multiple shoot proliferation as described by Gantait (2009).
Physical condition of in vitro culture: For the present study MS (Murashige and Skoog, 1962) basal medium (consisted of salts, vitamins and 3% sucrose) was used after solidifying with 0.7% (w/v) agar. Different growth regulators like α-naphthalene acetic acid (NAA) and N6-benzylamino purine (BAP) were added in variable concentrations to MS before adjusting pH to 5.7 and autoclaved at 1.06 kg cm-2, 121°C for 15 min. MS basal salts, agar, vitamins and growth regulators were obtained from SRL, India. The in vitro cultures in all growth stages were accomplished at 25±2°C temperature, 60% RH and 16 h photoperiod (using white fluorescent tubes) under a photosynthetic photon lux density (PPFD) of 30 μmol/m2/sec2.
In vitro maintenance of sustained culture: In vitro generated shootlets were subcultured for further multiple shoot proliferation in a sustainable manner. For sustained multiple shoot proliferation, the identified best performing multiple shooting medium i.e., MS supplemented with 2.5 mg L-1 of BAP was used. Five subcultures were performed at a 5 month interval over a period of 25 months in the same medium formulation. The performance of these subcultures was assessed on the basis of response, number and length of multiple shoots. Randomly selected propagules were assessed for clonally fidelity through ISSR.
Genomic DNA extraction and PCR amplification: Fresh young leaf samples were collected from three different sources such as mother plant; rhizomatous stem derived shootlets (considered as primary cycle of regeneration) and from the shootlets regenerated through fifth subculture (referred to as secondary explants) after 25 months. Genomic DNA was extracted from 80 mg tender leaves from each sample under study, according to the procedure described by Bhattacharyya and Kanta (1999). Extracted DNA samples were subjected to PCR (Polymerase Chain Reaction) amplification using 10 selected ISSR primers (Gantait et al., 2010). The 25 μL PCR mixture contained 40 ng DNA, 2.5 μL 10X Taq polymerase assay buffer, 3.5 μL 2.5 mM dNTPs, 0.5 U Taq DNA polymerase (all from Chromous Biotech Pvt. Ltd., India) and 200 ng of primer (Bangalore Genei Pvt. Ltd., India). PCR consisted of an initial denaturation at 94°C for 5 min followed by 35 cycles of 45 sec at 94°C, 45 sec at annealing temperature 90 sec at 72°C and final extension at 72°C for 7 min, 4°C for 5 min was done using Gene Amp PCR system 2400 (Applied Biosystems, USA). The annealing temperature was adjusted according to the Tm of the primer being used in the reaction. The amplified PCR products, along with 50 bp DNA ladder were resolved by electrophoresis on 1.5% agarose (SRL) gel in 1X TBE buffer stained with ethidium bromide (10 μg L-1 TBE buffer). Bands were scored and photographed on Gel Logic 200 trans-illuminator system (Kodak).
Statistical analysis: The potentiality of producing multiple shoots was the criteria to assess the difference between plantlets regenerated from primary explants and plantlets regenerated from secondary explants (without any reference to explants of origin) after 25 months. Then plantlets of both phases were screened through ISSR assay which revealed the genetic clonality of all the propagules. Complete Randomized Design (CRD) was followed for the in vitro culture experiments. The experiments were carried out in six replications including 20 explants in each and standard deviation was calculated. For data scoring the inoculant response to multiple shoot proliferation, number of shoot and shoot growth were considered as morphological competence. Data were subjected to Analysis of Variance (ANOVA). Significant difference among the treatments were tested by Duncan’s multiple range test (Duncan, 1955) at 5% level using WINDOWSTAT 7.5 (Indostat services, Hyderabad, India) (Gantait et al., 2010) software package. For ISSR profiles, the well-resolved and consistently reproducible amplified DNA fragments were scored in terms of their presence or absence. To detect the genetic purity, the resulting banding patterns were compared between DNA samples for each ISSR primer.
RESULTS AND DISCUSSION
Assessment of regeneration efficiency of long term culture: Morphological attributes: Successful bud induction (Fig. 1a) and shoot multiplication (Fig. 1b) was achieved within 50 days of culture. A novel attempt was made to favour mid-term conservation through sustained in vitro culture of multiple shoot of aloe. In vitro conservation as well as constant supply of quality propagules as and when necessary, was achieved through extending the subculture interval up to 5 months. There are two distinct advantages to maintain a steady supply of propagules from regenerated shootlets under prescribed MS media and acclimatization process as described. First, there was no need to start with a fresh explant. As a result time lag for culture establishment would be restrain. Moreover, each subculture resulted in an exponential increase in number of shoots (Ray et al., 2006). Finally, unbroken propagule supply can possibly be sustained over a period of time without genetic damage and so reduction of pressure on its natural population of concerned plant species can be achieved.
It was observed from the present data that plantlets regenerated after 1st subculture and plantlets from 5th subculture showed no significant difference in the response to shoot multiplication according to Duncan’s multiple range test (Duncan, 1955) as it was around 19-20 out of 20 inoculants in both cases (Table 1). The uniformity in number and length of multiple shoots was also confirmed by DMRT where these were more than 9.33-11.17 (Fig. 1c) and around 4 cm (Fig. 1d), respectively.
| Fig. 1: | In vitro culture of Aloe vera L. (a) Bud break, (b) Multiple shoot induction, (c) Shoot proliferation and (d) Sustained culture for in vitro conservation |
| Table 1: | Morphological performance of sustained in vitro multiple shoot culture of aloe |
| *Each MS medium was supplemented with 2.5 mg L-1 BAP. Data represent Mean±SD of 20 replicants per treatment in three repeated experiments. Means within columns separated by Duncan’s Multiple Range Test p = 0.05; Duncan, 1955 | |
No apparent variation was detected between in vitro generated clones and they were as good as their mother plant. The morphological efficiency was not hampered even after long-term sustained culture of 25 months. From the primary and secondary explants a similar multiple shoot proliferation was maintained. The sustained unaltered efficiency of in vitro multiple shoot culture of aloe supports the earlier report of Joshee et al. (2007) who observed the uniform regenerability of embryogenic calli for more than one year with regular subculture. Present study also resulted the production of a large number of multiple shoots through recycling the secondary cultures in 5 cycles for 25 months without losing regeneration efficiency.
Confirmation of genetic stability: ISSR assay: In the present study, 10 ISSR primers were used for the verification of the fidelity of in vitro generated clones among which IS-9, IS-10, IS-11 and IS-12 did not react with aloe DNA. Between remaining primers, IS-61, IS-63 and IS-65 displayed a positive interaction but failed to reproduce any major scorable band, whereas IS-6 (Fig. 2a), IS-7 (Fig. 2b) and IS-8 (Fig. 2c) showed positive reproducible bands (Table 2). Each of these three primers generated a unique set of amplified products with the size range of 250 bp in IS-8 to 1.95 kb in IS-6. The variation of the number of bands from each of these successfully used primers was from 4 (in IS-7) to 8 (in IS-8) per sample (Table 2). With an average of 5.6 bands per primer a total number of 102 reproducible monomorphic bands were scored from the clones including their mother. None of the primers showed any difference in banding pattern (Fig. 2).
In the present study, di-nucleotide SSRs motifs AG, GA, GT, TG, CT and CA were used among which three positive and reproducible primers (based on GA, GT and AG motif) amplified distinct scorable number of bands (Table 2). Interestingly, IS-8 based on AG motif amplified more number of bands thus resulting in more coverage of the genome. Similar results have been reported in other medicinal plants like Swertia chirayita (Joshi and Dhawan, 2007) and Allium ampeloprasum (Gantait et al., 2010).
| Fig. 2: | Agarose gel electrophoresis of ISSR fragments of in vitro regenerated clones (C1-C2 from primary culture, C3-C5 from secondary culture) with their mother (P) showing monomorphic bands generated by (a) primer IS-6, (b) primer IS-7 and (c) primer IS-8. Lane M-50 bp ladder |
| Table 2: | List of primers with their sequences, anchoring, annealing temperature, mode of reaction, number and size of amplified fragments |
Aloe being a diploid with 2n = 14, it can be assumed that the limited number of bands which are produced by these ISSR primers would partially cover the genome. However, none of the primers showed any difference in the banding pattern. Considering the detected uniform morphological competence of the primary and long-term secondary culture in vitro along with the displayed monomorphic banding pattern, it can be suggested that in vitro regenerated clones maintained their genetic integrity.
CONCLUSION
The results revealed the significance of the protocol reported in this study. Shoot tip explants maintained their regeneration frequency with morphogenetic competence even after prolonged in vitro culture. So, this protocol proves it potentiality to save this species from extinction and facilitate germplasm conservation. Considering these characteristics, we suggest that the developed strategy can be adopted for other endangered species also.