In India, industrial demand for raw materials is increasing rapidly with the rapid increase of aloe-based cosmetics. However, there is a substantial short fall of the supply of raw materials to meet the industrial demand, as seed production is limited. Vegetative propagation is being practiced for commercial plantation, as sexual reproduction is infrequent (Meyer and Staden 1991). The rate of vegetative propagation is very slow as only 3-4 offshoots are developing annually from a single plant, this reflects in the report of Bhattacharya and Hansda (2003), where, 641.5 tonnes shortage of raw materials was accounted in the year 2001-2002. To meet up the industrial demand during this investigation an attempt has been undertaken to establish an efficient and high throughput protocol to produce large number of propagules from a selected high biomass yielding genotype of A. vera.
Materials and Methods
A high biomass yielding genotype of Aloe vera (L.) was collected from the red laterite zone of Durgapur District, West-Bengal and another conserved genotype was collected from the ‘Veshaj Udyan’ garden of The Agri-horticulural Society, Alipore, Kolkata, India. The plants were grown at the net house of the Bose Institute garden. The young, 2-3 months old plants of approximately 4-6 cm length at 3-4 leaved stage were used as donor plants (Fig. 1a).
The plants were washed thoroughly under running tap water for 30 min followed by treatment with 1 gm% Bavistin solution for 1 h. These were then treated with the solution containing 0.3 gm% mercuric chloride and 1.5 g-l of Taxim for 20 min. All the traces of sterilants were removed by washing thoroughly with sterile distilled water. The older leaf tips of the surface sterilized plants were removed and the shoot meristem surrounded by juvenile leaves was aseptically divided into two equal halves and inoculated on the shoot induction medium (Fig. 1b). Thus, two explants were obtained from a single shoot.
Induction of multiple shoots
Sterilized explants were inoculated on Murashige and Skoog’s (MS, 1962) basal medium supplemented with different concentrations and combinations of plant growth regulators, namely, indoleacetic acid (IAA, 0.5, 1.0, 1.5, 2.0 and 2.5 mg l-1), N6-benzylaminopurine (BAP, 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0 mg l-1) and adenine sulphate (AdS, 25, 50 and 100 mg l-1). Sucrose (3.0 gm %) and PGRs were added in the solid agar medium prior to adjustment of pH at 5.8 and autoclaving. Cultures were kept under cool, fluorescent light (16 h photoperiod, 40m mol m-2 s-1 Philips, India) at 25 ± 1° C. Shoots were sub-cultured to fresh medium at regular interval of 28 days or otherwise whenever the medium turned brown. A total of 40 explants were used for each PGR treatment and data were recorded every 4th week of inoculation. Each set of experiments was repeated twice for each genotype.
A cluster of at least 3 shoots with no less than 3-4 leaves was used as the propagule for rooting. Full strength MS basal medium containing different concentrations ranging from 0.1 to 1.0 mg l-1) IAA was tested for root induction.
The rooted plants were carefully washed with water, treated briefly with 1.0 gm% Bavistin and transferred to pots containing sand. Precaution was taken to avoid application of excess water. After keeping the potted plants in net house for 7-10 d, these were transferred either to the field or in the pots containing soil.
Release of phenolic exudates was encountered during the initiation of the culture. The phenolic exudates hamper the establishment of in vitro culture. In this study browning problem was evaded by transferring the explant to the fresh medium in quick succession.
Swelling of the inoculated explants at the basal region and proliferation of the shoot was recorded within 15 days of inoculation (Fig. 1c). Development of multiple shoot-buds and proliferation of callus mass from the basal region was noted within 4 weeks (Fig. 1d). Among the various concentrations and combinations of PGRs tried, the highest number of adventitious shoots was obtained in the shoot induction medium containing 8.0-10.0 mg l-1 BAP, 1.0 mg l-1 IAA and 50.0 mg l-1 AdS, producing an average of 8 shoots (Table. 1) per explant after 8 weeks (Fig. 1e). Application of higher ratio (12:1) of BAP:IAA along with 100.0 mg l-1AdS resulted in the formation of etiolated shoot buds; white calli differentiation was noted with the progress of culture period. It was observed that moderately higher ratio of BAP:IAA combined with higher level of AdS (100.0 mg l-1) induced shoots with abnormally broad, thick, fleshy and pale green leaves. The clumps of stunted tuft of multiple shoots were virtually inseparable and with the progression of culture period callus differentiated along the margins of the leaves. These shoots failed to proliferate further even after lowering BAP: IAA ratio (data not shown). On the contrary, the number of shoots reduced to 6.0 per explant at the 8:1::BAP:IAA ratio with reduced level of AdS (25 mg l-1), but the regenerated shoots were healthy. While, adventitious shoot differentiation was not obtained at much lower ratios of BAP:IAA (4:1 or 2:1), irrespective of the concentration of AdS.
The roots were induced either in solid MS basal medium without PGRs or basal medium supplemented with 0.1 mg l-1 IAA. Roots were induced within two weeks of inoculation and efficient root system, required for the establishment in the soil, was developed within 4 weeks (Fig. 1f). Isolated single shoot failed to develop any roots in any other PGRs tried.
The rooted plantlets were separated carefully from the clusters and planted in the sand. The regenerants were successfully acclimatized within 4-6 weeks and then transferred to the soil (Fig 1g). Cent percent success was achieved in lab to field transfer of the culture regenerants. The in vitro raised plantlets were morphologically identical to the respective donor plants and did not show any apparent morphological abnormalities.
The intact shoot-tip meristem was used as explant in almost all the published work on A. vera tissue culture (Natali et al. 1990; Meyer and Staden 1991; Roy and Sarkar 1991; Liao et al. 2004; Aggarwal and Barna 2004). While, in the present study, a shoot tip meristem was divided into two halves to obtain two explants from a single meristem. The efficiency of wounded meristem for inducing multiple shoots is reasonably higher than the intact meristems (data not shown). This method also reduces the cost of multiplication and time for propagule development.
It was stated that tissue browning is a major hurdle for the successful establishment of Aloe culture (Roy and Sarkar 1991; Abrie and Staden 2001). Roy and Sarkar (1991) reported that browning was alleviated by supplementing PVP, an antioxidant, in the culture medium. While, Liao et al. (2004) have reported seldom browning of the explants of A. vera L. var. Chinensis (Haw.) Berger, when 600 mg l-1 PVP was added in the culture medium. In this protocol we have reduced the cost by limiting use of PVP in the culture medium and the explants were transferred in the fresh medium to surmount the browning problem as and when encountered.
Reviewing all the published work on adventitious shoot regeneration from the shoot-tip meristem of A. vera, it appeared that different PGRs at different concentrations produced multiple shoots and there is hardly any commonness in the reports. Meyer and Staden (1991) used only IBA, while Natali et al. (1990) and Roy and Sarkar (1991) reported success on medium containing 2,4-dichlorophenoxyacetic acid and kinetin. Aggarwal and Barna (2004) had used liquid MS medium supplemented with 1.0 mg l-1 BAP, 0.2 mg l-1 IBA and 10.0 mg l-1 citric acid to get maximum shoot multiplication. The differential PGR requirements for adventitious shoot differentiation from the same explant type indicate that there must be some genotypic differences of the A. vera used by the different group of researchers.
A micropropagation method has been established in A. vera var. Chinensis Liao et al. (2004), where suitability of PGRs and sucrose concentrations was determined by designing an orthogonal L9 (34) variation test. Researchers have claimed that sucrose was the most important factor for shoot bud induction followed by BAP and NAA has the least effectiveness.
The suitable ratio of cytokinin to auxin for the multiplication of the A. arborescence was determined as 10:1 by Wu (2000). In the present study multiplication of a high biomass yielding genotype of A. vera was obtained in BAP:IAA ratios ranging from 16:1 to 8:1, enriched with AdS either 50.0 mg l-1 or 100.0 mg l-1 (Table 1). Almost similar response was noted in another conserved genotype of A. vera (data not shown) suggesting either both of these are from the same germplasm stock or belongs to the same ecotype and such has similar response under in vitro condition. The methods of molecular genotyping would reveal the status of these two genotypes in future.
The success of any established in vitro protocol largely depends on the survival and growth performances of the propagules under ex vivo. Due to the succulent nature of A. vera dehydration problem has not been encountered during the hardening stage and cent percent survival was recorded after soil transfer, which establishes the suitability of the protocol. While, ~18 % mortality was reported earlier by Aggarwal and Barna (2004) on field transfer of A. vera regenerants.
It is estimated that from a single apical meristem > 256 propagules could be obtained within 24 weeks using the present micropropagation protocol (Table 2). An appropriate application of this protocol by scaling up might partially meet the shortfall of the propagules for commercial plantation and subsequent use in pharmaceutical and cosmetic industry.
Acknowledgements: The authors are grateful to the Secretary, Agri-horticulural Society, Alipore, Kolkata for supplying the research materials and to Dr. Malay Das for his constant help and guidance during the execution of the work.
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Legend of Figures
Fig. 1 a, b. Young shoot of Aloe vera donor, Bar = 1.2 cm (a), the basal part of the shoot splitted into two explants, Bar = 0.5 cm (b).
Fig. 1 c-e. Different stages of in vitro shoot regeneration from A. vera L. in solid MS medium supplemented with 8.0 mg l-1 BAP, 1.0 mg l-1 IAA and 50.0 mg/l AdS. Swelling of the base of the explant and proliferation of shoot meristem, Bar = 0.5 cm (c) multiple shoot-buds and callus differentiated from the base of the explant after 4 weeks of inoculation, Bar = 0.8 cm (d); elongation of in vitro regenerated shoot-buds and induction of new shoot buds from the meristematic tissue after 10 weeks, Bar = 1.0 cm (e).
Fig. 1f. Healthy root formed from the base of the shoots after 4 weeks of inoculation in root induction medium containing 1.0 mg l-1 IAA, Bar = 1.2 cm.
Fig. 1g. Hardened plant after 8 weeks of transfer to the soil.
Table 1: Mean number of shoots differentiated from a single explant after 8 weeks of inoculation on MS medium supplemented with different PGRs
Plant Growth Regulators (mg l-1 )
Number of shoots/explant*
8 ± 0.81
10 ± 0.86**
8 ± 0.32
6 ± 0.62
12 ± 0.61**
8 ± 0.12
* Mean ± SE
** Dwarf shoot with abnormally thick and broad leaves
*** Etiolated shoot buds that turned into callus with progress of culture time
Table 2. Schematic representation of adventitious shoot differentiation and respective time scale for each developmental stage<?xml:namespace prefix = v ns = "urn:schemas-microsoft-com:vml" /?> Young shoot (3-4cms) Medium Weeks 2 explants MS + 8.0 mg l - l BAP + 1.0 mg l – l IAA + 50.0 mg l – l AdS 8 shoots / explant = 16 shoots 8 32 explants MS + 8.0 mg l - l BAP + 1.0 mg l – l IAA + 50.0 mg l – l AdS 256 shoots MS + 1.0 mg l – l IAA 16 Rooted shoots Hardening 18 256 propagules ready for commercial plantation 24
Young shoot (3-4cms) Medium Weeks
2 explants MS + 8.0 mg l - l BAP + 1.0 mg l – l IAA + 50.0 mg l – l AdS
8 shoots / explant = 16 shoots 8
32 explants MS + 8.0 mg l - l BAP + 1.0 mg l – l IAA + 50.0 mg l – l AdS
256 shoots MS + 1.0 mg l – l IAA 16
Rooted shoots Hardening 18
256 propagules ready for commercial plantation 24