Callus formation was induced in stem segments from young axillary shoots grown on the underground rhizomatous stem. The use of polyvinylpyrrolidone (PVP) ...
Scientia Horticulturae, 47 ( 1991 ) 107-113
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Elsevier Science Publishers B.V., Amsterdam
In vitro regeneration and micropropagation of A l o e vera L. S.C. Roy and Aparajita Sarkar Centre of Advanced Study, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Calcutta-700019, India (Accepted for publication 6 December 1990)
ABSTRACT Roy, S.C. and Sarkar, A., 1991. In vitro regeneration and micropropagation of Aloe vera L. Scientia Hortic., 47:107-113. Rapid propagation by the formation of shoots from calli of Aloe vera was obtained in the present investigation. Callus formation was induced in stem segments from young axillary shoots grown on the underground rhizomatous stem. The use of polyvinylpyrrolidone (PVP) in the nutrient media reduced the secretion of phenolic substances from the explant. Murashige and Skoog's basal medium containing 1 mg 1- t 2,4-dichlorophenoxyacetic acid and 0.2 mg 1-1 kinetin gave the best callus induction. Shoots were initiated from the calli with reduced 2,4-D and increased kinetin concentration. Keywords: Aloe vera; micropropagation; tissue culture. Abbreviations: BAP=6-benzylamino purine; 2,4-D=2,4-dichlorophenoxyacetic acid; IAA=indole acetic acid; kn = kinetin; MS = Murashige and Skoog basal medium; NAA = a-naphthalene acetic acid; PVP = polyvinylpyrrolidone.
INTRODUCTION
One of the most extensive uses of tissue culture is the rapid clonal propagation of plants (Murashige, 1974, 1978; Vasil and Vasil, 1980). The technique is particularly useful for plants where the rate of multiplication is very slow. Plants belonging to the family Liliaceae have been cultured recently for their high medicinal and ornamental value (Vij et al., 1980). Aloe, a member of the tribe Aloineae of the Liliaceae, is cultivated in gardens for its unassuming succulent foliage as well as for Aloin, an important drug. Little work has been done on the in vitro culture of Aloe species because establishment of primary cultures is difficult owing to the secretion of phenolic substances by the explant. There is only one report on callus formation and plant regeneration from seed calli of Aloe pretoriensis (Groenewald et al., 1975 ). No work has been done so far with Aloe vera. The present investiga0304-4238/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
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tion was undertaken to develop an effective method for differentiation, regeneration and rapid multiplication of this species. MATERIALS AND METHODS
Plants o f Aloe vera were taken from the experimental garden of the Department of Botany, University of Calcutta. Axillary shoot segments (3-5 cm long) from underground nodes were taken as primary explants. Explant parts were sectioned into a suitable size (0.5-1 cm) and were surface sterilized with 0.1% mercuric chloride solution for 8-10 min, followed by six to eight washes in sterile distilled water. For induction, growth and maintenance of callus tissues, Murashige and Skoog's basal medium (MS) was used (Murashige and Skoog, 1962). To overcome browning of the explant and cultured tissues, different types of antioxidants including polyvinylpyrrolidone (PVP), ascorbic acid and activated charcoal at 0.5-1.0 mg 1-1 were used. The basal media were supplemented with different auxins (2,4-dichlorophenoxyacetic acid (2,4-D), tx-naphthalene acetic acid (NAA), indole acetic acid (IAA)) and cytokinins (kinetin (kn), 6-benzylaminopurine, (BAP). The pH of the media was adjusted to 5.6-5.8 in all cases. Explants were inoculated into culture tubes containing 25 ml medium and were subcultured after 28 days. The cultures were grown at 22-24°C with a relative humidity of 55-60% and under 2000 lx light intensity, using fluorescent tubes. For the vigorous growth of calli, three different supplements were added to the callus-inducing media: p-aminobenzoic acid (0.1 mg 1-1 ), casein hydrolysate ( 3 g 1- l ) and tyrosine ( 100 mg 1-1 ). For multiplication of shoots, calli were transferred to MS supplemented with different concentrations of kn (0, 0.5, 1, 2, 4 mg 1-1 ) with or without 2,4-D, NAA, IAA or BAP at concentrations of 0, 0.2, 0.5, 1, 2, 4 mg 1-1 RESULTS
For induction of callus tissues, the only positive response was observed in the case of young axillary shoot segments in MS (Fig. 1 ). The explants swelled within 15-20 days with concomitant browning of explants and media. Of all the antioxidants used, PVP ( 1 g 1- l ) was most effective in preventing browning of the media. A combination of 2,4-D at 1 mg 1-1 and kn at 0.2 mg 1-1 was most effective in callus induction (Fig. 2). Callus growth was not as vigorous in other combinations of 2,4-D and kn or in BAP. Of the three supplements added to the callus-inducing media, p-aminobenzoic acid (0.1 mg 1- ~) was found to be most effective and in inducing vigorous growth of the callus tissues.
REGENERATIONAND MICROPROPAGATIONOF ALOE
VERA
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Fig. 1. Young axillary shoot developing from underground rhizome used as explant.
Fig. 2. Growth of callus tissues in MS containing 1 mg 1- l of 2,4-D and 0.2 mg 1- ~ofkn.
Callus tissues showed formation of roots after 8 weeks on MS containing different concentrations of 2,4-D (0.02, 0.05, 0.1, 0.2 mg 1-1 ) without kn. These roots were formed from 20-25% of the calli after 20 days. Of the different concentrations of 2,4-D used, 0.02 mg 1-~ produced the highest number of roots per culture (Table 1 ). In addition to 2,4-D, NAA (0.05, 0.1 mg 1-1 ), IAA (0.05, 0.1 mg 1- i ) and BAP (0.05, 0.1 mg 1-1 ) also showed root formation, but few roots per culture. Roots were initially white but turned brown at maturity in each case. When the calli of 8 weeks growth were transferred to fresh media contain-
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TABLE 1 Effect of varying levels of 2,4-D on root initiation and growth. Values expressed as mean number of roots per culture + SE (each experiment was carried out with ten replicates and was repeated three times) and as mean root length from callus tissues (average of ten roots) + SE
MS + 2,4-D (mg 1-~ )
No of roots per culture
Length of roots (cm)
0.02 0.05 0.1 0.2
4.5+0.5 4.1+0.3 2.4+0.2 1.6+0.5
4.2+0.1 4.0+0.1 4.1+0.1 4.6+0.4
Fig. 3. Multiplication of shoots in media containing 2,4-D (0.02 mg 1-~ ) and kn ( 1 mg 1-~ ). Fig. 4. Formation ofplantlets.
ing 2,4-D (0.2 mg 1- ' ) and kn ( 1-4 mg 1-' ), shoot initiation occurred in 80% of the calli after 15 days. These shoots, however, failed to develop when maintained in the same media for over 4 weeks. In order to determine the optim u m concentrations of kn to initiate shoots, the media were modified with different concentrations of kn ( 0 - 4 mg 1- ' ), with or without 2,4-D ( 0 - 0 . 2 mg 1-1 ), N A A (0.02-0.1 mg 1-~ ), IAA (0.02-0.1 mg 1-~ ) and BAP ( 0 . 0 5 0.1 mg 1-~ ). Out of the different auxins tested, 2,4-D (0.02 mg 1-1 ) in combination with kn ( 1 mg 1- ' ) gave the highest rate of initiation (Figs. 3 and 4 ). About 10-15 new shoots were formed after 7 weeks (Table 2 ). When the shoots or plantlets were cultured separately in MS without any
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REGENERATION AND MICROPROPAGATIONOF ALOE VERA
TABLE 2 Effect of kn and 2,4-D on shoot organogenesis from call± grown in 2,4-D (0.2 mg 1- ~) and kn ( 1 mg l - t ) . The values are expressed as mean number of shoots per culture ± SE (each experiment was
carried out with ten replicates and was repeated three times) mean shoot length (average of 50 shoots) ± SE MS Supplemented with 2,4-D (mgl-'
kn (mgl-')
0 0 0 0 0 0.02 0.02 0.02 0.02 0.05 0.05 0.05 0.05 0.1 0.1 0.1 0.1 0.2 0.2 0.2 0.2
0 0.5 1 2 4 0.5 1 2 4 0.5 1 2 4 0.5 1 2 4 0.5 1 2 4
Fig. 5. Plantlet with roots.
No. of shoots per culture
Length of shoots (cm)
2.0±0.1 5.8±0.5 8.5±0.4 8.2±0.1 6.1±0.2 8.2±0.3 15.5±0.8 10.1±0.3 8.1±0.2 5.1±0.1 6.1±0.2 5.3±0.4 2.5±0.4 5.0±0.5 4.2±0.1 2.6±0.3 1.5±0.3 2.5±0.4 3.1±0.2 1.3±0.1 1.0±0.1
0.5±0.1 0.8±0.5 1.6±0.5 1.4±0.1 1.0±0.2 2.4±0.4 6.5±0.3 4.8±0.5 4.8±0.2 3.2±0.1 2.5±0.3 2.0±0.1 1.5±0.1 1.0±0.1 0.8±0.2 0.5±0.2 0.5±0.2 0.5±0.1 0.5±0.1 1.0±0.2 1.0±0.I
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added hormone, roots (three to six per plantlet) were developed after 5 weeks and well-developedroot systems were formed after 7 weeks (Fig. 5 ). Plantlets with well-developedroot systems were transferred directly to pots. These were initially kept in a shady place (4000 lx ) for hardening and acclimation of plants. After 1 month, the plants were kept in direct sunlight in their natural habitat. In some regenerated plants, after 7-8 months in the field, inflorescences were formed with yellowish orange flowers. DISCUSSION
Many plants, including Aloe, are rich in phenolic compounds. These products are known to inhibit the establishment of primary cultures. Other explants, except young stem segments, ceased to grow completely after the discharge of brown substances from the explants. The good response of the young segments in the present investigation might be a result of the fact that juvenile tissues tend to have a greater capacity for restoration (Murashige and Skoog, 1962 ) or produce less phenols. Wessels et al. (1976) succeeded in callus formation and subsequent regeneration of shoots from leaf tissues of Haworthia planifolia by using PVP in the culture media. Of the different auxins tested, 2,4-D (in combination with kn ) was found to give good callus growth in liUiaceous species (Wessels et al., 1976; Roy, 1980; Kar and Sen, 1985 ). 2,4-D in combination with kn was also found to be effective in initiating leaf callus ofNigella sativa and Vigna sinensis (Chand and Roy, 1981; Jha and Roy, 1982). The benefit ofp-aminobenzoic acid in supporting vigorous growth of calli was also noted in tobacco, Ruscus, Aloe pretoriensis and Agave by different workers (Groenewald et al., 1975; Durzan, 1984; Jha and Sen, 1985 ). The differentiation ofA. vera is dependent on the specific growth regulator balance in the medium. Shoot differentiation was found to be greatest when 2,4-D was reduced to 0.02 mg 1-1 and kn was increased to 1 mg 1- ~. Although kinetin alone promoted shoot initiation, it did not promote further development. These results suggest that in vitro culture may be used as a technique for rapid propagation ofA. vera.
REFERENCES Chand, S. and Roy, S.C., 1981. Induction of organogenesis in callus cultures ofNigella sativa L. Ann. Bot., 48: 1-4. Durzan, D.J., 1984. Special problems: Adult vs. juvenile explants. In: W.R. Sharp, D.A. Evand, P.V. Ammirato and Y. Yamada (Editors), Handbook of Plant Cell Culture. MacMillan, New York, pp. 47-63. Groenewald, E.G., Koeleman, A. and Wessels, D.C.J., 1975. Callus formation and plant regeneration from seed tissue of Aloe pretoriensis. Z. Pflanzenphysiol., 75: 270-272.
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Jha, T.B. and Roy, S.C., 1982. Effect of different hormones on Vigna tissue culture and its chromosomal behaviour. Plant Sci. Lett., 24:219-224. Jha, S. and Sen, S., 1985. In vitro regeneration ofRuscus hypophyllum plants. Plant Cell Tissue Organ Cult., 5: 79-87. Kar, D.K. and Sen, S., 1985. Effect of hormone on chromosome behaviour in callus cultures of Asparagus racernosus. Biol. Plant, 27: 6-9. Murashige, T., 1974. Plant propagation through tissue cultures. Ann. Rev. Plant Physiol., 25: 135-165. Murashige, T., 1978. Importance of plant tissue culture in agriculture. In: T.A. Thorpe (Editor), Proceedings of the 4th Intemational Congress on Plant Cell and Tissue Culture. University of Calgary, Alta, pp. 15-26. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant, 15: 473-497. Roy, S.C., 1980. Chromosomal variations in the callus tissues ofAllium tuberosum and A. cepa. Protoplasma, 102: 171-176. Vasil, I.K. and Vasil, V., 1980. Clonal propagation. Int. Rev. Cytol. (Suppl.), 11A: 145-173. Vij, S.P., Sharma, M. and Toor, I.S., 1980. Cytogenetical investigations into some garden ornamentals II. The genus Aloe. Cytologia, 45:515-532. Wessels, D.C.J., Groenewald, E.G. and Koeleman, A., 1976. Callus formation and subsequent shoot and root development from leaf tissue of Haworthia planifolia. Z. Pflanzenphysiol., 78: 141-145.
