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In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant
Saudi Journal of Biological Sciences (2013) 20, 63–68
King Saud University
Saudi Journal of Biological Sciences www.ksu.edu.sa www.sciencedirect.com
ORIGINAL ARTICLE
In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant Junaid Aslam a b
a,b
, Abdul Mujib
a,*
, Maheshwar Prasad Sharma
a
Cellular Differentiation and Molecular Genetics Section, Department of Botany, Hamdard University, New Delhi 110 062, India Department of Biotechnology, Jamia Hamdard (Hamdard University), New Delhi 110 062, India
Received 1 August 2012; revised 4 November 2012; accepted 6 November 2012 Available online 17 November 2012
KEYWORDS In vitro rooting; Dracaena sanderiana; Indoor ornamental; Plant regeneration; Shoot multiplication
Abstract A protocol has been developed for in vitro plant regeneration from a nodal explant of Dracaena sanderiana Sander ex Mast. Nodal explant showed high callus induction potentiality on MS medium supplemented with 6.78 lM 2,4-dichlorophenoxyacetic acid (2,4-D) followed by 46.5 lM chlorophenoxy acetic acid (CPA). The highest frequency of shoot regeneration (85%) and number of shoots per explant (5.6) were obtained on medium supplemented with 7.84 lM N6-benzylaminopurine (BA). Rooting was high on MS solid compared to liquid medium when added with 7.38 lM indole-3-butyric acid (IBA). Fifty percent of the roots were also directly rooted as microcuttings on soil rite, sand and peat mixture (1:1:1). In vitro and ex vitro raised plantlets were used for acclimatization. More than 90% of the plantlets was successfully acclimatized and established in plastic pots. Ex vitro transferred plantlets were normal without any phenotypic aberrations. ª 2012 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.
1. Introduction Dracaena sanderiana Sander ex Mast belongs to family Agavaceae, is known as Lucky Bamboo. It is distributed in tropical and subtropical open lands of India and Africa. The genus Dracaena is well known as an indoor ornamental. Some of the Dracaena species possess several medicinal properties and are used in curing a number of diseases. Spiroconazole-A, an active saponin compound obtained from Dracaena mannini * Corresponding author. Tel.: +91 971509146198; fax: +91 04 2646027. E-mail address: [email protected] (A. Mujib). Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
and Dracaena arborea exhibited antileishmanial, antimalarial, molluscicidal, fungicidal and bacteriostatic activities (Okunji et al., 1996). Dracaena draco produces several steroidal saponins, which showed cytostatic activity on Leukemia HL 60 cells (Mimaki et al., 1999; Yokosuka et al., 2000). Dracaena cochinensis extract is reported to improve the clotting process in mice significantly (Nong, 1997). The resin of some species is used as the substrate in xuejie (a Chinese drug) in China since long (Lin, 1994). Dragon’s blood, a red resin exuding from the stem of D. draco has been used frequently as a ‘‘herbal’’ remedy in traditional medicine (Bruck, 1999). The stem wood extract of Dracaena loureiri, a Thai plant is revealed to inhibit the estrogen effect by binding to the estrogen receptor (Ichikawa et al., 1997). Despite their medicinal and ornamental importance, not much work has been done in Dracaena species in in vitro conditions (Junaid et al., 2010) and mostly grows vegetatively.
1319-562X ª 2012 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.sjbs.2012.11.005
64 The vegetatively propagated plants are sensitive to several bacterial, fungal, viral and mycoplasmal diseases acquired from the air, soil and insect-vectors as a result of which their productivity declines (Anonymous., 1996), even mass propagation through seeds has many limitations like seed dormancy, low rate of germination and progeny variation in other plant species (Venkataramaiah et al., 1980; Chand and singh, 2004). To overcome these problems and fulfill the required demand it has necessitated restoring the productivity of plants by the use of plant tissue culture (Bhattacharjee, 2006), as in vitro micropropagation includes the rapid vegetative multiplication of valuable plant material for agriculture and forestry. Besides this, the in vitro technique is also widely used in the commercial field for the micropropagation of ornamental plants in large numbers; however, the process is regulated by the biochemical reserve, localized in specific organs (Thorpe, 1990; Mujib et al., 2004; Bhattacharjee, 2006). In the present investigation we have established a high regeneration protocol for the first time ever in D. sanderiana that could be used in future to enrich the ornamental industry. 2. Materials and methods 2.1. Plant material Healthy plants of D. sanderiana were collected from the Jamia Hamdard (Hamdard University, New Delhi, INDIA) herbal garden. Various explants (nodal 1 cm, internodal stem 1 cm, leaf 1 cm2, axillary buds 1 cm and roots 1 cm) were used as experimental material.
J. Aslam et al. concentrations of root inducing plant growth regulators (IBA, NAA, IAA); 0.0–9.86 lM indole-3-butyric acid (IBA), 0.0–10.73 lM a-naphthalene acetic acid (NAA), 0.0– 11.41 lM indole-3-acetic acid (IAA). Data were scored in terms of shoot development percentage (%), root number per shoot, root length (cm) and number of adventitious roots per shoot. 2.6. Culture conditions The pH of all the cultures was adjusted to 5.6–5.8 before autoclaving. The media were sterilized in an autoclave for 15 min at 121C; cultures were incubated at 25 ± 2C under a 16-h photoperiod with cool white fluorescent illumination (100 lmol m 2 s 1 PFD). 2.7. Ex vitro transplantation The in vitro developed plantlets were removed from the cultured vessels, transplanted in micro plastic pots (10 cm), containing autoclaved soil rite and sand (1:1), thoroughly covered with pored polythene bags to maintain high humidity. Plants were subsequently transferred to pots (15 cm) placed for one month at room temperature (25 ± 2 C) at a 16 h photoperiod with cool white fluorescent illumination (100 lmol m 2 s 1 PFD). For ex vitro rooting regenerated shoots were treated with IBA (7.38 lM) for 36 h before being transferred to pots (15 cm) containing a mixture of sand soil and peat (1:1:1) as described previously. 2.8. Statistical analysis
2.2. Surface sterilization Explants were rinsed 3–4 times with sterilized double distilled water, and surfaces disinfected for 10 min. in H2O2 (1%) solution. After rinsing, the explants were placed on sterilized blotting paper and finally placed on a callus induction medium.
The data on the effects of growth regulators on morphogenesis were analysed by one-way analyses of variance (ANOVAs). Values are means of five replicates from two experiments, and the presented mean values were separated using Duncan’s Multiple Range Test (DMRT) at p 6 0.05.
2.3. Callus induction
3. Results
After proper surface sterilization, the explants were placed on MS medium containing various auxin types and concentrations of 0.0–9.04 lM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.0–10.20 lM 2,4-5-triacetic acids (2,4-5-T), 0.0–52.0 lM chlorophenoxyacetic acid (CPA).
3.1. Callus induction
2.4. Regeneration and shoot multiplication Nodal callus masses (80–90 mg) were used for regeneration; nodal stem showed rapid callus initiation and vigorous callus growth compared to callus induced from other sources (data unpublished). Callus was cultured on MS medium fortified with various concentrations of cytokinins with a range of 0.0–8.96 lM N6-benzylaminopurine (BA) and 0.0–9.28 lM kinetin (Kn). Data were scored in terms of shoot development percentage (%), shoot number/nodal calli, shoot length and leaf number per shoot.
Of the various auxin types and explants used nodal stem (Fig. 1a) proved to be highly effective compared to others (data not shown) on MS medium supplemented with 2,4-D (6.78 lM) followed by CPA (46.5 lM). Explants varied in callus induction rate and percentage, however calli induced from all the sources were similar in morphological appearances; they were compact in nature and creamy in colour, although, it became black and necrotic at higher 2,4-D concentrations after the 9th week of culture. Table 1 shows a comparative account of nodal stem callus biomasses (fresh and dry wt.) under optimized concentrations of various auxins; 2,4-D (6.78 lM) was a highly promising concentration for high callus biomass production. 3.2. Callus regeneration /shoot multiplication
2.5. Rooting in solid and liquid MS medium Shoots that developed in regenerating medium were transferred on solid and liquid MS medium, added with various
Undifferentiated masses of callus (80–90 mg) were cultured on MS medium supplemented with BA (0.0–8.96 lM) and Kn (0.0–9.28 lM). Maximum response (in terms of shoot development
In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant
65
Fig. 1 In vitro micropropagation of Dracaena sanderiana. (a) Callus induction from nodal segment on MS medium supplemented with 2,4-D (6.78 (lM); (b and c) shoot multiplication in cytokinin added MS medium. (B) Kn (9.28 lM), (C) BA (7.84 (lM); (d) 8 week old culture before transfer to field conditions MS + BA (7.84 lM) + IBA (7.38 lM); (e and f) root induction from isolated single shoot in solid (e) and liquid (f) MS medium added with IBA (7.38 lM); (g) regenerated plantlets transferred to micropots in soil rite (100%).
percentage, shoot number, shoot length and leaf number) was noticed in culture fortified with BA (7.84 lM) (Fig. 1c and d). Although, Kn (9.28 lM) proved to be highly effective but in general was less (Fig. 1b) significant compared to BA. Table 2 shows the effect of BA and Kn on shoot regeneration.
3.3. Rooting A comparative study has been made to evaluate the effect of solid and liquid medium on rooting. Shoots that developed in regenerating medium were used for root induction and
66 Table 1
J. Aslam et al. Callus biomass (fresh and dry weight) growth in optimized auxin concentration. Data were scored up to 9 weeks of culture.
Optimized concentration of auxins (lM)
5 weeks old callus
7 weeks old callus
9 weeks old callus
Fresh wt. (gm)
Fresh wt. (gm)
Fresh wt. (gm)
a
2,4-D (6.78) CPA (46.5) 2,4-5-T (10.20)
Dry wt. (gm) a
0.22 0.15b 0.12c
0.78 0.62b 0.56b
a
Dry wt. (gm) a
1.25 0.88b 0.74c
0.35 0.27b 0.25b
a
2.69 2.00b 0.85c
Dry wt. (gm) 0.63a 0.46b 0.42b
Values are means ± standard deviation of 5 replicates from 2 experiments. Means with common letters within each column are not significantly different at p 6 0.05, according to Duncan’s Multiple Range Test (DMRT).
Table 2 Shoot regeneration in BA/KN supplemented MS medium. Nodal callus (80–90 mg) was cultured for regeneration. Data were scored after 6 weeks of culture. PGR (lM) BAP
Shoot number/callus mass
Shoot length (cm)
Leaf number
0.0g 2.8d 2.9d 5.3b 5.7a 5.0b 0.0g 1.8f 2.3e 2.8d 2.9d 3.3c
0.0h 2.1e 4.4b 7.5a 7.3a 3.9c 0.0h 1.2g 2.5d 4.4b 4.0c 1.5f
0.0i 5.7d 7.3c 7.6b 7.9a 4.9f 0.0i 4.3gh 5.0ef 5.2e 5.4e 4.2h
Kn
0.00 2.24 4.48 6.72 7.84 8.96 0.00 2.32 4.64 6.96 8.12 9.28
Values are means ± standard deviation of 5 replicates from 2 experiments. Means with common letters within each column are not significantly different at p 6 0.05, according to Duncan’s Multiple Range Test (DMRT).
transferred on MS medium, containing root inducing plant growth regulators. Table 3 shows a comparative account of root induction in solid and liquid media. Better response was observed in solid medium (Fig. 1e) when supplemented with IBA (7.38 lM), followed by NAA (10.73 lM); roots were high in number (14.6) with average length (48.6 mm) compared to their liquid counterpart (Fig. 1f). 3.4. Ex vitro transplantation Rooting was successfully induced within 5 weeks on IBA (7.38 lM) added MS medium. Rooted plantlets (Fig. 1e) were transferred in plastic pots (Fig. 1g) containing soil rite, kept at 25 ± 2C in the culture room, and covered with pored transparent polythene bags to reduce the humidity. But the survival frequency was moderate; thereafter the survival rate was enhanced by the application of a hardening procedure, where rooted shoots were transferred to half and quarter-strength liquid MS medium without plant growth regulators and were then transferred to plastic pots, containing sand and soil rite (1:1). Transferred plantlets showed normal morphological appearance. During hardening, shoots elongated and leaves turned green and expanded. Secondly, regenerated shoots were also rooted directly as microcuttings on an autoclaved mixture of sand and soil rite. The shoots’ treatment with 7.38 lM (IBA) for 36 h was found essential for in vitro rooting. The frequency of root formation was increased 85–94% by ex vitro rooting using microcutting procedures.
4. Discussion Propagation of plants through vegetative and sexual processes is well established in horticulture, however, micropropagation offers a rapid means of production of clonal plants in numbers, which can be used for afforestation and conservation of elite and rare germplasm and also for rapid multiplication of economically important plants (Bonga, 1982). In vitro culture may be initiated from the embryo, shoot apex, axillary bud and several other explants. In the present investigation in D. sanderiana various explants (node, internode, axillary bud, leaf and root) were used, of which nodal segments showed maximum callusing and high biomass on 2,4-D added MS medium. A similar promoting effect of 2,4D on callusing was earlier reported in the same species (Ilah et al., 2002). However, in Dracaena fragran young stem segments produced calli on medium supplemented with 2,4-D alone or in combination with BA (Vinterhalter, 1989). Additionally, in many other plants profuse callusing was observed on 2,4-D added medium (Khan et al., 2002). For cultured tissues, the requirement for exogenous hormones depends on the endogenous level of plant tissue which varies with organs, plant genotype and the phase of plant growth (Chand and Singh, 2004). Nodal explants along with high callus biomass also exhibited high regeneration potentiality compared to other tested explants. Pua et al. (1989) have reported in Brassica napus that the nodal segment was more responsive
In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant
67
Table 3 Root induction from individual shoot in solid and liquid media. MS medium was supplemented with various auxins. Data were scored after 5 weeks of culture. PGR (lM) IBA
After 5 weeks NAA
IAA
0.00 2.46 4.92 7.38 9.84 0.00 2.68 5.37 8.05 10.73 2.85 5.71 8.56 11.41
No. of root
Root length (mm)
Solid
Liquid
Solid
Liquid
0.0i 3.3g 5.3e 12.0a 7.0d 0.0i 1.7h 3.0g 5.0e 9.0b 0.0i 1.4hi 2.7gh 4.3f 8.3c
0.0h 2.0f 4.7c 9.6a 5.0b 0.0h 0.0h 0.0h 2.7e 4.5c 0.0h 0.0h 1.7g 2.0f 3.7d
0.0k 23.3c 24.5b 26.5a 14.6f 0.0k 6.3j 11.6h 16.6e 18.3d 0.0k 5.7j 9.0i 13.4g 17.0e
0.0h 3.0g 11.6b 15.0a 8.3c 0.0h 0.0h 0.0h 5.0e 7.3d 0.0h 0.0h 0.0h 4.2f 6.8d
Values are means ± standard deviation of 5 replicates from 2 experiments. Means with common letters within each column are not significantly different at p 6 0.05, according to Duncan’s Multiple Range Test (DMRT).
for callusing and regeneration compared to other explants. Out of the two cytokinins (BA and Kn), BA was more active during multiple shoot formation. The superiority of BA over Kn for multiple shoot formation was also demonstrated in other plants like Jetropha integerrima (Sujatha and Dhingra, 1993), Sapium sebiferum (Siril and Dhar, 1997), and Bombax ceiba (Chand and Singh, 1999, 2004). In contrast, kinetin was found to be better than 2iP and BA for promoting axillary shoot proliferation in Cephaelis ipecacuanha (Jha and Jha, 1989). However, shoot multiplication decreased with increasing BA concentrations. Similar effect was also noticed in many other plant systems (Ben Jouira et al., 1998; Biroscikova et al., 2004; Junaid et al., 2007a). In addition, among the used root inducing plant growth regulators, IBA alone or in combination with BA was found to be most effective for root induction particularly in the solid medium. Similar results were noticed in a number of other plant species (Tapati et al., 1995; Vandna et al., 1995; Khan et al., 2002; Biroscikova et al., 2004; Junaid et al., 2007a). In the present investigation, no major differences were observed in in vitro and ex vitro rooting, however, a moderate difference was seen in the survival rate’ that was due to the ex vitro developed healthy roots. Moreover, in vivo rooting offers several advantages over in vitro rooting as the former is cost effective and the roots structurally and functionally contain more root hairs than the latter (Debergh and Read, 1991; Preece and Sutter, 1991). Plantlets with healthy roots were transferred for acclimatization purposes, showing more than 90% survivability, they however, showed normal morphological appearance. Similar practice of transplantation was achieved in several other plant systems (Zhang and Davis, 1986; Debergh and Read, 1991; Preece and Sutter, 1991; Shibli and Smith, 1996; Kim et al., 1997; Dhar and Joshi, 2005; Junaid et al., 2007b).
5. Conclusion In conclusion, an efficient regeneration protocol for D. sanderiana has been established, which required 16 weeks from culture initiation to plant regeneration. All the plants transferred to the ex vitro conditions showed a high homogeneity without obvious morphological avoidance of somaclonal variation. Micropropagation would ensure a continuous supply of plants in limited time and space. This protocol will be helpful for rapid and large scale propagation, to enrich the ornamental industry. References Anonymous, 1996. In: The Wealth of India: A Dictionary of Indian Raw Materials and Industrial Products, vol. VIII. Council of Scientific and Industrial Research, New Delhi, pp. 303–305. Ben Jouira, H., Hassairi, A., Bigot, C., Dorion, N., 1998. Adventitious shoot production from strips of stem in the Dutch elm hybrid ‘Commelin’: plantlet regeneration and neomycin sensitivity. Plant Cell Tissue Organ Cult. 53, 153–160. Bhattacharjee, S.K., 2006. Advances in Ornamental Horticulture. Pointer, Jaipur 6, 2065, ISBN 81-7132-432-0. Biroscikova, M., Spisakova, K., Liptak, S., Pichler, V., 2004. Micropropagation of mature wych elm (Ulmus glabra Huds.). Plant Cell Rep. 22, 640–644. Bonga, J.M., 1982. Clonal propagation of mature trees: problems and possible solutions. In: Bonga, J.M. (Ed.), Tissue Culture in Forestry. Martinus Nijhoff Publishers, Dordrecht, pp. 249–271. Bruck, M., 1999. Dragon’s blood. A glance into the history of pharmacognosy. Bull. Soc. Sci. Med. 1, 96–101. Chand, S., Singh, A.J., 2004. In vitro shoot regeneration from cotyledonary node explants of a multipurpose leguminous tree,
68 Pterocarpus marsupium roxb. In Vitro Cell. Dev. Biol. Plant 40, 167–170. Chand, S., Singh, A.K., 1999. In vitro propagation of Bombex ceiba L. (Silkcotton). Silvae. Genet. 48, 313–317. Debergh, P.C., Read, P.E., 1991. Micropropagation. In: Debergh, P.C., Zimmerman, R.H. (Eds.), Micropropagation: Technology and Application. Kluwer Academic Publishers, Dordrecht, pp. 1– 13. Dhar, U., Joshi, M., 2005. Efficient plant regeneration protocol through callus for Saussurea obvallata (DC.) Edgew. (Asteraceae): effects of explant type, age and plant growth regulators. Plant Cell Rep. 24, 195–200. Ichikawa, K., Kitaoka, M., Taki, M., Takaish, i.S., Iijima, Y., Boriboon, M., Akiyama, T., 1997. Retrodihydrochalcones and homoisoflanes isolated from Thai medicinal plant Dracaena loureiri and their estrogen against activity. Planta Med. 63 (6), 648–656. Ilah, A., Junaid, A., Mujib, A., 2002. Biotechnology and future of an underused medicinal plant Dracaena. In: Khan, I., Khanum, A. (Eds.), Role of Biotechnology in Medicinal and Aromatic Plants. Ukkaz Publication, Hyderabad, pp. 192–206. Jha, S., Jha, T.B., 1989. Micropropagation of Cephaelis ipecacuanha Rich. Plant Cell Rep. 8, 437–439. Junaid, A., Mujib, A., Sharma, M.P., 2010. Cell and tissue culture of Dracaena sanderiana Sander ex Mast. – a review. Hamdard Med. 52 (4), 31–36. Junaid, A., Mujib, A., Bhat, M.A., Sharma, M.P., Samaj, J., 2007a. Somatic embryogenesis and plant regeneration in Catharanthus roseus. Biol. Plant 51 (4), 641–646. Junaid, A., Mujib, A., Sharma, M.P., Tang, W., 2007b. Growth regulators affect primary and secondary somatic embryogenesis in Madagaskar periwinkle (Catharanthus roseus (L) G. Don) at morphological and biochemical levels. Plant Growth Regul. 51, 271–281. Khan, M.R., Rashid, H., Quraishi, A., 2002. Effect of various growth regulators on callus formation and regeneration in Brassica napus Cv. Oscar. Pakistan J. Biol. Sci. 5 (6), 693–695. Kim, M.K., Sommer, H.E., Bongarten, B.C., Merkle, S.A., 1997. High frequency induction of adventitious shoots from hypocotyl segments of Liquidambar styraciflua L. by thidiazuron. Plant Cell Rep. 16, 536–540. Lin, A.P., 1994. Pharmacognostic studies on sanguis lignun Dracaena cochinensis. Zhongguo Zhong Yao Za Zhi 19 (11), 648–656. Mimaki, Y., Kuroda, M., Ido, A., Kameyama, A., Yokusuka, A., Sashida, Y., 1999. Steroidal saponin from the aerial parts of
J. Aslam et al. Dracaena draco and their cytostatic activity on HL 60 cells. Phytochemistry 50 (5), 805–813. Mujib, A., Cho, M.J., Predieri, S., Banerjee, S., 2004. In Vitro Application in Crop Improvement. Oxford IBH Publi. Co. Pvt. Ltd, New Delhi. Nong, X., 1997. Hemostatic effect of Dracaena cochinensis (Lour) S.C. Chen. Zhongguo Zhong Yao ZA Zhi 22 (4), 240–243. Okunji, C.O., Iwn, M.M., Jackson, J.E., Tally, J.D., 1996. Biological activity of saponins from two Dracaena species. Adv. Exp. Med. Biol. 404, 415–428. Preece, J.E., Sutter, E.G., 1991. Acclimatization of micropropagated plants to the greenhouse and field. In: Debergh, P.C., Zimmerman, R.H. (Eds.), Micro Propagation: Technology and Application. Kluwer Academic Publishers, Dordrecht, pp. 71–94. Pua, E.C., Mehra-Palta, A., Nagy, F., Chua, N.H., 1989. Transgenic plants of Brassica napus L. Biotechnology 5, 815–817. Shibli, R.A., Smith, M.A.L., 1996. Direct shoot regeneration from Vaccinium pahalae (ohelo) and Vaccinium myrtillus (bilberry) leaf explants. HortScience 31, 1225–1228. Siril, E.A., Dhar, U., 1997. Micropropagation of mature Chinese tallow tree (Sapium sebiferum Roxb.). Plant Cell Rep. 16, 637–640. Sujatha, M., Dhingra, M., 1993. Rapid plant regeneration from various explants of Jetropha integerrima. Plant Cell Tissue Organ Cult. 35, 293–296. Tapati, D., Mitra, G.S., Chatterjee, C., 1995. Micropropagation of Citrus sinensis var. Mosaambi – an important scion. Phytomorphology 45, 65–71. Thorpe, T.A., 1990. The current status of plant tissue culture. In: Bhojwani, S.S. (Ed.), Plant Tissue Culture: Application and Limitation. Elsevier Sc Publishing Company, New York, pp. 1–22. Vandna, M., Madhumati, P., Srivastava, P.S., 1995. In vitro micropropagation of Moringa pterygosperma. Phytomorphology 45, 253–261. Vinterhalter, D.V., 1989. In vitro propagation of green foliaged Dracaena fragrans Ker- Gwal. Plant Cell Tissue Organ Cult. 17, 13–20. Venkataramaiah, V., Prasad, S.V., Rajeswara, R.G., Swamy, P.M., 1980. Levels of phenolic acids in Pterocarpus santalinus L. Indian J. Exp. Biol. 18, 887–889. Yokosuka, A., Mimaki, Y., Sashida, Y., 2000. Steroidal saponin from Dracaena surculosa. J. Nat. Prod. 63 (90), 1239–1243. Zhang, Z.M., Davis, F.T., 1986. In vitro culture of Crape myrtle. HortScience 21, 1044–1045.
King Saud University
Saudi Journal of Biological Sciences www.ksu.edu.sa www.sciencedirect.com
ORIGINAL ARTICLE
In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant Junaid Aslam a b
a,b
, Abdul Mujib
a,*
, Maheshwar Prasad Sharma
a
Cellular Differentiation and Molecular Genetics Section, Department of Botany, Hamdard University, New Delhi 110 062, India Department of Biotechnology, Jamia Hamdard (Hamdard University), New Delhi 110 062, India
Received 1 August 2012; revised 4 November 2012; accepted 6 November 2012 Available online 17 November 2012
KEYWORDS In vitro rooting; Dracaena sanderiana; Indoor ornamental; Plant regeneration; Shoot multiplication
Abstract A protocol has been developed for in vitro plant regeneration from a nodal explant of Dracaena sanderiana Sander ex Mast. Nodal explant showed high callus induction potentiality on MS medium supplemented with 6.78 lM 2,4-dichlorophenoxyacetic acid (2,4-D) followed by 46.5 lM chlorophenoxy acetic acid (CPA). The highest frequency of shoot regeneration (85%) and number of shoots per explant (5.6) were obtained on medium supplemented with 7.84 lM N6-benzylaminopurine (BA). Rooting was high on MS solid compared to liquid medium when added with 7.38 lM indole-3-butyric acid (IBA). Fifty percent of the roots were also directly rooted as microcuttings on soil rite, sand and peat mixture (1:1:1). In vitro and ex vitro raised plantlets were used for acclimatization. More than 90% of the plantlets was successfully acclimatized and established in plastic pots. Ex vitro transferred plantlets were normal without any phenotypic aberrations. ª 2012 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.
1. Introduction Dracaena sanderiana Sander ex Mast belongs to family Agavaceae, is known as Lucky Bamboo. It is distributed in tropical and subtropical open lands of India and Africa. The genus Dracaena is well known as an indoor ornamental. Some of the Dracaena species possess several medicinal properties and are used in curing a number of diseases. Spiroconazole-A, an active saponin compound obtained from Dracaena mannini * Corresponding author. Tel.: +91 971509146198; fax: +91 04 2646027. E-mail address: [email protected] (A. Mujib). Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
and Dracaena arborea exhibited antileishmanial, antimalarial, molluscicidal, fungicidal and bacteriostatic activities (Okunji et al., 1996). Dracaena draco produces several steroidal saponins, which showed cytostatic activity on Leukemia HL 60 cells (Mimaki et al., 1999; Yokosuka et al., 2000). Dracaena cochinensis extract is reported to improve the clotting process in mice significantly (Nong, 1997). The resin of some species is used as the substrate in xuejie (a Chinese drug) in China since long (Lin, 1994). Dragon’s blood, a red resin exuding from the stem of D. draco has been used frequently as a ‘‘herbal’’ remedy in traditional medicine (Bruck, 1999). The stem wood extract of Dracaena loureiri, a Thai plant is revealed to inhibit the estrogen effect by binding to the estrogen receptor (Ichikawa et al., 1997). Despite their medicinal and ornamental importance, not much work has been done in Dracaena species in in vitro conditions (Junaid et al., 2010) and mostly grows vegetatively.
1319-562X ª 2012 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.sjbs.2012.11.005
64 The vegetatively propagated plants are sensitive to several bacterial, fungal, viral and mycoplasmal diseases acquired from the air, soil and insect-vectors as a result of which their productivity declines (Anonymous., 1996), even mass propagation through seeds has many limitations like seed dormancy, low rate of germination and progeny variation in other plant species (Venkataramaiah et al., 1980; Chand and singh, 2004). To overcome these problems and fulfill the required demand it has necessitated restoring the productivity of plants by the use of plant tissue culture (Bhattacharjee, 2006), as in vitro micropropagation includes the rapid vegetative multiplication of valuable plant material for agriculture and forestry. Besides this, the in vitro technique is also widely used in the commercial field for the micropropagation of ornamental plants in large numbers; however, the process is regulated by the biochemical reserve, localized in specific organs (Thorpe, 1990; Mujib et al., 2004; Bhattacharjee, 2006). In the present investigation we have established a high regeneration protocol for the first time ever in D. sanderiana that could be used in future to enrich the ornamental industry. 2. Materials and methods 2.1. Plant material Healthy plants of D. sanderiana were collected from the Jamia Hamdard (Hamdard University, New Delhi, INDIA) herbal garden. Various explants (nodal 1 cm, internodal stem 1 cm, leaf 1 cm2, axillary buds 1 cm and roots 1 cm) were used as experimental material.
J. Aslam et al. concentrations of root inducing plant growth regulators (IBA, NAA, IAA); 0.0–9.86 lM indole-3-butyric acid (IBA), 0.0–10.73 lM a-naphthalene acetic acid (NAA), 0.0– 11.41 lM indole-3-acetic acid (IAA). Data were scored in terms of shoot development percentage (%), root number per shoot, root length (cm) and number of adventitious roots per shoot. 2.6. Culture conditions The pH of all the cultures was adjusted to 5.6–5.8 before autoclaving. The media were sterilized in an autoclave for 15 min at 121C; cultures were incubated at 25 ± 2C under a 16-h photoperiod with cool white fluorescent illumination (100 lmol m 2 s 1 PFD). 2.7. Ex vitro transplantation The in vitro developed plantlets were removed from the cultured vessels, transplanted in micro plastic pots (10 cm), containing autoclaved soil rite and sand (1:1), thoroughly covered with pored polythene bags to maintain high humidity. Plants were subsequently transferred to pots (15 cm) placed for one month at room temperature (25 ± 2 C) at a 16 h photoperiod with cool white fluorescent illumination (100 lmol m 2 s 1 PFD). For ex vitro rooting regenerated shoots were treated with IBA (7.38 lM) for 36 h before being transferred to pots (15 cm) containing a mixture of sand soil and peat (1:1:1) as described previously. 2.8. Statistical analysis
2.2. Surface sterilization Explants were rinsed 3–4 times with sterilized double distilled water, and surfaces disinfected for 10 min. in H2O2 (1%) solution. After rinsing, the explants were placed on sterilized blotting paper and finally placed on a callus induction medium.
The data on the effects of growth regulators on morphogenesis were analysed by one-way analyses of variance (ANOVAs). Values are means of five replicates from two experiments, and the presented mean values were separated using Duncan’s Multiple Range Test (DMRT) at p 6 0.05.
2.3. Callus induction
3. Results
After proper surface sterilization, the explants were placed on MS medium containing various auxin types and concentrations of 0.0–9.04 lM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.0–10.20 lM 2,4-5-triacetic acids (2,4-5-T), 0.0–52.0 lM chlorophenoxyacetic acid (CPA).
3.1. Callus induction
2.4. Regeneration and shoot multiplication Nodal callus masses (80–90 mg) were used for regeneration; nodal stem showed rapid callus initiation and vigorous callus growth compared to callus induced from other sources (data unpublished). Callus was cultured on MS medium fortified with various concentrations of cytokinins with a range of 0.0–8.96 lM N6-benzylaminopurine (BA) and 0.0–9.28 lM kinetin (Kn). Data were scored in terms of shoot development percentage (%), shoot number/nodal calli, shoot length and leaf number per shoot.
Of the various auxin types and explants used nodal stem (Fig. 1a) proved to be highly effective compared to others (data not shown) on MS medium supplemented with 2,4-D (6.78 lM) followed by CPA (46.5 lM). Explants varied in callus induction rate and percentage, however calli induced from all the sources were similar in morphological appearances; they were compact in nature and creamy in colour, although, it became black and necrotic at higher 2,4-D concentrations after the 9th week of culture. Table 1 shows a comparative account of nodal stem callus biomasses (fresh and dry wt.) under optimized concentrations of various auxins; 2,4-D (6.78 lM) was a highly promising concentration for high callus biomass production. 3.2. Callus regeneration /shoot multiplication
2.5. Rooting in solid and liquid MS medium Shoots that developed in regenerating medium were transferred on solid and liquid MS medium, added with various
Undifferentiated masses of callus (80–90 mg) were cultured on MS medium supplemented with BA (0.0–8.96 lM) and Kn (0.0–9.28 lM). Maximum response (in terms of shoot development
In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant
65
Fig. 1 In vitro micropropagation of Dracaena sanderiana. (a) Callus induction from nodal segment on MS medium supplemented with 2,4-D (6.78 (lM); (b and c) shoot multiplication in cytokinin added MS medium. (B) Kn (9.28 lM), (C) BA (7.84 (lM); (d) 8 week old culture before transfer to field conditions MS + BA (7.84 lM) + IBA (7.38 lM); (e and f) root induction from isolated single shoot in solid (e) and liquid (f) MS medium added with IBA (7.38 lM); (g) regenerated plantlets transferred to micropots in soil rite (100%).
percentage, shoot number, shoot length and leaf number) was noticed in culture fortified with BA (7.84 lM) (Fig. 1c and d). Although, Kn (9.28 lM) proved to be highly effective but in general was less (Fig. 1b) significant compared to BA. Table 2 shows the effect of BA and Kn on shoot regeneration.
3.3. Rooting A comparative study has been made to evaluate the effect of solid and liquid medium on rooting. Shoots that developed in regenerating medium were used for root induction and
66 Table 1
J. Aslam et al. Callus biomass (fresh and dry weight) growth in optimized auxin concentration. Data were scored up to 9 weeks of culture.
Optimized concentration of auxins (lM)
5 weeks old callus
7 weeks old callus
9 weeks old callus
Fresh wt. (gm)
Fresh wt. (gm)
Fresh wt. (gm)
a
2,4-D (6.78) CPA (46.5) 2,4-5-T (10.20)
Dry wt. (gm) a
0.22 0.15b 0.12c
0.78 0.62b 0.56b
a
Dry wt. (gm) a
1.25 0.88b 0.74c
0.35 0.27b 0.25b
a
2.69 2.00b 0.85c
Dry wt. (gm) 0.63a 0.46b 0.42b
Values are means ± standard deviation of 5 replicates from 2 experiments. Means with common letters within each column are not significantly different at p 6 0.05, according to Duncan’s Multiple Range Test (DMRT).
Table 2 Shoot regeneration in BA/KN supplemented MS medium. Nodal callus (80–90 mg) was cultured for regeneration. Data were scored after 6 weeks of culture. PGR (lM) BAP
Shoot number/callus mass
Shoot length (cm)
Leaf number
0.0g 2.8d 2.9d 5.3b 5.7a 5.0b 0.0g 1.8f 2.3e 2.8d 2.9d 3.3c
0.0h 2.1e 4.4b 7.5a 7.3a 3.9c 0.0h 1.2g 2.5d 4.4b 4.0c 1.5f
0.0i 5.7d 7.3c 7.6b 7.9a 4.9f 0.0i 4.3gh 5.0ef 5.2e 5.4e 4.2h
Kn
0.00 2.24 4.48 6.72 7.84 8.96 0.00 2.32 4.64 6.96 8.12 9.28
Values are means ± standard deviation of 5 replicates from 2 experiments. Means with common letters within each column are not significantly different at p 6 0.05, according to Duncan’s Multiple Range Test (DMRT).
transferred on MS medium, containing root inducing plant growth regulators. Table 3 shows a comparative account of root induction in solid and liquid media. Better response was observed in solid medium (Fig. 1e) when supplemented with IBA (7.38 lM), followed by NAA (10.73 lM); roots were high in number (14.6) with average length (48.6 mm) compared to their liquid counterpart (Fig. 1f). 3.4. Ex vitro transplantation Rooting was successfully induced within 5 weeks on IBA (7.38 lM) added MS medium. Rooted plantlets (Fig. 1e) were transferred in plastic pots (Fig. 1g) containing soil rite, kept at 25 ± 2C in the culture room, and covered with pored transparent polythene bags to reduce the humidity. But the survival frequency was moderate; thereafter the survival rate was enhanced by the application of a hardening procedure, where rooted shoots were transferred to half and quarter-strength liquid MS medium without plant growth regulators and were then transferred to plastic pots, containing sand and soil rite (1:1). Transferred plantlets showed normal morphological appearance. During hardening, shoots elongated and leaves turned green and expanded. Secondly, regenerated shoots were also rooted directly as microcuttings on an autoclaved mixture of sand and soil rite. The shoots’ treatment with 7.38 lM (IBA) for 36 h was found essential for in vitro rooting. The frequency of root formation was increased 85–94% by ex vitro rooting using microcutting procedures.
4. Discussion Propagation of plants through vegetative and sexual processes is well established in horticulture, however, micropropagation offers a rapid means of production of clonal plants in numbers, which can be used for afforestation and conservation of elite and rare germplasm and also for rapid multiplication of economically important plants (Bonga, 1982). In vitro culture may be initiated from the embryo, shoot apex, axillary bud and several other explants. In the present investigation in D. sanderiana various explants (node, internode, axillary bud, leaf and root) were used, of which nodal segments showed maximum callusing and high biomass on 2,4-D added MS medium. A similar promoting effect of 2,4D on callusing was earlier reported in the same species (Ilah et al., 2002). However, in Dracaena fragran young stem segments produced calli on medium supplemented with 2,4-D alone or in combination with BA (Vinterhalter, 1989). Additionally, in many other plants profuse callusing was observed on 2,4-D added medium (Khan et al., 2002). For cultured tissues, the requirement for exogenous hormones depends on the endogenous level of plant tissue which varies with organs, plant genotype and the phase of plant growth (Chand and Singh, 2004). Nodal explants along with high callus biomass also exhibited high regeneration potentiality compared to other tested explants. Pua et al. (1989) have reported in Brassica napus that the nodal segment was more responsive
In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant
67
Table 3 Root induction from individual shoot in solid and liquid media. MS medium was supplemented with various auxins. Data were scored after 5 weeks of culture. PGR (lM) IBA
After 5 weeks NAA
IAA
0.00 2.46 4.92 7.38 9.84 0.00 2.68 5.37 8.05 10.73 2.85 5.71 8.56 11.41
No. of root
Root length (mm)
Solid
Liquid
Solid
Liquid
0.0i 3.3g 5.3e 12.0a 7.0d 0.0i 1.7h 3.0g 5.0e 9.0b 0.0i 1.4hi 2.7gh 4.3f 8.3c
0.0h 2.0f 4.7c 9.6a 5.0b 0.0h 0.0h 0.0h 2.7e 4.5c 0.0h 0.0h 1.7g 2.0f 3.7d
0.0k 23.3c 24.5b 26.5a 14.6f 0.0k 6.3j 11.6h 16.6e 18.3d 0.0k 5.7j 9.0i 13.4g 17.0e
0.0h 3.0g 11.6b 15.0a 8.3c 0.0h 0.0h 0.0h 5.0e 7.3d 0.0h 0.0h 0.0h 4.2f 6.8d
Values are means ± standard deviation of 5 replicates from 2 experiments. Means with common letters within each column are not significantly different at p 6 0.05, according to Duncan’s Multiple Range Test (DMRT).
for callusing and regeneration compared to other explants. Out of the two cytokinins (BA and Kn), BA was more active during multiple shoot formation. The superiority of BA over Kn for multiple shoot formation was also demonstrated in other plants like Jetropha integerrima (Sujatha and Dhingra, 1993), Sapium sebiferum (Siril and Dhar, 1997), and Bombax ceiba (Chand and Singh, 1999, 2004). In contrast, kinetin was found to be better than 2iP and BA for promoting axillary shoot proliferation in Cephaelis ipecacuanha (Jha and Jha, 1989). However, shoot multiplication decreased with increasing BA concentrations. Similar effect was also noticed in many other plant systems (Ben Jouira et al., 1998; Biroscikova et al., 2004; Junaid et al., 2007a). In addition, among the used root inducing plant growth regulators, IBA alone or in combination with BA was found to be most effective for root induction particularly in the solid medium. Similar results were noticed in a number of other plant species (Tapati et al., 1995; Vandna et al., 1995; Khan et al., 2002; Biroscikova et al., 2004; Junaid et al., 2007a). In the present investigation, no major differences were observed in in vitro and ex vitro rooting, however, a moderate difference was seen in the survival rate’ that was due to the ex vitro developed healthy roots. Moreover, in vivo rooting offers several advantages over in vitro rooting as the former is cost effective and the roots structurally and functionally contain more root hairs than the latter (Debergh and Read, 1991; Preece and Sutter, 1991). Plantlets with healthy roots were transferred for acclimatization purposes, showing more than 90% survivability, they however, showed normal morphological appearance. Similar practice of transplantation was achieved in several other plant systems (Zhang and Davis, 1986; Debergh and Read, 1991; Preece and Sutter, 1991; Shibli and Smith, 1996; Kim et al., 1997; Dhar and Joshi, 2005; Junaid et al., 2007b).
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