Short communication
Micropropagation of
Crossandra infundibuliformis
(L.) Nees
S. Girija, A. Ganapathi
*, G. Vengadesan
Department of Biotechnology, School of Life Sciences, Bharathidasan University, Thiruchirappalli 620024, Tamil Nadu, India
Accepted 16 March 1999
Abstract
Multiple shoots were induced from axillary and apical buds ofCrossandra infundibuliformis(L.) on MS medium containing BAP and kinetin individually. The maximum number of multiple shoots were obtained from MS medium containing BAP 1 mgÿ1. The subcultures were done on fresh
medium with the same composition and the number of multiple shoots increased during subculture of both types of explant. At the end of the second subculture, development of 45± 49 shoots per explant was noticed from the axillary bud culture, whereas apical buds produced 40±43 shoots per explant. Maximum elongation was obtained on the same medium but during subsequent subcultures. Elongated shoots were harvested and transferred to MS medium containing IBA 1.0 mgÿ1
for root induction. Rooted plantlets were transplanted to soil.#1999 Elsevier Science B.V. All rights reserved.
Keywords: Micropropagation; Apical bud; Axillary bud;Crossandra
1. Introduction
Commercial floriculture has become a potential money spinner for the developing countries. The total area under floriculture in the world is about
Abbreviations: BAP, Benzylamino purine; GA3, Gibberellic acid; 2,4-D, 2,4-dichloroacetic acid;
IAA, Indole-3-acetic acid; NAA,a-naphthaleneacetic acid; IBA, Indole-3-butyric acid
* Corresponding author. Tel.: +91-431-660-386; fax: +91-431-660-245
E-mail address:[email protected] (G. Vengadesan)
20 000 ha (Rao et al., 1996). Crossandra infundibuliformis is an important horticultural plant and belongs to the family Acanthaceae. In India, the plant is considered an aphrodisiac; it is ornamental and bears red flowers which have a high value in the Indian flower market. Recently, a promising triploid cultivar of Crossandra has been released and there is a great demand from farmers for planting material. Unlike the earlier (wild) cultivars,these cultivar can flower throughout the year, irrespective of the season. As they do not produce seeds, the only way of propagation is through cuttings. Propagation by conventional methods necessarily limits the rate of output and makes the end product expensive. Micropropagation is being used extensively for the rapid clonal propagation of many fruits, nuts and ornamental trees (Zimmerman, 1985). It also enables rapid clonal propagation and hastens the availability of planting material (Zimmerman, 1981). A preliminary study onCrossandrawas made by Bonke et al. (1983), but with limited success. However, none of the above studies optimized conditions for effective micropropagation of Crossandra. For commercial micropropagation an efficient multiplication system is essential (Watad et al., 1995). In order to meet the demand an attempt has been made to evolve mass multiplication through in vitro culture. The method described here would have a great advantage for the rapid multiplication ofCrossandra.
2. Materials and methods
Rooted cuttings were obtained from the Horticulture college and Research Institute, Tamil Nadu Agriculture University, Coimbatore, Tamil Nadu, India. Prior to explant collection, the plants were sprayed with fungicide (Bavistin 2%) to prevent fungal contamination. The apical and axillary buds (0.5±1.0 cm) were collected from 8-year old plants, then they were washed several times with distilled water and suspended for 15±20 min in sodium hypochlorite (0.5%) solution and subsequently washed five to six times in sterile water to remove the sterilant. The explants were cultured on MS (Murashige and Skoog, 1962) medium containing 3% sucrose, 0.7% agar (Hi- media, Mumbai, India) with different concentrations of BAP (0.5±2.5 mgÿ1) and kinetin (0.5±2.5 mgÿ1) in test tubes (25 mm150 mm). For subculturing, shoot clusters were transferred to 150 ml Erlenmeyer flasks containing 25 ml of same fresh medium at intervals of 15 days. Shoots which attained 4±5 cm in height were transferred to MS medium supplemented with different auxins (NAA, IAA and IBA) at various concentrations (0.5±2.5 mgÿ1
) to test their rooting efficiency.
were transferred to the field. The pH of the media was adjusted to 5.8 prior to autoclaving at 1.06 kg cmÿ2 (1218C) for 15 min. Cultures were maintained at 2528C under a 16 h photoperiod (2000 lux) provided by `cool white' fluorescent lamps. There were 24 explants per treatment and the experiment was repeated thrice. Data were analysed using analysis of variance and Duncan's multiple range test to separate the means.
3. Results and discussion
3.1. Multiple shoot induction from apical and axillary buds
Multiple shoots were induced from apical and axillary buds. The percentage of response varied with the type of growth regulator used, its concentration and the nature of the explant. BAP and kinetin in MS medium showed variable responses. BAP was more effective than kinetin in inducing multiple shoots. A similar result was reported in banana using MS basal medium supplemented with 2.25 mgÿ1 BAP (Smith and Drew, 1990). The maximum number of shoots per explant (4±5 shoots) were induced from axillary buds (Fig. 1a) and (2±3 shoots) from apical buds (Fig. 1b) on MS medium when supplemented with 1.0 mgÿ1
BAP, whereas kinetin (1 mgÿ1) induced 2±3 shoots per explant. In contrast to the cytokinins, auxins viz IAA, NAA, IBA and 2,4-D, were ineffective in shoot induction and this was also corroborated inAcacia nilotica (Singh et al., 1993). After 2 weeks the shoot clusters were subcultured on fresh medium with the same composition for proliferation.
3.2. Shoot proliferation and elongation
Fig. 1. (a) Multiple shoot formation from axillary bud explants, (b) Multiple shoot induction from apical bud explants, (c) and (d) Shoot proliferation from axillary and apical bud explants, (e) In vitro rooting of microshoot, (f) Hardened plant.
Effect of BAP and kinetin on multiple shoot induction from axillary and apical bud explants ofCrossandra
Hormone Conc. mgÿ1
Initial culture I Subculture II Subculture
% of culture response No of shoot/explant No of shoot/explant No of shoot/explant Mean shoot length
ax ap ax ap ax ap ax ap ax ap
BAP
0 ± ± ± ± ± ± ± ± ± ±
0.5 89.6abc 90.0b 2.4c 2.3c 10.4c 10.0b 26.3d 22.7d 3.0d 2.5e
1.0 100a 100a 4.5a 3.2a 23.5a 22.4a 48.7a 42.5a 4.5a 4.9a
1.5 93.6ab 86.0bc 3.7b 3.0ab 19.5ab 20.1a 48.7ab 38.3b 4.3ab 4.1b
2.0 73.0d 68.0d 2.4c 2.1c 19.7ab 19.7a 42.0c 26.8c 3.9c 3.4c
2.5 47.3e 29.3e 1.1d 0.5d 14.3b 17.8a 22.9e 22.4e 3.1d 3.0d
Kinetin
0 ± ± ± ± ± ± ± ± ± ±
0.5 82.3ab 76.3ab 1.8c 1.7a 9.0e 8.9e 19.3e 14.4e 3.4c 3.0c
1.0 87.1a 82.6a 3.0a 2.6a 20.7a 20.0a 42.6a 38.0a 4.4a 4.0a
1.5 75.6abc 73.3abc 2.8ab 2.3a 19.5b 18.7b 42.2ab 35.2b 4.0b 3.4b
2.0 62.6c 52.3d 2.0c 1.7a 17.0c 17.0c 37.1c 34.4c 3.5bc 3.0c
2.5 47.3d 30.0e 1.8c 1.3b 14.2d 17.9d 24.4d 24.4d 3.0d 3.0c
axaxillary bud, apapical bud.
Mean within a colum followed by the same letter are not significantly different at 5% level according to Duncan's multiple range test. Data were collected at the end of each subculture.
S.
Girija
et
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Scientia
Horticultur
ae
82
(1999)
331±337
economical. However, from an economic point of view, it is advisable to transplant clusters of shoots to a medium that allows high rates of proliferation and good elongation as observed in the present investigation. The maximum proliferation and optimum elongation (4±5 cm) of shoots was achieved when the shoot clusters were transferred to the same fresh medium. There was a three-fold increase in multiplication rate in both the explants at the end of second subculture. In kinetin supplemented medium, more proliferation of shoots was observed with axillary buds (average of 42 shoots per explant) than with apical buds (average of 38 shoots per explant).
3.3. Rooting of shoots
The shoots which attained 4±5 cm height were harvested from the BAP fortified medium and transferred to MS medium supplemented with different concentrations of (0.5±2.0 mgÿ1) NAA, IAA and IBA for rooting. Root induction efficiency of auxins is well established (D'Silva and D'Souza, 1992). In the present study, the shoots developed callus at the basal cut end when cultured on MS medium fortified with IAA/NAA at various concentrations (0.5±2.5 mgÿ1). The shoots produced roots at all concentrations of IBA (0.5±2.5 mgÿ1
) tested (Table 2), but the highest efficiency of rooting was observed in 1.0 mgÿ1 IBA. From a single shoot, development of 6±8 roots per explant was noticed in IBA fortified medium at the end of the 15th day of culture (Fig. 1e).
3.4. Transplantation
The rooted plants (9±10 cm in length) could be taken out gently from the vessels and washed to remove adhered agar and traces of the medium to avoid
Table 2
Effect of IBA on root induction
Hormone conc. mgÿ1
% of culture response No of root/shoot Mean root length IBA
ax ap ax ap ax ap
0 ± ± ± ± ± ±
0.5 80d 78b 6.8d 6.7d 4.9e 5.1e
1.0 100a 100a 9.6a 9.4a 7.6a 7.1ab
1.5 91ab 94ab 8.9b 8.1b 7.5ab 7.2a
2.0 84c 86ab 7.5c 7.3c 7.1abc 6.9c
2.5 61e 62c 6.7d 6.4e 5.8d 5.8d
apapical bud; axaxillary bud.
Mean within a column followed by the same letter are not significantly different at 5% level according to Duncan's multiple range test.
Data were collected after 15 days of culture.
contamination. A final wash was given in distilled water for 10 min as suggested by Tisserat (1984). They were then transferred to plastic vessels containing sterile sand and vermiculite (1 : 1) (Fig. 1f) and after a week they were transferred to the pot. Hardening of potted plants for 15 days in a growth chamber was found essential. There was 60% survival of plants derived from axillary buds whereas 52% from apical buds when rooted plantlets were transferred from pot to field conditions. Plants transferred to the field have established themselves in the soil and are growing well.
Acknowledgements
I gratefully acknowledge Mr. Sridharan, Edumalai, Thiruchirappalli, who donated the planting material. The senior author is thankful to CSIR for providing financial assistance in the form of a Senior Research Fellowship.
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