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South African Journal of Botany 86 (2013) 46–50

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Rapid in vitro micropropagation of praecox

Ponnusamy Baskaran, Johannes Van Staden ⁎

Research Centre for Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Scottsville 3209,

article info abstract

Article history: In vitro micropropagation and acclimatization for the ornamental Agapanthus praecox, are reported. The influ- Received 6 March 2012 ence of different growth regulators on multiplication from shoot-tip explants of A. praecox was inves- Received in revised form 19 November 2012 tigated. Prolific shoot multiplication (47.3±1.96 per explant) was achieved on Murashige and Skoog Accepted 28 January 2013 (MS) medium supplemented with 22.2 μM benzyladenine (BA), 2.9 μM indole-3-acetic acid (IAA), and Available online 6 March 2013 4.5 μM thidiazuron (TDZ). Shoots were rooted on half-strength MS basal medium supplemented with μ μ Edited by NJ Taylor 5.7 M IAA and 2.5 M 2-isopentenyladenine (2iP) with 11.3±0.78 roots per shoot. The in vitro-raised were established successfully in a 1:1 (v/v) vermiculite:sand mixture when maintained in a greenhouse with Keywords: 100% survival. The elongated shoots (more than 5 cm in length) were treated for rooting and acclimatization Agapanthus praecox in a moistened (5.7 μM IAA and 2.5 μM 2iP) vermiculite:sand (1:1 v/v) mixture, first in the misthouse and In vitro regeneration then in the greenhouse. Rooting and acclimatization was achieved simultaneously (100%) in the misthouse Micropropagation which was followed by greenhouse cultivation. This system can be used for rapid mass clonal propagation Ornamental plant of A. praecox, for conservation strategies, commercial production, gene transformation studies and to produce Plant growth regulators phytomedicines. © 2013 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction buds of A. africanus (Supaibulwatana and Mii, 1997), and somatic em- bryogenesis from callus cultures of A. praecox spp. orientalis (Suzuki et The genus Agapanthus includes several ornamental species such as al., 2001a; Wang et al., 2012), Agrobacterium-mediated transformation Agapanthus africanus, Agapanthus inapertus, and Agapanthus praecox. of A. praecox ssp. orientalis (Suzuki et al., 2001b) and regeneration These plants, endemic to southern Africa, have tuberous, fleshy root- from callus-derived protoplasts of A. praecox ssp. orientalis (Nakano et stocks. A. praecox is an evergreen with medicinal and ornamental uses al., 2003) have also been achieved. In the present study, we report and is commonly called Agapanthus, African lily or lily of the Nile. This pe- plant regeneration through an in vitro micropropagation system from rennial plant has recently become popular as a potted plant, for landscap- shoot-tip cultures of A. praecox Wild. ing, and as a cut flower because of its beautiful, blue–violet or white flowers (Mor et al., 1984). The breeding of A. praecox is by seed or division 2. Materials and methods of rhizomes. The plants hybridize freely with each other. Seed germinates within six to eight weeks in late summer–autumn. Commercially avail- 2.1. Plant material and in vitro germination able Agapanthus plants consist mostly of heterozygous seedlings due to the lack of an efficient vegetative propagation method of the cultivars Mature seeds of A. praecox were collected from the Botanical (Supaibulwatana and Mii, 1997). Hence, an alternative mass propagation Garden, University of KwaZulu-Natal, Pietermaritzburg, South Africa. tool for this economic species would be very useful. The seeds were washed under running tap water for 10 min to re- Micropropagation techniques offer a viable alternative and innova- move loose dirt, then disinfected with a drop of Tween 20 in 100 ml tive method of asexual propagation. Micropropagation is widely used of distilled water for 2 min and then decontaminated with the more for rare, endangered and medicinal plants for conservation and com- effective sterilizing agent of aqueous mercuric chloride [0.1% (w/v)] mercial propagation (Pattnaik and Chand, 1996). In order to apply in for 10 min, as sodium hypochlorite was ineffective. The seeds were vitro micropropagation for clonal propagation, it is a prerequisite to de- then washed five times with sterile distilled water whereafter they velop a shoot-tip meristem-to-plant system. In the genus Agapanthus, were soaked in 100 ml liquid MS (Murashige and Skoog, 1962) medi-

however, no studies on in vitro micropropagation have been reported. um, supplemented with 5 μM gibberellic acid (GA3) for 1 h. Control There are however, several reports describing plant regeneration seeds were soaked in 100 ml distilled water. Seeds were then inocu- through callus cultures and somatic embryogenesis from young flower lated on MS basal medium containing 20 g l−1 sucrose and 8 g l−1 agar, and maintained in the culture room at 25±2 °C in the dark ⁎ Corresponding author. Fax: +27 332605897. for 48 h. Thereafter, seeds were placed in a culture room (25±2 °C) E-mail address: [email protected] (J. Van Staden). with cool-white fluorescent lights (OSRAM L 58 W/740, Germany)

0254-6299/$ – see front matter © 2013 SAAB. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.sajb.2013.01.008 P. Baskaran, J. Van Staden / South African Journal of Botany 86 (2013) 46–50 47 at a photosynthetic photon flux density (PPFD) of 40 μmol m−2 s−1 IAA+2.5 μM 2iP respectively) vermiculite:sand mixture (1:1 v/v) under a 16 h light period. Shoot-tip explants (±15 mm in length) for ex vitro rooting and acclimatization. Shoots irrigated with water were generated from 30-day-old in vitro seedlings. devoid of PGR were used as controls. The potted shoots were maintained in an environmentally controlled misthouse (20±5 °C) 2.2. Propagation of shoots and rooting of in vitro-propagated shoots for 7 days or transferred to the greenhouse (25±2 °C under natural light conditions and a midday PPFD of 950±50 μmol m−2 s−1). In Aseptic shoot-tip explants (±15 mm in length) were cultured on the misthouse, a high pressure misting system was used to maintain MS medium containing 30 g l−1 sucrose, 8 g l−1 agar and different relative humidity between 90 and 100%. The average midday PPFD – μ −2 −1 plant growth regulators (PGRs) alone or in combination for in the misthouse was 90 100 mol m s under natural light con- high-frequency rapid shoot multiplication. The precise concentration ditions. After 7 days, the potted shoots were successfully transferred and combinations of PGRs are indicated in Tables 1 and 2. In all exper- and established in the greenhouse. The survival percentage was iments, the chemicals used were of analytical grade (Biolab, South recorded after 10 weeks. Africa; Oxoid, England and Sigma, USA). The pH of the medium was adjusted to 5.8 with 0.1 N NaOH and/or 0.1 N HCl before gelling 2.5. Statistical analysis with 8 g l−1 agar and autoclaving at 121 °C for 20 min. The cultures were maintained at 25±2 °C under a 16 h light period and PPFD of Data were collected after 12 weeks of culture for shoot multiplication 40 μmol m−2 s−1 provided by cool white fluorescent light. and 8 weeks for rooting experiments. All experiments were repeated Proliferating shoots (above 3.0 cm in length) were isolated from three times with 50 explants per treatment. The data were subjected shoot-tip explants on shoot multiplication medium, and transferred to one-way analysis of variance (ANOVA) using SPSS version 18.0 for − − fi to half-strength MS basal salts plus 10 g l 1 sucrose and 8 g l 1 Windows (Chicago, USA). Signi cantly different means were separated agar and different plant growth regulators (PGRs) alone or in combina- using Duncan's multiple range test (p=0.05). tion for high-frequency rapid root induction. The precise concentration and combinations of PGRs are indicated in Table 3. Media devoid of 3. Results and discussion growth regulators were used as controls. The pH of the media were ad- fl justed to 5.8 with 0.1 N NaOH and/or 0.1 N HCl prior to autoclaving at 3.1. In uence of PGRs on shoot regeneration 121 °C for 20 min. All cultures were maintained at 25±2 °C under − − cool-white fluorescent light at a PPFD of 40 μmol m 2 s 1 with a Seeds germinated (approximately 90%) better after a GA3 presoaking 16 h light period. treatment than the control. GA3 and presoaking treatments have been recommended to break dormancy and enhance germination of other monocotyledonous species (Hilhorst and Karssen, 1992; Akman, 2009). 2.3. Acclimatization of in vitro plantlets Shoot-tip explants excised from 30-day-old seedlings of A. praecox (Fig. 1A) were cultured on MS medium with different cytokinins All rooted shoots were removed gently from the rooting medium [adenine sulfate (Ads), kinetin (Kin), 2-isopentenyladenine (2iP), meta- after 8 weeks and transferred to terracotta pots (95×120 mm, topolin (mT), meta-topolin riboside (mTR), thidiazuron (TDZ) and 500 ml) containing a 1:1 (v/v) vermiculite:sand mixture and irrigated benzyladenine (BA)] at low concentrations (2.7–5.4 μM) to evaluate with water every second day. These plantlets were maintained in the the potency of cytokinins in shoot multiplication. Benzyladenine greenhouse (25±2 °C under natural light conditions and a midday supported better shoot regeneration (10.5 shoots per explant) than the PPFD of 950±50 μmol m−2 s−1) for acclimatization ex vitro. other cytokinins (Table 1 and Fig. 1B). It was also more effective on shoot multiplication with several other plant species such as A. africanus, 2.4. Ex vitro rooting and acclimatization Ocimum americanum, Ocimum sanctum and Eclipta alba (Kim and Kim, 1999; Pattnaik and Chand, 1996; Baskaran and Jayabalan, 2005). In this The regenerated shoots more than 5.0 cm in length were dipped study, shoot regeneration was significantly increased with TDZ and Kin in distilled water supplemented with 5.7 μM IAA, 10.7 μM NAA, (Table 1 and Fig. 1C). MS medium containing 2iP was most effective for 9.8 μM IBA and 5.7 μM IAA+2.5 μM 2iP individually for 30 min. The shoot elongation with a mean length of 8.2 cm after 12 weeks (Table 1 shoots were then planted individually in moistened (50 ml of water and Fig. 1D). For the remainder of this study, BA was selected as the containing 5.7 μM IAA; 10.7 μM NAA; 9.8 μM IBA and 5.7 μM cytokinin of choice. Higher BA concentrations (>13.3 μM) significantly increased shoot Table 1 regeneration, but reduced shoot length (Table 1). Christopher and Effect of plant growth regulators on adventitious shoot and root regeneration of Rajam (1994) reported the highest shoot number with high levels of Agapanthus praecox. BA. Since the young shoot apex is an active site for auxin biosynthesis, Plant growth Shoots [explant−1] Shoot Roots [explant−1] Root length shoot-tip explants require high levels of cytokinins for shoot prolifera- regulator [μM] [#] length [cm] [#] [cm] tion (Christopher and Rajam, 1994). A high number of shoots (20 shoots μ Control 0 0 0 0 per explant) was obtained on 22.2 MBAafter12weeks(Table 1). In- MS+5.4 Ads 4.2±0.32gh 4.0±0.22cd 2.9±0.27b 5.8±0.26b creasing the concentration of BA (31.1 μMand44.4μM) in the medium MS+4.6 Kin 5.8±0.36fg 4.5±0.28cd 2.3±0.37bc 2.4±0.23d significantly decreased shoot number (Table 1). MS+4.9 2iP 1.8±0.24i 8.2±0.47a 5.5±0.34a 11.6±0.70a Root growth in terms of number and length were recorded from MS+4.1 mT 3.7±0.39h 3.4±0.28d 3.6±0.32b 4.1±0.19c shoots in different PGR-containing media after 12 weeks in culture. MS+2.7 mTR 3.4±0.30h 3.8±0.25d 3.0±0.26b 3.6±0.26c MS+4.5 TDZ 7.1±0.61ef 5.9±0.32b 0.0±0.00 0.0±0.00 When using BA and TDZ no roots were produced. Using 2iP, mT and MS+4.4 BA 10.5±0.67d 6.3±0.67b 0.0±0.00 0.0±0.00 mTR induced rooting (Table 1 and Fig. 1E); with no significant differ- MS+13.3 BA 14.6±0.76b 4.9±0.35c 0.0±0.00 0.0±0.00 ence observed between mT and mTR. The highest root number (5.5) MS+22.2 BA 20.0±0.98a 4.5±0.41cd 0.0±0.00 0.0±0.00 and length (11.6 cm) were recorded on 2iP-containing medium MS+31.1 BA 12.5±1.01c 4.1±0.36cd 0.0±0.00 0.0±0.00 MS+44.4 BA 8.4±0.65e 3.8±0.44d 0.0±0.00 0.0±0.00 (Table 1). Rooting stimulatory effects of cytokinins were also reported for other plants (Chin, 1982; Baskaran and Jayabalan, 2005). In this The data were recorded after 12 weeks of culture. Values are mean±standard error study, TDZ-induced white-greenish compact callus at the base of shoots (SE). Results are means±SE for three experiments (n=3). Means followed by same letters in each column are not significantly different (p=0.05) using Duncan's after 8 weeks of culture and these calli later initiated shoot buds multiple range test. (Fig. 1F). Similar effects were also observed for other species (Preece 48 P. Baskaran, J. Van Staden / South African Journal of Botany 86 (2013) 46–50

Table 2 Effect of plant growth regulator combinations on adventitious shoot and root regeneration of Agapanthus praecox.

Plant growth regulator [μM] Shoots [explant−1] [#] Shoot length [cm] Roots [explant−1] [#] Root length [cm]

MS+22.2 BA+5.7 IAA 22.9±0.94d 6.0±0.49ab 1.9±0.23a 2.6±0.21a MS+22.2 BA+4.9 IBA 18.1±0.85de 5.1±0.38ab 1.5±0.21a 2.0±0.27a MS+22.2 BA+5.4 NAA 20.0±0.67d 4.5±0.43bc 1.8±0.37a 2.3±0.25a MS+22.2 BA+4.5 TDZ 26.4±0.72c 4.3±0.39cd 0.0±0.00 0.0±0.00 MS+22.2 BA+4.6 Kin 21.1±0.87d 6.3±0.42a 0.6±0.42b 0.5±0.43b MS+22.2 BA+4.1 mT 20.6±0.92d 4.9±0.33bc 0.0±0.00 0.0±0.00 MS+22.2 BA+2.7 mTR 19.2±0.74de 5.0±0.42ab 0.0±0.00 0.0±0.00 MS+22.2 BA+2.9 IAA+4.6 Kin 31.4±1.17b 5.7±0.45ab 0.0±0.00 0.0±0.00 MS+22.2 BA+2.9 IAA+4.5 TDZ 47.3±1.96a 4.0±0.24d 0.0±0.00 0.0±0.00 MS+22.2 BA+2.9 IAA+4.1 mT 29.8±0.78b 5.0±0.43ab 0.0±0.00 0.0±0.00

The data were recorded after 12 weeks of culture. Values are mean±standard error (SE). Results are means±SE for three experiments (n=3). Means followed by same letters in each column are not significantly different (p=0.05) using Duncan's multiple range test. et al., 1991; Magyar-Tabori et al., 2010). Yaacob et al. (2012) reported the medium enhanced adventitious shoot proliferation and produced that TDZ was least effective for somatic embryogenesis of A. praecox healthy shoots. In contrast, synergistic effects of BA and cinnamic acid ssp. minimus. Somaclonal, genetic or epigenetic variation may be combinations were observed in adventitious shoot regeneration in A. reflected in morphological, biochemical or molecular characteristics of africanus (Kim and Kim, 1999). the regenerated plants obtained from calli treated with PGRs. This var- iation appears in in vitro culture systems with low risk of genetic instability (Schoofs, 1992), as organized meristems are generally more 3.3. Effect of PGRs on rooting of shoots and acclimatization resistant to genetic changes (Shenoy and Vasil, 1992). There are numer- ous conflicting reports on the incidence of somaclonal variation in Excised shoots were rooted on half-strength MS medium with micropropagated plants via shoot-tip culture (Schoofs, 1992; Swarna different types of auxin. The promotory effect of reducing the salt and Ravindhran, 2012). The transgenic plants of A. praecox ssp. orientalis concentration of MS on in vitro rooting of shoots has been described derived from embryogenic calli showed morphological variations in several reports (Skirvin et al., 1980; Baskaran and Jayabalan, in vegetative and floral organs (Mori et al., 2007). Some plants 2005). Of the three auxins tested at different concentrations, 5.7 μM regenerated from callus cultures of various Agapanthus genotypes with- IAA had a better effect on root induction than IBA and NAA (Table 3 out transformation showed similar somaclonal variations as those ob- and Fig. 1I). In contrast, optimal root induction was achieved on served in the transgenic Agapanthus plants (Mori et al., 2007). The MS medium supplemented with 0.1 mg l−1 NAA in A. africanus acclimatized plants in the current study did not exhibit morphogenetic (Supaibulwatana and Mii, 1997). Higher concentrations of IAA variations; however, molecular studies need to be used for confirmation (>11.4 μM), NAA (>10.7 μM) and IBA (>9.8 μM) reduced the of genetic fidelity. number and lengths of roots but induced mild callus on roots. In the present study, IAA (5.7 μM) and NAA or IBA or Ads or 2iP combi- 3.2. Influence of PGR combinations on shoot regeneration nations were more effective in induction of number of root and their length (Table 3). The highest number of roots (11.3 roots per shoot) Various combinations and concentrations of PGR were examined and root length (14.8 cm) were obtained with a IAA (5.7 μM) and 2iP for high-frequency rapid shoot multiplication from shoot-tip ex- (2.5 μM) combination after 8 weeks of culture (Table 3 and Fig. 1J). plants. The beneficial effects of combinations of cytokinins and auxins Mild callus was formed on roots with these combinations. on shoot regeneration have been reported in other plants (Tsay et al., The in vitro-rooted shoots were removed from all cultures after 1989; Sultana and Bari Miah, 2003). Combinations of BA and IAA, BA 8 weeks. These plantlets were then successfully acclimatized (100%) and IBA, BA and NAA, BA and TDZ, BA and Kin, BA and mT and BA and in a vermiculite–soil mixture (1:1 v/v) in the greenhouse (25±2 °C) mTR significantly improved the number of shoots (Table 2). Kinetin under natural light conditions with a midday PPFD of 950± and NAA combinations are effective for direct shoot regeneration from 50 μmol m−2 s−1 (Fig. 1K). young flower buds of A. africanus (Supaibulwatana and Mii, 1997). Combinations of BA and IAA or IBA or NAA or Kin induced roots oc- Table 3 curred simultaneously in shoot formation. Similarly, Kin and NAA com- Effect of plant growth regulators on root induction by in vitro shoots of Agapanthus bination showed the highest percentage of root induction, which was praecox. independent of shoot induction, in A. africanus (Supaibulwatana and Plant growth regulator Roots Root length μ μ fi − Mii, 1997). A combination of 22.2 M BA and 4.5 M TDZ signi cantly [μM] [shoot 1] [#] [cm] improved shoot proliferation (26.4 shoots per explant) with an average μ Half-MS+5.7 IAA 6.9±0.38b 6.4±0.46d length of 4.3 cm after 12 weeks (Table 2). Combinations of 22.2 MBA, Half-MS+11.4 IAA 4.2±0.36de 4.3±0.50ef 2.9 μM IAA and Kin (4.6 μM), TDZ (4.5 μM) or mT(4.1μM) significantly Half-MS+17.1 IAA 3.2±0.33ef 3.7±0.42f increased number of shoots but the shoot lengths differed (Table 2 and Half-MS+5.4 NAA 2.9±0.23f 4.5±0.32ef Fig. 1G). The highest number of shoots (47.3 shoots per explant) was Half-MS+10.7 NAA 5.5±0.34c 5.4±0.30de Half-MS+16.1 NAA 3.5±0.27ef 6.2±0.39d obtained with a combination of BA, IAA and TDZ after 12 weeks of cul- Half-MS+4.9 IBA 3.0±0.25ef 4.3±0.19ef ture (Table 2 and Fig. 1H). BA, IAA and TDZ combinations produced Half-MS+9.8 IBA 5.2±0.31cd 3.8±0.16f compact white-greenish callus at the base of the shoots. This callus Half-MS+14.8 IBA 3.0±0.26ef 3.2±0.26f later turned deep green and shoot buds were initiated after 12 weeks Half-MS+5.7 IAA+2.7 NAA 7.6±0.50b 8.7±0.63c of culture. Similar results were also reported in Psoralea corylifolia Half-MS+5.7 IAA+2.5 IBA 7.2±0.34b 9.3±0.66c Half-MS+5.7 IAA+2.7 Ads 8.1±0.46b 12.5±0.46b (Baskaran and Jayabalan, 2009). High concentrations of BA and NAA Half-MS+5.7 IAA+2.5 2iP 11.3±0.78a 14.8±0.80a combinations have been reported to yield positive results in the forma- The data were recorded after 8 weeks of culture. Values are mean±standard error tion of embryogenic callus from A. praecox ssp. minimus leaf and root ex- (SE). Results are means±SE for three experiments (n=3). Means followed by same plants (Yaacob et al., 2012). The present investigation showed that letters in each column are not significantly different (p=0.05) using Duncan's long-term exposure to combinations of PGRs (cytokinins and IAA) in multiple range test. P. Baskaran, J. Van Staden / South African Journal of Botany 86 (2013) 46–50 49

Fig. 1. Effect of plant growth regulators on shoot regeneration and plant formation from shoot-tip explants of A. praecox. (A) in vitro seedlings on MS basal medium containing 20 g l−1 sucrose and 8 g l−1 agar (Bar, 5 mm), (B) production of adventitious shoots on MS medium containing 4.4 μMBA(Bar, 5 mm), (C) multiple shoots and roots developing from shoot-tip explants on MS medium with 4.6 μM Kin after 12 weeks of culture (Bar, 2 mm), (D) shoot multiplication with roots on MS medium with 4.9 μM 2iP (Bar, 4 mm), (E) shoot multiplication with roots on MS medium containing 4.1 μM mT(Bar, 7 mm), (F) shoot multiplication with white-greenish compact callus on MS medium with 4.5 μM TDZ (Bar, 6 mm), (G) production of adventitious shoots on MS medium with 22.2 μM BA, 2.9 μM IAA and 4.6 μM Kin (Bar, 8 mm), (H) production of adventitious shoots on MS medium with 22.2 μM BA, 2.9 μM IAA and 4.5 μM TDZ (Bar, 9 mm), (I) rooting of shoots on MS medium with 5.7 μM IAA (Bar, 8 mm), (J) prolific rooting from shoots on MS medium with 5.7 μM IAA and 2.5 μM 2iP (Bar, 6 mm), (K) acclimatized plants in the greenhouse (Bar, 10 mm).

3.4. Ex vitro rooting and acclimatization ornamental plant makes the system very attractive. The protocol de- veloped is cheap and can be applied for large-scale micropropagation In vitro shoots (above 5.0 cm in length) were rooted and hardened for germplasm conservation and genetic transformation of A. praecox. simultaneously in a vermiculite–soil mixture (1:1 v/v). This reduced the cost and the time of micropropagation of A. praecox. Approxi- Acknowledgements mately 3–5 roots per shoot with an average length of 5–7 cm were obtained with 5.7 μM IAA, 10.7 μM NAA and 9.8 μM IBA respectively, Financial support by the National Research Foundation (NRF), Pre- after 10 weeks; whereas, about 7–9 roots per shoot of 8–10 cm in toria and the University of KwaZulu-Natal is acknowledged. length were obtained with a 5.7 μM IAA and 2.5 μM 2iP combination. The ex vitro rooting and acclimatization were achieved concurrently with 100% success in the misthouse followed by greenhouse condi- References tions. On the other hand, when in vitro shoots were transferred directly to the greenhouse 70% ex vitro rooting (7–9 roots per shoot Akman, Z., 2009. Effects of GA3 and kinetin pre-sowing treatments on seedling emergence – and seedling growth in wheat under saline conditions. Journal of Animal and Veterinary of 8 10 cm length) and hardening was achieved after 10 weeks. Advances 8, 362–367. The present investigation showed that successful ex vitro rooting Baskaran, P., Jayabalan, N., 2005. An efficient micropropagation system for Eclipta alba — a and hardening required short-term exposure to misthouse conditions valuable medicinal herb. In Vitro Cellular and Developmental Biology — Plant 41, – before transfer to the greenhouse. 532 539. Baskaran, P., Jayabalan, N., 2009. An improved protocol for adventitious shoot regeneration and plant formation in Psoralea corylifolia. Scientia Horticulturae 123, 283–286. 4. Conclusions Chin, C.K., 1982. Promotion of shoot formation in Asparagus in vitro by ancimidol. Hortscience 17, 590–591. fi Christopher, T., Rajam, M.V., 1994. In vitro clonal propagation of Capsicum spp. Plant We report an ef cient in vitro technique for the rapid multiplica- Cell, Tissue and Organ Culture 38, 25–29. tion of A. praecox. Shoot-tips cultured on MS medium containing BA Hilhorst, H.W.M., Karssen, C.M., 1992. Seed dormancy and germination, the role of (22.2 μM), IAA (2.9 μM) and TDZ (4.5 μM) combinations induced abscisic acid and gibberellins and the importance of hormone mutants. Plant Growth Regulation 11, 225–238. high numbers of shoots accompanied by ex vitro rooting. The possibil- Kim, S.Y., Kim, M.S., 1999. Clonal propagation of Agapanthus by floral tissue culture. The ity of mass propagation of A. praecox as a commercial medicinal and Journal of The Korean Society of International Agriculture 11, 294–297. 50 P. Baskaran, J. Van Staden / South African Journal of Botany 86 (2013) 46–50

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