In vitro regeneration of Cyrtanthus species: ornamental plants with medicinal benefits
B. Ncube, J. F. Finnie & J. Van Staden
In Vitro Cellular & Developmental Biology - Plant ISSN 1054-5476 In Vitro Cell.Dev.Biol.-Plant DOI 10.1007/s11627-014-9652-y
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Author's personal copy In Vitro Cell.Dev.Biol.—Plant DOI 10.1007/s11627-014-9652-y
MICROPROPAGATION
In vitro regeneration of Cyrtanthus species: ornamental plants with medicinal benefits B. Ncube & J. F. Finnie & J. Van Staden
Received: 4 March 2014 / Accepted: 14 October 2014 / Editor: John Forster # The Society for In Vitro Biology 2014
Abstract Cyrtanthus (Amaryllidaceae) is a genus of perennial geophytes, endemic to the southern African region. Destructive and indiscriminate harvesting of bulbs for medicinal and ornamental purposes has led to intensive decimation of the populations of most of these species in their natural habitats. The aim of this study was to develop in vitro regeneration systems for Cyrtanthus contractus, Cyrtanthus guthrieae, and Cyrtanthus obliquus using twin-scale explants from mature bulbs. Twin scales from the three species were cultured on solid Murashige and Skoog (MS) medium with different concentrations of 6-benzyladenine (BA) and αnaphthaleneacetic acid (NAA) under 16/8-h light/dark conditions at 25±2°C. The best shoot induction responses were obtained on MS medium containing 4.4 μM BA+1.1 μM NAA (3.1 shoots/explant) for C. contractus and C. guthrieae and 6.7 μM BA+2.7 μM NAA for C. obliquus. When the best shoot induction medium for each species was investigated for the effect of cytokinins on shoot organogenesis, using different concentrations of BA, kinetin (Kin), meta-topolin (mT), zeatin (ZT), and thidiazuron (TDZ), the medium that produced the best shoot induction for C. guthrieae also produced the highest number of shoots per explant, whereas the highest numbers of shoots per explant were obtained with 10 μM TDZ for C. contractus and 10 μM BA for C. obliquus. Superior quality shoots in all three species were obtained from MS medium supplemented with Kin and mT. Regenerated shoots were rooted successfully on half- and full-strength MS medium without plant growth regulators, transferred to organic soil mix, and successfully acclimatized in the greenhouse. The micropropagation protocols described provide rapid and B. Ncube : J. F. Finnie : J. Van Staden (*) Research Centre for Plant Growth and Development, School of Life Sciences, University of KwaZulu-Natal Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa e-mail:
[email protected]
cost-effective methods for in vitro propagation for application to the conservation and domestication of Cyrtanthus species. Keywords Cyrtanthus species . Cytokinins . In vitro regeneration . Shoot organogenesis
Introduction The genus Cyrtanthus Aiton belongs to the Amaryllidaceae family and has about 60 species. The genus is endemic to the southern and eastern parts of Africa and is distributed across all provinces in South Africa (Reid and Dyer 1984). It is the most diverse in terms of floral color, morphology, and orientation of any genus in the family, and this has seen numerous species attracting great ornamental value in the floriculture industry (Du Plessis and Duncan 1989). Virtually every member of the genus exhibits some degree of horticultural potential but, due to their rather delicate nature and the difficulty in cultivating many of the species, their greatest potential lies in their use as container plants (Duncan 1990). Based on visual appearances only, Cyrtanthus species have been cited as most likely candidates for having commercial potential as potted plants on an international market (Reinten et al. 2011). The variety of colors and diversity of floral forms make Cyrtanthus an obvious choice for cut flower production. Daly and Henry (2009) reported potted geophytes as winterblooming house plants that are in demand for the USA market and cited Cyrtanthus as among these. The species are grown commercially in South Africa, New Zealand, the UK, and the Netherlands (Clark et al. 2000; Duncan 2010). Cyrtanthus flowers are cultivated as cut flowers in New Zealand, with a number of hybrids developed annually to meet demand. They are usually grown either in the field till flowering or sold as flowering-grade bulbs (Van Epenhuijsen 2005). The red or scarlet type of Cyrtanthus elatus is grown in New Zealand for
Author's personal copy NCUBE ET AL.
both the domestic and export markets. The economic value of the species is not limited to ornamental potential, but many are used and traded in traditional medicine (Du Plessis and Duncan 1989; Mander 1998). The great surge of public interest in the use of plants in the wild assumes that the plant resources will be available on a continuing basis. However, for some species, little effort has been made to ensure this, especially considering the threats posed by increasing demand, a fast increasing human population, and extensive destruction of natural habitats. A significant portion of the plants used for medicinal purposes is harvested from the wild, with little or no regulatory control. This practice renders the majority of plants subject to overharvesting and hence the risk of extinction. The dire consequences of unsustainable harvesting of these biological resources are evident today on a global scale (Hamilton 2004). Habitat loss is one of the greatest threats to Amaryllidaceae plants in South Africa, where 59 species are endangered or vulnerable and 58 species are threatened (Snijman 2004). The vulnerability of species to commercial collection depends on the plant parts used and how they are collected. The collection of underground parts for medicinal purposes, as is the case with Cyrtanthus spp., is liable to inflict more damage, leading to intensive decimation of the populations in natural habitats. Destructive and indiscriminate harvesting of plants from the wild is a major concern, particularly in developing countries, where commercial harvesting has become a source of livelihood for many people living in rural areas (Williams et al. 2000). In South Africa, for example, most of the Cyrtanthus species used for both horticultural and medicinal purposes are harvested from the wild. As a result, their populations have been put under immense pressure in the wild. This has seen Cyrtanthus guthrieae L. Bolus and Cyrtanthus obliquus (L.f.) Ait. being placed under the National Red List of South African plants as endangered (EN) species (Raimondo et al. 2009). The situation is exacerbated for C. guthrieae because its bulbs tend to be solitary and reproduce less often by offset formation and for C. obliquus because the generation time is very long and often requires 5– 10 years from seed to flowering even under favorable natural conditions. A species is placed in the endangered category when the best available evidence indicates that it meets any of the five International Union for Conservation of Nature (IUCN) criteria for an endangered bioresource and is therefore facing a very high risk of extinction in the wild. Although, under the same listing, Cyrtanthus contractus N.E.Br. is regarded as of least concern (LC), the high demand for these slow-growing geophyte species for medicinal, horticultural, and other uses makes this group of plants regarded as threatened plant species that require conservation priority (Raimondo et al. 2009). Given the demand for a continuous and uniform supply of ornamental plants and the consequent accelerating depletion
of wild resources, increasing plant populations through cultivation would be an important strategy for meeting this growing demand (Uniyal et al. 2000). The seeds of Cyrtanthus species are papery and have limited viability (Duncan 2002). Members of the family have long generation times and can take up to 15 seasons to begin flowering (Du Plessis and Duncan 1989). Propagation by conventional methods is therefore a challenge for the survival of the species in the wild. Large-scale production through in vitro propagation offers a means of ensuring continuous availability of such economically important species on the market. From the genus Cyrtanthus, only a few of a total of about 60 species have established micropropagation protocols, in spite of the fact that many species in the genus are severely threatened in the wild (Raimondo et al. 2009). In this study, we evaluated the in vitro regeneration of three Cyrtanthus species (C. contractus, C. guthrieae, and C. obliquus) as a step toward their conservation.
Materials and methods Plant material and culture conditions. The primary explants were twin scales obtained from mature Cyrtanthus plants growing in the University of KwaZulu-Natal Botanical Gardens, Pietermaritzburg, South Africa. A twin scale is a fragment of a basal plate with at least two partial scales. The bulbs were washed in tap water to remove soil, and the brown outermost scales were peeled away to expose the fresh white inner scales. Peeled and trimmed bulbs were surfacedecontaminated using 70% (v/v) ethanol for 2 min followed by 1% (w/v) Benlate® for 15 min, then 0.2% (w/v) mercuric chloride (HgCl2) solution supplemented with a few drops of Tween 20® for 15 min and rinsed three times in sterile distilled water. The rinsed bulbs were then cut in half and soaked in 0.2% (w/v) mercuric chloride with a few drops of Tween 20® for 10 min, after which they were again rinsed three times in sterile distilled water. Twin scales joined by 5 mm of the basal plate were excised from these segments and placed adaxial side down onto sterilized Murashige and Skoog medium (MS; Murashige and Skoog 1962). The MS medium was supplemented with 0.1 g L−1 myo-inositol, 8 g L−1 bacteriological agar (Bacteriological agar-Oxoid Ltd., Basingstoke, Hampshire, England), and 3% (w/v) sucrose and different combinations and concentrations of 6-benzyladenine (BA) and αnaphthalene acetic acid (NAA), in 40-mL culture tubes. The pH of the medium was adjusted to 5.8 prior to autoclaving at 121°C and 103 kPa for 20 min. Cultures were maintained in a growth room with cool white fluorescent tubes (Osram® L 58W/640, Munich, Germany) at 16-h photoperiod, with a photosynthetic photon flux density (PPFD) of 60 μmol m−2 s−1 at 25±2°C. Each treatment was repeated twice with each replicate consisting of 24 twin-scale explants.
Author's personal copy IN VITRO REGENERATION OF CYRTANTHUS SPECIES
In vitro shoot induction. The surface-decontaminated twinscale explants measuring approximately 10×10 mm were cut in half longitudinally and placed in culture tubes (100× 25 mm, 40 mL volume) containing 12 mL of modified MS medium supplemented with different ranges of concentrations of BA and NAA under continuous darkness and 16/8-h light/ dark conditions. In vitro shoot multiplication. Five cytokinins (CKs), BA, meta-topolin (mT), kinetin (Kin), thidiazuron (TDZ), and zeatin (ZT) were used to evaluate shoot multiplication and in vitro plant growth at three concentrations (1.0, 5.0, and 10.0 μM) in a completely randomized design. A set of cultures without plant growth regulators (PGRs) served as the control. In addition, the media that gave the highest numbers of shoots for each species in the shoot induction experiments were included in the treatments. Each treatment was repeated twice with 24 explants per replicate. The combined results from the two separate experiments were used for data analysis. The number of shoots per explant, the fresh and the dry masses of the shoots, and the lengths of the shoots were recorded after 8 wk of culture. Effects of photoperiod on shoot multiplication. Based on the results obtained from the preceding experiment, shoot explants of each species were excised from the media with high shoot proliferation rates and were cultured on the same medium. The cultures were divided into two, each with 40 replications, and placed under continuous light and 16/8-h light/ dark conditions, respectively, at 25°C. Shoot multiplication frequency and shoot fresh and dry mass, together with shoot length, were recorded after 8 wk of culture. In vitro rooting of regenerated shoots. In vitro regenerated shoots from the optimized shoot multiplication media were evaluated for rooting. The shoots were transferred to half- and full-strength MS medium without any growth regulator supplement. Each treatment was repeated twice with each replicate consisting of 24 individually cultured shoot explants. The data from the two replicates were combined for data analysis. Data on root induction (%), number of roots and root length, and fresh mass as well as dry mass were recorded after 4 wk in culture. Ex vitro acclimatization of rooted plant. In vitro-rooted plants were potted in plastic containers in a mixture of sand-soilvermiculite (1:1:1; v/v/v) and maintained in a mist house with high relative humidity (90–100%) for 3 d. The midday photosynthetic photon flux density (PPFD) in the mist house was 30–90 μmol m−2 s−1 with simulated natural photoperiod conditions. The plants were then transferred to the greenhouse set at 25°C and midday PPF of approximately 1,000 μmol m−2 s−1.
Statistical analysis. Percentage data were arcsine transformed before being subjected to one-way analysis of variance (ANOVA) using SPSS software for Windows (IBM SPSS®, version 21.0, Chicago, IL). Significantly different means (P≤ 0.05) were further separated using Duncan’s multiple range test (DMRT) and/or least significant difference (LSD).
Results and discussion Shoot induction. The number of shoots initiated per explant in each of the three species varied significantly among the different auxin and cytokinin combinations (Fig. 1). Although shoot induction occurred in almost all the treatments in this study, twin-scale explants cultured on MS medium supplemented with a PGR combination of 4.4 μM BA and 1.1 μM NAA produced the highest number of shoots/explant, 2.6 and 6.3, for C. contractus and C. guthrieae, respectively, each with a regeneration frequency above 80%. On the other hand, for C. obliquus, the highest shoot induction rate (1.9 shoots/ explant) was recorded in slightly higher PGR levels (6.7 μM BA+2.7 μM NAA). The lower requirement of PGRs for shoot induction in C. contractus and C. guthrieae may suggest that the two species contain higher endogenous levels of PGR to induce shoot formation compared to C. obliquus (Maesato et al. 1994). The interactive effect of an exogenous supplement of auxins and CK on shoot morphogenesis in cultured explants is exerted through the alteration of their endogenous balance within the explant (Bajguz and Piotrowska 2009). An optimally adjusted balance between the two PGRs shifts morphogenic development of cultured cells significantly toward shoot development. Increasing the concentrations of BA and NAA resulted in a corresponding increase in adventitious shoot production. One possible explanation for this phenomenon is that the increased exogenous NAA may be upregulating CK activity, e.g., by promoting metabolic activation through N-glycosylation of BA (Avalbaev et al. 2012) and hence shoot organogenesis. The biosynthesis and cellular level balance of endogenous CK is fine-tuned by internal and external factors such as endogenous auxins and inorganic nitrogen sources, which appear to be important in linking nutrient signals and morphogenetic responses (Sakakibara 2006). The highest number of shoots as well as the percentage of explants producing shoots in Cyrtanthus loddigesianus and Cyrtanthus speciosus was achieved with high doses of NAA and BA (5.37 and 8.8 μM, respectively; Angulo et al. 2003). These results are consistent with those observed by others researching Cyrtanthus species (McAlister et al. 1998; Morán et al. 2003; Hong and Lee 2012), other members of the Amaryllidaceae (Slabbert et al. 1993; Vishnevetsky et al. 2003), and bulbous species from other families (Cheesman et al. 2010). While a number of research findings are
Author's personal copy NCUBE ET AL. 3.5
Figure 1 Effects of the interaction of BA and NAA on the shoot induction frequency on C. contractus, C. guthrieae, and C. obliquus after 8 wk of culture. Bars with different letters are significantly different (P=0.05) according to DMRT.
C. contractus
0 µM NAA 0.5 µM NAA 1.1 µM NAA 2.7 µM NAA
d
3.0
d 2.5
d
cd
d
2.0
cd
cd cd
1.5
c
1.0
c b
b ab
ab
0.5
a
a
a
a a 0.0
Number of shoots per explant
C. guthrieae
de
d
6
d c
d
c c
4
b b
2
b
b
b
b
ab
b
b b
a a a 0 2.0
d
C. obliquus
c c
bc 1.5
b
c
b b
b
b b
1.0
b a a
0.5
a
a
a a 0.0
0
consistent with this observed trend on bulb explants, several others have yielded results in sharp contrast. For example, Slabbert and Niederwieser (1999), Fennell (2002), and Rice et al. (2011) reported the highest shoot and bulblet induction, respectively, from twin scales cultured on MS medium free of exogenous PGR, findings that further support the significant role of the endogenous content of phytohormones and their interplay with the exogenous supply in regulating morphogenic parameters in plant cells. Unfortunately, ascertaining exogenous phytohormone levels is difficult and thus seldom analyzed during micropropagation. Although most shoots in all three species were derived from the basal plates, some explants, particularly those of C. contractus and C. guthrieae, produced shoots directly from
1.1
4.4 6.7 BA concentration (µM)
8.9
the twin scales (Fig. 2A). Anatomical observations on bud regeneration in Nerine bowdenii revealed that buds form on twin-scale explants that contained basal plate tissue as a result of outgrowth of preexisting axillary meristems and the regeneration of adventitious buds in the axils of the scales (Vishnevetsky et al. 2003). A similar mechanism of shoot regeneration to that described for N. bowdenii could be assumed to have been responsible for the shoot induction observed in this study. The number of shoots produced from axillary buds is limited to the number of preexisting meristems placed in each culture. A greater potential for multiplication exists with adventitious buds as shoots may arise from any part of the explant (Zimmerman 1993), an attribute that offers promising potential for further manipulation of Cyrtanthus
Author's personal copy IN VITRO REGENERATION OF CYRTANTHUS SPECIES Figure 2 In vitro culture of Cyrtanthus spp. and ex vitro acclimatization. (A) In vitro shoot development on the bulb scales of C. contractus after 4 wk in culture. (B) Shoot organogenesis from C. contractus callus derived from media supplemented with 10 μM Kin. (C) Visually superior quality C. obliquus plantlets derived from 5-μM mT MS medium supplement after 8 wk in culture. (D) Thin pale C. obliquus plantlets derived from 1-μM ZT MS medium supplement after 8 wk. (E) Shoot multiplication of C. guthrieae on MS growth medium supplemented with 4.4 μM BA+1.1 μM NAA. (F) Rooting response of C. guthrieae shoots on PGR-free full-strength MS medium after 8 wk in culture. (G) In vitro-rooted C. obliquus plants that were successfully acclimatized in the greenhouse after 8 wk. Scale bar 10 mm.
scale explants for improved shoot regeneration. The conclusion drawn from this study is that the uptake of BA and NAA from the medium by the twin-scale explants of the three Cyrtanthus species was essential for the shoot organogenesis process.
Effect of plant growth regulators on shoot organogenesis. Different types and concentrations of CK treatments differentially influenced shoot proliferation in the three Cyrtanthus species (Table 1). With the exception of 1.0 μM Kin on C. contractus and all the ZT treatments on C. guthrieae, shoot
Author's personal copy NCUBE ET AL. Table 1 Effect of plant growth regulators on in vitro growth and development of Cyrtanthus spp. shoots after 8 wk in culture Plant species
Cytokinin
(μM)
No. of shoots/explant
Fresh mass (mg)
Dry mass (mg)
Shoot length (cm)
C. contractus
BA
1.0 5.0 10.0 1.0 5.0 10.0 1.0 5.0 10.0 1.0 5.0 10.0 1.0 5.0 10.0
3.4±0.13cd 5.7±0.25f 5.9±0.21fg 2.0±0.16ab 2.4±0.13b 3.5±0.20cde 1.5±0.13a 3.1±0.11c 3.4±0.13cd 3.1±0.17c 3.8±0.13de 7.5±0.30h 3.7±0.16de 4.0±0.14e 6.3±0.15g 6.8±0.21g 1.9±0.15ab
151.1±4.75fg 160.4±2.70g 125.0±3.85de 275.8±5.95i 192.9±7.76h 97.8±5.39b 130.2±3.04e 124.5±2.88de 94.4±3.64b 114.1±5.08cd 104.8±7.85bc 74.5±5.30a 162.0±3.22g 144.5±3.44f 180.1±3.48h 183.8±4.62h 185.7±3.36h
13.8±0.69ef 14.5±0.73f 11.0±0.85d 19.4±1.45h 17.8±1.28gh 7.6±0.87abc 11.1±1.15d 9.6±1.06bcd 7.5±0.74ab 10.3±0.95cd 10.0±0.30bcd 6.3±0.43a 16.0±0.58fg 15.2±0.57g 11.4±0.81de 9.8±1.09bcd 18.0±0.88gh
7.9±0.66bc 5.7±0.53ab 5.0±0.34a 11.0±0.60d 6.8±0.76abc 7.0±0.51abc 7.9±0.49bc 6.6±0.33abc 6.1±0.39abc 7.1±1.23abc 5.4±0.58a 6.4±0.93abc 5.8±0.73ab 4.8±0.54a 8.2±1.12c 6.2±0.52abc 12.7±0.74d
1.0 5.0 10.0 1.0 5.0 10.0 1.0 5.0 10.0 1.0 5.0 10.0 1.0 5.0 10.0
10.4±0.29g 9.0±0.13f 6.5±0.17d 6.9±0.18de 7.0±.018de 7.4±0.20e 3.3±0.23c 3.5±0.15c 3.7±0.22c 14.0±0.16h 15.0±0.16i 10.0±0.20g 2.2±0.14ab 1.7±0.18a 2.0±0.14ab 15.8±0.14i 2.3±0.22b 2.1±0.26abcd 3.1±0.23ef
121.1±2.73bc 123.3±2.50c 94.5±3.93a 141.2±3.49fg 136.1±3.06def 112.3±4.94b 149.5±3.43hij 146.7±2.64g 123.5±3.73c 157.6±2.43hi 126.4±3.61cd 158.8±2.63i 148.0±2.77gh 131.0±3.23cde 139.7±2.89efg 123.4±2.53c 193.1±3.60j 125.4±3.41efg 120.3±3.44efg
10.6±1.01cd 7.9±0.61ab 6.8±0.23a 10.8±0.48cde 12.9±0.59efg 11.4±0.67cde 14.2±1.10gh 13.6±0.61fgh 11.2±0.56cde 9.5±0.55bc 8.1±1.12ab 11.1±0.50cde 11.8±0.58def 12.0±0.72def 13.0±0.64efg 10.0±0.68bc 15.5±0.45h 8.6±0.59ef 9.0±0.45defg
10.1±0.55de 8.9±0.50cde 6.6±0.48abc 10.6±0.84e 9.0±0.65cde 7.7±0.65abcd 9.2±0.95de 7.8±0.76bcd 9.4±0.92de 8.3±1.02bcde 8.0±1.33bcde 5.4±0.48a 9.1±0.71cde 7.9±0.67bcd 6.1±0.39ab 5.8±0.42ab 10.2±0.96de 5.7±0.58ab 5.2±0.80a
4.0±0.24g 2.5±0.26cde 2.5±0.28cde 2.6±0.19cde 1.9±0.18abc 1.9±0.18abc 2.4±0.23bcd 3.4±0.20fg 3.5±0.28fg 3.6±0.26fg 1.6±0.19a 2.6±0.23cde 3.6±0.33fg
87.1±3.43b 93.3±3.48bc 75.0±2.01a 84.7±3.01b 126.6±4.17efg 118.3±2.68de 92.7±2.95bc 129.8±4.16fg 134.1±1.87gh 140.4±4.76hi 99.2±2.29c 100.7±4.22c 133.4±3.61gh
6.7±0.22ab 6.9±0.23abc 5.8±0.25a 7.0±0.52abc 10.1±0.38fgh 9.7±0.30efgh 7.8±0.19bcd 9.5±0.63efg 10.4±0.70gh 9.4±0.92efg 8.7±0.18def 7.8±0.29bcd 9.4±1.11efg
4.8±0.64a 6.8±0.73ab 6.5±0.44ab 7.0±0.76abc 9.1±0.50c 7.8±1.01bc 6.3±0.65ab 5.4±0.87ab 5.3±1.02a 5.3±0.43a 6.4±0.78ab 6.1±0.36ab 5.3±0.93a
mT
Kin
TDZ
ZT
4.4 μM (BA) 1.1 μM (NAA) Control C. guthrieae
BA
mT
Kin
TDZ
ZT
4.4 μM (BA) 1.1 μM (NAA) Control C. obliquus BA
mT
Kin
TDZ
ZT 6.7 μM (BA) 2.7 μM (NAA)
1.0 5.0 10.0 1.0 5.0 10.0 1.0 5.0 10.0 1.0 5.0 10.0 1.0 5.0
Author's personal copy IN VITRO REGENERATION OF CYRTANTHUS SPECIES Table 1 (continued) Plant species
Cytokinin
Control
(μM)
No. of shoots/explant
Fresh mass (mg)
Dry mass (mg)
Shoot length (cm)
10.0
2.7±0.18de 1.6±0.17ab
114.1±1.95d 146.9±2.81i
8.3±0.40cde 11.0±0.35h
5.4±0.96a 9.1±0.60c
Mean values±standard error (n=24) in the same column with different letter(s) for each species indicate a significant difference (P≤0.05) based on DMRT
multiplication increased at all CK concentrations compared to the PGR-free controls in all three species. When the concentration of BA was increased from 1.0 to 10.0 μM, the number of shoots per explant decreased in C. guthrieae, whereas they increased in C. contractus and C. obliquus. The highest numbers of shoots per explant from the media supplemented with different types and concentrations of CKs were recorded at 10.0 μM TDZ for C. contractus (7.5), 5.0 μM TDZ for C. guthrieae (15.0; although insignificantly lower than for 4.4 μM BA+1.1 μM NAA [Fig. 2E]), and 10.0 μM BA for C. obliquus (4.0). Thidiazuron, a substituted phenylurea, has proven to be a highly effective PGR in various morphogenetic and embryogenic responses of different plant species (Mithila et al. 2001; Jones et al. 2007; Arshad et al. 2012; Baskaran et al. 2012). Characteristically, TDZ is a cheap and potent CK, the high activity of which may be attributed partly to its extreme stability in plant tissues (Mok et al. 2000). Its mode of action may be affected either directly or indirectly, by regulating endogenous CK biosynthesis and/or metabolism (Mok et al. 2000). In sugar beet, TDZ-induced somatic embryogenesis occurred on the abaxial surface of cotyledons at a concentration of 0.5 mM and was found to be less genotypedependent than BA (Zhang et al. 2001). The superior effects of TDZ over other CKs on shoot proliferation of C. contractus and C. guthrieae are consistent with previous findings from other plant species and suggest that it may be advantageous to use TDZ instead of BA in the tissue culture of these plant species. A mixture of auxin/CK (1.1 μM NAA/4.4 μM BA), however, proved to be an excellent PGR media supplement for shoot proliferation compared to any of the tested CKs applied alone on C. guthrieae (Table 1). The combination resulted in the highest shoot frequency for this species, although it was not significantly different from the 5.0 μM TDZ treatment. The synergistic effect of auxins with CKs on shoot multiplication has been reported in studies of other Cyrtanthus species (Angulo et al. 2003; Morán et al. 2003). However, none of these studies investigated the effects of different CKs on shoot morphogenesis of Cyrtanthus species. The role of PGRs on the physiological and morphogenic developmental patterns of cultured cells has been inferred mostly from the effects of exogenous applications, often with poor understanding of their mechanisms of action. Nordström et al. (2004) argued that auxin controls CK biosynthesis via the specific activation
of IPT5 and IPT7 genes in Arabidopsis (Miyawaki et al. 2004) and that most auxin-resistant mutants also show changes in their CK sensitivity (Nordström et al. 2004; Ioio et al. 2008). On the other hand, Pernisová et al. (2009) showed that auxin-induced organogenesis is modulated by CK through regulating the efflux of auxin distribution between cells. In their in vitro study system on the mechanism underlying the onset of organogenesis, the authors found that auxin is responsible for organogenesis in hypocotyl explants. They, however, demonstrated that auxin distribution is differentially affected by CK and that CK modulates organogenesis through inhibition of polar auxin transport (Pernisová et al. 2009). This interdependent hormonal network gets integrated at multiple levels, such as during auxin signaling, metabolism, and carrier-dependent distribution, with the resulting positive morphogenic developments. Following this logic, the same mechanism of action and the integrated effects could be assumed in the excellent shoot proliferation demonstrated by a treatment combination of NAA and BA in C. guthrieae compared to media with single CK applications. Despite reports emphasizing the superior ability of mT to affect shoot proliferation, in vitro morphogenesis, and plantlet quality, when compared with other aromatic CKs in a number of plant species (Bogaert et al. 2004; Bairu et al. 2007; Moyo et al. 2012), its ability to affect the growth and development of shoots in three Cyrtanthus species in this study was inferior compared with the other PGRs tested. On the other hand, related studies report that Kin, even at high concentrations, yielded comparatively low adventitious shoot regeneration (Beck and Caponetti 1983), results that agree with the findings of this study. However, visually superior quality shoots (in terms of plant texture, stem thickness, and vigor) were obtained from treatments with mT and Kin (Fig. 2C, D) in all three species (data not shown for other two species), although their effect on shoot multiplication was significantly lower than for BA and TDZ. In addition to producing good quality plantlets, Kin also exhibited some auxin-like activity by inducing rooting and callus on C. contractus and C. guthrieae plantlets as well as shoot organogenesis from callus in C. contractus (Fig. 2B). Formation of lateral roots represents one of the examples of postembryonic de novo organogenesis in plants. Recent studies (Laplaze et al. 2007; Péret et al. 2009) suggest potential involvement of CKs in the regulation of lateral root formation, a phenomenon that could have facilitated this
Author's personal copy NCUBE ET AL.
process in the two species with Kin treatment in this study (data not shown). The shoots produced from TDZ treatments were of uniformly good quality, although they were not as robust as those obtained from mT or Kin treatments. However, the inexpensive nature of TDZ, its stability in plant tissues, and the number and quality of shoots induced in C. guthrieae make it a preferred choice compared to the other tested CKs for this plant species. Although there were no distinct trends observed on shoot biomass and shoot length in relation to the type and concentration of CKs, there seemed to be a general trade-off between shoot proliferation rates and other growth parameters in all three species. Control treatments, most of which had lower shoot multiplication frequencies, recorded the highest shoot biomasses and shoot lengths. In C. guthrieae, the medium containing 5.0 μM TDZ induced a sevenfold higher number of shoots (15) than the control (2.3), but the average length of the shoots did not differ significantly between the two treatments (Table 1). The high CK activity and positive response of these and other species (mostly woody species) to TDZ have established it as among the most important CKs for in vitro manipulation of plant species. Effect of photoperiod on shoot organogenesis. The regulation of photomorphogenetic processes of plant tissues in culture together with plant growth and quality is largely determined by light parameters provided in the culture environment (Economou and Read 1987). Light duration is among the important environmental factors that affect plant growth and development in culture (Murashige 1974). The results of the morphogenetic parameters of Cyrtanthus shoots investigated at two different photoperiods (16 h or continuous light) are presented in Table 2. In all three species, the number of shoots produced per explant and shoot length were higher in explants cultured under a 16-h photoperiod. Shoot fresh and dry weights were, however, significantly higher in explants maintained under continuous light conditions, suggesting an improved dry matter accumulation, possibly as a result of activated CK biosynthesis or its photo-oxidation to a desirable
endogenous PGR ratio (Kamada et al. 1995; Kozai and Kubota 2005). Consistent with these findings was the significant increase in stem diameter, leaf area, and number of leaves in potato plantlets cultured in vitro under long photoperiods and high PPF levels found by Kozai et al. (1995). In the same experiment, longer photoperiods also led to an increase in fresh and dry weights with an enhanced root growth. The influence of photoperiod on various morphogenic parameters, however, varies from species to species. Debergh and Maene (1977) reported an improved shoot multiplication frequency in shoot tip cultures of Pelargonium species maintained under continuous light, while a decrease in shoot number, length, and quality was recorded in Rhododendron species under similar photoperiodic conditions (Economou and Read 1987). Light is thought to exert these effects in plant tissue culture by either inducing the activation of CK biosynthesis and/or its degradation, with the subsequent increase/decrease in the endogenous levels of CK (Tapingkae and Taji 2000). In vitro rooting and ex vitro acclimatization. In vitro regenerated shoots from the optimized shoot multiplication experiments rooted effectively after 4 wk of culture when transferred to full- and half-strength MS media without PGRs (e.g., Fig. 2F). The competent ex vitro acclimatization of in vitro regenerated plants within the shortest possible time is a critical step in the conservation of any plant species. Based on the results from the multiplication experiments, regenerated shoots were rooted individually on half- and full-strength MS medium without PGRs. Figure 3 shows the effects of the growth media on the root growth parameters (root induction frequency, mean number of roots per shoot, and root length). Mean number of roots and root lengths were slightly higher in shoots cultured on full-strength MS in all three species, although they were not significant from those maintained on half-strength MS. The root induction frequency was more than 75% for all three Cyrtanthus species on either media, suggesting that both can be used for efficient in vitro rooting of the species.
Table 2 Effect of photoperiod on in vitro shoot morphogenesis of Cyrtanthus spp. Species
Treatment
C. contractus 10 μM TDZ C. guthrieae 4.4 μM BA/1.1 μM NAA C. obliquus 10 μM BA
No. of shoots/explant
Shoot length (cm)
Fresh weight (mg)
Dry weight (mg)
16/8-h (light/dark)
24-h light
16/8-h (light/dark)
24-h light
16/8-h (light/dark)
24-h light
16/8-h (light/dark)
24-h light
7.9±0.9b 15.4±1.0b 4.0±0.2a
5.3±0.6a 11.5±0.8a 3.1±0.7a
5.8±0.8b 5.8±0.4a 4.7±1.1a
3.1±0.4a 4.7±0.6a 3.2±0.3a
178.8±2.7a 123.4±2.5a 87.1±3.4a
183.7±1.6a 131.1±3.1b 93.4±2.1b
9.3±1.1a 10.1±0.8a 6.7±0.2a
11.2±0.7a 13.3±0.5b 10.2±1.0b
Mean values±standard error (n=24) in the same row with different letter(s) are significantly different (P≤0.05) based on LSD
Author's personal copy IN VITRO REGENERATION OF CYRTANTHUS SPECIES
Conclusions Micropropagation protocols for C. contractus, C. guthrieae, and C. obliquus were successfully established from mature bulb scales. Owing to the declining populations of the three Cyrtanthus species in the wild, the developed procedures can be used for rapid propagation to meet commercial demands and in conservation programs, taking on average 16 wk from explant preparation to acclimatization. A combination of BA and NAA as MS medium supplements proved to be an effective PGR combination with significantly higher shoot induction and multiplication frequencies for C. contractus and C. guthrieae. The highest number of shoots per explant was obtained on MS medium supplemented with 4.4 μM BA/ 1.1 μM NAA for C. guthrieae, 10 μM TDZ for C. contractus, and 10 μM BA for C. obliquus. Treatments with TDZ as a supplement to the MS medium also exhibited superiority over treatments with other CKs for shoot multiplication and other morphogenic attributes on C. contractus and C. guthrieae. The fact that 5.0 μM TDZ produced statistically similar shoot proliferation effects to those of a combination of NAA and BA for C. guthrieae makes TDZ an attractive option for the rapid propagation of this species. No PGR pretreatment was necessary for in vitro rooting before acclimatizing the regenerated plantlets for all three species. The high number of plantlets that are obtained with the proposed micropropagation procedures represents an excellent alternative to conventional horticultural propagation methods. Acknowledgments The University of KwaZulu-Natal and National Research Foundation of South Africa are gratefully acknowledged for financial assistance.
References
Figure 3 Effect of half- and full-strength MS media on rooting and root parameters of three Cyrtanthus spp. Error bars with similar letters for each species indicate insignificant difference (P≤0.05) based on the t test.
Agar was carefully washed from the in vitro-rooted plantlets before transferring to soil, and they acclimatized and established successfully in the greenhouse (Fig. 2G) with a more than 95% survival rate. There were no observable morphological abnormalities in all species. The high rooting frequency and number of roots per shoot produced on PGR-free medium in the three Cyrtanthus species, with subsequent high survival rate upon ex vitro acclimatization, make these regeneration protocols particularly attractive economically in that they reduce labor and production costs.
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