Abstract: In this research, we aimed to establish an effective in vitro propagation protocol for Hypericum heterophyllum Vent., an endemic Turkish species. The seeds were surface sterilised and transferred to Murashige and Skoog basal medium supplemented with BA (benzyladenine, 0.1, 1 mg L-1) and 2,4-D (2,4-dichlorophenoxyacetic acid, 0.1, 1 mg L-1) under the photoperiod (8/16 h light/dark period, 1200 μmol m-2s-1). Culturing seeds on MS basal medium supplemented with 1 mg L-1 BA and 0.1 mg L-1 2,4-D resulted in greenish and compact callus induction. MS basal medium supplemented with 4.4 mg L-1 BA was used for shoot induction, while the same basal medium supplemented with 1 mg L-1 IAA (indolacetic acid) was employed for the rooting. Regenerated plants were easily acclimatized in greenhouse conditions.
Introduction
Medicinal plants have always had an important place in the therapeutic armoury of mankind. Up to 80 % of populations in developing countries are totally dependent on plants for their primary health care. And despite the remarkable progress in synthetic organic chemistry of the twentieth century, over 25% of prescribed medicines in industrialised countries derive directly or indirectly from plants (Newman et al., 2000). Especially, Hypericum species have been used as traditional medicinal plants due to their wound-healing (Yazaki and Okuda, 1990), bactericide (Ishiguro et al., 1998), anti-inflammatory (Dias et al., 1998), diuretic and sedative properties (Holz and Ostrowiski, 1987) for last two hundred years. In particular, extracts of Hypericum perforatum are now widely used in Europe as drugs for the treatment of depression (Brutovska et al., 2001). The Hypericum genus of Guttiferae is represented in Turkey by 89 species of which 43 are endemic. Hypericum heterophyllum Vent. existing in dry clearings in Pinus thickets, etc., 1200-1600 m from northern Turkey is one of these endemic Hypericum species. Its stem is 10-30 cm; leaves on main stem are 5-13 mm, narrowly elliptic-oblong to linear, those on short axillary strobiloid shoots 0.5 mm, broadly ovoid, densely crowded. Sepals 2-3 mm, oblong to lanceolate, acute, entire. Petals 5-8 mm, oblanceolate. Capsule 6-8 mm, ovoid cylindric to cylindric (Davis, 1988). This plant is a source of medicinal compounds and well known with its antifungal activity (Cakir et al., 2004).
Many rare or/and endemic plant species are known to respond well to sterile in vitro conditions, including a nutrient rich medium and also phytohormone supply (Fay, 1992; Pence, 1999) and recent studies have demonstrated that in vitro culture is an option for multiplication of Hypericum species such as H. foliosum (Moura, 1998), H. brasiliense (Cardoso and Oliveira, 1996; Abreu et al., 2003), H. canariense (Mederos, 1991), H. maculatum (Kartnig and Brantner, 1990) and H. perforatum (Cellarova et al., 1992; Murch et al., 2000; Pretto and Santarem, 2000; Bezo and Stefunova, 2001; Santarem and Astarita, 2003, Zobayed et al., 2004, Ayan et al., 2005). In vitro culture has also been reported as a very useful tool for both studying and producing economically important secondary metabolites of the genus (Yazaki and Okuda, 1990; Dias et al., 1998; Kirakosyan et al., 2000, 2001; Bais et al., 2002; Zobayed and Saxena, 2003). Therefore, the objective of the present study was to establish an efficient in vitro propagation protocol for this endemic species.
Materials and Methods
Plant Materials
The seeds of H. heterophyllum were obtained from Hypericum plants
growing wild in Trabzon province of Turkey. Plant materials were identified
by Dr. Hasan Korkmaz, Department of Biology, University of 19 Mayis, Samsun-Turkey.
The 2 months-old seeds were surface sterilized in 75% ethanol for 2 min and
subsequently in a 15% (v/v) sodium hypochlorite solution (commercial sodium
hypochlorite with 53% active constituent) for 20 min, followed by rinsing in
sterile distilled water three times.
Callus Initiation
Surface sterilized seeds were cultured in media supplemented with different
culture constituents in magenta boxes. The callus initiation medium contained
MS salts and vitamins (Murashige and Skoog, 1962). The pH was adjusted to 5.8
before autoclaving at 121°C for 20 min. Plant growth regulators BA and 2,4-D
and their concentrations (0.1 and 1 mg L-1), selected from previous
studies, were tested (Table 1). Medium without plant growth
regulators was used as control. Cultures were kept under the photoperiod (8/16
h light/dark period, 1200 μmol m-2s-1) at 26±2°C
for 12 weeks with two sub cultures. Ten seeds were cultured per 25 mL magenta
boxes with four replications. The frequency of callus induction and callus fresh
weight was determined 12 weeks after culture initiation.
Shoot Induction and Multiplication
For shoot induction, calli were transferred to shoot induction medium containing
MS salt and vitamins and 4.4 mg L-1 BA, pH: 5.8 and kept under the
same conditions employed in callus initiation for 6 weeks. The number of shoots
per treatment was recorded at 7 weeks after transferring the calli for shoot
induction medium.
Rooting
To induce roots, elongated shoots (1-2 cm) were excised and transferred
to MS medium supplemented with 1 mg L-1 IAA, pH: 5.8. Four shoots
were placed in 25 mL magenta boxes per treatment. Data were recorded after 4
weeks of culture.
After removal from magenta boxes, the rooted plantlets were washed with tap water to remove rooting medium debris. The young plants were transplanted in boxes containing soil and sand mixture (2:1) autoclaved at 120°C for 20 min under non-sterile conditions and gradually exposed to ambient humidity.
| Table 1: | The Media used for callus induction |
After 10 days, the acclimated plants were transferred to greenhouse, maintained under partial shade and irrigated daily. The percentage of survival was recorded at 2 week-intervals.
Results and Discussion
Callus Initiation
Callus induction and proliferation are useful tools for the studies concerning
both micropropagation and biosynthesis of natural products in Hypericum genus
(Pretto and Santarem, 2000). However, few species within this genus have been
reported to produce callus in response to different in vitro culture
conditions such as H. erectum (Yazaki and Okuda, 1990), H. brasiliense
(Cardoso and Oliveira, 1996), H. foliosum (Maciel and Moura, 2000),
H. maculatum, H. tomentosum, H. bithynicum, H. glandulosum,
H. balearicum (Kartnig et al., 1996), H. linarioides,
H. lydum, H. origanifolium, H. venustum and H. scabrum
(Ayan and Çırak, 2005). In the present study, callus formation was
observed only in the MS basal medium supplemented with 1 mg L-1 BA
and 0.1 mg L-1 2,4-D. The calli were greenish in color and compact
(Fig. 1a). Mean values of frequency of callus induction and
callus fresh weight were 43.47% and 44.8 mg/callus, respectively. The results
are in accordance with those of previous studies reporting BA and 2,4-D as callus
inducing agents in H. erectum (Yazaki and Okuda, 1990), H. brasiliense
(Cardoso and Oliveira, 1996) and H. perforatum (Pretto and Santarem,
2000; Bezo and Stefunova, 2001; Santarem and Astarita, 2003; Ayan et al.,
2005). But, it should be noted that callus induction in this endemic species
in response to in vitro culture is poor when compared to that of aforesaid
species of Hypericum. At this point, it will be useful to try other growth
regulators for enhancing callus induction.
Shoot Induction
When calli were cultured on medium supplemented with 4.4 mg L-1
BA, a very intensive shoot induction was observed (Fig. 1b)
and number of shoots per callus was 69.43. Many studies concerning shoot induction
and multiplication in H. perforatum L. have reported an average 30 to
50 shoots, depending on the factors evaluated (Ayan et al., 2005; Santarem
and Astarita, 2003).
| Fig. 1: | (a) Callus formation from seeds cultivated on MS + 1 mg L-1 BA + 0.1 mg L-1 2,4-D. (b) Shoot induction in BA containing medium from callus induced on MS + 1 mg L-1 BA + 0.1 mg L-1 2,4-D. (c) Rooting of shoots cultured on the medium containing 1 mg L-1 IAA. (d) A regenerated plant in greenhouse conditions (Bars represent 1 cm) |
We reported a procedure allowing the production of up to 65 shoots per callus in this new species of Hypericum.
BA, alone or in combination with NAA, are known as an effective shoot inducing agents in different species (Blakesley and Constantine, 1992). For H. perforatum, BA was found to be the most efficient in promoting shoot regeneration when leaf (Pretto and Santarem, 2000) or excised seedling parts (Cellarova et al., 1992) were used as explant. Similarly, BA can be used together with NAA in shoot induction from anther-originated callus (Kirakosyan et al., 2000).
Rooting
IBA (indolebutyric acid), IAA and NAA (naphthaleneacetic acid) have been
used commonly to improve rooting in different Hypericum species (Pretto
and Santarem, 2000). In H. canariense, rooting was obtained in the presence
of either IBA or NAA (Mederos, 1991). But, the two growth regulators were fully
ineffective in inducing rooting in shoots of H. foliosum (Moura, 1998).
IAA and IBA were the most effective for rooting in H. perforatum (Cellarova
and Kimakova, 1999).
In this study, elongated shoots were rooted successfully in the presence of 1 mg L-1 IAA (Fig. 1c) and the number of roots per shoot was 8.49. The result is similar that of Ayan et al. (2005) reporting 7-12 roots per shoot in response to IAA in H. perforatum. Regenerated plants were transferred to non-sterile conditions for acclimatization and to conditions which supply progressively lower humidity levels. After 10 days, 30% of regenerants was recorded up to 100 % of survival (Fig. 1d).
Conclusions
Conservation of endemic species by in vitro techniques has been an attractive topic for plant biotechnologist. The present study, it is the first time we have described an effective model concerning micropropagation of H. heterophyllum. If the multiplication of selected clones is required, surface sterilized seeds can be appropriate explants. Once induced, calli are transferred to BA containing medium for shoot induction and shoots can be rooted on MS medium supplemented with 1 mg L-1 IAA. Thus, regenerated plants can be obtained after five months. This is the first report on H. heterophyllum and the poor callus formation and low level of acclimatized regenerants require further in vitro studies on this endemic species.