VEJSADOVÁ HANA
Silva Tarouca Research Institute for Landscape and Ornamental Gardening, Publ. Res. Inst. Průhonice Objective of this work was to find the regeneration way of in vitro explants in two grapevine rootstocks ‘SO-4’ and ‘Craciunel 2’. The petiole segments and leaf blades of in vitro cultures were used as initial explants. After 10 weeks of explant incubation on modified MS medium, combination of thidiazuron (TDZ) and benzyladenine (BA) significantly stimulated organogenesis initiation. The blades regenerated in greater rate at 1 mg.l−1 TDZand 4 mg.l−1 BA compared to petiole segments in both vine rootstocks. Somatic embryogenesis induction was significantly influenced by type and growth regulator combination. Auxin ß-naphthoxyacetic acid (NOA) with cytokinin TDZ showed as key factors of embryogenic structure formation in all tested explants of both grapevine rootstocks. Significantly positive effect of cytokinin BA on somatic embryo initiation was not found.
Key words: grapevine, rootstocks, ‘SO-4’, ‘Craciunel 2’, organogenesis, somatic embryogenesis
INTRODUCTION
Shoot organogenesis and in vitro somatic embryogenesis have been achieved from various explants of different
Vitis species and cultivars (Bouquet and Torregrosa, 2003). The somatic embryogenesis was induced in anthers
(Stamp and Meredith, 1988) or ovules (Gray and Mortensen, 1987) but rarely from leaf segments (Robacker, 1993, Cutanda et al., 2008). Somatic embryogenesis was also observed in callus initiated from tendrils of greenhouse grown grapevine cultivars (Salunkhe et al., 1997).
The leaves and petioles as explant sources are often harvested from in vivo cultivated plants, a surface sterilization is required and the explants are often threatened with bacteria contamination. Torregrosa and Bouquet (1996) found high level of regeneration in the youngest leaves excised from axillary shoots in proliferating nodal cultures of several Vitis x Muscadinia hybrids. Robacker (1993) demonstrated a greater somatic embryogenesis incidence in immature petioles than in leaf laminae of cultivars 'Regale' and 'Fry' muscadine grapes (Vitis rotundifolia Michx.).
In this report, shoot regeneration of leaf explants was studied in in vitro grown leaves of grapevine rootstocks ‘SO-4’ and ‘Craciunel 2’.
MATERIAL AND METHODS
In grapevine rootstocks ‘SO-4’ (SO4) and ‘Craciunel 2’ (Cr2), the leaves were excised from axillary shoots produced by one subculture on WPM (Lloyd and McCown, 1980) medium without growth regulators. Medium was supplemented with 2.5 g.1-1 activated charcoal, 25 g.1-1 sucrose, vitamins B5 and 8 g.l-1 agar (Phytoagar,
Duchefa). The explants – dark green leaves were obtained from top and central part of in vitro cultures. Modified MS (Murashige and Skoog, 1962) medium with a full concentration of salts was used as regeneration medium. This medium contained a mix of vitamins, 40 g.1-1 sucrose, 7 g.l-1 agar (Microagar, Duchefa) and combination of
auxin ß-naphthoxyacetic acid (NOA) with cytokinins thidiazuron (TDZ) and benzyladenine (BA), Table 1. The blades were cut through using scissors and cultured (abaxial side down) on regeneration medium. The petioles were dissected into segments cca 5 mm long and placed horizontally on the medium. The explants were cultivated in Petri dishes in the thermostat (23±2°C) at darkness.
The cultures were assessed after 10 weeks. For each treatment, 30 explants were used. The experiment was repeated two-times. Data were statistically evaluated by analysis of variance (ANOVA) and means by Duncan’s test (P=0.05).
De novo formed shoots were cultivated in the Erlenmeyer flasks (100 ml) with tinfoil covers in a growth room
maintained at a temperature of 23/19°C (day/night) with 16 h photoperiod and a photon fluence rate of 55 µmol.m-2.s-1 provided by cool white fluorescent tubes. Maintenance WPM medium (Duchefa) contained 2.5 g.1-1
activated charcoal, 25 g.1-1 sucrose, 8 g.l-1 agar (Phytoagar, Duchefa) and 1.5 mg.l-1 BA with indolylacetic acid
(IAA) in concentration of 0.1 mg.l-1
RESULTS AND DISCUSSION
The ability of explant morphogenesis induction is highly depended on the particular genotype of various Vitis species, cultivars or hybrids and on certain culture conditions (Bouquet and Torregrosa, 2003). In this work, two grapevine rootstocks ‘SO-4’ (SO4) and ‘Craciunel 2’ (Cr2) were used in the presented culture conditions. After 10 weeks, combination of TDZ and BA without auxin stimulated shoot organogenesis in leaf explants – the blades regenerated in greater rate at 1 mg.l−1 TDZ+ 4 mg.l−1 BA compared to petiole segments in both grapevine rootstocks (Table 1).
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Table 1: Effect of NOA, TDZ and BA treatments on organogenesis and somatic embryogenesis induction in leaf explants after 10 weeks
NOA TDZ BA Explants with shoot bud regeneration (%)
Explants with embryogenic structure formation (%) mg.l−1 ‘SO4’ ‘Cr2’ ‘SO4’ ‘Cr2’ BL PE BL PE BL PE BL PE – 0.5 2 11.3d 8.5d 20.8b 15.6c – – – – – 1 4 16.3c 10.2d 27.3a 20.9b – – – – 4 0.5 – – – – – 60.2c 72.4b 32.5e 47.2d 4 1 – – – – – 70.5b 80.1a 30.6e 50.7d 4 0.5 2 – – – – 58.7c 73.5b 34.7e 48.6d 4 1 4 – – – – 69.3b 86.8a 29.9e 45.5d
BL – blade explant; PE – petiole explant (30 explants/treatment). The values represent the means of two repeated experiments. Numbers followed by the same letter within one column do not differ significantly at P=0.05 (Duncan’s test).
Induction of embryogenic structures was exclusively enhanced by auxin NOA. In ‘SO4’, the percentage of blade and petiole explants forming embryogenic structures increased significantly to 70–87% with addition of 1 mg.l−1 TDZ and 4 mg.l−1 BA. In ‘Cr2’ explants, embryogenic tissues appeared in reduced rate – on average 30% in blades and 48% in petioles.
The process of somatic embryogenesis usually requires the involvement of auxin in the induction media with or without subsequent removal of explant from auxin media while cytokinins generally induce shoot formation on casus and support growth in tissue and organ cultures (Fellman et al., 1987). Robacker (1993) used auxin 2,4- dichlorophenoxyacetic acid (2,4-D) and cytokinin BA for embryogenic mass induction in immature leaf laminae and petioles of 'Regale' and 'Fry' muscadine grapes (Vitis rotundifolia). In Vitis rupestris, Stamp and Meredith (1988) found the embryogenic tissue initiation directly from the explant rather from the callus. Likewise, in the present experiments, the white somatic embryos were observed on the blade disrupted (dissected) surface and on the petiolar stub without callus formation. Whereas, in V. vinifera, somatic embryogenesis was induced by 5 µM 2,4-D and 1 µM BA (Cutanda et al., 2008), in rootstocks ‘SO-4’ and ‘Craciunel 2’, key factors of somatic embryogenesis induction of leaf explants were auxin NOA and cytokinins TDZ with or without BA.
CONCLUSION
After 10 weeks of explant incubation on modified MS medium, combination of thidiazuron (TDZ) and benzyladenine (BA) significantly stimulated organogenesis initiation. The blades regenerated in greater rate at 1 mg.l−1 TDZ and 4 mg.l−1 BA compared to petiole segments in both vine rootstocks. Somatic embryogenesis induction was significantly influenced by type and growth regulator combination. Auxin ß-naphthoxyacetic acid (NOA) with cytokinin thidiazuron (TDZ) showed as key factors of embryogenic structure formation in both
grapevine rootstocks.
Acknowledgment: This work was financial supported by the Ministry of Agriculture of the Czech Republic
(Project No. QH91214).
REFERENCES
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Adresa autora:
RNDr. Hana Vejsadová, CSc., Výzkumný ústav Silva Taroucy pro krajinu a okrasné zahradnictví,v.v.i., CZ-25243 Průhonice, Czech Republic, [email protected]
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