Dianthus Caryophyllus L. Is one of the valuable commercial cut flowers around the world. Due to its excellent storage quality, wide range of forms, ability to withstand long distance transportation, and remarkable ability to rehydrate after continuous shipping, it is preferred by growers to roses and chrysanthemums in several fl over-exporting countries. In Vitro micropropagation technique is widely used in ornamental crops. The global cut flower market by the introduction of new improved cultivars is maintaining. Standard carnation generally propagating through stem cuttings.
However, this method is slow and rely on collecting and cutting mother plants. To strengthen the conservation, development, and utilization of this plant resource, it is important to establish an efficient in vitro micropropagation and plant regeneration protocol. The genetic variability within carnation is relatively poor; therefore, the breeding potential for new flower colors and patterns as well as resistance to biotic and abiotic stresses is also very limited. Carnation is a vegetatively propagated plant, which further reduces its genetic pool availability. The modern cell and molecular techniques could be regarded as an alternative and an additional complementation tool to the classical improvement methods of carnation. A few articles for tissue culture and plant regeneration protocol via adventitious shoot regeneration exists for carnation, but because of variation among the cultivars of this plant, set and optimize for all of varieties is necessary.
In this study, we established efficient protocols for high-frequency regeneration via two pathways direct and indirect shoot organogenesis from some leaf explants carnation cultivars. Although callus-mediated shoot formation is not only a useful method for plant propagation but also a powerful tool for plant genetic improvement (such as in vitro mutagenesis and gene transformation), germplasm conservation, and production of useful compounds. We also investigated the effects of different types and concentrations of plant growth regulators and media tightness for Overcoming to Hyperhydricity shoot produced.
Young leaf explants were taken from six species of carnation cultivars. We cutting the leaves into 0.5 to 0.7 cm2 explants and inoculated adaxial-side down on MS basal medium (Murashige and Skoog 1962). The pH of MS basal medium, which contained 30 g L−1 sucrose, was adjusted to 5.8 before being solidified with 0.7% (w/v) agar .We used from sterile Motherhood plant that they established at the MS media Containing PGR (Kin 1 mg/l and Naa .1 mg/l). Leaf explant planted in 80 mm petri and after callus induction transfer to Culture jars (10 cm high and 6 cm in diameter) were placed in an air-conditioned culture room at 25 ± 2°C with a 16-h photoperiod under 100-μM m−2 s−1 fluorescent light. These culture conditions were identical for all experiments.
Leaf segments adaxial-side down on MS medium supplemented with different concentration of 2.4,D (0, 0.2, 0.5, 1, 2, 3 mg/L) , NAA (0, 0.2, 0.5, 1, 2 mg/L) and BA (0, 0.5, 1 mg) in factorial combination were inoculated. After 3 weeks, callus driven from leaf explant transferred to MS media supplemented with different concentration of BA (1, 2, 3 mg/L), NAA (0, 0.2, 0.5, 1 mg/L) and adenine sulfate (0, 20, 40 and 60 mg/l). Among the PGRs tested, GA3 was filter-sterilized through a 0.24-μm filter and added after autoclaved medium had cooled while 2,4-D, BA, indole-3-butyric acid (IBA), and NAA were added prior to autoclaving at 121°C for 15 min.
For direct regeneration, we used different combination of Kin, BA, NAA, adenine sulfate, casein hydrolysate and AgNo3.
Shoots with 2–3 leaves that had reached 2–3 cm in height on MS medium supplemented with 2.0 μMBA and 0.5 μMNAAwere isolated from multiple shoot clusters, cut off at the base, and ransferred to half-strength MS medium supplemented with 0.5 μM IBA, 0.5 μM NAA, 0.5 μM IBA, and 0.5 μM NAA or no PGRs. After culture for a total of 30 days on these media, root formation was investigated (Table 4). A total of 270 rooted plantlets with 4–5 leaves and 3–4 cm tall were removed from jars representing the above four treatments. The agar was gently washed off roots in tap water. Plantlets were transplanted to square plastic trays (50cm×40 cm× 10 cm) containing three substrates: sand, loessal clay, and sand: loessal clay
Each treatment contained seven explants per culture jar and tree jars per treatment. The data were reported as mean±SD (standard deviation). Percentage values were arcsine transformed prior to analysis. Means were statistically analyzed by one-way analysis of variance (ANOVA), and treatment means were considered to be significantly different from controls by Duncan’s multiple range test at P≤ 0.05 using SPSS v. 19.0 (IBM, New York, NY).
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