Tissue Culture And Cell Activity Studies Of Carnation (Dianthus Caryophyllus Linn.) Cv. Grenadin

Author

NORFARIZA BINTI A. KADIR

Date

2001

Keyword

Dianthus caryophyllus LinN cv. Grenadin, carnation, tissue culture, cell activity studies, cytological studies, polyploid cells

Abstract

Cellular behaviour in primary roots of standard length 11.15 ± 0.33 mm from four-day-old seedlings of Dianthus caryophyllus Linn. cv. Grenadin were studied in vivo and in vitro. Parameters such as mitotic index (MI), chromosome count, the ploidy level, the mean cell and nuclear areas and the cell doubling time (Cdt) were determined from 2 mm segments of root tips. The MI value of the root segment decreased when transferred from in vivo to Murashige and Skoog (MS) medium supplemented with 2.0 mg/1 NAA. The mean chromosome count were generally stable during 6 months in culture period with 2n=2x=30 within the range of   25-33 chromosomes per cell.   The ploidy level increased with high percentage of polyploid cells during 6 months in culture. Most of the cells also accumulated in G2-phase of the cell cycle. The mean cell and nuclear areas were stable for the initial 2 weeks and then dropped after 4 weeks until 6 months in culture, The cell doubling time increased to 96.07 h in 3 weeks culture while Cdt in vivo was only 66.11 h. In tissue culture technique, nodal stem and shoot were the most responsive explants to regenerate multiple shoots compared to stem, leaf and root explants. Combination of 0.5 mg/1 NAA and 1.5 mg/1 BAP, pH 5.8, 30 g/1 sucrose, 8 g/1 agar, temperature 25 ± 1 °C and photoperiod of 16 h light and 8 h darkness were suitable for carnation regeneration. Multiple shoots were also, produced when explants from shoot and nodal stem were cultured in liquid medium, 1/3 MS medium supplemented with 30 g/1 sucrose. The concentration of 30 g/1 sucrose could prevent vitrification problems which often happened in carnation in vitro. Multiple shoots were able to regenerate better on MS medium compared to Nistch and Nistch (NN) medium. The presence of NAA hormone was more capable of inducing roots compared with IAA and 2, 4-D. BAP hormone obviously could enhance the frequency of shoot regenerations than 2iP, kinetin and zeatin. Acclimatization was successfully done and the plantlets obtained could survive in the glasshouse. Shoot and nodal stem explants on MS supplemented with '2.0 mg/1 NAA could produce multiple shoots. Cytological studies on these regenerative tissues had been done and compared with non-regenerative tissues from root, leaf and stem explants. The MI value in roots of both tissues were high, i.e., 36-43%. But then, the MI decreased during culture. The mean chromosome was more or less 28.3 chromosomes per cell. The differences between regenerative and non-regenerative tissues wrere the percentage of S-phase and G2-phase of the cell cycle. In non-regenerative tissues, the percentage of S-phase and G-2 phase were higher. The percentage of polyploid cells in regenerative and non-regenerative tissues were obviously lower than in vivo cells. Nevertheless, the regenerative tissues had more polyploid cells compared to non-regenerative tissues. The mean cell and nuclear areas of regenerative tissues obtained were larger than non-regenerative. The Cdt of regenerative tissues, e.g., shoot was only'62.8 h and non-regenerative tissues , e.g., stem was 155.8 h.  Perhaps, the ability of regenerative tissues to divide faster than non-regenerative explained their ability to produced multiple shoots. The interesting fact about regeneration of Dianthus caryophyllus Linn. cv. Grenadin is that although high ploidy level was detected in regenerative cells, the tissues could still regenerate into complete plants. It shows that this species maintained the polyploid cells from in vivo to in vitro plantlets. The regenerants were successfully transferred to the soil.