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Callus Induction and Plantlet Regeneration from Shoot Tip Explants in Tomato (solanum Lycopersicum L.)

Meherunnesa Papry
Department of Horticulture, Patuakhali Science and Technology University, Bangladesh

S. M. Ahsan
Department of Horticulture, Patuakhali Science and Technology University, Bangladesh

Md. Jamil Hossain Biswas
Department of Entomology, Bangladesh Agricultural University, Mymensingh Bangladesh

Soleh Akram
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh Bangladesh

Maria Akter Sathi
Department of Horticulture, Patuakhali Science and Technology University, Bangladesh

Abstract/ Executive Summary:

The experiment was conducted on in vitro regeneration of tomato at the Plant Biotechnology Laboratory from May 2013 to April 2014, Department of Horticulture, Patuakhali Science and Technology University, Patuakhali. The objective was to develop an efficient regeneration protocol in tomato through callus induction for subsequent plantlet regeneration from shoot tip explants. Seeds were inoculated on MS medium where germination rate was 78.4%. The shoot tips of in vitrocultured seedlings were used as explants. Different concentration and combination of growth regulators were added to MS medium to observe their efficacy on callus induction, shoot initiation and root formation. Shoot tip explants cultured on MS medium fortified with 2 mg/L BAP gave the highest number of shoots (3.25) at 45 DAC. Among the concentrations of PGRs, 0.25 mg/L IAA produced the highest length (4.685 cm) of plantlets, number(5.25) of leaves and fresh weight (0.7130 g) of plantlets with the shoot tips explants at 45 DAC. The concentration of 0.5 mg/L IAA produced the highest number (23.50) of roots/plantlet, length (8.004 cm) of roots at 45 DAC, from the same explants. The highest survival rate of in vitro regenerated plantlets in the pot was 70.00 % with the shoot tips explants.

Keywords: Culture medium, Tomato, Callus, Plantlet, Explants

Location: Plant Biotechnology Laboratory of the Department of Horticulture, Patuakhali Science and Technology University, Patuakhali

Start Date: 00-05-2013

End Date: 00-04-2014

Program Area: Variety and Species

Commodity: Tomato

Objectives:

Objective of the study was to develop an efficient regeneration protocol in tomato through callus induction for subsequent plantlet regeneration from shoot tip explants.

Methodology:

The present research work was conducted at the Plant Biotechnology Laboratory, Department of Horticulture, Patuakhali Science and Technology University. The seeds were collected from the Regional Horticulture Research Station (RHRS), Lebukhali, Patuakhali. Winter variety of BARI tomato-14 was used as the plant material. Shoot tip from in vitro grown tomato plants were cultured on MS medium and used as explants. pH of the medium was adjusted to 5.8 with 0.1 N NaOH or 0.1 N HCI. All the media were autoclaved for 20 minutes with 15 psi at 121°C. Collected tomato seeds were washed in tap water and surface sterilized with 70% ethanol for five minutes with vigorous shaking followed by washing with sterile distilled water, surface disinfected with Sodium hypochlorite 5.25% for 10 minutes and rinsed 4-5 times with sterile water. Then they were washed with Tween 20 for 2-3 minutes and rinsed with sterilized water till the foam was completely removed. The surface sterilized seeds were then allowed to soak overnight to break dormancy. Then the seeds were placed in test tubes containing 10 ml MS medium and later transferred to growth room at 25 ± 1°C temperature under 16 hours photoperiod with a light intensity 1500 lux and relative humidity 60-70%. Sterilized seeds were placed onto seed germination medium in test tubes. In each test tube, 4 seeds were inoculated. The culture was then incubated in incubation room till the germination of seeds. It was noticed that seeds started growing in dark and later they were transferred to light. Thirty days old seedlings were used as the source of explants. Callus proliferation: The seedlings raised in vitro culture were used as the source of shoot tip explants. Excised shoot tips were placed on the sterile culture medium with various concentration and combination of BAP (1, 2, 3 mg/L) and NAA (0.25, 0.5 mg/L) and subsequent fresh weight and dry weight of the callus and changes in colors were recorded visually after 15, 30 and 45 DAC. Dry weight of the callus: The calli were kept in an oven (Model no.: NIIVE FN-400) for drying for 72 hours at 50^oC after taking fresh weights. After 72 hours, dried calli were weighed. Subculture of the callus for shoot regeneration: When the calli turned into green to yellow color, those were removed aseptically. The pieces were again cultured on freshly prepared medium supplemented with 0, 0.5, 1, 2 and 3 mg/L BAP for shoot induction from callus and subsequent fresh weight and number of shoot were recorded after 15, 30 and 45 DAC. Number of shoots/plantlet: The number of shoots emerged in each cultured bottle was calculated by counting the number of shoots emerged. The data were recorded at 15 days of interval up to 45 days of culture and the means were calculated. In vitro plantlet regeneration with leaves: Initially 1.5 cm of plantlets were transferred to ½ strength MS media containing 0.0, 0.1, 0.25, 0.50, 1.0 mg/L IAA and average length of plantlets and number of leaves per plantlet were counted at 15, 30 and 45 DAC. Subculture of the shoots for root induction: The sub cultured calli continued to proliferate and differentiated into shoots. When these shoots grew about 2-3 cm in length, those were rescued aseptically from the bottle and were separated from each other and again cultured on freshly prepared half strength MS medium containing 0.0, 0.1, 0.25, 0.50, 1.0 mg/L IAA supplements for root induction. Plantlets of the 5-7 cm length with well developed roots were removed from culture vessel with the forceps and transferred into a small pot containing prepared soil. The plantlets established within 5 to 7 days and the polythene bags were removed. Statistical analysis: Data collected on different parameters under study were statistically analyzed to ascertain the significance of the experimental results. The Analysis of Variance was performed and means were compared by Least Significant Difference (LSD) test for interpretation of results. The significance of the difference between the pair of means was evaluated using MSTAT-C computer package programs.

Source of Information: International Journal of Business, Social And Scientific Research, Volume: 4, Issue: 1, Page: 50-59, 2015

Article Link (Online Journal):

Funding Source:

1. Government

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Results of these experiments show the influence and importance of growth regulators on the number of shoots and roots regenerated from tomato shoot tips explants. The in-vitromorphogenic responses of invitro cultured plant tissues are therefore affected by the different components of the culture media, especially by concentration of growth hormones. These responses are also dependent on cultivar and explants types.

Knowledge Management: Journal