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In Vitro Regeneration of Popular Tobacco Varieties of Bangladesh from Leaf Disc

M. A. RAHMAN
Department of Biotechnology, Bangladesh Agricultural University

M. A. ALAM
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh

M. R. HOSSAIN
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh

A. HOSSAIN
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh

R. AFROZ
Scientific Officer
Bangladesh Agricultural Research Institute, Gazipur

Abstract/ Executive Summary:

Regeneration ability of five Nicotiana varieties viz., Virginia, Jati, Motihari, CC Bengal and Sumatra were investigated via callus induction using leaf discs. Explants were cultured on MS medium supplemented with different concentrations and combinations of plant growth regulators. Callus formation frequency was 67.20%. Among the varieties used, Motihari induced the highest percentage (97.50%) of callus followed by Jati (92.50%) in 2.0 rng/L Kinetin and 2.0 mg/L IAA. Shoots were induced from calli cultured on the same medium. Maximum shoot formation from leaf discs was 82.50% on medium supplemented with 2.0 mg!L Kinetin and 2.0 mg!L IAA. It was also revealed from this study that Motihari was the best variety for callus formation and subsequent plantlet regeneration which is a pre-requisite for vector mediated transformation for varietal improvement of Nicotiana species. The rooting response of regenerated shoots was observed by using 1/ 2 MS medium with IBA (0.0, 0.5, and 1.0 mg/L). The highest root formation was found in Motihari (90%) with 1/2 MS medium supplemented with 0.5 mg/L IBA. After that regenerated plantlets with plenty of roots were transferred successfully to pots and subsequently to the field.

Keywords: Tobacco, Nicotiana, in vitro regeneration, callus induction, plantlet regeneration, leaf disc, phytohormone.

Location: Mymensingh

Start Date: 23-02-2004

End Date: 21-12-2004

Program Area: Variety and Species

Commodity: Tobacco

Objectives:

To standardize the different hormonal concentrations for tobacco callus ogenesis and plantlet regeneration using leaf disc to observe the callus induction potentiality in five tobacco cultivars.

Methodology:

The seed materials of five tobacco cultivars viz., Virginia, Jati, Motihari, CC Bengal, and Sumatra were used in this study. Different culture media were used in the present investigation for various purposes. MS (Murashige and Skoog, 1962) medium containing 1.0, 1.5, 2.0, 2.5, and 3.0 mg/L kinetin in association with 2.0 mg/L was used for callus induction and subsequent plantlet regeneration. Hormone free 1 MS medium and 1 MS medium with IDA (0.5 and 1.0 mg/L) were used to observe the rooting responses of regenerated shoots.When the plantlets became 5-8 em in height with sufficient root system, they were taken out from the vials. Medium attached to the roots was gently washed out running tap water. The plantlets were then transplanted to pot containing potting mixture mentioned above. Immediately after transplantation, the plants alon g with the pots were covered with moist polythene bag to prevent dcsccat.on. 'To reduce sudden shock, the pots were kept in a growth room for 7- 15 days u der .c ntrolled environments. The interior of the polythene bags was sprayed With distllled water at every 24 hrs to maintain high humidity around the plantIets. At the same time, plantlets were also nourished with Hogland's solution. After two to three days, the polythene bags were gradually perforated to expose the plants to natural environment. The polythene bags were completely removed after fifteen days when the plantlets appeared to be self sustainable. At this stage, the plantlets were placed in natural environment for 3- 10 hours daily. Finally, after 15-20 days, they were transferred to the field.

Source of Information: Bangladesh J. Agril. Res. 35(1) : 125-134, March 2010

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

After satisfactory development in root system, the small plantlets were removed from the culture media and transplanted in small plastic pots containing sterile soil, sand, and cowdung in a 1:2:1 ratio in growth chamber for proper hardening of the plantlets (Plate-5) and finally those were planted in earthen pots (Plate 6). Gradually, the plantlets were adapted to the soil. The survival rate of plantlets of Virginia, Motihari, Jati, CC Bengal, and Sumatra were 75.00, 77.77, 71.42, 62.50 and 57.14% in the pots and 83.33, 85.00, 80.00, 60.00, and 50.00% in the soil, respectively. The survived plants then showed vigorous growth with proper leaf development. Comparative survivability of regeneration obtained from leaf discs of five genotypes of Nicotiana after transferring them in pot and in soil are shown in the Table 5.A wide range of variation m in vitro regeneration potentiality of the genotypes was observed on different treatment combinations on MS media. Induced calli ranged from 35-100%. Average callusing was the highest in Motihari (77.50%) followed by Jati (72.50%), Virginia (67.50%), CC Bengal (57.50%), and Sumatra (48.50%). Callusing was also highest (84.50%) in MS + 2.0 mg!L Kinetin+ 2.0 mg/L IAA (T₃) and the lowest in MS + 1.0 mg/L Kinetin + 2.0 mg!L IAA (T₁ ). Responses of shoot formation of different genotypes were different. Among the investigated genotypes, the regeneration performances were found better in Motihari (87.50%) followed by Jati (77.50%) in T₃ (MS + 2.0 mg/L Kinetin + 2.0 mg/L IAA). Therefore, considering callus induction, shoot regeneration and rooting performances, Motihari was found more potential for in vitro regeneration than the other four varieties.

Knowledge Management: Journal