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Varietal Improvement and Multiplication of Tea Through Tissue Culture Techniques

Shah Kamal Lanchue Haque
Senior Scientific Officer
Botany Division Bangladesh Tea Research Institute Srimongal-3210

Dr. M A Samad
Senior Scientific Officer
Bangladesh Institute of Nuclear Agriculture P.O. Box - 4, Mymensingh

Abstract/ Executive Summary:

Shoot tip and stem segment explants of nine tea (Camellia sinensis) clones were cultured in vitro. Regeneration of plantlets from shoot tip explant was observed in MS media supplemented with 1.0 mg/l Kn + 1.0 mg/l IAA, 1.5 mg/l Kn + 1.5 mg/l IAA and 1.0 mg/l BAP + 1.0 mg/l IAA. Plantlets obtained in regenerating media were subsequently transferred to rooting media containing several combinations of ½ MS IBA and IAA but no root initiation was observed.

Callus was obtained from stem segment when MS media were supplemented with 2 mg/l 2,4-D, 1 mg/l 2,4-D + 300 CH and 1.5 mg/l 2,4-D + 300 CH. Higher concentrations of 2,4-D were found to inhibit callus induction. The calli thus obtained were transferred to regenerating media containing several combinations of BAP, NAA and Kinetin but no organogenesis was observed.

Callus induction from the anthers of clone BT2 and BT6 was found when cultured in MS nutrient salts and vitamins of N6 medium supplemented with 1.5 mg/l 2,4-D + 2.0 mg/l BAP; 1.5 mg/l 2,4-D + 2.5 mg/l BAP; 1.0 mg/l 2,4-D + 2.0 mg/l Kn and 1.5 mg/l 2,4-D + 2.5 mg/l Kn.

Keywords: In vitro, microspores, calli, anthers and tea

Location: Bangladesh Tea Research Institute, Srimangal-3210, Moulvibazar and Bangladesh Institute of Nuclear Agriculture, Mymensingh

Start Date: 01-11-1997

End Date: 31-10-2000

Program Area: Variety and Species

Commodity: Tea

Objectives:

Rapid multiplication of the superior tea plants which have high yielding characteristics as well as better cup quality.
Selection of desirable plants through screening by exploiting somaclonal variation.
Development of homozygous diploid plants derived through anther culture.

Methodology:

Shoot tips and stem segments of nine BTRI released clones were taken as source of in vitro propagation. Shoot tips about 5 mm were cut. The explants were surface sterilized by successive treatments with 70% ethanol for 2 minutes followed by the treatment of 0.2% mercuric chloride solution for 15 minutes. Then the treated explants were rinsed with sterile distilled water 4-5 times and aseptically placed in 30 × 150 mm test tubes each containing 15 ml of the MS medium with different concentration of hormone supplements.

On the other hand, stem segments were collected from the growing shoots of tea plants (between the 4^th-8^th leaf) and were cut into small pieces, the length of which was about 2 mm. the explants were then surface sterilized by agitating with a solution of 7% calcium hypochloride and 0.01% Tween-20 for 15 minutes. Then the explants were rinsed in sterile distilled water changing it 4-5 times. The explants were placed in MS medium with different combination of growth hormones.

Flower buds of clones BT1, BT2, BT3, BT4, BT5, BT6 and BT10 were collected for anther culture. Buds were washed in running water for one hour and soaked in 0.1% Tween-20 for 2-3 minutes under gentle shaking. Buds were then rinsed repeatedly with distilled water to remove detergent and treated with 0.1% Mercuric chloride solution for 15 minutes and finally, washed 3-4 times with distilled water again. Anther was excised carefully from the buds with the help of a needle taking all precautions so that filaments are totally excised. Anthers containing pollen in the mid and late uninucleate stage were plated in the test tubes for callus induction. Five to seven anthers were inoculated in each tube containing the MS salt and N6 vitamins. 6% sucrose and different concentrations of auxins and cytokinins.

Source of Information: Tea Journal of Bangladesh, Vol. 37 (1&2), 2001; Tea Journal of Bangladesh, Vol. 38 (1&2), 2002 and Review 1983-2008, Bangladesh Tea Research Institute, Srimongal-3210.

Article Link (Online Journal):

Funding Source:

1. Bangladesh Agricultural Research Council

Budget: 729,000.00

Total Budget (Tk.) : 729,000.00

Achievement/Findings:

Multiple shoot regeneration was found from clones BT2, BT6 and BT10 when MS + 1.0 mg/l BAP + 1.0 mg/l IAA was used. Regeneration percentage was higher in BT2 followed by BT10 and BT6. Regeneration was not found in case of clone BT1 and BT5.

Callus formation from the stem culture of clones BT1, BT2, BT5, BT6 and BT10 was recorded when MS media was supplemented with 2.0 mg/l 2,4-D. The range of callus formation was 8% to 30%. The percentage of callus formation was the highest in BT2 while it was the lowest in case of BT1.

Uninucleate stage of the pollen was found to be best materials for haploid calli induction. Callus formation was also observed from the plated anthers of clones BT2 and BT6 when 1.5 mg/l 2,4-D + 2.0 mg/l BAP; 1.5 mg/l 2,4-D + 2.5 mg/l BAP; 1.0 mg/l 2,4-D + 2.0 mg/l Kn and 1.5 mg/l 2,4-D + 2.5 mg/l Kn were used. The range of callus formation was15 to 20%. Higher concentration of 2,4-D was found to inhibit callus induction.

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