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In Vitro Propagation of Banana (musa Sp. Cv. Kanthali): an Endogenous Variety of Bangladesh with Controlling Bacterial Contaminations

Sarder Nasir Uddin
Biotechnology & Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh.

Soubir Titov
Biotechnology & Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh.

Md. Abdul Wadud
Biotechnology & Genetic Engineering Discipline, Khulna University, Khulna-9208, Bangladesh.

Abstract/ Executive Summary:

Shoot tips of a traditional table banana [Musa sp. cv. Kanthali (Genome AAB)] of Bangladesh were evaluated for in vitro propagation. Initial surface sterilization (with 0.1% HgCl₂ for 12 min) of shoot tips was successful but microbial contamination (mostly bacteria) at the rhizomatous base of the explants were observed within 6-15 days after inoculation which eventually killed 85% inoculated explants. So, for contamination free culture establishment explants were soaked in two broad spectrum antibiotics namely ampicillin and gentamicin. Cent percent contamination free cultures were established by soaking the explants in 400 mg L^-1 ampicillin or 200 mg L^-1 gentamicin for 1 h. Antibiotic treated explants were found to be full contamination free but failed to regenerate after 3 weeks of culture. But some of them absorbed media for up to 2nd subculture and showed swelling of explants and some color changes from pale white to light/deep green. Finally, a few days after 3rd subculture, no growth of explants was observed and all treated explants eventually started to die. Among the untreated alive explants the best medium for single shoot development was MS + 4.0 mg L^-1 BA + 0.5 mg L^-1 Kn + 15% CW and average time required for shoot development was 18-21 days. But the regeneration percentage was very low (30%). The best medium for shoot multiplication was MS + 4.0 mg L^-1 BA + 2.0 mg L^-1 IAA + 15% CW and average time required for production of multiple shoots from single shoot was 40-45 days. Multiplication rate was also too low (40%) and only average 3-4 shoots were formed. Finally, in vitro proliferated shoots produced roots with maximum frequency (90%) in half strength of MS medium fortified with 0.5 mg L^-1 IBA.

Keywords: Banana, Kanthali, In-vitro, Shoot tip, Antibiotic

Location: Batiaghata Thana, Khulna

Start Date: 00-00-2005

End Date: 00-00-2005

Program Area: Variety and Species

Commodity: Diseases, Banana, Diseases, Banana

Objectives:

To study traditional table banana (Musa sp. Cv. Kanthali) for in vitro Propagation.

Methodology:

Banana cv. Kanthali (Genome AAB), a leading traditional cultivar of Bangladesh was used as the source material for culture establishment. The shoot tips along with a portion of rhizomatous tissue were used. The plants were collected from a village named Amtola in Batiaghata Thana, Khulna and was very near from Khulna University campus in early of 2005. The explants were prepared by removing the outer layer of tissues from suckers with a clean knife. The pale white tissue blocks containing the shoot tip and rhizomatous bases were surface sterilized with 0.1 % HgCI₂ for 12 min. Microbial contamination (mostly bacteria) at the rhizomatous base of the explants was observed within 6-15 days after inoculation which eventually killed 85% inoculated explants. So, for contamination free culture establishment explants were soaked in two broad spectrum antibiotics namely ampicillin and gentamicin. Cent percent contamination free cultures were established by soaking the explants in 400 mg L^-1 ampicillin or 200 mg L^-1 gentamicin for 1 h. After the treatment with antibiotics the shoot tips of banana were inoculated in MS medium with varying concentrations of hormonal supplements for single shoot regeneration. But all of them completely failed to regenerate. But some of them absorbed media for up to 2nd subculture and showed swelling of explants and some color changes from pale white to light/deep green Finally a few days after 3rd subculture, no growth of explants was observed and all treated explants eventually stared to die. From the untreated alive explants, in all concentrations of BA tissue swelling and ball like structures were formed. But the control didn't respond at all. Highest percentage (40%) of single shoot was formed when explants were cultured on MS + 4.0 mg L^-1 BA + 0.5 mg L^-1 Kn + 15% CW and the average length of shoot was 4.5±0.37 cm. These single shoots were further subculture on different concentrations of cytokinin (BA), auxin (IAA) and 15% CW for multiplication of regenerated shoots. The growth rate was very slow and 40-45 days after inoculation only highest 3-4 shoots were produced in MS medium containing 4.0 mg L^-1 BA + 2.0 mg L^-1 IAA + 15% CW. Finally, in vitro proliferated shoots produced roots with maximum frequency (90%) in half strength of MS medium fortified with 0.5 mg L^-1 IBA. All inoculations and aseptic manipulations were carried out in a laminar air flow cabinet. The micro-air-flow was switched on 30 min. ago before use the area. At the time of working the cabinet was cleaned with 90% ethanol to reduce the chances of contamination. The instruments like scalpels, forceps, needles etc. were sterilized by an alcoholic dip and flaming method inside the laminar air flow chamber. Other requirements like Petri dish, bottles, conical flask, cotton, distilled water etc. was sterilized by steam sterilization method. Before the onset of inoculation hands were cleaned thoroughly by soap and then by spraying 99% absolute ethyl alcohol. Surgical operations were carried out taking all possible care to ensure contamination free condition.

Source of Information: American Journal of Plant Physiology, Year: 2006, Volume: 1, Issue: 2, Page No.: 169-176, ISSN 1557-4539

Article Link (Online Journal): DOI: 10.3923/ajpp.2006.169.176

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Shoot tips of banana (Musa sp. cv. Kanthali) were evaluated for in vitro propagation. The initial surface sterilization (with 0.1 % HgCl₂ for 12 min) was successful but microbial contamination at the base of the explant was observed within 6-15 days after inoculation which eventually killed 85% inoculated explants. So, for contamination free culture establishment explants were soaked in 200 mg L^-1 gentamicin or 400 mg L^-1 ampicillin for 1 h. Antibiotic treated explants found to be full contamination free but failed to regenerate after 3 weeks of culture. But some of them absorbed media for up to 2^nd subculture and showed swelling of explants and some color changes from pale white to light/deep green. Finally a few days after 3rd subculture, no growth of explants was observed and all treated explants eventually stared to die. Among the untreated alived explants the best medium for single shoot development was MS + 4.0 mg L^-1 BA + 0.5 mg L^-1 Kn + 15% CW and average time required for shoot development was 18- 21 days. But the regeneration percentage was very low. The best medium for shoot multiplication was MS + 4.0 mg L^-1 BA + 2.0 mg L ^-1 lAA + 15% CW and average time required for production of multiple shoots from single shoot were 40-45 days. Multiplication rate was also too low and only average 3-4 shoots were formed in that particular concentration. The in vitro proliferated shoots produced roots with maximum frequency in half strength of MS medium fortified with 0.5 mg L ^-1 IBA. The interest in Musa sp. cv. Kanthali both for domestic and export sales would be increased in future. This banana cultivar has a restricted distribution (only southern part of Bangladesh) which has serious ecological implications as all plant material is harvested from wild populations. It is essential that an appropriate method to expedite introduction to cultivation be determined. The success of in vitro propagation methods reported that the experimental plant can be tissue cultured with further plant growth regulators experimentation. Successful micropropagation will be of major benefit to the Agricultural Biotechnology making conventional breeding method unnecessary thus greatly assisting in the conservation of this unique Bangladeshi plant.

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