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Endogenous Bacterial Contamination During in Vitro Culture of Table Banana : Identification and Prevention

Ummey Habiba
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Sharmin Reza
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Mihir Lal Saha
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

M. R. Khan
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Syed Hadiuzzaman
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Abstract/ Executive Summary:

The best medium for single shoot development to obtain contamination free culture of the table bananas Musa sapientum cv. chini champa and sagar was MS + 4.0 mg/l BAP + 1.0 mg/l Kn. Average time required for shoot development was 15 - 21 days. The best medium for shoot multiplication was MS + 4.0 mg/l BAP + 2.0 mg/l IAA + 13% CW and average time required for production of multiple shoots from single shoot was 40 - 45 days. Half strength MS + 2.0 mg/l IBA was best for root induction in the regenerated shoots. Although initially surface sterilization was successful, microbial contamination at the base of the explant was observed within 7 - 15 days after inoculation which eventually killed the explants. Endogenous microorganisms, mostly bacteria, were observed under microscope. Altogether seven bacterial strains were isolated from the contaminated culture. Four of them were found to be gram positive and the rest were gram negative. The gram positive strains were Cellulomonas uda, C. flavigena, Corynebacterium paurometabolum and Bacillus megaterium. The gram negative strains were identified as Klebsiella sp., Erwinia cypripedii and Pseudomonas sp. All the strains were found to be susceptible to gentamicin. Cent per cent contamination free cultures were obtained by soaking the explants in 160 mg/l gentamicin for one hr and 40 min.

Keywords: Endogenous bacteria, In vitro culture, Table banana, Prevention

Location: Vawal Modhupur, Trishal, Mymensingh and from the botanical garden of Curzon Hall, Dhaka University, Dhaka.

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Banana

Objectives:

To isolation and characterization of these endogenous bacteria associated with the rhizome of banana and measure to control them.

Methodology:

The plants were collected from Vawal Modhupur, Trishal, Mymensingh and from the botanical garden of Curzon Hall. 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% HgCl₂ for 15 min. To determine the presence of endogenous bacteria transverse section of rhizome was observed under the microscope. In order to isolate the endogenous bacteria, surface sterilized explants were inoculated in MS medium. The developed bacterial contaminants were transferred to nutrient agar (NA) medium. All the isolated contaminants were purified by serial dilution technique. To test the CS the Kirby-Bauer method was followed. Mueller-Hinton agar and seven antibiotic disks viz. ampicillin, cephradin, chloramphenicol, gentamicin, vancomycine, tetracycline, doxycycline were used. Disks containing antibiotics were placed after inoculation of the test organisms. The inoculated plates were incubated at 37_C for 24 hrs. The developed inhibition zone around the disks were measured. In this test, the surface sterilized explants were immersed in screened antibiotics (ampicillin, gentamicin and tetracycline) for different durations of time to ensure contamination free cultures. These decapitated shoots were incised vertically and placed in MS medium with different concentrations of auxin and cytokinin. The materials were subcultured at 30 days interval in the same fresh medium for the production of multiple shoots. The regenerated shoots were cultured in rooting media containing half strength MS supplemented with different concentrations of IBA (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/l). After rooting the plantlets were transferred to small polythene bags containing loamy soil and cowdung (1 : 1) for hardening under cover. Finally the plantlets were transferred to the field.

Source of Information: Plant Tissue Cult. 12(2) : 117-124, 2002 (December)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The explants treated with antibiotic were cultured in MS supplemented with different concentrations of BAP and Kn. The best medium for single shoot development was MS + 4.0 mg/l BAP + 1.0 mg/l Kn and average time required was 15 - 21 days (Table 4). For the production of multiple shoots regenerated single shoots were cultured in MS with different concentrations of auxin and cytokinin. The highest number of shoots were produced on MS supplemented with 4.0 mg/l BAP + 2.0 mg/l IAA + 13 % CW and 5.0 mg/l BAP + 2.0 mg/l IAA + 2.0 mg/l IBA (Table 5). When MS medium with 5.0 mg/l BAP + 2.0 mg/l IAA + 2.0 mg/l IBA was used, a higher intensity of rooting was observed. As a result multiplication rate was decreased after subculture. Therefore, MS + 4.0 mg/l BAP + 2.0 mg/l IAA + 13 % CW was selected for the production of multiple shoots. 100 % healthy roots were induced from half strength MS with 2.0 mg/l IBA. The regenerated plantlets were successfully established in the soil. The gram negative strains were identified as Klebsiella sp., Erwinia cypripedii and Pseudomonas sp. All the strains were found to be susceptible to gentamicin. Cent percent contamination free cultures were obtained by soaking the explants in 160 mg/l gentamicin for one hr and 40 min.

Knowledge Management: Journal