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Somatic Embryogenesis of Musa Sp. Cv. Kanthali Using Floral Bud Explants

Sarder Nasir Uddin
Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna, Bangladesh.

Soubir Titov
Biotechnology and Genetic Engineering Discipline, Khulna University, Khulna, Bangladesh.

Abstract/ Executive Summary:

A study was undertaken to examine banana (Musa sp. cv. Kanthali) floral bud apex as an alternative source material for in vitro propagation as because huge number of explants die due to microbial contamination in case of shoot tip explants. Contamination free cultures were established by treating the floral bud explants with 0.1% HgCl₂ for 6 min. For reducing phenolic compounds secretion, explants were treated with 0.125% potassium citrate: citrate solution. Compact, white/greenish white callus was formed in different amount at all concentrations of BA+Kn+IAA+15% CW and BA+Kn+IBA+15% CW from terminal floral bud explants after 3 weeks of culture. All were again subcultured at same medium and after another 30-35 days at 2.0 mg L^-1 BA+2.0 mg L^-1 Kn+2.0 mg L^-1 IAA+15% CW some callus showed embryogenic structure.

Keywords: Musa sp. cv. Kanthali, Floral bud apex, Antioxidant, Callus, Somatic embryo

Location: Amtola of Batiaghata Upazila, Khulna

Start Date:

End Date:

Program Area: Quality and Nutrition

Commodity: Banana

Objectives:

To establish in vitro rapid clonal propagation of banana Musa sp. cv. Kanthali from its floral bud apices.

Methodology:

Banana cv. Kanthali (Genome AAB), a Cavendish type leading traditional cultivar of southern part of Bangladesh was the investigating subject. The source material used for culture establishment was banana floral bud explants. The plant materials were collected from a village named Amtola in Batiaghata Thana, Khulna and was very near from Khulna University campus. Terminal floral apices of banana were collected from mature plants after they produced all possible hands. The terminal bud was cut from the peduncle and the bracts with their associated hands of male flowers were removed in a stepwise manner until they became too small to remove by hand. Working with a dissecting microscope, scalpel and forceps, the remaining bracts and minute hand of flowers were removed until the rounded growing point was exposed. The floral apex and approximately 1 cm long subtending peduncle tissue were excised. The surface sterilization procedure began with dissection of explant material into manageable units. Stem sections containing axillary buds and immature inflorescences were treated by initially removing the small leaflets and cleaning away surface detritus under running tap water for 1 to 2 min. A plastic vessel (130x320x120 mm) was used for treatments with sterilant solution. Sterilization was undertaken for 6 min using 0.1% (w/v) HgCl₂. Explants were transferred to a separate vessel for the washing phase in three changes of sterile distilled water. One of the most common problems associated with the in vitro establishment of many monocotyledonous and woody species is the deleterious effects of oxidized phenols. The results of the detection of phenolic compounds experiment clearly indicate that Musa sp. cv. Kanthali has phenolic compounds present in inflorescence tissue. This experiment provides a simple technique for detecting the presence of phenolic compounds in banana inflorescence tissue. This procedure assists in early detection of phenols and assists in preparation of explant source material to reduce injury associated with phenolic oxidation. Antioxidants containing citrate and ascorbate reduce browning of tissue by detoxifying these free radicals. The results provide evidence that K-C: C combination is a useful antioxidant for explant preparation for Musa sp. cv. Kanthali. Musa sp. cv. Kanthali stems are susceptible to tissue browning and elimination or minimization of this process is an essential prerequisite to successful culture establishment. The extracted buds were placed in petri dishes containing an antioxidant wash of 0.125% potassium citrate: citrate (K-C: C in a ratio of 4:1 w/w) solution. A concentrated stock of the antioxidant wash was filter sterilized (with 0.22 μM disposable filter) and frozen in 10 mL units until required. The concentrate was later thawed and further diluted with SDW to give the final 0.125% concentration. Petri dishes (90 x 14 mm) were filled with sufficient antioxidant solution to fully cover the explants. Peduncle sections were cut into discs under the antioxidant solution to minimize browning during initial preparation. Each explant was placed in a test tube containing 20 mL of media after five min in the antioxidant treatment. After antioxidant treatment the floral bud explants of banana were cultured on MS solid medium supplemented with cytokinins, auxins and coconut water for initiating vegetative growth. After 3 weeks of culture compact, white/greenish white callus was formed more or less at all treatments. All were again sub cultured at same medium and after another 30-35 days at a specific concentration some callus showed embryo genic structure but others remained unchanged. These were observed at the conclusion of this research and were not able to be further analyzed.

Source of Information: Journal of Plant Sciences, 2(1): 35-44, 2007, ISSN 1816-4951.

Article Link (Online Journal): DOI: 10.3923/jps.2007.35.44; URL: http://scialert.net/abstract/?doi=jps.2007.35.44

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The inflorescence tissues of experimental plant was high in phenolic compounds. The oxidation of tissues was severe and proved deleterious to all tissues in the initial stages of explant preparation. For successful explant establishment, a wide range of cytokinin and auxin combinations were investigated. The floral bud apices were cultured for 3 weeks on MS basal medium supplemented with various concentrations and combinations of cytokinins, auxin and additives. Hard, compact, white/greenish white callus was formed in different amounts at all concentrations of BA+Kn+IAA+15% CW. All were again subcultured at same medium and after another 30-35 days at 2.0 mg L^-1 BA+2.0 mg L^-1 Kn+2.0 mg L^-1 IAA+15% CW some callus showed embryo genic structure.

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