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Optimizing the Initial Steps of Immature Endosperm Culture of Seeded Banana (musa Sapientum L.) Cultivar Bhutia of Bangladesh

P.R. Paul
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

A.H.K. Robin*
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

M.R. Hossain
Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Abstract/ Executive Summary:

The local Bangladeshi banana varieties, possessing similar brix percentage to that of commercial varieties, grow well under adverse conditions with minimum care but are less popular due to the presence of seed. Endosperm culture of seeded banana can produce triploid seedless varieties which can be cultivated commercially in unsuited environments with less agricultural inputs. The current study was conducted to optimize the initial steps of endosperm culture using the immature endosperm of seeded banana cultivar Bhutia. Young fruits at various stages were collected from the local banana gardens to find out suitable developmental stage of endosperm for culture. Endosperms of juvenile fruits at 25 days age, exhibited jelly state, was selected for culture because endosperm explants at that age survived the most in MS medium. It was observed that non-treated explants produced larger calli comparatively quickly than that of cold-treated explants. Largest calli (0.41 cm) within shorter time period (27 days after inoculation) was produced in MS medium additionally supplemented with 0.5 ppm 1-Naphthaleneacetic acid (NAA) besides 0.5 ppm 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 ppm Kinetin (Kn). The produced calli gradually became blackish in appearance and higher ascorbic acid content (480 mg/100g) was observed in blackish calli. Avoiding the blackening of calli derived from endosperm of seeded banana would be a challenge to establish a successful triploid production protocol in future.

Keywords: Musa sapientum L., Seeded banana, Endosperm culture, Seedlessness, Triploid, Callus blackening, Cold treatment

Location: At the tissue culture laboratory of the Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh, Bangladesh

Start Date: 00-04-2013

End Date: 00-10-2014

Program Area: Resource Development and Management

Commodity: Banana

Objectives:

The present study was planned to optimize the initial steps of culturing endosperm of a localseeded banana variety of Bangladesh.

Methodology:

The present study was carried out during the period from April, 2013 to October, 2014 at the tissue culture laboratory of the Department of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh, Bangladesh. To compare fruit size, brix percent, percent pulp occupied by seeds in seeded banana variety ‘Bhutia’ with the two most popular varieties: ‘Sagor’ and ‘Sabri’, five ripe and randomly chosen fruits were collected from the local market. Individual fruit peel, pulp and seed were collected and their dry weights were recorded. The brix percentage was also measured using a refractometer. This comparative study of the fruit size, seed content and brix percentage was conducted to show that the seeded banana has immense potential in terms of its quality as compared to the existing popular seedless varieties of Bangladesh. Plant material: Seeded green bananas (local name: Bhutia kola, kola is the Bengali name of Banana) at 15, 20, 25 and 30 days of age (days after flowering) were collected to isolate immature endosperm. The seeded banana was used to optimize initial steps of endosperm culture. Developing endosperm at 15, 20 and 25 days of fruit age was cultured in MS medium to observe their survivability. The fruits of 15, 20, 25 and 30 days of age were cross-sectioned and the developmental stages of endosperm were visually observed. Cold treatment of banana: A set of 10 banana fruits were kept at 4 ^o C for 72 hours while another set of 10 fruits were non-treated. Media preparation: Two different hormone compositions such as: i) MS medium (Murashige and Skoog, 1962) containing 0.5 ppm 2,4-D + 0.5 ppm Kn (Treatment 1) and ii) MS medium containing 0.5 ppm 2,4-D + 0.5 ppm NAA + 0.5 ppm Kn (Treatment 2) were used for culturing endosperm of seeded banana. Sterilization of explants: Surface sterilization of seeded banana fruits was carried out under a Laminar Air Flow Cabinet. Fruits were rinsed in 70% ethanol for 2 minutes. Ethanol treated banana was immersed into 0.1% HgCl2 solution for 3 min followed by 3 rinses in sterile distilled water to remove traces of HgCl2 which would be toxic to the explants if kept for longer duration. Inoculation and culture of endosperm: The selected seeded bananas were cut into slices to expose the developing seeds. Endosperm at jelly-state was aseptically removed from banana seeds using a scalpel and a fine forceps and inoculated into vials each containing 25 mL MS medium with desired hormone concentrations. The inoculated culture vials were kept at 25^o C temperature, 1000 lux light intensity with a 16:8 hours day:night cycle. A convenient relative humidity and restricted access were also maintained in the culture room. The endosperms under incubations were monitored regularly and any contaminations were removed immediately. The experiment was conducted following a completely randomized design with 10 replicates for two hormone compositions for both cold-treated and non-treated explants. Days to callus formation, size of callus, colour of callus and percent survival of callus were recorded. Estimation of ascorbic acid: Ascorbic acid content of five replicates of each of the the blackish callus produced from the endosperm culture of seeded banana and the white calli produced from the garlic was measured (Plummer, 1971). Whitish calli was chosen as ‘control’ considering that those were not suffering from any oxidation whereas it was assumed that calli blackening was because of oxidative stress produced during culture. Statistical analysis: Data were analyzed using MINITAB 15 statistical software package (Minitab Inc., State College, Pennsylvania, USA). To find out variations among varieties, treatments imposed on explants, hormonal compositions and ascorbic acid content in calli separate one-way analysis of variance (ANOVA) was conducted. Tukey’s pairwise comparisons were conducted when mean difference was significant.

Source of Information: Journal of Applied Horticulture, 18(1): 34-38, 2016

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The study revealed that ‘jelly like’ endosperm explants from around 25 days old seeded banana fruits survived the best in MS culture medium. NAA supplemented MS medium produced calli of larger diameter comparatively quickly compared to the medium lacking NAA. Calli became blackish in appearance probably due to oxidative stress as indicated by higher ascorbic acid content in blackish calli compared to white calli. Furhter research is needed to avoid blackening of callus which is a challenge for establishing a complete triploid plantlet regeneration protocol through endosperm culture of seeded banana.

Knowledge Management: Journal