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In Vitro Regeneration and Molecular Characterization of Some Varieties of Lycopersicon Esculentum Mill. in Bangladesh

M. Billah
Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia-7003, Bangladesh

T. A. Banu
Plant Tissue Culture Section, Biological Research Division, Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka 1205, Bangladesh

M. Islam
Plant Tissue Culture Section, Biological Research Division, Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka 1205, Bangladesh

N. A. Banu
Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia-7003, Bangladesh

S. Khan
Plant Tissue Culture Section, Biological Research Division, Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka 1205, Bangladesh

S. Akter
Plant Tissue Culture Section, Biological Research Division, Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka 1205, Bangladesh

A. Habib*
Plant Tissue Culture Section, Biological Research Division, Bangladesh Council of Scientific and Industrial Research (BCSIR), Dhaka 1205, Bangladesh

Abstract/ Executive Summary:

An efficient regeneration protocol was established for two varieties (BARI tomato-9 and BARI tomato-15) of tomato (Lycopersicon esculentum Mill.) using three explants namely cotyledonary node, cotyledonary leaf and hypocotyls. Among the three explants, maximum number of shoots was produced from cotyledonary leaf explants of BARI tomato-15 on MS with 2.0 mg/l BAP and 0.5 mg/l IAA. In this combination of BAP and IAA 86%, on an average, cotyledonary leaf explants showed regeneration response 14.12 shoots/explants. Explants from hypocotyl showed best results in MS medium with 2.0 mg/l BAP and 0.2 mg/l IAA in both the varieties. In case of cotyledonary node, BARI tomato-15 showed 6.0 shoot/explant on MS with 2.0 mg/l BAP and 1.0 mg/l IAA. Molecular characterization of total ten varieties of tomato in Bangladesh was done by using six arbitrary oligonucleotide RAPD primers. A total of 140 bands were produced where the highest genetic distance (0.6769) was found between BARI tomato-3 and Mintoo tomato and lowest distance (0.1035) was observed between BARI tomato-7 and BARI tomato-8. This result will be useful for designing future breeding programs.

Keywords: Lycopersicon esculentum; Leaf explants; Multiple shoot; RAPD analysis

Location: Biotechnology and Genetic Engineering, Islamic University, Kushtia-7003, Bangladesh

Start Date:

End Date:

Program Area: Development of Host and Medicinal Plants

Commodity: Medicinal Plants

Objectives:

The present study was conducted to test an efficient regeneration protocol and the genetic diversity of the local tomato varieties of Bangladesh using RAPD markers.

Methodology:

In this study, two varieties of tomato namely, BARI tomato-9 and BARI tomato-15 were used for in vitro regeneration. While for molecular characterization, 10 other varieties namely, BARI tomato-2, BARI tomato-3, BARI tomato-7, BARI tomato -8, BARI tomato -9, BARI tomato-14, BARI tomato-15, Mintoo tomato, Delta tomato and Sawsan tomato were used. For the preparation of explants surface sterilized procedure were followed according to the protocol describe by Khan et al. (2017). The sterilized seeds were then transferred in autoclaved cotton soaked bottle for in vitro germination and growth of seeds. Different explants such as cotyledonary leaf, cotyledonary node and hypocotyls were excised from 8-10 days old seedling and inoculated on MS (Murashige and Skoog, 1962) media containing BAP, Kn and IAA, singly or in combinations for in vitro regeneration of shoots. Cultures were sub-cultured to fresh media regularly, at an interval of three to four weeks for maintenance. All cultures were maintained under fuorescent light of 20,000-25,000 lux intensity on a 16/8 (light/dark) hours at 25 ± 2°C. For induction of roots, regenerated shoots (2.5 – 4.0 cm long) were excised and transferred to MS and half strength MS medium with 3% sucrose without hormonal supplements. The plantlets with well-developed root system were transplanted in sterilized soil in small pots. For RAPD analysis genomic DNA were isolated from fresh leaves of the 10 tomato varieties using CTAB method (Doyle and Doyle 1987). DNA is quanti?ed by 0.8% (W/V) agarose gel electrophoresis and spectrophotometer (Analylikjena, Specord 50, Germany), respectively (Naz et al. 2013). Six RAPD primers such as OPA-1, OPA-4, OPA-5, OPA-10, Primer-6, and Primer-12 were used for RAPD analysis of 10 tomato varieties. Polymerase chain reactions (PCRs) were performed in 25 µl of reaction mixture containing Taq buffer A 10x (10 mM Tris-HCl with1.5 mM MgCl2) 2.5 µl, primer (10 µM) 1.0 µl, dNTPs mix (10 mM) 0.5 µl, Taq DNA polymerase (5 U/µl) 0.2 µl, Template DNA(25 ng) 2 µl and sterile de-ionized distilled water 18.8 µl. DNA amplification is carried out in Thermal cycler and applications products are separated by horizontal electrophoresis in agarose with ethidium bromide (10 mg/ml). DNA bands were observed on UV-transilluminator and photographed by a gel documentation system (ms major science UVDA). The photographs were critically discussed on the basis of presence (1) or absence (0), size of bands and overall polymorphism of bands. The scores obtained using all primers in the RAPD markers analysis were then pooled for constructing a single data matrix. This was used for estimating polymorphic loci, Nei’s (1972) gene diversity, genetic distance (D) and constructing a UPGMA (Unweighted Pair Group Method of Arithmetic Means) dendrogram among the germplasm using computer program “POPGENE 32” (Version 1.32).

Source of Information: Bangladesh J. Sci. Ind. Res. 54(2), 117-124, 2019

Article Link (Online Journal): DOI: https://doi.org/10.3329/bjsir.v54i2.41667

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

From the results obtained in the present investigation it can be concluded that this regeneration protocol is simple, reproducible and genotype independent. This optimized regeneration protocol can be effciently used for Agrobacterium mediated genetic transformation in tomato. To characterize the tomato variety of Bangladesh using PCR based RAPD primers, this information would be helpful for future breeding program as well as patenting each variety to prevent varietal piracy.

Knowledge Management: Journal