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Callus Induction and Efficient Plant Regeneration in Cucumber (cucumis Sativusl.)

Rubaiya Jesmin
Dept.of Genetics and Plant Breeding, Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU), Gazipur-1706, Bangladesh

M Abdul Khaleque Mian
Dept.of Genetics and Plant Breeding, Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU), Gazipur-1706, Bangladesh

Abstract/ Executive Summary:

A reliable and reproducible protocol is established to get healthy and well formed callus from juvenile explants of cucumber. The sterilized seeds of cucumber cultivar were cultured on MS basal medium (Murashige and Skoog, 1962). The seeds germinated after 7 days of culture with 24 hours dark photoperiod. Explants from germinated seedlings were cultured on MS medium supplemented with individual treatments of different auxins (2,4-dichloro-phenoxyacetic acid (2,4-D), α naphthalene acetic acid (NAA)) or cytokinins (benzyl aminopurine (BAP)). Plant parts such as leaves, stems and cotyledons were used as source of explants. Callus were initiated from leaves, stems and cotyledons after 4 weeks of culture. The optimum medium for callus induction from leave, stem and cotyledon explants was MS medium supplemented with 0.5 mg/L BAP added with 1.0 mg/L NAA. The highest percentage of callus was obtained from stem explants (89.0 ± 0.75 %) followed by leave (79.05 ± 3.28%) and cotyledon (74.43 ± 1.30 %) explants. Maximum callus induction in stems (73.05 ± 2.1%) was obtained in 1.mg/l concentration of BAP. Incorporating 2,4-D in the callus induction media promoted slow callus growth and low quality callus compared to that produced on media containing NAA and BAP. Callus induced on media containing 2,4-D was friable and yellow in color. This protocol can promote the application of tissue culture technology to facilitate the genetic transformation of this species.

Keywords: Cotyledon, Naphatalene acetic acid, Benzylaminopurine, Propagation, Explants

Location: Dept.of Genetics and Plant Breeding, Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU), Gazipur-1706, Bangladesh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Cucumber

Objectives:

The present work was undertaken to establish an efficient and reproducible protocol for callus induction from stem, leaf and cotyledon explants of commercially important slicing cucumber Hatiyakira.

Methodology:

Seed sources and sterilization procedures: The seeds of cucumber cultivar Hatiyakira, were obtained from Horticultural Centre, BSMRAU. Seeds were rinsed under running tap water for thirty minutes and then rinsed twice with distilled water. Subsequently, the seed coat was removed. After that, the seeds were surface sterilized with 70% ethanol for 30 seconds respectively followed by rinsing with sterilized distilled water for five times followed by 0.1% HgCl2 for 5 minutes. Finally, the seeds were rinsed three times with sterile distilled water. Rinsing of seeds with 70 % ethanol was done in the laminar flow (under aseptic conditions). Culture procedures:The sterilized decoated seeds were then placed on MS basal medium solidified with agar for germination. Culture were incubated in dark photoperiod at 26°C. Seeds were germinated after 7 days of culture. Prior to germinating, cultures were transferred to cool-white-fluorescent light room and incubated at 25 ± 1°C with 16 hours light and 8 hours dark photoperiod. Explant isolation and media composition: Leaf, stem, and cotyledon explants excised from in vitro plantlets containing were then placed on MS medium containing 30 g/L sucrose supplemented with various concentrations of 2, 4-D (2, 3, 5 mg/L), BAP separately (0.5, 1.0, 1.5 mg/L) and in a combinations of BAP and NAA (0.5 + 1.5, 0.5 + 1.0, 1.0 + 0.5 mg/L, respectively). Media were solidified with 8 gram agar and sterilized in an autoclave for 20 minutes at 121°C at 15 psi pressure. Culture conditions: The pH of media was adjusted to 5.8 prior to autoclaving. All cultures at 25±1^oC under white fluorescent light of 40-60 μmol m^-2s^-1 intensity for 16 hours light/8 hours dark photoperiod were incubated. Experimental layout: The experiments were laid out in completely randomized design (CRD) with at least three replications. All data obtained were statistically analyzed using Microsoft Excel. The data gathered from the experiments were analyzed according to mean percentages and analysis variance (ANOVA) at 5% level of significance. Total number of explants in each treatment was 60. The experiments was conducted twice.

Source of Information: J. Biosci. Agric. Res. 09(02): 796-803

Article Link (Online Journal): DOI: 10.18801/jbar.090216.96

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Micro propagation and callus induction of Cucumis sativus L. was successfully obtained. In recent years, many investigations have been performed in establishing reliable regeneration protocol for important vegetable crops primarily because of both primary and essential steps for facilitating gene introduction and crop improvement.The present experiment showed that an effective protocol for micropropagation of Cucumis sativus depends on various key factors like choice of explants, surface sterilization, growth regulators and their combination at different concentrations.It is concluded that the manipulation of culture conditions using various combinations and concentrations of growth hormones and other adjuvants can provide a reproducible protocol and reduce the high costs of hybrid seed production. Stem explants has been identified as the more regenerative explants for induction of callus. Formation of callus of stems, cotyledons and leaves respectively in MS medium supplemented with 0.5 mg/L BAP added with 1.0 mg/L NAA was showed the best result. Studies of CucumissativusL.clonal propagation and callus induction could also be efficiently adapted for other crops in future research.

Knowledge Management: Journal