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Influence of Benzyl Amino Purine on Shoot Production Through in Vitro Shoot Tip Culture of Cucumber

Dr. S. M. Faisal
Senior Scientific Officer
RARS, BARI, Hathazari, Ctg., Bangladesh

Dr. Md. Shahidul Haque
Professor
Dept. of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Dr. K. M. Nasiruddin
Professor
Dept. of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Multiple shoot production ability of some commercial cucumber varieties were assessed. Four varieties of cucumber such as Shila, Green Field, Shital and shahi-50 and four BAP concentrations such as 0.0, 1.0, 3.0 and 5.0 mg/l were tested against this investigation. Different cucumber varieties and BAP concentrations showed significant effects in all the parameters studied. All the varieties regenerated maximum number of explants when they were cultured on Murashige and Skoog media supplemented with BAP @ 3.00 mg/l which was followedby the BAP @ 5.00 mg/l. Variety Green Field took the shortest time (6.11 days) for shoot initiation when it was cultured on BAP @ 3.00 mg/l enriched Murashige and Skoog media. Variety Green Field produced the highest number of multiple shoots regardless of BAP concentrations except control at second subculture but at third subculture all the varieties produced more than double number of multiple shoots regardless of BAP concentrations except BAP free media.

Keywords: Multiple shoot, in vitro, shoot tip, Subculture, Cucumber

Location: The experiment was carried out at the Biotechnology Laboratory, Department of Biotechnology, Bangladesh Agricultural University (BAU), Mymensingh, Bangladesh.

Start Date: 30-06-2005

End Date: 31-07-2008

Program Area: Variety and Species

Commodity: Cucumber

Objectives:

The present study was carried out with the objectives to establish a suitable and develop a reproducible protocol for in vitro regeneration of some cucumber varieties and to asses the multiple shoot production ability of popular cucumber varieties in Bangladesh.

Methodology:

There were 2 factors in this experiment. Factor A consisted of four cucumber varieties and factor B consisted of four concentrations of benzyl amino purine (BAP). Four cucumber varieties such as Shila, Green Field, Shital and Shahi-50 and four BAP concentrations @ 0.0 (Control), 1.0, 3.0 and 5.0 mg/l were used. So total number of treatments were 16. Each treatment consisted of 6 test tubes/vials. The experiment was carried out in a Completely Randomized Design (CRD)with three replications. A nutrient medium for plant regeneration usually consists of organic and inorganic salts, irons, a carbon source, some vitamins and growth regulators. In this study Murashige and Skoog (1962) medium was used as basal medium for plant regeneration. Shoot tip explants were collected from seeds of four cucumber varieties which were germinated in vitro in several separate petridishes. For establishing the plants in the media only the tender and active growing shoot tips having 1.00-1.25 cm long were prepared to use as explants. To maintain aseptic condition, precautions were taken in every step of works. All inoculations and aseptic manipulations were carried out in a laminar airflow cabinet. It is usually started half an hour before use. The cabinet was wiped with 70% ethyl alcohol (C2H5OH) to reduce the chances of contamination. The inoculating instruments like scalpels, forceps etc. were sterilized. Other required materials like distilled water, hard papers etc. were sterilized by autoclave. Hands were properly washed with soap before starting work in laminar airflow cabinet. During operation, hands and cabinet base were rubbed with 70% ethyl alcohol frequently for maintaining clean condition. To obtain possible contamination free condition in clean bench proper care was taken during explant preparation. Shoot tips were prepared inside the laminar airflow cabinet using a fine sterile forcep and scalpel. The Successful shoot formation became evident when small green fresh leaves began to emerge. Developed tiny leaves, were transferred into fresh media containing the same hormonal concentration for further proliferation and development of shoots. First subculture was carried out at 25 days after shoot (DAS)initiation. Second and third subcultures were carried out at 50 and 75 DAS initiation, respectively. Multiple shoots were counted at second and third subculture only. The regenerated multiple shoots were carefully removed from the culture tubes/vials and placed on a sterile hard paper. Each shoot was cut from the basal end and was transferred to new media containing concentrations of BAP for further multiple shoot induction. Data on number of explants regenerated, days to shoot initiation, percentage of explants regenerated, number of multiple shoots per explant at second and third subculture were recorded. The data were analyzed using MSTAT-C statistical software. Differences among the means were compared following Duncan’s Multiple Range Test (DMRT) at A5% level of significance.

Source of Information: Malaysian Journal of Science 30(3): 163-170 (2011)

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) : N/A

Achievement/Findings:

The primary goals of the in vitro propagation of vegetable crops include; 1) production of large number of plantlets from the species in which plant development from seed is difficult, 2) clonal propagation of large number of genetically identical plantlets, 3) production of virus-free plant material and 4) crop improvement through various techniques of genetic modification (Torres, 1989). To produce disease-free plants, meristem tip should be cultured. Meristem tips of 0.2 - 0.5 mm most frequently produce virus-free plants and this method is referred to as “meristem tip culture”. Actively growing shoot tips are recommended for their culture because of their strong growth potential and low virus concentration (Chawla, 2002). For large scale multiple shoot production in cucumber shoot tip culture on MS medium supplemented with BAP @ 3.0 mg/l was found most effective. To obtain large scale multiple shoots of cucumber these findings may be helpful for researchers/users who are working on in vitro culture and genetic transformation of cucumber.

Knowledge Management: Journal