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In Vitro Propagation of Embryonic Explants of Satkara (citrus Macroptera Montrouz)

M. A. Jahan
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology Sylhet-3114, Bangladesh

M.K. Saifullah
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology Sylhet-3114, Bangladesh

M.R. Chowdhury
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology Sylhet-3114, Bangladesh

S. Begom
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology Sylhet-3114, Bangladesh

M. S. Hossain
Department of Genetic Engineering and Biotechnology, University of Rajshahi, Rajshahi-6205, Bangladesh

Abstract/ Executive Summary:

Citrus macropter is a wild or semi wild Citrus species grow well in hill-forest. The present investigation was conducted to study the effects of auxin and cytokinin on callus induction from embryonic explants of Citrus macroptera Mont. The embryonic explants produced semi-hard greenish and creamy-white callus and adventitious buds within 4-5 week of culture. The highest percentage of callus is produced in media containing 2.0 mgl^-1 2,4-D+1.5 mgl^-1 KIN were used. The rate of adventitious shoot regeneration from callus was also influenced different concentration of BA in MS medium. The highest shoot regeneration rate was observed in embryogenic callus on MS media supplemented with 1.5 mgl^-1 P BA. Best rooting achieved within 28 days after the isolated adventitious shoots were transferred to a rooting media containing 1.0 mgl^-1 P IBA. In vitro cultured plantlets transfer to pot soil and successfully acclimatized with natural condition.

Keywords: In vitro propagation, Auxin, Cytokinin, Callus, Citrus macroptera Mont

Location: Plant Genetic Engineering Laboratory of Genetic Engineering and Biotechnology Department, Shahjalal University of Science & Technology, Sylhet

Start Date:

End Date:

Program Area: Crop-Soil-Water Management

Commodity: Satkara

Objectives:

To study the effects of auxin and cytokinin on callus induction from embryonic explants of Citrus macroptera Mont.

Methodology:

This research work was conducted at Plant Genetic Engineering Laboratory of Genetic Engineering and Biotechnology Department, Shahjalal University of Science & Technology, Sylhet, Bangladesh. Mature embryos of C. macroptera were used as explants in the experiment. Ripen C. macroptera was collected from Bandar bazar of Sylhet city, Bangladesh. Seeds separated and dried in the sun for the study. Preparation of explants: At first seed coats were removed manually to obtain embryos. Then embryos were exposed to sterilizing agents for varying durations, either before or after removing the embryo sec to identify the ideal surface sterilization conditions. Embryos were treated with 70% ethanol for 2-3 minutes, followed by few drop of tween-20 for 5 minutes, then with 0.1% Mercuric Chloride solution for 5 minutes then washed with sterilized distilled water for several times. The embryos were then placed on the sterilized petriplate having sterile filter papers with the help of forceps to remove excess water. Embryo sac removed carefully with sterile forceps before inoculation. Embryos were then placed individually in culture tubes. The culture tubes were then incubated 27±2°C temperature under florescent light of 16 hours photoperiods. Callus induction: Callus induction was initiated in 25 x 150 mm culture tubes containing 10 ml of MS medium containing 3% sucrose and solidified with 0.7% agar having different concentrations of callus inducing growth regulators in different combination. Ten explants were used for each treatment and thirty test tubes were inoculated in each time. Visual observations were taken every three days and the effect of different treatments was quantified on the basis of percentage of explants showing response for callus induction. Shoot regeneration: Shoot regeneration was performed in 25 x 150 mm culture tubes containing 10 ml of MS medium containing 3% sucrose and solidified with 0.7% agar, having different concentrations of shoot regenerating hormones. In the case of shoot regeneration from callus, a healthy green portion of callus was taken and cut into pieces, and these pieces were then placed on a shoot regeneration medium. Visual observations were taken every three days and the effect of different treatments was quantified on the basis of percentage of calli showing response for shoot regeneration. Rooting of regenerated shoot: Rooting was performed in 25 x 150 mm culture tubes containing 10 ml of MS medium containing 3% sucrose and solidified with 0.7% agar, having different concentrations of root regenerating hormone IBA individually. Visual observations were taken every three days and the effect on different shoots was quantified on the basis of percentage of shoots showing response for rooting. Data collection and analysis: Weekly visual observation of culture was made and frequency of culture showing callus, shoot and root formation was recorded. Completely randomized design has been used in each experiment. The data pertaining to shooting and rooting per culture were analyzed using Microsoft Office Excel 2007. Transfer to the soil: Rooted plantlets transferred to plastic pots filled with 2:1 sterilized garden soil and compost. The potted plants (one plantlet per pot) were then put into large polythene bags (25 cm × 15 cm) to maintain high humidity. Open portion of the large bag was made airtight and kept them in growth chamber under artificial illumination. Within 7-9 days the covering bags were finally removed. Potted plants were acclimated with natural environment.

Source of Information: Int. J. Sustain. Crop Prod. 7(1):19-24, (2012)

Article Link (Online Journal):

Funding Source:

1. Government

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In vitro propagation of Satkara (Citrus macroptera Mont.) were conducted with a view to develop an ideal protocol for large scale production of as well as detrermination of the most suitable medium compositions for the best response, standardization of growth regulators for maximum callus induction, shoot proliferation and root induction in vitro from embryonic explants. BA and KIN used as cytokinin on the other hand 2,4-D and NAA used as auxin. Our results show that up to 87.5% of embryonic explants can produce calli on MS with 2.0 mgl^-12,4-D combination with 1.5 mgl^-1KIN which can produce maximum (40%) shoots on MS with BA 1.5 mgl^-1. Rooting can be induced in up to 80% of these shoots by using MS medium supplemented with 1.0 mgl^-1 IBA. Data generated in this study could be practically useful for efficient in vitro propagation of this plant.

Knowledge Management: Journal