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Development of Mutants in Sugarcane Through Callus Culture

Kuasha Mahmud
Biotechnology Division, Bangladesh Sugarcane Research Institute, Ishurdi?6620, Bangladesh

KM Nasiruddin
Biotechnology Division, Bangladesh Sugarcane Research Institute, Ishurdi?6620, Bangladesh

MA Hossain
Biotechnology Division, Bangladesh Sugarcane Research Institute, Ishurdi?6620, Bangladesh

L Hassan
Biotechnology Division, Bangladesh Sugarcane Research Institute, Ishurdi-6620, Bangladesh

Abstract/ Executive Summary:

The effects of different concentrations (0.0 M, 0.01 M, 0.02 M and 0.03 M for 24 hours treatment) of chemical mutagen like, ethyl methanesulphonate (EMS) on callus derived from sugarcane var. Isd 37, Isd 39 and Isd 40 were investigated for in vitro plant regeneration. The highest number of shoots (58.33%) was reduced over control of Isd 40 followed by Isd 37 (57.62%) and Isd 39 (the lowest 56.06%), respectively due to the use of EMS for treating calli. The regeneration of mutant plantlets was achieved from EMS (0.01 M) treated calli obtained from leaf sheath explants on MS supplemented with 2, 4-D (3 mg/l) and 10% coconut water. Most of the mutant plants were developed from Isd 37. The developed mutant plants showed variation in tiller number, internode, green leaf, millable cane and stalk colour from the source variety of Isd 37.

Keywords: Sugarcane, Callus, EMS, Mutants

Location: The Biotechnology Laboratory, Bangladesh Sugarcane Research Institute (BSRI), Ishurdi, Pabna, Bangladesh

Start Date: 00-09-2010

End Date: 00-01-2011

Program Area: Crop-Soil-Water Management

Commodity: Sugarcane

Objectives:

To induce variation in local varieties of sugarcane through the application of mutagens using in vitro techniques.

Methodology:

The experiment was conducted at the Biotechnology Laboratory, Bangladesh Sugarcane Research Institute (BSRI), Ishurdi, Pabna, Bangladesh during the period from September 2010 to January 2011 to investigate in vitro plant regeneration capability of BSRI released variety such as, Isd 37, Isd 39 and Isd 40. The leaf sheath explants collected from 8 - 10 months old field-grown sugarcane from BSRI experimental field were used for this study. The explants were kept in a beaker and treated with 3% (w/v) Savlon for 5 - 6 minutes with constant shaking and washed thoroughly with distilled water for 3 - 4 minutes. The explants were then treated with 1% HgCl₂ and followed by washing 3 - 4 times with sterile distilled water. Explants (approximately 1 cm × 0.5 cm) were prepared in laminar airflow cabinet from sterilized leaf sheath segments. Besides, pH of the medium was adjusted to 5.7 and agar (0.6%) was added in the medium. Media was sterilized by autoclaving at 1.2 Kg cm^-2 pressure at 121ºC for 30 minutes. Cultures were incubated at 25 ± 2ºC and kept 16 hrs under fluorescent tube light. The experiment was laid out in Complete Randomized Design (CRD). There were three replications and ten test tubes of each treatment. The data were statistically analyzed following Factorial Completely Randomized Design (CRD). MS with coconut water (10%) containing 3 mg/l of 2,4-D was prepared for callus induction and treated with EMS (0, 0.01, 0.02, 0.03 M for 24 hours). Treated calli were transferred to MS including 2,4-D and then transferred into shooting medium. Calli obtained from 2,4-D (3 mg/l) were treated with different concentrations of mutagen. Treated calli were aseptically transferred to fresh medium containing the BAP (3 mg/l) for proliferation and development of shoots. After inoculation of callus for 20 days on shooting medium, small green leaves began to emerge. Callus with tiny leaves were transferred to fresh medium containing the same concentration of BAP for further proliferation of shoots. Days required for shoot initiation were recorded from the beginning of shoot formation. Moreover, number of shoots per callus, length per shoot, shoot initiation percentage and also increased/decreased shoot (%) for mutagen application were recorded following inoculation on shooting medium. After sufficient amount of shoot formation, the shoots were aseptically transferred onto fresh medium containing NAA (5 mg/l) for development of roots. Days required for root initiation, the number of roots per shoot, length of roots and increased/decreased number of root (%) due to mutagen treated were recorded after inoculation of shoots in the rooting medium. Rooted plantlets were acclimated and transplanted to polybag and then to field, respectively.

Source of Information: Plant Tissue Cult. & Biotech. 26(1): 123-130, 2016 (June)

Article Link (Online Journal): DOI: 10.3329/ptcb.v26i1.29773

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Various responses were observed among the shoots and roots derived from EMS treated callus derived from the leaf sheath explants from different varieties of sugarcane. The lowest numbers of days required for shoot and root initiation (7.83 and 6.25, respectively) were recorded from the variety Isd 37 while similar time (6.25 days) was also required for root initiation by Isd 40 and it was maximum (9.41 and 7.83 days, respectively) in Isd 39. On the other hand, both the varieties Isd 37 and Isd 40 produced significantly the maximum shoots per callus (4.25) while Isd 39 produced the maximum roots per shoot (3.91). Similarly, Isd 39 showed the minimum shoots per callus (3.00) while both the varieties Isd 37 and 40 produced similar (3.50) roots per shoot. No shoots and roots were produced in 0.02 and 0.03 MEMS. Among other treatments, without EMS required least time for shoot and root initiation (10.89 and 7.22 days, respectively) than 0.01 EMS (23.78 and 19.67 days, respectively). Similarly, medium without EMS showed significantly the highest number of shoots per callus (10.67) and roots per shoot (11.11) than 0.01 EMS (4.66 and 3.44, respectively). This finding is in agreement with the results of Gahukar et al. (2000) who reported that EMS affected the regeneration efficiency (RE) where RE significantly decreased in increasing EMS level.

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