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Production of Chromosomal Variant in Garlic Using Colchicine Under in Vitro Condition

M. A. Haque
Dept. of Genetics and Plant Breeding, Bangladesh Agricultural University, Mymensingh, Bangladesh

Abstract/ Executive Summary:

The experiment was conducted to create variants by using colchicine in clones of Garlic. Three garlic strains (G-101, G- 102 and G-103) were used from which Root tips were cultured in MS medium supplemented with various concentrations (0.5.1.0,1.5,2.0 and 2.5 mg L^-1) of 2, 4-0 and fixed concentration (30 g L^-1) of sucrose for callus induction. The somatic chromosome number of the three garlic strains in the cells of callus were recorded as 2n = 16. No deviation of the chromosomal counting was observed excluding total length. Calli were suspension cultured with liquid medium added different concentrations of colchicine for different periods. Colchicine concentrations were five levels (0.05, 0.1, 0.2, 0.3 ppm and control and treatment periods were 3.4, 6, 8 and 10 hours of which 6 hr treatment gave better response showed chromosomal variations in all the three Garlic strains. The heterogeneity of chromosome number in all the three garlic trains were a striking phenomenon. Tetraploid (80%, 90 % and 85% tetraploid cells in G-101, G-102 and G-103 strains respectively) and hypertctraploid condition (2n >36) was a common to all the garlic strains.

Keywords: Allium, Variant, Colchicine, In vitro

Location: Bangladesh Agricultural University, Mymensingh, Bangladesh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Garlic

Objectives:

To study karyological changes during callus induction form garlic root explants and the influence of colchicine application along with hormonal composition of the medium on the changes in questions.

Methodology:

Cloves of garlic strain G101, G-102 and G-103 were surface-sterilized by immersion, first in 70% ethanol for 30s and then in 1.0 g/L sodium hypochlorite solution containing 'Tween 20' (2 drops/100 ml solution). Cloves were agitated continuously in the sodium hypochlorite solution for 20 mins. and then rinsed three times with sterile distilled water. Root tips measuring 2-3 mm were excised aseptically. Five explants were cultured per culture bottle (5.7 x 10.2 cm) containing 40 ml medium. The culture medium consisted of MS (Murashige and Skoog, 1962) salts and vitamins, 30 gl/i sucrose and 8.0 g/L agar. The explants Were placed on an induction medium supplemented with various concentrations (0.5, 1.0, 1.5, 2.0, and 2.5 mg/l) of 2,4-dichlorophenoxyacetic acid (2,4-0). Each treatment consisted of three replications and each replication consisted of 20 explants. The percentage of explants forming embryogenic callus and the percentage of calli with somatic embryos were recorded after 6 and 15 weeks of culture, respectively. Some root tip derived calli of the three strains of Garlic were subjected to colchicine treatment. When a subculture was a bit greenish. about 3^rd or 4^th day of subculturing, it was put under the laminar airflow cabinet, kept under completely submerged condition in freshly prepared 0.05% colchicine solution. The duration of colchicine treatment given in several subculture pieces was 3 hrs, 6 hrs and 10 hrs. After the treatment was over, the calli were washed in distilled water for three times and then returned to fresh culture vessel containing basal medium. All activities were done under the laminar airflow cabinet. Following colchicine treatment, minute greenish portion of the callus was processed on 2^nd or 3^rd day for mitotic slide preparation. MBN treatment and hydrolysis in 10% HCI was avoided. Mordanting and staining was done as and when required. Observation was made for the effect of colchicine, if any, as regards chromosome number and nuclear size. Proportion of the occurrence of bigger size nuclei was checked IS days after treatment and subsequently IS days after subculturing following treatment. Rate of growth of the treated calli was checked subsequently through visual observation. Treated calli, presumed to have colchicine action were subcultured and maintained usually. A very few number of roots that were spharable cultured from tissue culture regenerates were processed for slide preparation. Photom icrography of the chromosome plates was done form permanent and temporary slides. Photomicrographs of the selected chromosome plates were taken with the aid of olympus research microscope model BX40 using plan 100X objective. Fujicolour 100 ASA DX Film was used. Film processing and printing was made from commercial centres.

Source of Information: Progress. Agric. 14(1 & 2) : 57-60, 2003, ISSN 1017-8139

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The chromosomal instability occurs in primary callus cultures of Allium sativum very early and it is controlled to a certain extent by hormonal composition of the medium. However, Colchicine concentrations have a remarkable effect on the origin of polyploid as well as aneuploid in root tip cells of garlic. Further work is in progress to determine the exact mode of the origin of the chromosomal variations.

Knowledge Management: Journal