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Genetic Variation under the Influence of Long Term Culture Expressed Salt Tolerant Variant in Sorghum

A. M. Hassanein1, A.M. Ahmed2, D. M. Soltan1
1 Central lab. of Genetic Engineering, Faculty of Science, Sohag University, POB 82524 Sohag, Egypt.
2 Botany Department, Faculty of Science, Assuit University, Assuit, Egypt.

    Regeneration frequency, shoot formation, phenotypic variation, selection for salt-stress tolerance and gene expression were studied in sorghum under the influence of long term culture. ………etc.

 Key words: gene expression, izoenzymes, somaclonal variation, Sorghum bicolor, tissue culture.
Correspondence: AM Hassanein, Central lab. of Genetic Engineering, Faculty of Science, Sohag University, 82524 Sohag, Egypt. E-mail: hassaneinam@yahoo.com

    Conventional methods of sexual hybridization followed by variant selection have been used over the centuries for genetic improvement of plant species. …..etc.
The use of molecular technique as a tool for detection of genetic instability is described in the literature since molecular technique can characterize somaclonal variation with greater precision and lesser effort than traditional techniques depending on karyological and phenotypic analysis (Raimondi et al. 2001; Hossain et al. 2003; Hao et al. 2004; Hassanein 2004). Change of isoenzyme banding patterns could reflect gene expression or even gene changes; therefore, they can be used to study development, differentiation and plant variation (Tanksley and Orton 1983).

Materials and Methods:
Preparation of plant materials:
Sorghum seeds were surface-sterilized in 5% commercial bleach solution for 10 min followed by 5 min treatment in 75% (v/v) ethanol. …. etc.
Test for shoot formation on MS medium:
    During the third, 15th and 28th subculture, 30 clumps (each with about 15 buds) of each line were transferred to MS basal medium without phytohormones to study the ability of buds to develop shoots. After three weeks, the number of clumps forming shoots and the number of shoots/explant were counted. Shoot clumps failed to form shoots upon their subculture on growth regulator-free medium were separated into individual buds and subcultured twice on shoot multiplication medium. Consequently, the progeny of each clump were subjected again for shoot formation on growth regulator-free medium (three successive subcultures, two weeks each). The number of clumps failed to form any shoot were counted and considered as a mutant.

Test for NaCl tolerance:

    During the 3th, 15th and 26th subculture, 30 clumps of each line with about 15 buds of each clump were transferred to basal MS medium supplemented with 325 mM NaCl. … etc.

Detection of albino mutant:
Buds obtained from successive subcultures on bud multiplication medium were subjected to basal MS medium with or without 325 mM NaCl to study …. etc.

Can be divided to several subtitles if it simplifies the text
   Tables should be included at the end of the manuscript. Table legend should be as the following:
Table 1: Effect of number of subculture on bud formation, number of buds, shoot development and induction of stress tolerance

Number of subculture

Culture period

Bud multiplication frequency (%)

Number buds

Frequency of shoot formation (%)

Number of shoots

Frequency of stress tolerance(%)



15 ± 1


15± 1





15± 1


12± 1.5





12± 2


2± 0.3


It is better to use sophisticated statistical analysis.

Fig.1. Shoot tip multiplication on MS medium supplemented with different growth regulators.
As previously mentioned.

Brewer G. J. Introduction to Isoenzyme Techniques. Academic Press, New York, San Francisco, London, 1970.
Hassanein A. M. Somatic embryogenesis of cucumber (Cucumis sativus L) using seed cuttings obtained from pre-mature fruit. Plant Biotechnology 20: 275-281, 2003.