The Effect of Different 2, 4-D Doses on Callus Induction and Chromosomal Structure in Maize (Zea Mays L.)
Abstract
This research in which the matured embryos of 10 registered common corn varieties grown in Turkey (Pioneer 31N27, Pioneer 31P41, Pioneer 3223, Pioneer 34N24, ADA 8924, DKC 6022, BC 666, TECTOR, ADA 523 and HELEN) were used as plant materials was carried out in Biotechnology Laboratory, Department of Field Crops, Faculty of Agriculture, Ankara University, Turkey. In the stage of in vitro of this research which was arranged in vitro and in vivo media, the effect on callus induction and plant regeneration in 6 different varieties of 2,4-D doses (0, 2, 4, 6, 8 and 10 mg/l) through callus culture was investigated. In the stage of in vivo, samples from the root tips composed of the callus of all maize varieties were taken, and the changes observed in the chromosomal numbers in maize lines were evaluated. As a result of the microscopic analyses of the root tips obtained through the application of 2,4-D auxin in different corn varieties in different doses, changes were observed in the chromosomal numbers of Pioneer 31N27 (2n=18), Pioneer 3223 (2n=19) and Pioneer 34N24 (2n=19) maize varieties. Mitotic anomaly observed through the application of 2,4-D was established as aneuploidy (the change of the chromosomal number in genome) and it was found to form as the decrease of the chromosomal number (hipoploidy). According to the data found, the dose of 2,4-D to be used in the transgenic maize producing studies to be utilized in Turkey was determined to be 2 mg/l providing the highest callus formation ratio but causing no chromosomal deviations.
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Introduction
Maize (Zea mays L.) (2n=2x=20), which is grown widely in the world, industrialized and because it has rich nutrients, is very valuable and is a field plant which has usage varieties for both human and animal nutrition. Maize is the grain which has the highest yield in all cool and warm climate grain in the world and which uses the solar energy best.
Maize is used both in human nutrition directly and in the industry of starch, glucose, oil and dry food as raw material and it is a very renewable energy source. Most of the maize product in the world is used as animal food [1].
As well as clasical studies of breeding nowadays, significant developments were established also in related studies with genetically modified maize obtained through various methods such as particle bombardment [2], [3], [4] and Agrobacterium infection [5], [6].
The first molecular-marker maize map was published in 1986 [7]. It contains 116 loci and in this mapping, random genomic clones like cDNA and probes were used. Genetic linkage maps of maize consist of thousands of Classical Mutation Loci, Restriction Fragment Length Polymorphism (RFLP), Simple Sequence Repeat (SSR), Quantitative Trait Loci (QTL) and Expressed Sequence Tags (EST) markers [8], [9], [10], [11].
Plant biotechnology studies, which started in the 1990s in Turkey focused on tissue culture at the beginning and began to entegrate with molecular biology and genetical technics in the 2000s. Most of the researchers related with plant biotechnology are on tissue culture. The plant regeneration from tissue culture of mazie was first reported in 1975 [12].
The auxin, a group of natural growth regulators, support rooting, cell development and callus induction. The widely used auxin group hormone to support embryogenesis is a pestiside, 2,4-Dichlorophenoxy-acetic acid (2,4-D). 2,4-D chemical is frequently preffered to start callus induction. However, a chemical agent 2,4-D has a powerful immunotoxic effect. 2,4-D increases the risk of autoimmune reaction and causes an increase in the alergenic reaction. In addition to above-mentioned effects, 2,4-D is known to have teratogenic effects.
Conclusion
In our research, data related with callus, the ratio of shoot induction and callus weigth were obtained. In the 10 maize varities studied the highest callus induction was observed in HELEN (100%) maize variety threated with 2 mg/l 2,4-D and the variety of the maize in which callus weight was the most was established to be DKC 6022 (2.85 g) threated with 2 mg/l 2,4-D.
In this research, at the end of the high dose application of 2,4-D, the variation of chromosomal number in Zea mays, that is, chromosomal abberrations occurred. Such mitotic abnormalities were reported to have been caused from the effect of the pesticide on DNA. The toxic effects of the pesticides on different living systems were reported with a lot of studies.
For this reason, this commonly-used 2,4-D chemical must be used carefully in high doses because it may be fatal in several organisms including human.
In conclusion, different 2,4-D doses were applied to different origion maize varieties commonly planted in Turkey and the most effective but chromosomally the most harmless callus induction protocol was determined. This protocol can also be used in the studies to produce transgenic maize in Turkey in the future.