Effect of Different Concentrations of Colchicine and Oryzalin on Cytological and Histological Traits of Crepis capillaris with and without B Chromosome in vitro
A Dissertation Submitted to the Council of the
College of Agriculture, University of Duhok
in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy in
Agricultural Sciences- Horticulture
Payman Aziz Abdullah Zibari
B.Sc. Agricultural Sciences/ Horticulture
University of Duhok
M.Sc. Agricultural Sciences/ Horticulture
(Plant Tissue Culture)
University of Duhok
Dr. Mosleh M. S. Duhoky
September, 2013 A.D. Gelawej, 2713 K Zolqiada, 1434 A.H.
These experiments were conducted at Tissue Culture Laboratory/ Faculity of Agriculture and Forestory/ University of Duhok during the period from January 2011 to May 2013.
The objectives of this study were to study the effects of different concentrations of two chemical substances (colchicine and oryzalin) on chromosome number of Crepis capillaris (without and with 2B chromosomes) through long term callus culture until regeneration of plants from the callus, to investigate the effectivity of different concentrations of these two chemicals in polyploidy induction and which one of them can increase the ploidy level in long-term callus culture, and to determine the number of chromosomes in the produced plants, as well as to study the effects of these two chemicals on the histological analysis of the callus during different passages.
The highest rate of callus cell mitotic index was recorded by Crepis capillaris with 2B chromosomes in root and callus explants before treatment as compared with Crepis capillaris without B chromosome. After treatment, interaction between Crepis capillaris with 2B chromosomes and 0.15 mM of colchicine and shaking it for ten days recorded high rate of callus cell mitotic index in passage four (6.36%); however, in the case of interaction between Crepis capillaris without B chromosome and 55 µM of oryzalin and shaking it for ten days recorded high rate of mitotic index in passage four (6.09%).
For chromosomes analysis, Crepis capillaris, without B chromosome treated with 55 µM of oryzalin for ten days inscribed high polyploidy percentage at the level of 8n=24 (16.40%) in first passage, also the same treatment had the same effect on increasing polyploidy (%) in passage fifteen (43.74%). Furthermore, interaction between Crepis capillaries with 2B chromosomes and 0.15 mM of colchicine and shaking it for ten days consummated the high rate of polyploidy (%) in the second passage at the level of 4n=12 (52.18%) and the same variety with all colchicne concentrations and shaking it for five and ten days had significant influences on increasing of polyploidy percentage on passage fifteen.
In case of Crepis capillaris callus without B chromosome, it is clear from our results that frequency of cell polyploidization in callus lines without B chromosome decreased gradually after oryzalin and colchicine treatments and in passage 7 after treatment the callus become completely diploid, however, in case of Crepis capillaris callus with 2B chromosomes, the callus remained producing an increase of polyploidy cells passage after passages.
In this study, it is clear that oryzalin was more effective in increasing MI % and polyploidy levels as compared with the other treatments. Oryzalin showed higher affinity to plant microtubules than colchicine.
Callus originated from Crepis capillaris leaf explants without B chromosome was white to yellow, soft and grew very well as compared with callus originated from Crepis capillaris leaf explants with 2B chromosomes. All the callus of Crepis capillaris without B chromosome treated with different concentrations of colchicine and oryzalin produced plantlets which were easily rooted in rooting media; however, the chromosome number was checked for all callus derived plant roots and all the regenerated plants had only the diploid number of chromosomes. Histological analysis of Crepis capillaris without B chromosome treated with different concentrations of colchicine and oryzalin showed that regeneration of Crepis capillaris callus could be through organogenesis or somatic emberyogenesis. Again treatment of Crepis capillaries callus regenerated from leaf explants with 2B chromosomes had different cell arrengment as compared with callus derived from Crepis capillaris leaf explants without B chromosome. Treatment of Crepis capillaris calli with 2B chromosomes with different concentrations of colchicine and oryzalin did not result in regeneration of any plants.