الفهرس 
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Abstract
This study was carried out in the Laboratory of Tissue Culture, in Centre of Genetic Engineering and Biotechnology, Faculty of Agriculture, Ain Shams University, Shoubra El-Keima, Egypt. Molecular DNA manipulations were carried out in the Laboratory of Gene Expression and Regulation Technologies (GERT), Agricultural Genetic Engineering Research Institute (AGERI), Agricultural Research Centre (ARC), Giza, Egypt, during the period from 2012-2017.
Four cultivars of stevia were provided by the Sugar Crops Research Institute (SCRI), Agricultural Research Centre (ARC). Cultivars Spantia, Shou2A3, China, and High Sugar were tested to optimize in vitro propagation. Four different combinations of growth regulators treatments were investigated [6-benzylamino purine (BAP) + Kinetin (Kin) (0.25 + 0.25 mg/l); Forchloefenuron (Cppu) + Kin (0.25 + 0.25 mg/l); Cppu+ Kin (0.5+0.25 mg/l); and the control medium (growth regulators-free)]. Out of the different media components, the growth regulators-free medium produced the best performance of explants. The analysis of variance showed that:-
1- The control treatment was the most significant for all traits except the number of branches.
2- China cultivar showed consistent results at the morphological and chemical traits assessment.
3- The cultivar Spantia was comparatively characterized for low level of stevioside (322.49 mg/100 g) using the HPLC analysis and was characterized by using the SCoT-6 (200 bp), SCoT-10 (450 bp), SCoT-16 (590 bp), and SCoT-36 (1000 bp).
4- The cultivar Shou2A3 was comparatively characterized as middle-low levels of stevioside (457.71mg/100 g) using HPLC data and was identified by using the SCoT-10 (1000 bp), SCoT-16 (1000 bp), SCoT-32 (220, 380, 900, and 1200 bp), SCoT-35 (850 bp), and SCoT-36 (220 bp).
5- The cultivar High Sugar was comparatively characterized for middle-high level of stevioside (634.32 mg/100 g) using the HPLC analysis.
6- The cultivar China was comparatively characterized for the highest level of stevioside (699.80 mg/100 g) using the HPLC analysis and was also identified by using the SCoT-32 (260 bp), SCoT-36 (650 bp), and SCoT-44 (1600 bp).
7- The China cultivar showed high level of stevioside (634.32 mg/100 g); based on the HPLC results and high level band concentration 346.7 ng/µl on gene expression analysis.
8- The comparative megalignment sequence analysis, between the different SrKA13H genes were isolated from the four stevia cultivars in the current study, Specific sequence features represented by many SNP and indels were very characteristic to each stevia cultivar. If any mutations occur, there may be an increase or decrease in steviol glycoside, or it can be used as a marker, which requires future studies.
9- As for gene expression analysis, China cultivar showed high level of SrKA13H RNA transcript that was 590.8bp with band concentration of 346.7 ng/µl.
10- from extraction of steviol glycoside, HPLC analysis and gene expression analysis, it is evident that China cultivar which was consistent at the morphological, chemical and molecular trait assessments to be superior to the other tested cultivars and could be selected as candidate cultivar for mass production of stevioside.
This study has successfully validated the SCoT markers as a new molecular tool to differentiate between stevia cultivars. Moreover, cultivar-specific markers can further be applied for genotyping and association studies of quality traits that require stevioside and rebaudioside-A in stevia.