ASSESSMENT OF GENETIC DIVERGENCE, STEVIOSIDE AND REBAUDIOSIDE A CONTENTS AND THE EFFECTS OF GAMMA IR- RADIATION ON THE PERFORMANCE OF STEVIA ( Stevia rebaudiana BERTONI) GENOTYPES.

Abstract

Stevia rebaudiana Bertoni is a perennial and sweet herb native to the highlands of Paraguay belongs to family Asteraceae. Seeds of five stevia varieties were treated with 0, 10, 15 and 20 K rad doses of gamma rays from Co60 Indian gamma cell source (Dose rate: 1,323KGy/h) to evaluate the effects of gamma irradiation on stevia genotype performance. Stevioside and rebaudioside A contents and genetic diversity among the nonrelated stevia varieties were assessed utilizing HPLC analysis and molecular markers respectively. The leaves dry weight as the most important agronomic trait was decreased significantly with increase gamma-ray dose in all studied varieties except China1 and Shou2 at 10 K rad and Sughigh at 20 K rad that was increased from 23.84 to 28.84 g, 31.38 to 36.25 g and from 32.88 to 40.75 g, respectively. The obtained results showed the existence of considerable amounts of genetic diversity among the five tested varieties with EST-SSRs, SSR, ISSR, and ISJ primers, suggesting the potential use of these markers and in detecting molecular diversity among stevia genotypes. Wide range diversity was found between Spanti and China1 varieties. However, the close pair of genotypes was China1 and Shou2. These results confirm the nearby origin of the genetic background of China1 and Shou2. The overall clustering was largely based on origin and /or genetic background. HPLC analysis of stevioside and rebaudioside A contents in stevia leaves revealed significant differences among the studied genotypes. A large amount of stevioside value was recorded by Shou2 variety (43.2 mg/500mg with 8.64%), while the lowest amount value was recorded by Eg1 (2.533 mg/500mg with 0.51%). The results demonstrated the power of molecular techniques used in distinguishing high stevioside content genotypes from lower content ones.
Stevia rebaudiana Bertoni is a perennial and sweet herb native to the highlands of Paraguay belongs to family Asteraceae (Brandle and Rose, 1992). It was first discovered by a Swiss botanist, Moises Santiago Bertoni in 1888 and exploit even since. Its genus contains about 150 species of herbs and shrubs (Abdulameer et. l., 2018). Commonly known names are honey leaf plant, sweet herbs, and sweet leaf. The leaves of Stevia contain glycoside diterpenes such as rebaudioside A, B, C, D, E, F, M, stevioside, steviol bioside, dulcoside A and dulcoside C which are estimated to be 200-350 times sweeter than sucrose with free calories (Ghaheri et al, 2017). In addition to non-caloric sweetener, stevia have many other therapeutic benefits such as anti-microbial (Debnath, 2008), anti-fungal (Silva et al., 2008), anti-inflammatory (Gupta et al, 2013), anti-diarrheal (Takahashi et al, 2001), anti-tumour (Jayaraman et al, 2008), anti-virus (Kedik et al., 2009), anti-hyperglycemic (Benford et al, 2006), anti-hypertensive (Hsieh et al., 2003), and immunomodulatory effects (Chatsudthipong and Muanprasat, 2009).
In the early 1970s, stevioside known as the major sweetener present in leaf and stem tissue by Japanese consortium formed to commercialize stevioside and stevia extracts (Brandle and Rosa, 1992). Stevioside and rebaudioside A are the major components of the total glycoside content. The stevioside component is believed to give a somewhat bitter aftertaste with a "licorice" taste while rebaudioside A is more pleasant sweet taste with no bitter aftertaste (DuBois, 2000, Yadav et al., 2011 and Chiew et al., 2016).
The mutation is one of the breeding methods in several crops to improve crop quality and bioactive metabolites production such as in rice, maize, and cassava breeding for low amylase, protein contents improvement, and high amylase content, respectively, (Chiew et al., 2016). Mutations can be induced by chemicals or various types of ionizing radiation (X-rays, gamma rays, neutrons, UV light, etc.) (Jain, 2010 and Ali et al., 2015). Gamma rays and x-rays are the most widely used mutagens in mutation breeding (Mba, 2013).
Genetic diversity estimation between different genotypes using molecular marker techniques is very import in breeding programs (Sharma et al., 2016). Molecular fingerprinting is a useful tool for several applications. Several molecular marker techniques are available now to assess the diversety between several genotypes such as Random Amplified Polymorphism (RAPD), Simple Sequence Repeats (SSR), Inter-Simple Sequence Repeats (ISSR), Single Nucleotide Polymorphism (SNP) (Williams et al., 1990, Schulman, 2007 and Sharma et al, 2016). Therefore, the present investigation was undertaken to evaluate the effects of gamma irradiation on some stevia genotypes, the assessment of genetic diversity among tested varieties using molecular marker techniques and to evaluate stevioside and rebaudioside A contents using HPLC analysis.