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Abstract
Leaves of green nightshade (Solanum viride Solander ex Forst. f.) Family Solanaceae were used as a source of explants to start micropropagation. Leaves were successfully sterilized using the treatment of sodium hypochlorite (NaOCl) at 1.0% that showed the highest percentage of survival without any contamination. The highest fresh weight of callus/explant was recorded when the leaf segments were cultured on MS media which contained 0.5 mg/l BA and 1.5 mg/l NAA. Interestingly, some treatments recorded direct organogenesis (direct shoots and roots) from the leaf explants. The highest direct shoot formation was recorded with MS medium supplemented with 2 mg/l BA alone. However, it was clear that all obtained shoots with the responded treatments were vitrified. Therefore, in a trial to obtain unvitrified shoots as indirect organogenesis through callus formation and differentiation, all indirect obtained shoots were vitrified except some shoots (4.7%) obtained on MS medium supplemented with 1.0 mg/l BA alone. These unvitrified shoots were successfully acclimatized in soil mixture of sand and peatmoss (1:2, v: v) that showed the best growth and percentage of survival (95%). Sixteen unvitrified shoots were produced through cell line technique on recommended media. These media were, MS medium supplemented with 0.5 mg/l BA + 1.5 mg/l NAA for callus formation, and MS medium which contained 1.0 mg/l BA for callus differentiation. The effect of number of subcultures and the clone type on the growth and development of these clones was evaluated by subculturing the sixteen clones for five subcultures on MS medium supplemented with 2.0 mg/l BA. Subculture number 3 significantly showed the highest shoot number, leaf number and fresh weight/plantlet compared to all other subcultures. Concerning the effect of the clone type, clones No. 9 and 10 showed the highest response in the same parameters mentioned above. The obtained plantlets of all clones were successfully acclimatized. However, during the acclimatization, plantlets of clones No. 11 and 10 showed the highest responses of survival percentage (95 and 92.33%, respectively), and clones No. 9 and 10 showed high responses in the other growth parameters (shoot and leaf number/plantlet and plantlet height). Results of the rest of this study showed that, the highest yield of total glycoalkaloids (244.6 mg/g) was recorded with clone No. 10 followed by clone No. 9 when compared with the mother plant and other clones. The HPLC analysis showed four main components (solamargine, solasonine, solanine and solasodine). Interestingly, clone No. 2 showed an increase in solamargine and solanine percentage (27.32 and 30.33 %, respectively) when compared with all other clones including the mother plant. The highest percentage of solasonine (74.18 %) was obtained with clone No. 8, and solasodine (14.31 %) was recorded with clone No. 4. SDS-PAGE of leaf proteins, isozymes (peroxidase and polyphenol oxidase), randomly amplified polymorphic DNA using PCR (RAPD-PCR) and inter simple sequence repeat using PCR (ISSR-PCR) were used to assess the genetic diversity of Solanum viride plants under investigation (mother plants and its 16 clones). All these systems confirmed that, all the sixteen clones were different from the mother plant. Moreover, most of these clones showed some differences between each other. Accordingly, recommendation can be raised as to produce unvitrified plantlets as new clones of green nightshade (Solanum viride Solander ex Forst. f.) through cell line technique