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Abstract
The main objectives of this investigation were: 1) Establishment of callus and cell suspension cultures of Sisymbrium irio. 2) Development of a low cost in vitro technique for establishment of callus and cell suspension cultures of Sisymbrium irio by substituting vitamins and synthetic phytohormones in tissue culture medium by Spirulina aqueous extract for minimizing the use of the expensive components of this medium. 3) Phytochemical investigation of callus and cell suspension cultures of Sisymbrium irio. 4) Evaluating the antioxidant capacity of the established cultures. 5) Screening of the antimicrobial activities of suspension cells extracts to clarify their medicinal and biological importance. This extract stimulated growth and delayed senescence of the calli and could successively replace both vitamins and phytohormones in MS medium. The biomass could be immobilized successfully using calcium alginate beads.analysis of immobilized biomass revealed an increase of limonene and palmitic acid percentage up to 7 and 8 folds, respectively and inhibition of other volatile constituents productivity. The different extracts from sisymbrium irio cultures showed significant antimicrobial activities against fungi and gram positive bacteria and to less extent against gram negative bacteria . chloroform and petroleum ether extracts were the most potent antimicrobial fractions. For induction of Sisymb1’ium i1’io callus different explants were cultured on MS medium supplemented with auxin (2,4-D or NAA) combined with I<n at different concentrations. The highest callus formation was obtained from seed. hypocotyl explants on medium contained 2,4-D at 2 mg/l plus I<n at either 0.5 or 1 mg/1. Whereas, the optimal phytohormonal combination for growth and maintenance ofhypocotyl derived calli was 1 mg/I 2,4-0 with 0.5 mg/I I(n. The effect of Spi1’ulin.a aqueous extract on growth of Sisymb1’ium irio callus and cell suspension cultures was investigated. This extract stimulated growth and delayed senescence of the calli and could successively replace both vitamins and phytohormones in MS medium. chromatographic analysis of SAE using IIPL.C confirmed’ the presence of high concentrations of vitamins and phytohormones. Biochemical analysis of Sisymbrium irio callus and cell suspension cultures revealed that, SAE-treated cultures had higher protein and nitrogen contents than the control cultures (untreated cultures). Moreover. SAE enhanced total phenolic and flavonoid contents as well as the antioxidant activities 0 r the treated cultures. Whereas, in all treatments; the addition of SAE to the mediJ resulted in elevated decline in the activities of the antioxidant enzymes. GLC analysis of the fatty acids in the cultures indicated a more pronounced presence of unsaturated fatty acids in SAE-treated cultures than untre81ed culture’._ Additionally several compounds were isolated for the first time li’om ;/1 ”;/m cultures of Sisymbr.i,uni irio including lupeol acetate, Jupeol and a-amyrin in addition to a mixture of ~-sitosterol, stigmasterol and their glucosides. Moreover, 5,4’-di-hydroxy, 6, 7, 8, 3’-tetramethoxy flavone.