Adventitious rooting of proliferating shoots is controlled by many physical and chemical factors including phenolic and flavonoid compounds. Chalcone synthase (CHS) is an important enzyme in allocating significant amounts of carbon in the phenypropanoid pathway into the biosynthesis of flavonoid compounds. In this study, the expression of CHS gene during rhizogenesis of in vitro-grown shoots of two genotypes of walnut, ‘Sunland’ and ‘Howard’, was investigated using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The amounts of flavonoids, flavonols, proanthocyanidins and auxin in these cultures were also determined. Patterns of CHS gene expression in different genotypes of shoots undergoing rhizogenesis were associated with observed flavonoids content. Similar observations were also noted for flavonol and proanthocyanidin contents in ‘Howard’ walnut. Overall, a high frequency of rhizogenesis was accompanied with low flavonoid content in shoots. These observed relationships suggested that chalcone synthase is involved in control of flavonoid biosynthesis and in promoting rhizogenesis in walnut microshoots.

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