In Vitro Shoot Regeneration and Polyploid Induction from Leaves of Hypericum Species

Abstract

Hypericum L. H2003-004-016 is a complex hybrid among Hypericum frondosum Michx., Hypericum galioides Lam., and Hypericum kalmianum L. and exhibits valuable ornamental characteristics, including compact habit, bluish green foliage, and showy flowers. Inducing polyploidy may further enhance the ornamental traits of this hybrid and provide new opportunities for hybridizing with other naturally occurring polyploid Hypericum sp. In this study, in vitro shoot regeneration and treatment of regenerative callus with the dinitroaniline herbicide oryzalin (3,5-dinitro-N4,N4-dipropylsufanilamide) were investigated as a means of inducing allopolyploidy. First, in vitro regeneration was optimized for callus and shoot induction by culture of leaf explants on medium supplemented with benzylamino purine (BA) or meta-topolin (mT) at 5, 10, or 15 μM in combination with indoleacetic acid (IAA) at 0, 1.25, 2.5, or 5 μM. Both BA and mT treatments successfully induced regenerative callus and shoots. Multiple regression analysis estimated maximum regenerative callus (94%) and shoot induction (18 shoots per explant) in medium supplemented with 5 μM BA and 3.75 μM IAA. In the second part of the study, exposure of regenerative callus to oryzalin at 0, 7.5, 15, 30, 60, or 90 μM for durations of 3, 6, or 9 d was investigated for polyploid induction. There was no survival for any of the calli in the 60- or 90-μM oryzalin treatments, but calli subjected to the other treatments exhibited some survival and polyploid induction. Duration had no effect on callus survival or ploidy level, but oryzalin concentration was a significant factor in both. The greatest percentage (44%) of polyploids was induced with 30 μM oryzalin. Spontaneous chromosome doubling was observed in 8% of control explants receiving no oryzalin treatment.