Propagation, micropropagation and cryopreservation of Buxus hyrcana Pojark., an endangered ornamental shrub

Buxus hyrcana Pojark. is an important ornamental shrub, widely used for landscape and gardens. This ornamental shrub is a critically endangered species due to invasion of some pathogens. Rooting of this species is slow using propagation technique such as cutting. This study was carried out in three sections: 1) the effect of indole-3-butiyric acid (IBA) and ɑ-naphthalene acetic acid (NAA) on rooting of semi-hardwood cuttings in greenhouse condition; and 2) the effect of 6-benzylaminopurine (BAP), IBA and NAA on micropropagation in tissue culture condition and 3) cryopreservation of shoot tip by encapsulation-dehydration technique. For the first experiment, the basal region of the cutting was treated with various IBA and NAA solutions: 0 (free of IBA), 500, 1000, 2000 and 3000 mg l− 1. The 1000 mg l− 1 IBA along with 1000 mg l− 1 NAA induced the largest number of root (8.700/plantlet). For the second experiment, shoot tip dissected from 2-years-old pot-grown plants was cultured on Murashige and Skoog (MS) medium supplemented with 50 combinations of BAP, IBA and NAA at concentrations of 0.00, 0.50, 1.00, 1.50 and 2.00 mg l− 1. Here, we present a new protocol for sterilization of shoot tip. This shoot tip sterilization protocol minimized bacterial and fungal contamination. Multiple shoot formation (6.433/plantlet) was obtained through the use of 1.00 mg l− 1 BAP. Also, the optimum plant growth regulators (PGRs) combination for the largest number of root (7.633/plantlet) was 0.50 mg l− 1 BAP plus 1.00 mg l− 1 NAA. Plantlets were hardened-off and transferred to soil for further growth. About 95% of the acclimatized plantlets were established successfully. Regenerated plantlets were morphologically identical. For the third experiment, B. hyrcan shoot tip was cryopreserved by direct immersion in liquid nitrogen (LN) using encapsulation-dehydration method. After rapid rewarming, encapsulated and non-encapsulated shoot tips were cultured on MS medium enriched with 0.00, 0.50, 1.00, 1.50 and 2.00 mg l− 1 of BAP, IBA and NAA for rapid germination and growth. The highest percentage of re-growing the cryopreserved shoot tip was 60% for those grown on medium containing 0.50 mg l− 1 BAP along with 1.50 mg l− 1 NAA. No survival was observed for non-encapsulated shoot tip after storage in LN and through cultivation on regeneration medium.