Establishment of contaminant-free perennial plants in vitro

Abstract

Perennial plant tissue cultures are established by disinfecting field or greenhouse-grown plant parts and transferring them to sterile medium in vitro. Typically, shoots harvested from field or greenhouse-grown plants are placed in water, either to force growth from dormant branches or to maintain them until ready for explanting. In spite of extreme care, 90 to 100% contamination rates in newly established in vitro cultures are not unusual. Experience has identified several routine procedures that reduce contamination, such as minimizing the amount of time a stem cutting is maintained in water before being explanted, adjusting pH of the medium to a more acidic condition, and using pH neutralized bleach to sterilize instruments during subculture. Other methods to reduce contamination include establishing field-grown plants in a greenhouse where inoculum levels can be better controlled, trellising vining plants to get them off the soil, avoiding wetting foliage, and selecting vigorous explants that are not in contact with soil.