Establishment of contaminant-free perennial plants in vitro

Perennial plant tissue cultures are established by disinfecting field or gr eenhouse-grown plant parts and transferring them to sterile medium in vitro . Typically, shoots harvested from field or greenhouse-grown plants are pla ced in water, either to force growth from dormant branches or to maintain t hem until ready for explanting. In spite of extreme care, 90 to 100% contam ination rates in newly established in vitro cultures are not unusual. Exper ience 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 condit ion. and using pH neutralized bleach to sterilize instruments during subcul ture. Other methods to reduce contamination include establishing field-gl o wn plants in a greenhouse where inoculum levels can be better controlled, t rellising vining plants to get them off tie soil, avoiding wetting foliage, and selecting vigorous explants that are not in contact with soil.