Optimizing a Protocol for Sterilization and in vitro Establishment of Vegetative Buds from Mature Douglas Fir Trees

Abstract

A successful tissue culture initiation step often begins with effective explant sterilization. To improve douglas fir bud culture initiation, five sterilization treatments (20% bleach, 100% bleach, 3 second flaming, 5 second flaming, and self-extinguishing flaming) were evaluated for their effectiveness on winter and spring bud sterilization. The 20% and 100% bleach treatments resulted in the highest percentage of healthy bud cultures (>90% for winter buds). Spring buds showed a higher level of contamination with 20% bleach sterilization (36%) than did winter buds (1%). Successful sterilization was also achieved by flaming, but bud injury was observed. Increased flaming time caused a decrease in the percentage of healthy actively growing buds. The percentage of healthy bud cultures after 3 second flaming, 5 second flaming and self-extinguishing-flaming (9 to 14 s) were 66%, 59%, and 10% respectively. In addition, sterilization by either approach required subsequent bud dissection to remove the outer scales; otherwise most buds were lost to contamination. When sterilization was followed by bud dissection, contamination rates for winter buds were <2% for all treatments. After successful sterilization and culture initiation, bud expansion was the highest (50% to 98%) in the presence of low concentrations of BA (0 to 0.045 μmol·L–1), while high concentrations of BA (0.448 to 4.527 μmol·L–1) reduced bud expansion (0% to 60%), but promoted bud multiplication.