Infection of Pinus radiata with Agrobacterium rhizogenes and long-term growth of detached hairy roots in vitro

Bergmann BA, Dukes J, Stomp A-M. 1997. New Zealand Journal of Forestry Science 27(1): 11-22.

Infection of Pinus radiata D.Don with Agrobacterium rhizogenes strains A4 and R1601 was demonstrated through greenhouse inoculation of seedlings and verification of opine production in gall tissue. Frequencies of seedlings exhibiting gall formation after epicotyl stem inoculation was 4% for strain A4 and 7% for strain R1601 (compared to 59% for the positive control inoculation with A. tumefaciens strain 542). Adventitious shoots of 22 P. radiata clones, micropropagated from seeds of the same bulk seedlot from which the seedlings had been grown, differed significantly in susceptibility to A4, R1601, and 542 when inoculated in vitro. In vitro shoots were more susceptible to both A. rhizogenes strains but less susceptible to A. tumefaciens 542 than seedlings inoculated in the greenhouse. The highest frequency of shoots forming a gall and/or hairy roots was observed in a different P. radiata clone for each Agrobacterium strain: 22% for A4, 44% for R1601, 58% for 542. Agrobacterium rhizogenes strain R1601 was superior to A4 for infection of P. radiata shoots grown in vitro. Gall production and hairy roots were observed in 11% and 1%, respectively, of P. radiata shoots 12 weeks after in vitro inoculation with A. rhizogenes strain R1601. Hormone autotrophic, bacteria-free cultures were established from stem segments that possessed tissue proliferating from Agrobacterium inoculation wound sites. Hairy roots formed directly from the wound site as early as 8 weeks after inoculation. Though opines were difficult to detect in callus tissues resulting from R1601 inoculations, the likelihood of detection was increased if the sample included roots. Cultures of detached hairy roots maintained on quarter strength modified LePoivre medium reached an average length of 19.5 cm after 1 year and were shown to continue opine expression throughout that time. A greenhouse rooting experiment provided no evidence that rooting of P. radiata tissue culture shoots was improved by inoculation with A. rhizogenes R1601.