Radiata pine (Pinus radiata D. Don) is a species of considerably economic importance in New Zealand and other southern-hemisphere countries. Large-scale vegetative propagation of some "desirable" clones may be limited if they do not respond to traditional root inducing treatments such as exposure to indole-3-butyric acid (IBA), a root forming plant growth regulator.

Agrobacterium rhizogenes has been used to enhance adventitious root formation in a number of plants (Hatta et al. 1996). This bacterium transfers its transfer-DNA (T-DNA) which is a portion of the large plasmid called the root-inducing plasmid (pRi) to susceptible plant cells where the T-DNA, if integrated into the nuclear genome of the plant cell, will encode genes that direct the synthesis of auxin (indole-3-acetic acid) and / or increase the sensitivity of the transformed plant cells to auxin (Hatta et al. 1996). The endogenous production of auxin and / or an increase in auxin sensitivity can lead to the formation of hairy roots at the site of infection.

This study reported the effects of two strains of A. rhizogenes on root formation in radiata pine. In particular, the potential for a good cultured hypocotyl system for investigations of adventitious root formation in pines has been demonstrated.

Materials and Methods

Bacterial culture

Two strains of virulent Agrobacterium rhizogenes, A4T (Dommisse et al. 1990) and LBA9402 (kindly provided by Dr. D. Clapham, Swedish University of Agricultural Sciences, Uppsala) were used in this work. The bacteria were cultured either on solid YM medium (Life Technologies, Gaithersburg, MD, USA) or in liquid YM medium at 26ºC.

Growth of seedlings

Radiata pine seeds were surface-sterilized in 70% (v/v) ethanol for 30 s, rinsed briefly in sterile water; then soaked in 50% (v/v) of a commercial bleach (containing 31.5 g/liter active sodium hypochlorite) for 30 min before being rinsed thoroughly with sterile water. The sterilized seeds were sown in autoclaved vermiculite in tissue culture jars, and stratified in a cold room (4ºC) for one week. The jars were then maintained in a warm dark room (26ºC) till seedling emerged before transfer to a plant growth room at 22ºC with continuous lighting at 80 µmol · m^-2 · s^-1 . Uniform seedlings, which were about 1 mm in diameter and with 2.5-3 cm long hypocotyls, were selected for use as cuttings. All experiments here were on seedling materials grown aseptically unless indicated otherwise.

Inoculation of hypocotyl segments

Hypocotyl segments of about 1 cm long, two or three from each seedling, were randomly selected and inserted with the cut end proximal to the root into ½ MS (Murashige and Skoog, 1962) medium (containing 20 gL^-1 sucrose and 8 gL^-1 agar) in Petri dishes. To the top cut-surface of the segments, a colony of the appropriate agrobacterial strain was inoculated with a loop, and then the Petri dishes were placed in a plant growth room as described above. The segments without inoculation with the bacteria were designated as controls. The segments were either kept in the IBA (indole-3-butyric acid)-free medium or transferred to the above medium supplemented with 4.4 uM IBA at day 7 for 1 week before they were transferred back to the growth regulator-free medium.

Inoculation of adventitious shoots

Three year-old greenhouse-grown seedlings were pruned to remove the top 5-7 cm of growth and after 4 months the newly emerged adventitious shoots with w3 3-5 nodes were used for bacterial inoculation as follows. The adventitious shoots were injected in vivo with approximately 20 µl of the bacterial suspension using a syringe fitted with a 24G needle at the site of 10 - 15 cm from the tops of the adventitious shoots. One week later, the shoots were removed from the plants at about 0.5 cm below the injection site, and inserted upright in tap-water saturated vermiculite in small containers (22 cm × 15 cm × 12 cm) that were covered with a plastic film and then placed in a plant growth room as described above. Control shoots were treated in the same way except that the bacterial suspension was replaced with liquid YM medium.

Results and Discussion

The cultured hypocotyl segments after co-cultivation with strain LBA9402 could produce a significantly greater rooting percentage and mean root number compared with those treated with A4T. Moreover, both the rooting percentage and mean root number in the treatments with the bacterium could be enhanced by the supplement with IBA in the medium. However, no root formation resulted in cultured hypocotyl segments without inoculation with A. rhizogenes, regardless whether IBA was present or absent in the medium.

Strain LBA9402 was used to induce roots in adventitious shoots because it seemed to be more effective than A4T in the preliminary root induction experiments. Eight weeks after inoculation, more adventitious shoots inoculated with LBA9402 rooted than control shoots did (54.8 and 8.0%, respectively).

The cultured hypocotyl segments described here is a good system for the study of adventitious root induction in pines by A. rhizogenes. Since the process of root formation can be easily manipulated under highly defined experimental conditions, and the rooting response could be relatively high, this makes this system attractive for further investigations into the mechanism of root induction by A. rhizogenes in pines. Strain LBA9402 significantly increased rooting percentage and root number in adventitious shoots. It seems worthwhile to screen additional A. rhizogenes strains for efficient root induction in pines. This application, possibly combined with other treatments, appears to be useful for vegetative propagation of radiata pine clones that are difficult to root.

References

DOMMISSE, E.M.; LEUNG, D.W.M.; SHAW, M.L. and CONNER, A.J. Onion is a monocotyledonous host for Agrobacterium. Plant Science, 1990, vol. 69, no. 2, p. 249-257.

HATTA, M.; BEYL, C.A.; GARTON, S. and DINER, A.M. Induction of roots on jujube softwood cuttings using Agrobacterium rhizogenes. The Journal of Horticultural Science, 1996, vol. 71, no. 6, p. 881-886.

MURASHIGE, T. and SKOOG, F. A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiologia Plantarum, 1962, vol. 15, p. 473-497.