Development of a laboratory protocol for the micropropagation of Monterey pines (Pinus radiata), Año Nuevo stand
Monterey pine (Pinus radiata), a native tree to California and two Mexican islands, is important both ecologically and economically. Outside native stands, Monterey pines are grown for landscaping in California and on plantations around the world. Pitch canker, a disease caused by the fungus Gibberella circinata Nirenberg & O'Donnell (Fusarium circinatum Nirenberg and O'Donnell) is threatening the survival of Monterey pines. The disease currently affects Monterey pines in many parts of the world including the native stands. No effective chemical or biological control is available but some Monterey pines show resistance to the disease. The purpose of this project was to develop a working protocol for producing genetic clones of the resistant pines through micropropagation. These genetic clones will be used for outplanting in places outside the native stands for ornamental and plantation purposes. This project analyzes the results of ten trials with varied parameters and bases the final protocol on the parameters used in the trial that induces the growth of new shoots. The final protocol developed in this project describes, step-by-step, the media preparation for the initiation, plant material collection, surface sterilization of plant material, plating in media and initiation of shoots on explants. The protocol calls for collecting shoot tips with hardened buds that have not yet elongated, then washing the shoot tips in sterile water with Tween 20 for 15 minutes. The shoots tips are then surface sterilized in a 50% bleach solution for 20 minutes. The explants are broken into disks (to minimize damage to the cells) by inserting the tip of a scalpel and tilting it slightly. The initiation media shown to induce growth consists of ½ strength LePoivre basal salt mixture, 5mg/L benzylaminopurine, 3% sucrose and 0.8% agar and is adjusted to a pH of 5.7, then autoclaved for 20 minutes. The explants are inserted into solidified media and incubated in a growth chamber programmed for 16 hours of light and 8 hours of dark with temperatures of 27ºC and 22ºC and light irradiance of 80µEm-2s-1. After 1 month the protocol calls for transferring the growing shoots to elongation media with full LP basal salts and transferring every month. When the number of desired shoots has been reached the forthcoming protocol for rooting can be followed.