| Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) is a widely cultivated timber and Christmas tree species. The Christmas tree industry plays an important role in agriculture, both within Pennsylvania as well as across the nation. According to the 2007 census of agriculture, Pennsylvania was ranked as the 4th state in the nation in terms of Christmas tree production. Many conifer species have been utilized for Christmas tree production, generating valuable revenue for growers and retailers. The overall goal of this micropropagation project is to develop a true to type clonal propagation system to alleviate the cost of tree to tree variation by conventional seedling propagation. Eventually, developed techniques can be transferred to Pennsylvania Christmas tree growers willing to utilize these techniques to enhance their profits. This study addresses three goals associated with Douglas-fir micropropagation which include (1) to understand and characterize the demography and phenology of collected explant materials, (2) to investigate factors associated with the culture vessels to ensure proper tissue culture practices, and (3) to assess specific objectives for shoot multiplication and rooting. When explant demography and phenology were evaluated, the size of inner vegetative bud was found to be highly correlated with later explant height growth in vitro. Explant growth performance in vitro was not only influenced by genotype but also bud collection season, bud collection position, bud type, and donor age. To understand the physical factors associated with micropropagation, the effects of medium pH and hyperhydricity on explant growth dysfunction in vitro were evaluated. Prolonged culturing was found to decrease medium pH and reduce explant weight growth over time. The addition of 2-(N-morpholino) ethanesulfonic acid (MES), a medium pH stabilizer, resulted in better maintenance of medium pH over time, but did not necessarily alter explant growth potential. Additionally, moisture condensation inside the culture vessel was found to be correlated with explant hyperhydricity. A shortened culture time within the petri dish vessel and subsequent transfer to larger vessels with greater head-air space was found to reduce the occurrence of hyperhydricity of explants. To define efficient explant multiplication and rooting protocols, a wide range (0--204.8 mg/L) of benzylaminopurine (BA) concentrations and rooting conditions were evaluated with Douglas-fir. For efficient induction of multiple shoots from bud explants of both juvenile and mature genotypes, the optimal BA concentration was found to be 6.4--51.2 mg/L at 2--3 week induction time. For mature genotypes, shoot organogenesis could also be induced by applying the range of 6.4--204.8 mg/L BA. Rooting of Douglas-fir micropropagated explants is feasible by incubation in half-strength mDCR medium containing 3 mg/L naphthaleneacetic acid (NAA) with 3-day complete darkness followed by 3-day complete light at 20ºC. However, the efficiency of rooting was low and will require further investigation. By understanding these influential factors and their roles in explant induction and development in vitro, healthy and vigorous shoots could be produced with which to facilitate further investigations. These findings may contribute to an efficient micropropagation system, that will allow Christmas tree growers to avoid losses from variation from seedlings in the future. |
