David D. Ellis

Expression of inducible angiosperm promoters in a gymnosperm,Picea glauca (white spruce)

Electrical discharge particle acceleration was used to test the transient expression of numerous inducible angiosperm promoters in a gymnospermPicea glauca (white spruce). Promoter expression was assayed in three different tissues capable ofin vitro regeneration, zygotic embryos, seedlings and embryogenic callus. The promoters tested include the light-inducibleArabidopsis and soybean ribulose-1,5-bisphosphate small subunit promoters and a maize phosphoenolpyruvate carboxylase promoter; a soybean heat-shock-inducible promoter, a soybean auxin inducible promoter and a maize alcohol dehydrogenase promoter. Promoters were cloned into a promoter-less expression vector to form a promoter-β-glucuronidase-nopaline synthase 3′ fusion. A similar construct was made using the cauliflower mosaic virus 35S (CaMV 35S) promoter as a control. All promoters were expressed in white spruce embryos, yet at levels lower than CaMV 35S. In addition, in the embryos the heat-shock and the alcohol dehydrogenase promoters showed inducible expression when given the proper induction stimulus. In seedlings, expression of all promoters was lower than in the embryos and expression was only inducible with the heat-shock promoter in the cotyledons. Of the tissues tested, the expression level of all promoters was lowest in embryogenic callus. Interestingly, the expression of the β-glucuronidase gene in embryogenic callus was restricted to the proembryonal head cells regardless of the promoter used. These results clearly demonstrate the use of particle bombardment to test the transient expression of heterologous promoters in organized tissue and the expression of angiosperm promoters in a gymnosperm.

Transformation of white spruce and other conifer species byAgrobacterium tumefaciens.

Studies of the ability ofAgrobacterium to transform white spruce (Picea glauca), Engelmann spruce (P. engelmanni), Sitka spruce (P. sitchensis) and Douglas-fir (Pseudotsuga menziesii) showed frequencies of gall formation from 0–80% depending upon the strain ofAgrobacterium, and the conifer species. Thirty sixA. tumefaciens strains and oneA. rhizogenes strain were tested on 6 month old white spruce seedlings. NineA. tumefaciens strains induced gall formation on more than 50% of the inoculated trees and at greater than 10% of the inoculated sites. One strain, B2/74 gave rise to galls at 28% of the inoculated sites on white spruce and induced the highest overall frequency of gall formation on all the conifer species tested. Relative frequency of gall formation was consistent among species, although the overall frequency was much higher on Douglas-fir. Of the well characterized strains for which disarmed derivatives are available only A281 (carrying the supervirulent tumor inducing plasmid, pTiBo542) gave efficient transformation. Stable integration of T-DNA encoded genes has been confirmed by the expression of opine synthesis and hormone autonomous growth. The transfer and long-term stable expression of kanamycin resistance and firefly luciferase activity using binary vector systems was also achieved.

LEAF ONTOGENY INFLUENCES LEAF PHENOLICS AND THE EFFICACY OF GENETICALLY EXPRESSED Bacillus thuringiensis cry1A(a) d- ENDOTOXIN IN HYBRID POPLAR AGAINST GYPSY MOTH

We tested the hypothesis that ontogenetic variation in leaf chemistry could affect the efficacy of genetically expressed Bacillus thuringiensiscry1A(a) d-endotoxin, and thus provide spatial variation in (1) foliage protection and (2) selective pressures that could delay the resistance of folivores. Our model consisted of clonal hybrid Populus plants (NC5339). Consumption of foliage and relative growth rates of gypsy moth, Lymantria dispar (L.) increased, and phenolic glycoside concentrations decreased, as leaves from transformed plants containing the cry1A(a) d-endotoxin and nontransformed plants matured from leaf plastochron index (LPI) 1–6. Feeding and growth rates were negatively correlated with phenolic glycosides in both transformed and nontransformed foliage. The presence of the B. thuringiensisd-endotoxin was at most, additive to the effect of the phenolic glycosides. Feeding and growth rates were positively correlated with condensed tannins in transformed foliage, but there was no relationship with condensed tannins in nontransformed foliage. The results indicate that the presence of foliar allelochemicals of poplar can enhance the effectiveness of genetically expressed B. thuringiensisd-endotoxin against gypsy moth larvae. However, the spatial variation in gypsy moth performance in response to the combination of foliar allelochemicals and d-endotoxin was not greater than the effect of ontogenetic variation in foliar allelochemicals alone. These results suggest that for this important pest, foliage protection may be obtained without genetically engineered defenses, and instead, by relying on ontogenetic and clonal variation in allelochemicals. The benefits of combining novel resistance mechanisms with natural ones will depend upon the specific folivores adaptation to natural resistance mechanisms, such as allelochemicals. Moreover, some of the greatest benefits from transgenic resistance may arise from the need to protect trees from multiple pests, some of which may not be deterred by, or may even prefer, allelochemicals that confer protection from a few species.

Immunocytochemical localization of taxol in Taxus cuspidata

The supply of taxol, a valuable anticancer compound, depends completely on the extraction of taxol from Taxus brevifolia (Pacific yew) plants. Although taxol is found in virtually all parts of the plant, nothing is known about the localization of taxol within the cells or tissues of the plant. Portions of T. cuspidata stems were chemically fixed, dehydrated in ethanol, and then embedded in standard resins. In these tissues, immunolocalization using polyclonal antitaxol antiserum indicates that taxol is associated with vacuolar tannin inclusions in axial phloem parenchyma cells. Little or no label is bound over the cell walls of any cell type. However, chemical preparation causes diffusion of taxol, which results in an erroneous localization of taxol. In contrast, using cryotechniques and a water-soluble melamine resin (Nanoplast), taxol is localized almost exclusively in the cell walls of phloem, vascular cambium, and xylem. Our method yields insight into the problems associated with the intra- and extracellular localization of lipophilic plant secondary compounds. It also offers an alternative tissue-preparation protocol that could be useful for the localization of other plant metabolites.

Transgene Expression in Spruce and Poplar: From the Lab to the Field

As the development of transformation systems progresses in annual crop plants, one troubling theme has emerged: there is extreme variation in transgene expression, particularly between plants derived from individual transformation events even within the same petri dish. If such variation in transgene expression occurs in annual crops, what variation can be expected in long-lived perennial crops, such as trees? To answer this question, we have studied the expression over time, both in the lab and in the field, of the uidA gene controlled by an enhanced CaMV 35S promoter in transformants from two Populus hybrids and several transformed lines of Picea glauca. Based on MUG assays for s-glucuronidase (GUS) activity, there is a general trend: the highest and least variable GUS activity occurs in vitro and the most variable GUS activity is in field grown transgenics. Detailed analysis of GUS activity in three different developmental stages of leaves during two growing seasons indicate that uidA expression in young leaves of hybrid poplar NC5339 (P. alba x P. grandidentata) was highly variable and that in older fully expanded leaves, GUS activity was relatively stable during the season and from year to year. In contrast, older leaves in another hybrid poplar, NM6 (P. nigra x P. maximowiczii) had higher GUS activity relative to the younger leaves. In spruce, GUS activity in needles was only detected during needle elongation, yet activity in the stems persists throughout the season. In all transgenics in the field, GUS activity was highest early in the spring just after bud break, then activity rapidly dropped. The suppression of uidA expression has been noted in a limited number of transgenic lines in both spruce and poplar. After three years in the field, uidA expression has not been restored in any transgenic lines that had uidA expression suppressed. Using a wound-inducible promoter (pinII) to control the uidA gene, evidence suggests that year-to-year stability of transgene expression in poplar is not unique to CaMV 35S. It is interesting to note that the overall level of GUS activity in two consecutive seasons was similar within a species and that despite seasonal variability, predictable yearly trends in transgene expression may be possible in woody perennials.

Application of Somatic Embryogenesis to Tree Improvement in Conifers

ABSTRACT Somatic embryogenesis (SE) offers advantages in tree breeding due to the genetic gain improvements that can be realized through selection and production of elite (clonal) lines. Additionally, it is a platform through which value-added traits can be introduced via genetic engineering. At CellFor considerable effort has been directed towards the improvement of SE protocols for coniferous species, with significant focus placed on development in vitro. Recent progress includes rapid bulk-up of cultures via liquid culture, production of desiccation-tolerant embryos in bioreactors and direct delivery of somatic seed to the nursery. Additionally, transgenic spruce, loblolly pine and radiata pine have been developed via SE, with the targets including enhanced cellulose synthesis, lignin modification and control of key developmental pathways As a tool for tree improvement, conifer SE circumvents the infrastructure and time constraints that are inherent to production of improved seed via seed orchards. The application of SE in this context necessitates the development of a deployment strategy that addresses genetic diversity and the genetic gain achieved. Approaches, based on the principles of population genetics, have been developed to optimize both the initial selection strategy and subsequent deployment of clonal lines. Since 1995, CellFor and its predecessor companies have implemented commercial programs for spruce (interior; Sitka), pine (loblolly; radiata; slash) and Douglas-fir. Annual volume production in excess of 1 million seedlings has been achieved.

Light regimes used in conifer tissue culture

Light has a major influence on the growth, development and morphogenesis of plants. In an in vitro environment, where conditions are manipulated to optimize a given response, careful consideration should be given to light quantity and intensity as well as the photoperiod. Unfortunately, all too often the light regime used for a given microculture situation is based either on (1) incomplete information of the optimum in vitro requirements for the species and tissue with which one is working or (2) the conditions that currently exist or are available in a particular research setting. Often a compromise must be also made when choosing a light regime to satisfy a wide range of species within a single growth area.

Establishment of a Shoot Culture System for Larix Decidua

One limiting aspect in the micropropagation of many conifers is the inability to establish a sustained shoot culture based on axillary bud proliferation. In contrast, all successful commercial micropropagation systems of dicotyledonous woody plants, are based on the establishment of such a shoot culture system. Most conifer microculture systems rely on the one time proliferation of adventitious buds from embryonic tissue, with very few species being capable of reliable and sustained axillary bud proliferation from these adventitious buds.

A Transient Assay for Heterologous Promoter Activity in Picea Glauca

One important aspect of the successful genetic engineering of trees is the proper and coordinated expression of introduced foreign genes in the tree. To date, few studies have focused on the controlled expression of foreign genes in forest trees. Using electrical discharge particle acceleration, the expression of several heterologous promoters were tested in Picea glauca (white spruce) embryos, seedlings and embryogenic callus. Promoters tested include: Arabidopsis and soybean ribulose-l,5-bisphosphate small subunit (rbcS) promoters, a maize phosphoenolpyruvate carboxylase (PEP) promoter, a soybean heat shock promoter, a maize alcohol dehydrogenase (ADH) promoter and a soybean auxin inducible promoter. All promoters were used to drive the β-glucuronidase marker gene and expression was determined both with and without the respective induction stimulus required for maximal promoter activity in the source plant. Furthermore, the induction stimuli were also tested to determine the effects of the stimuli on the expression of a cauliflower mosaic virus 35s promoter. While β-glucuronidase gene expression in white spruce embryos was detected from all promoters tested, inducible gene expression was observed only with the heat shock and ADH promoters. Expression in seedlings and embryogenic callus indicate that this method for transient assays allows testing not only of promoter activity but also of tissue and/or cell specific expression.