David T. Webb

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.

Characterization of immature embryos of interior spruce by SDS-PAGE and microscopy in relation to their competence for somatic embryogenesis

SDS-PAGE analysis of total proteins from cotyledonary embryo explants reveals that their competence to form somatic embryos is limited to a specific stage of development prior to the accumulation of storage proteins. When protein profiles of embryo explants of different open pollinated families from the same collection date are compared, there is a close relationship between the absence of storage proteins and their ability to produce embryogenic callus. In addition, the appearance of storage proteins in embryos from subsequent collections is associated with their loss of competence. Light microscopy combined with staining for total protein demonstrates that competent immature embryos have cotyledons but do not contain protein bodies.

Characterization and tissue-regulated expression of genes involved in pineapple (Ananas comosus L.) root development

Abstract This study was initiated to increase the molecular genetic resources available for pineapple ( Ananas comosus L.) breeding and biotechnology. We differentially screened a root cDNA library for mRNAs that are preferentially present in the roots. Over 300 randomly selected cDNAs from the root library were categorized into 14 classes based on their expression characteristics. About 41% of the clones were classified as constitutive, being expressed about equally in roots, fruit and aerial tissues, while 23% of the clones were expressed higher in roots than other tissues. Twenty-four unique clones were sequenced and preliminarily identified based on sequence homology. Genes involved in lignin biosynthesis, cell wall biogenesis, and responses to stress or pathogens were highly represented. Six mRNAs (encoding a germin-like protein, mannose-binding lectin, two lipoxygenases, xyloglucan endotransglycosylase and peroxidase) were verified to be preferentially present in roots by analyzing in detail their abundance during field-grown plant and fruit development. Analysis of mannose-binding lectin gene expression via tissue-prints and micro-dissection of roots showed that its transcript was predominantly present in the protoderm. The occurrence of stress/pathogen-related mRNAs in healthy tissues is discussed in terms of processes that allow CAM plants to adapt to extreme environments. The molecular methods and genetic resources developed in this work will facilitate isolation of root-specific promoters, physical mapping, marker-assisted breeding, genomic studies and bioengineering for pineapple improvement.

Novel thigmomorphogenetic responses in Carica papaya: touch decreases anthocyanin levels and stimulates petiole cork outgrowths.

BACKGROUND AND AIMS Because of its rapid growth rate, relative ease of transformation, sequenced genome and low gene number relative to Arabidopsis, the tropical fruit tree, Carica papaya, can serve as a complementary genetic model for complex traits. Here, new phenotypes and touch-regulated gene homologues have been identified that can be used to advance the understanding of thigmomorphogenesis, a multigenic response involving mechanoreception and morphological change. METHODS Morphological alterations were quantified, and microscopy of tissue was conducted. Assays for hypocotyl anthocyanins, lignin and chlorophyll were performed, and predicted genes from C. papaya were compared with Arabidopsis touch-inducible (TCH) and Mechanosensitive channel of Small conductance-like genes (MscS-like or MSL). In addition, the expression of two papaya TCH1 homologues was characterized. KEY RESULTS On the abaxial side of petioles, treated plants were found to have novel, hypertrophic outgrowths associated with periderm and suberin. Touched plants also had higher lignin, dramatically less hypocotyl anthocyanins and chlorophyll, increased hypocotyl diameter, and decreased leaf width, stem length and root fresh weight. Papaya was found to have fewer MSL genes than Arabidopsis, and four touch-regulated genes in Arabidopsis had no counterparts in papaya. Water-spray treatment was found to enhance the expression of two papaya TCH1 homologues whereas induction following touch was only slightly correlated. CONCLUSIONS The novel petiole outgrowths caused by non-wounding, mechanical perturbation may be the result of hardening mechanisms, including added lignin, providing resistance against petiole movement. Inhibition of anthocyanin accumulation following touch, a new phenotypic association, may be caused by diversion of p-coumaroyl CoA away from chalcone synthase for lignin synthesis. The absence of MSL and touch-gene homologues indicates that papaya may have a smaller set of touch-regulated genes. The genes and novel touch-regulated phenotypes identified here will contribute to a more comprehensive view of thigmomorphogenesis in plants.

Zygotic embryogenesis in Anthurium (Araceae).

Morphological, anatomical, and histochemical aspects of zygotic embryogenesis by Anthurium andraeanum Lind. were investigated from 4 to 24 wk postpollination. Anatomical features were correlated with morphology of the spadix and capacity of embryos to germinate in vitro. Development from a single-cell zygote to fully mature seed takes 24 wk. The suspensor was two ranked and obvious during the early stages of embryogeny. It was apparent by week 8, substantial until week 14, and diminished rapidly until its absence by week 22. Differentiation of the shoot apex, cotyledon, and protoderm occurs at 14 wk. The embryo starts to derive nutrition from the endosperm at this time, and germination of cultured ovules reached 56%. By 20 wk the shoot apex had visible leaf primordia and the root apex was clearly defined. The cotyledon was well developed and surrounded the shoot tip. The storage of protein and starch was at its greatest in the endosperm and embryo. Furthermore, 100% germination of cultured ovules and embryos occurred at 20 wk and thereafter. Fully mature embryos at 24 wk are green and contain protoxylem elements.

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.