Brent H. McCown

Agrobacterium mediated transformation and regeneration of Populus

SummaryA plant transformation and regeneration system has been developed for Populus species. Leaf explants, from stabilized shoot cultures of a Populus hybrid NC-5339 (Populus alba x grandidentata), were co-cultivated with Agrobacterium tumefaciens on a tobacco nurse culture. Both oncogenic and disarmed strains of A. tumefaciens harboring a binary vector which contained two neomycin phophotransferase II (NPT II′) and one bacterial 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase (aroA) chimeric gene fusions were used. Shoots did not develop when leaf explants were co-cultivated with the binary disarmed strain of A. tumefaciens. However, transformed plants with and without the wild type T-DNA were obtained using an oncogenic binary strain of A. tumefaciens. Successful genetic transformation was confirmed by NPT II′ enzyme activity assays, Southern blot analysis and immunological detection of bacterial EPSP synthase by Western blotting. This is the first report of a successful recovery of transformed plants of a forest tree and also the first record of insertion and expression of a foreign gene of agronomic importance into a woody plant species.

Using next-generation sequencing approaches to isolate simple sequence repeat (SSR) loci in the plant sciences

The application of next-generation sequencing (NGS) technologies for the development of simple sequence repeat (SSR) or microsatellite loci for genetic research in the botanical sciences is described. Microsatellite markers are one of the most informative and versatile DNA-based markers used in plant genetic research, but their development has traditionally been a difficult and costly process. NGS technologies allow the efficient identification of large numbers of microsatellites at a fraction of the cost and effort of traditional approaches. The major advantage of NGS methods is their ability to produce large amounts of sequence data from which to isolate and develop numerous genome-wide and gene-based microsatellite loci. The two major NGS technologies with emergent application in SSR isolation are 454 and Illumina. A review is provided of several recent studies demonstrating the efficient use of 454 and Illumina technologies for the discovery of microsatellites in plants. Additionally, important aspects during NGS isolation and development of microsatellites are discussed, including the use of computational tools and high-throughput genotyping methods. A data set of microsatellite loci in the plastome and mitochondriome of cranberry (Vaccinium macrocarpon Ait.) is provided to illustrate a successful application of 454 sequencing for SSR discovery. In the future, NGS technologies will massively increase the number of SSRs and other genetic markers available to conduct genetic research in understudied but economically important crops such as cranberry.

Stable transformation of Populus and incorporation of pest resistance by electric discharge particle acceleration.

Three different target tissues (protoplast-derived cells, nodules, and stems) and two unrelated hybrid genotypes of Populus (P. alba x P. grandidentata ‘Crandon’ and P. nigra ‘Betulifolia’ x P. trichocarpa) have been stably transformed by electric discharge particle acceleration using a 18.7 kb plasmid containing NOS-NPT, CaMV 35S-GUS, and CaMV 35S-BT. Four transformed plants of one hybrid genotype, NC5339, containing all 3 genes were recovered and analyzed. Two expressed GUS and one was highly resistant to feeding by 2 lepidopteran pests (the forest tent caterpillar, Malacosoma disstria, and the gypsy moth, Lymantria dispar.) Pretreatment of the target tissues, fine-tuning of the bombardment parameters, and the use of a selection technique employing flooding of the target tissues were important for reliable recovery of transformed plants.

Resorption Protection. Anthocyanins Facilitate Nutrient Recovery in Autumn by Shielding Leaves from Potentially Damaging Light Levels

The resorption protection hypothesis, which states that anthocyanins protect foliar nutrient resorption during senescence by shielding photosynthetic tissues from excess light, was tested using wild-type (WT) and anthocyanin-deficient mutants of three deciduous woody species, Cornus sericea, Vaccinium elliottii (Chapmn.), and Viburnum sargentii (Koehne). WT Betula papyrifera (Marsh) was included to compare the senescence performance of a species that does not produce anthocyanins in autumn. Plants were subjected to three environmental regimes during senescence: an outdoor treatment; a 5-d high-stress (high light and low temperature) treatment followed by transfer to a low-stress environment and a low-stress treatment that served as control. In the outdoor treatment, the appearance of anthocyanins in senescing leaves of WT plants was concomitant with the development of photo-inhibition in mutant plants of all three anthocyanin-producing species. In the high-stress environment, WT plants maintained higher photochemical efficiencies than mutants and were able to recover when transferred to the low-stress environment, whereas mutant leaves dropped while still green and displayed signs of irreversible photooxidative damage. Nitrogen resorption efficiencies and proficiencies of all mutants in both stressful treatments were significantly lower than the WT counterparts. B. papyrifera displayed photochemical efficiencies and nitrogen resorption performance comparable with the highest of the anthocyanin-producing species in all three senescing environments, indicating a photoprotective strategy divergent from the other species studied. These results strongly support the resorption protection hypothesis of anthocyanins in senescing leaves.

Culture and regeneration of Populus leaf protoplasts isolated from non-seedling tissue

Abstract Leaf protoplasts of Populus alba L. × P. grandidentata Michx. (NC-5339) were isolted from shoot cultures of non-seedling origin and cultured through plant regeneration. Complete protoplast development was dependent on providing a stress-free culture environment which included eliminating ammonium, agar, exudate build-up, and light during the culture period. Contact with a solid surface appeared to stimulate development and thus the protoplasts were cultured in a liquid floating-disc system in which they adhered to the fibers of a polyester screen. Protoplasts exhibited a slow, staged development which resulted in cell division 6 weeks following protoplast isolation. The resulting colonies proliferated rapidly and rooted spontaneously. Shoot regeneration occurred when the protoplast-derived calli were exposed to thidiazuron, and such shoots could be readily rooted. This is the first report of reproducible plant regeneration from leaf protoplasts of non-seedling origin of a tree species.

Recovery of plants from leaf protoplasts of hybrid-poplar and aspen clones

Leaf protoplasts were isolated from shoot cultures of two hybrid poplar clones (Populus alba × P. grandidentata ‘Crandon’, NC-5339 and P. nigra ‘Betulifolia’ × P. trichocarpa, NC-5331) and the Upright European Aspen (P. tremula ‘Erecta’) and were cultured in contact with screen discs floated in liquid medium. Protoplast culture was influenced by the growth medium of the source shoot cultures, the protoplast purification procedure, the plating density, and the presence or absence of a coconut water and casein hydrolysate supplement added to the culture medium. The protoplast-derived cells divided more quickly and with higher incidence than previously reported for hybrid poplars. Shoots were regenerated from the protoplast-derived calli and were maintained as shoot cultures. Plants were developed from microcuttings rooted ex vitro and were grown-on in the greenhouse and field.

Special symposium: In vitro plant recalcitrance recalcitrance of woody and herbaceous perennial plants: Dealing with genetic predeterminism

SummaryAs a general, long-lived perennial plants probably present the most challenging obstecles to the researcher, breeder or propagator utilizing microculture as a tool. These challenges appear during all stages of the microculture process, but are probably most resplendent during the stabilization phase. What may be particularly frustrating is that much of this ‘recalcitrance’ is genetically driven and is thus difficult to control by environmental and nutritional manipulations in microculture. Perennials have complex seasonal cycles and life cycles, which complicate control of their growth in microculture. As shoot cultures have provided useful tools for overcoming these limitations, the inability to establish stabilized shoot cultures is a major form of recalcitrance. Plants having seasonal growth dynamics dominated by strong episodic or determinant shoot growth are some of the most recalcitrant species because stabilized shoot cultures cannot be readily generated. In some cases, episodic growth may be tied closely to phase state and can thus be controlled by manipulating phase; nevertheless, adequate controls have not been identified for many problematic plants. Another trait contributing to recalcitrance of perennials is the relatively slow growth rate in microculture. Slow growth complicates such procedures as selection of transformed tissues. The high phenolic content of many perennial tissues can interfere with the efficacy of transgenic traits such as β-glucuronidase. Developmentally determined growth characteristics such as plagiotropism may persist through all stages of microculture and complicate the recovery of commercially useful micropropagules. Although some technical approaches can occasionally circumvent immediate microculture limitations, general solutions await the development of a deeper understanding of physiological bases of such genetically predetermined phenomena.

Nodule Culture: A Developmental Pathway with High Potential for Regeneration, Automated Micropropagation, and Plant Metabolite Production from Woody Plants

Nodules are independent, spherical, dense cell clusters which form a cohesive unit and display a consistent internal cell/tissue differentiation. At a minimum, three cell types (meristematic cells, plastid-dense parenchyma, and vascular elements) and two cell layers (epidermal and internal cortex/vascular) can be distinguished in nodules of poplar. Although nodules have been randomly observed by many researchers working with a myriad of plant species, they most commonly are seen in cultures of woody plant species being differentiated from dedifferentiated cells. We have developed liquid culture systems in which nodules are the predominant structures. Such cultures grow via nodule enlargement (a three-stage process) and nodule multiplication (via two general pathways). In general, nodules display a high capacity for plant/organ regeneration via organogenesis. The nodular developmental pathway parallels that of the embryogenic developmental pathway; a theoretical comparison of the two pathways as bridges between totipotency and competence is discussed. Although the research is still very preliminary, nodule cultures have apparent applications in regeneration strategies, automated micropropagation, and in vitro phytochemical production.

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.

A Comparison of Source Tissue for Protoplast Isolation from Three Woody Plant Species

Summary Greenhouse-grown and shoot-cultured stock plants from 2 woody plant genera (Betula and Rhododendron) were compared as source material for protoplast isolation. Shoot cultures provided higher yields/gram source tissue and enhanced viability of isolated protoplasts. Successful isolation depended on stock culture age and culture environment, with best results from young (20-30-day-old) shoot cultures. Additional advantages of the use of shoot cultures for protoplast research, especially on woody species, are discussed.