Joel Hague

Transgenic perennial biofuel feedstocks and strategies for bioconfinement

The use of transgenic tools for the improvement of plant feedstocks will be required to realize the full economic and environmental benefits of cellulosic and other biofuels, particularly from perennial plants. Traits that are targets for improvement of biofuels crops include herbicide resistance, pest, drought, cold and salt tolerance, nutrient use efficiency, altered cell wall composition and improved processing and end-use characteristics. However, controlling gene flow is a major issue and there is no regulatory experience with perennial plants as dedicated biofuels feedstocks. Bioconfinement of transgenes is thus an obvious regulatory and biosafety objective to the release and commercialization of transgenic bioenergy feedstocks. In this article, we review bioconfinement strategies that target pollen or seeds that can be applied to perennial plants used as biofuels. These include male sterility, integration of transgenes into plastid genomes, removal of transgenes in pollen and seeds, transgene expression in vegetative organs for harvest before appearance of reproductive structures or gene use restriction technologies.

Selectable marker independent transformation of recalcitrant maize inbred B73 and sorghum P898012 mediated by morphogenic regulators BABY BOOM and WUSCHEL2

Key messageDiscriminatory co-expression of maizeBBMandWUStranscriptional factor genes promoted somatic embryogenesis and efficientAgrobacterium-mediated transformation of recalcitrant maize inbred B73 and sorghum P898012 genotypes without use of a selectable marker gene.AbstractThe use of morphogenic regulators to overcome barriers in plant transformation is a revolutionary breakthrough for basic plant science and crop applications. Current standard plant transformation systems are bottlenecks for genetic, genomic, and crop improvement studies. We investigated the differential use of co-expression of maize transcription factors BABY BOOM and WUSCHEL2 coupled with a desiccation inducible CRE/lox excision system to enable regeneration of stable transgenic recalcitrant maize inbred B73 and sorghum P898012 without a chemical selectable marker. The PHP78891 expression cassette contains CRE driven by the drought inducible maize RAB17M promoter with lox P sites which bracket the CRE, WUS, and BBM genes. A constitutive maize UBIM promoter directs a ZsGreen GFP expression cassette as a reporter outside of the excision sites and provides transient, transgenic, and developmental analysis. This was coupled with evidence for molecular integration and analysis of stable integration and desiccation inducible CRE-mediated excision. Agrobacterium-mediated transgenic introduction of this vector showed transient expression of GFP and induced somatic embryogenesis in maize B73 and sorghum P898012 explants. Subjection to desiccation stress in tissue culture enabled the excision of CRE, WUS, and BBM, leaving the UBIM::GFP cassette and allowing subsequent plant regeneration and GFP expression analysis. Stable GFP expression was observed in the early and late somatic embryos, young shoots, vegetative plant organs, and pollen. Transgene integration and expression of GFP positive T0 plants were also analyzed using PCR and Southern blots. Progeny segregation analysis of primary events confirmed correlation between functional GFP expression and presence of the GFP transgene in T1 plants generated from self pollinations, indicating good transgene inheritance. This study confirms and extends the use of morphogenic regulators to overcome transformation barriers.

Gene Flow in Genetically Engineered Perennial Grasses: Lessons for Modification of Dedicated Bioenergy Crops

This chapter was initiated from a symposium and workshop on the scientific advances, issues and bioethics of gene confinement in genetically modified grasses conducted on 13–14 May 2005 at Yale University in New Haven, CT. Genetic modification of dedicated bioenergy crops such as switchgrass will play a major role in crop improvement for a wide range of beneficial traits specific to biofuels. One obstacle that arises regarding transgenic improvement of perennials used for biofuels is the propensity of these plants to be open pollinated, with the undesirable capacity of outcrossing to non-transgenic and wild relative species. We examine previous work on pollen-mediated and seed-mediated gene flow of genetically modified grasses, in particular herbicide resistant traits, relevant to gene flow in grasses providing a perspective on the implementation of this technology for improvement of perennial bioenergy crops.

Control of sexuality by the sk1-encoded UDP-glycosyltransferase of maize.

The maize silkless 1 gene encodes a UDP-glycosyltransferase that protects pistils from elimination by jasmonic acid signaling. Sex determination in maize involves the production of staminate and pistillate florets from an initially bisexual floral meristem. Pistil elimination in staminate florets requires jasmonic acid signaling, and functional pistils are protected by the action of the silkless 1 (sk1) gene. The sk1 gene was identified and found to encode a previously uncharacterized family 1 uridine diphosphate glycosyltransferase that localized to the plant peroxisomes. Constitutive expression of an sk1 transgene protected all pistils in the plant, causing complete feminization, a gain-of-function phenotype that operates by blocking the accumulation of jasmonates. The segregation of an sk1 transgene was used to effectively control the production of pistillate and staminate inflorescences in maize plants.

Genetic Modification in Dedicated Bioenergy Crops and Strategies for Gene Confinement

Genetic modification of dedicated bioenergy crops is in its infancy; however, there are numerous advantages to the use of these tools to improve crops used for biofuels. Potential improved traits through genetic engineering (GE) include herbicide resistance, pest-, drought-, cold- and salt-tolerance, lower inputs, compositional alterations, addition of cellulases and other biofuels-specific traits such as increased biomass yields and increased photosynthetic efficiencies. To achieve these goals on an agricultural scale, these improvements must meet regulatory standards for release into the environment. In most cases, these criteria will probably require gene confinement strategies to prevent gene flow into wild and non-transgenic populations. Here, we consider the options for prevention or mitigation of gene flow in genetically modified (GM) biofuels crops.

Genomic Characterization of Interspecific Hybrids and an Admixture Population Derived from Panicum amarum × P. virgatum

Switchgrass (Panicum virgatum L.) and its relatives are regarded as top bioenergy crop candidates; however, one critical barrier is the introduction of useful genetic diversity and the development of new cultivars and hybrids. Combining genomes from related cultivars and species provides an opportunity to introduce new traits. In switchgrass, a breeding advantage would be achieved by combining the genomes of intervarietal ecotypes or interspecific hybrids. The recovery of wide crosses, however, is often tedious and may involve complicated embryo rescue and numerous backcrosses. Here, we demonstrate a straightforward approach to wide crosses involving the use of a selectable transgene for recovery of interspecific [P. virgatum cv. Alamo × Panicum amarum Ell. var amarulum or Atlantic Coastal Panicgrass (ACP)] F1 hybrids followed by backcrossing to generate a nontransgenic admixture population. A nontransgenic herbicide‐sensitive (HbS) admixture population of 83 F1BC1 progeny was analyzed by genotyping‐by‐sequencing (GBS) to characterize local ancestry, parental contribution, and patterns of recombination. These results demonstrate a widely applicable breeding strategy that makes use of transgenic selectable resistance to identify and recover true hybrids.

Transformation of Recalcitrant Sorghum Varieties Facilitated by Baby Boom and Wuschel2

Most reliable transformation protocols for cereal crops, including sorghum (Sorghum bicolor L. Moench), rely on the use of immature embryo explants to generate embryogenic callus cells that are then transformed using Agrobacterium- or particle-bombardment-mediated DNA delivery. Subsequent to DNA transfer, most protocols rely on selectable markers for the recovery of stably transformed callus that is then regenerated to produce T0 plants. However, these protocols require specific genotypes that are innately capable of efficient embryogenic callus initiation. Here, we describe a system that makes use of the differential expression of the morphogenic regulators Baby Boom (Bbm) and Wuschel2 (Wus2) to achieve transformation in varieties of sorghum typically recalcitrant to standard transformation methods.

Morphogenic Regulator‐Mediated Transformation of Maize Inbred B73

Maize B73 is a reference genome and has long been a major resource for genetics and molecular biology research. We have developed an efficient B73 transformation protocol by enabling somatic embryogenesis through differential co-expression of maize morphogenic regulators BBM and WUS2. We describe a successful protocol that utilizes Agrobacterium tumefaciens strain AGL1 harboring binary vector PHP78891 that comprises a BBM and WUS2 expression cassette as well as a green fluorescent protein (GFP) reporter cassette. The PHP78891 vector also contains, within the T-DNA region, a CRE/lox recombination system flanking the CRE/BBM/WUS2 co-expression cassette driven by the desiccation inducible RAB17 promoter that allows removal of the BBM/WUS2 cassette. Introduction and co-expression of BBM and WUS2 induced direct somatic embryogenesis (SE) in non-regenerable maize B73 from immature embryo explants. Removal of the CRE/BBM/WUS2 cassette is essential to allow regeneration to fertile plants. The GFP expression cassette outside the lox excision sites is retained in the transgenic plant genome, allowing subsequent phenotypic analysis of calli and regenerated transgenic events. This transformation system enables a selectable marker-free transformation process by taking advantage of BBM/WUS2-induced SE as a developmental selection system.

In situ embryo rescue for generation of wide intra- and interspecific hybrids of Panicum virgatum L.

Summary Wide crosses have been used for decades as a method for transferring novel genetic material and traits in plant breeding. Historically, many products of wide crosses require tedious and inefficient surgical embryo rescue prior to embryo abortion to recover single plantlets. We have utilized transgenic switchgrass (Panicum virgatum L. cv Alamo) as a pollen donor in conjunction with antibiotic or herbicide selection for recovery of intra‐and interspecific F1 crosses by using developing ovules from the female parent and selecting for embryogenic cultures derived from the in situ immature embryo. Using this approach, several intravarietial crosses were generated between transgenic Alamo and the switchgrass varieties Kanlow, Blackwell and Cave‐in‐Rock as well as an interspecific cross with Atlantic coastal panicgrass. This procedure selected F1 embryogenic callus produced from the developing embryo contained within isolated immature ovules. Several clonal plants were successfully regenerated from each cross. Southern blot, PCR, phenotypic analyses and genomic analysis confirmed F1 hybrids. Using genotyping‐by‐sequencing shows the hybridization of the recovered plants by determining the ratio of transgressive markers to total compared markers between parents and their potential offspring. The ratio of transgressive markers to total compared markers was significantly lower between parents and their predicted offspring than between parents and offspring unrelated to them. This approach provides the possibility to move useful transgenes into varieties that are recalcitrant to direct transformation which can be optionally segregated thus useful to create new hybrids, as well as recovery of wide crosses that are either difficult or impossible using traditional techniques.