Kimberly Nelson

Controlling transgene escape in GM creeping Bentgrass

Trait improvement of turfgrass through genetic engineering is important to the turfgrass industry and the environment. However, the possible transgene escape to wild and non-transformed species raises ecological and commercial concerns. Male sterility provides an effective way for interrupting gene flow. We have designed and synthesized two chimeric gene constructs consisting of a rice tapetum-specific promoter (TAP) fused to either a ribonuclease gene barnase, or the antisense of a rice tapetum-specific gene rts. Both constructs were linked to the bar gene for selection by resistance to the herbicide glufosinate. Agrobacterium-mediated transformation of creeping bentgrass (cv Penn A-4) with both constructs resulted in herbicide-resistant transgenic plants that were also 100% pollen sterile. Mendelian segregation of herbicide resistance and male sterility was observed in T1 progeny derived from crosses with wild-type plants. Controlled self- and cross-pollination studies showed no gene transfer to non-transgenic plants from male-sterile transgenic plants. Thus, male sterility can serve as an important tool to control transgene escape in bentgrass, facilitating the application of genetic engineering in producing environmentally responsible turfgrass with enhanced traits. It also provides a tool to control gene flow in other perennial species using transgenic technology.

FLP-mediated site-specific recombination for genome modification in turfgrass

To develop molecular strategies for gene containment in genetically modified (GM) turfgrass, we have studied the feasibility of using the FLP/FRT site-specific DNA recombination system from yeast for controlled genome modification in turfgrass. Suspension cell cultures of creeping bentgrass (Agrostis stolonifera L.) and Kentucky bluegrass (Poa pratensis) were co-transformed with a FLP recombinase expression vector and a recombination-reporter test plasmid containing β-glucuronidase (gusA) gene which was separated from the maize ubiquitin (ubi) promoter by an FRT-flanked blocking DNA sequence to prevent its transcription. GUS activity was observed in co-transformed cells, in which molecular analyses indicated that FLP-mediated excision of the blocking sequence had brought into proximity the upstream promoter and the downstream reporter gene, resulting in GUS expression. Functional evaluation of the FLP/FRT system using transgenic creeping bentgrass stably expressing FLP recombinase confirmed the observation in suspension cell culture. Our results indicate that FLP/FRT system is a useful tool for genetic manipulation of turfgrass, pointing to the great potential of exploiting the system to develop molecular strategies for transgene containment in perennials.

Controlling Transgene Escape in Genetically Modified Grasses

Trait improvement of turfgrass through genetic engineering is important to the turfgrass industry and the environment. However, the possibility of transgene escape to wild and non-transformed species raises commercial and ecological concerns. Male sterility provides an effective way for interrupting gene flow. We have designed and synthesized two chimeric gene constructs consisting of a rice tapetum-specific promoter (TAP) fused to either a ribonuclease gene barnase, or the antisense of a rice tapetum-specific gene rts. Both constructs were linked to the bar gene for selection by resistance to the herbicide glufosinate. Using Agrobacterium-mediated transformation, we have successfully introduced those gene constructs into creeping bentgrass (cv Penn-A-4), producing a total of 219 stably transformed individual events. Tapetum-specific expression of barnase or antisense rts gene did not affect the vegetative phenotype compared with the control plants, and male-sterile flowers were obtained with both constructs. Microscopic studies confirmed the failure of mature pollen formation in male-sterile transgenics. Mendelian segregation of herbicide tolerance and male sterility has been observed in T1 progeny derived from crosses with wild-type plants. Male sterility in transgenic grasses provides the best tool to evaluate gene flow in genetically modified perennial plants and should facilitate the application of genetic engineering in producing environmentally responsible grasses with enhanced traits.

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.

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.

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.