Biolistics-mediated transformation of hornworts and its application to study pyrenoid protein localization.

Hornworts are a deeply diverged lineage of bryophytes that are sister to mosses and liverworts. Hornworts have an array of unique features that can be leveraged to illuminate not only the early evolution of land plants, but also alternative paths for nitrogen and carbon assimilation via cyanobacterial symbiosis and a pyrenoid-based CO2-concentrating mechanism (CCM), respectively. Despite this, hornworts are one of the few plant lineages with limited available genetic tools. Here we report an efficient biolistics method for generating transient-expression and stable transgenic lines in the model hornwort, Anthoceros agrestis. An average of 569 (± 268) cells showed transient expression per bombardment, with green fluorescent protein expression observed within 48-72 hours. A total of 81 stably transformed lines were recovered across three separate experiments, averaging six lines per bombardment. We followed the same method to transiently transform nine additional hornwort species, and obtained stable transformants from one. This method was further used to verify the localization of Rubisco and Rubisco activase in pyrenoids, which are central proteins for CCM function. Together, our biolistics approach offers key advantages over existing methods as it enables rapid transient expression and can be applied to widely diverse hornwort species.

Accelerating gametophytic growth in the model hornwort Anthoceros agrestis.

Premise: Hornworts belong to a unique lineage of bryophytes with critical traits for elucidating the evolution of land plants; however, the development of functional genetic tools for hornworts has been hampered by their relatively slow gametophytic growth. Methods: To identify the external factors that influence the development of hornwort gametophytes and potentially augment their growth, we evaluated the contributions of several culture medium components on the axenic gametophytic growth of Anthoceros agrestis, a model hornwort. A streamlined growth assay utilizing semiautomated image analysis was developed to rapidly quantify and compare tissue development spanning four weeks of culture on solidified medium. Results: The addition of sucrose, ammonium nitrate, activated charcoal, pH buffering, and growth regulators (2,4-dichlorophenoxyacetic acid, 6-benzylaminopurine, and thidiazuron) affected gametophyte tissue survival, growth patterns, and the rate of thalli growth. Subsequently, an optimized medium composition and growth regimen for accelerating A. agrestis gametophytic growth were formulated, which at four weeks of culture increased the tissue wet weight by 2.1- to 8.5-fold compared with other previously utilized hornwort growth media. Discussion: Our protocol for generating vigorous starting material and accelerated tissue regeneration is pertinent for advancing gene function characterization and genome editing in hornworts.

An Agrobacterium-mediated stable transformation technique for the hornwort model Anthoceros agrestis.

Despite their key phylogenetic position and their unique biology, hornworts have been widely overlooked. Until recently there was no hornwort model species amenable to systematic experimental investigation. Anthoceros agrestis has been proposed as the model species to study hornwort biology. We have developed an Agrobacterium-mediated method for the stable transformation of A. agrestis, a hornwort model species for which a genetic manipulation technique was not yet available. High transformation efficiency was achieved by using thallus tissue grown under low light conditions. We generated a total of 274 transgenic A. agrestis lines expressing the ß-glucuronidase (GUS), cyan, green, and yellow fluorescent proteins under control of the CaMV 35S promoter and several endogenous promoters. Nuclear and plasma membrane localization with multiple color fluorescent proteins was also confirmed. The transformation technique described here should pave the way for detailed molecular and genetic studies of hornwort biology, providing much needed insight into the molecular mechanisms underlying symbiosis, carbon-concentrating mechanism, RNA editing and land plant evolution in general.

Charting the genomic landscape of seed-free plants.

During the past few years several high-quality genomes has been published from Charophyte algae, bryophytes, lycophytes and ferns. These genomes have not only elucidated the origin and evolution of early land plants, but have also provided important insights into the biology of the seed-free lineages. However, critical gaps across the phylogeny remain and many new questions have been raised through comparing seed-free and seed plant genomes. Here, we review the reference genomes available and identify those that are missing in the seed-free lineages. We compare patterns of various levels of genome and epigenomic organization found in seed-free plants to those of seed plants. Some genomic features appear to be fundamentally different. For instance, hornworts, Selaginella and most liverworts are devoid of whole-genome duplication, in stark contrast to other land plants. In addition, the distribution of genes and repeats appear to be less structured in seed-free genomes than in other plants, and the levels of gene body methylation appear to be much lower. Finally, we highlight the currently available (or needed) model systems, which are crucial to further our understanding about how changes in genes translate into evolutionary novelties.

Overexpression of the Transcription Factor GROWTH-REGULATING FACTOR5 Improves Transformation of Dicot and Monocot Species.

Successful regeneration of genetically modified plants from cell culture is highly dependent on the species, genotype, and tissue-type being targeted for transformation. Studies in some plant species have shown that when expression is altered, some genes regulating developmental processes are capable of triggering plant regeneration in a variety of plant cells and tissue-types previously identified as being recalcitrant to regeneration. In the present research, we report that developmental genes encoding GROWTH-REGULATING FACTORS positively enhance regeneration and transformation in both monocot and dicot species. In sugar beet (Beta vulgaris ssp. vulgaris), ectopic expression of Arabidopsis GRF5 (AtGRF5) in callus cells accelerates shoot formation and dramatically increases transformation efficiency. More importantly, overexpression of AtGRF5 enables the production of stable transformants in recalcitrant sugar beet varieties. The introduction of AtGRF5 and GRF5 orthologs into canola (Brassica napus L.), soybean (Glycine max L.), and sunflower (Helianthus annuus L.) results in significant increases in genetic transformation of the explant tissue. A positive effect on proliferation of transgenic callus cells in canola was observed upon overexpression of GRF5 genes and AtGRF6 and AtGRF9. In soybean and sunflower, the overexpression of GRF5 genes seems to increase the proliferation of transformed cells, promoting transgenic shoot formation. In addition, the transformation of two putative AtGRF5 orthologs in maize (Zea mays L.) significantly boosts transformation efficiency and resulted in fully fertile transgenic plants. Overall, the results suggest that overexpression of GRF genes render cells and tissues more competent to regeneration across a wide variety of crop species and regeneration processes. This sets GRFs apart from other developmental regulators and, therefore, they can potentially be applied to improve transformation of monocot and dicot plant species.

Transient Transformation Using Particle Bombardment for Gene Expression Analysis.

Transient transformation or transient expression results in rapid and fleeting gene expression. This approach is often used as a first-tier screening tool for evaluation of components that affect gene expression. Here, we describe the use of particle bombardment of lima bean cotyledons with constructs containing the green fluorescent protein (gfp) coding region for evaluation of promoter components that influence gene expression. Although this approach is conceptually quite simple, this lima bean transient expression system may not work well, if our methods and notes are not carefully read and followed. Our laboratory has successfully optimized this method over the past 10 years, resulting in a transient expression system, which works like no other that we have seen.

Establishment of in vitro soybean aphids, Aphis glycines (Hemiptera: Aphididae): a tool to facilitate studies of aphid symbionts, plant-insect interactions and insecticide efficacy.

BACKGROUND: Studies on plant-insect interactions of the soybean aphid, Aphis glycines (Matsumura), can be influenced by environmental fluctuations, status of the host plant and variability in microbial populations. Maintenance of aphids on in vitro-grown plants minimizes environmental fluctuations, provides uniform host materials and permits the selective elimination of aphid-associated microbes for more standardized controls in aphid research. RESULTS: Aphids were reared on sterile, in vitro-grown soybean seedlings germinated on plant tissue culture media amended with a mixture of antimicrobials. For initiation and maintenance of in vitro aphid colonies, single aphids were inoculated onto single in vitro seedlings. After three rounds of transfer of 'clean' aphids to fresh in vitro seedlings, contamination was no longer observed, and aphids performed equally well when compared with those reared on detached leaves. The addition of the insecticides thiamethoxam and chlorantraniliprole to the culture medium confirmed uptake and caused significant mortality to the in vitro aphids. The use of the antimicrobial mixture removed the associated bacteria Arsenophonus but retained Buchnera and Wolbachia within the in vitro aphids. CONCLUSION: The in vitro aphid system is a novel and highly useful tool to understand insecticidal efficacy and expand our knowledge of tritrophic interactions among plants, insects and symbionts. © 2016 Society of Chemical Industry.