Apple CRISPR-Cas9-A Recipe for Successful Targeting of -like Genes in Domestic Apple.

Fruit trees and other fruiting hardwood perennials are economically valuable, and there is interest in developing improved varieties. Both conventional breeding and biotechnology approaches are being utilized towards the goal of developing advanced cultivars. Increased knowledge of the effectiveness and efficiency of biotechnology approaches can help guide use of the CRISPR gene-editing technology.

Variation in floral form of CRISPR knock-outs of the poplar homologs of and after FT heat-induced early flowering.

Plant migration and gene flow from genetically modified or exotic trees to nearby lands or by crossing with wild relatives is a major public and regulatory concern. Many genetic strategies exist to mitigate potential gene flow; however, the long delay in onset of flowering is a severe constraint to research progress. We used heat-induced FT overexpression to speed assessment of the expected floral phenotypes after CRISPR knockout of poplar homologs of the key floral genes, and .

Knockout of floral and meiosis genes using CRISPR/Cas9 produces male-sterility in Eucalyptus without impacts on vegetative growth.

spp. are widely cultivated for the production of pulp, energy, essential oils, and as ornamentals. However, their dispersal from plantings, especially when grown as an exotic, can cause ecological disruptions.

Genetic Containment for Molecular Farming.

Plant molecular farming can provide humans with a wide variety of plant-based products including vaccines, therapeutics, polymers, industrial enzymes, and more. Some of these products, such as Taxol, are produced by endogenous plant genes, while many others require addition of genes by artificial gene transfer. Thus, some molecular farming plants are transgenic (or cisgenic), while others are not.

RNAi Suppression of Gives Stable Floral Sterility, and Reduced Growth Rate and Leaf Size, in Field-Grown Poplars.

The central floral development gene (, whose mutation leads to striking changes in flowering and often sterility, is commonly expressed in non-floral structures; however, its role in vegetative development is poorly understood. Sterility associated with suppression of expression is an attractive means for mitigating gene flow by both seeds and pollen in vegetatively propagated forest trees, but the consequences of its suppression for tree form and wood production are unclear. To study the vegetative effects of RNAi suppression of , we created a randomized, multiple-year field study with 30-40 trees (ramets) in each of two sterile gene insertion events, three transgenic control events, and a wild-type control population.

Overexpression of SHORT VEGETATIVE PHASE-LIKE (SVL) in Populus delays onset and reduces abundance of flowering in field-grown trees.

The spread of transgenes and exotic germplasm from planted crops into wild or feral species is a difficult problem for public and regulatory acceptance of genetically engineered plants, particularly for wind-pollinated trees such as poplar. We report that overexpression of a poplar homolog of the floral repressor SHORT VEGETATIVE PHASE-LIKE (SVL), a homolog of the Arabidopsis MADS-box repressor SHORT VEGETATIVE PHASE (SVP), delayed the onset of flowering several years in three genotypes of field-grown transgenic poplars. Higher expression of SVL correlated with a delay in flowering onset and lower floral abundance, and did not cause morphologically obvious or statistically significant effects on leaf characteristics, tree form, or stem volume.

Genetic containment in vegetatively propagated forest trees: CRISPR disruption of LEAFY function in Eucalyptus gives sterile indeterminate inflorescences and normal juvenile development.

Eucalyptus is among the most widely planted taxa of forest trees worldwide. However, its spread as an exotic or genetically engineered form can create ecological and social problems. To mitigate gene flow via pollen and seeds, we mutated the Eucalyptus orthologue of LEAFY (LFY) by transforming a Eucalyptus grandis × urophylla wild-type hybrid and two Flowering Locus T (FT) overexpressing (and flowering) lines with CRISPR Cas9 targeting its LFY orthologue, ELFY.

RNAi of AGAMOUS genes in sweetgum alters reproductive organ identity and decreases fruit persistence.

Sweetgums (), members of the family Altingiaceae (Altingiales), have inflorescences and floral organs that are distinctive in structure compared with other angiosperms in which the roles of floral homeotic genes have been studied. To begin to understand the role of AGAMOUS (AG)-a floral homeotic gene that has a major role in stamen and carpel development-in development of the monosexual flowers of sweetgum, we used RNAi to reduce the expression of two members of the subfamily. Because suppression should induce floral sterility, RNAi might also provide a tool to mitigate the risks of invasiveness-and to reduce the production of its nuisance fruits or allergenic pollen-when sweetgum is used as an exotic shade or forest tree.

RNA interference suppression of AGAMOUS and SEEDSTICK alters floral organ identity and impairs floral organ determinacy, ovule differentiation, and seed-hair development in Populus.

The role of the floral homeotic gene AGAMOUS (AG) and its close homologues in development of anemophilous, unisexual catkins has not previously been studied. We transformed two RNA interference (RNAi) constructs, PTG and its matrix-attachment-region flanked version MPG, into the early-flowering female poplar clone 6K10 (Populus alba) to suppress the expression of its two duplicate AG orthologues. By early 2018, six out of 22 flowering PTG events and 11 out of 12 flowering MPG events showed modified floral phenotypes in a field trial in Oregon, USA.

Strategies for Engineering Reproductive Sterility in Plantation Forests.

A considerable body of research exists concerning the development of technologies to engineer sterility in forest trees. The primary driver for this work has been to mitigate concerns arising from gene flow from commercial plantings of genetically engineered (GE) trees to non-GE plantations, or to wild or feral relatives. More recently, there has been interest in the use of sterility technologies as a means to mitigate the global environmental and socio-economic damage caused by the escape of non-native invasive tree species from planted forests.

Variation in Mutation Spectra Among CRISPR/Cas9 Mutagenized Poplars.

In an effort to produce reliably contained transgenic trees, we used the CRISPR/Cas9 system to alter three genes expected to be required for normal flowering in poplar (genus ). We designed synthetic guide RNAs (sgRNAs) to target the poplar homolog of the floral meristem identity gene, (), and the two poplar orthologs of the floral organ identity gene (). We generated 557 transgenic events with sgRNA(s) and the Cas9 transgene and 49 events with Cas9 but no sgRNA, and analyzed all events by Sanger Sequencing of both alleles.

Reproductive modification in forest plantations: impacts on biodiversity and society.

1000 I. 1000 II. 1001 III.

Virus-Induced Flowering: An Application of Reproductive Biology to Benefit Plant Research and Breeding.

Virus-induced flowering combines fundamental research in reproductive biology with efficient tools for manipulating gene expression in nonmodel systems to accelerate discovery and breeding.

Transgenic Suppression of AGAMOUS Genes in Apple Reduces Fertility and Increases Floral Attractiveness.

We investigated the ability of RNA interference (RNAi) directed against two co-orthologs of AGAMOUS (AG) from Malus domestica (domestic apple, MdAG) to reduce the risks of invasiveness and provide genetic containment of transgenes, while also promoting the attractiveness of flowers for ornamental usage. Suppression of two MdAG-like genes, MdMADS15 and MdMADS22, led to the production of trees with highly showy, polypetalous flowers. These "double-flowers" had strongly reduced expression of both MdAG-like genes.

FT overexpression induces precocious flowering and normal reproductive development in Eucalyptus.

Eucalyptus trees are among the most important species for industrial forestry worldwide. However, as with most forest trees, flowering does not begin for one to several years after planting which can limit the rate of conventional and molecular breeding. To speed flowering, we transformed a Eucalyptus grandis × urophylla hybrid (SP7) with a variety of constructs that enable overexpression of FLOWERING LOCUS T (FT).

Recruitment of PLANT U-BOX13 and the PI4Kβ1/β2 phosphatidylinositol-4 kinases by the small GTPase RabA4B plays important roles during salicylic acid-mediated plant defense signaling in Arabidopsis.

Protection against microbial pathogens involves the activation of cellular immune responses in eukaryotes, and this cellular immunity likely involves changes in subcellular membrane trafficking. In eukaryotes, members of the Rab GTPase family of small monomeric regulatory GTPases play prominent roles in the regulation of membrane trafficking. We previously showed that RabA4B is recruited to vesicles that emerge from trans-Golgi network (TGN) compartments and regulates polarized membrane trafficking in plant cells.

Arabidopsis Myosin XI-K Localizes to the Motile Endomembrane Vesicles Associated with F-actin.

Plant myosins XI were implicated in cell growth, F-actin organization, and organelle transport, with myosin XI-K being a critical contributor to each of these processes. However, subcellular localization of myosins and the identity of their principal cargoes remain poorly understood. Here, we generated a functionally competent, fluorescent protein-tagged, myosin XI-K, and investigated its spatial distribution within Arabidopsis cells.