An efficient protoplast-based genome editing protocol for species.

CRISPR-Cas technologies allow for precise modifications in plant genomes and promise to revolutionize agriculture. These technologies depend on the delivery of editing components into plant cells and the regeneration of fully edited plants. In vegetatively propagated plants, such as grape, protoplast culture provides one of the best avenues for producing non-chimeric and transgene-free genome-edited plants.

A GRF-GIF chimeric protein improves the regeneration efficiency of transgenic plants.

The potential of genome editing to improve the agronomic performance of crops is often limited by low plant regeneration efficiencies and few transformable genotypes. Here, we show that expression of a fusion protein combining wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) substantially increases the efficiency and speed of regeneration in wheat, triticale and rice and increases the number of transformable wheat genotypes. GRF4-GIF1 transgenic plants were fertile and without obvious developmental defects.

In vitro gene expression and mRNA translocation from transformed walnut (Juglans regia) rootstocks expressing DsRED fluorescent protein to wild-type scions.

An in vitro grafting method was developed for examining gene translocation from rootstock to scion in walnut. Results showed the DsRED gene itself was not translocated but expressed mRNA was. Grafting is widely used in plants, especially in fruit and nut crops.

Advancing Crop Transformation in the Era of Genome Editing.

Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process.

A red fluorescent protein (DsRED) from Discosoma sp. as a reporter for gene expression in walnut somatic embryos.

An improved scorable marker was developed for somatic embryo transformation. This method is more reliable than GFP and provides more efficient embryo selection than β-glucuronidase assays (GUS, MUG). Reporter genes are widely used to select transformed cells and tissues.

Symbiotic rhizobia bacteria trigger a change in localization and dynamics of the Medicago truncatula receptor kinase LYK3.

To form nitrogen-fixing symbioses, legume plants recognize a bacterial signal, Nod Factor (NF). The legume Medicago truncatula has two predicted NF receptors that direct separate downstream responses to its symbiont Sinorhizobium meliloti. NOD FACTOR PERCEPTION encodes a putative low-stringency receptor that is responsible for calcium spiking and transcriptional responses.

Evidence that resistance in squash mosaic comovirus coat protein-transgenic plants is affected by plant developmental stage and enhanced by combination of transgenes from different lines.

Three transgenic lines of squash hemizygous for the coat protein genes of squash mosaic virus (SqMV) were shown previously to have resistant (SqMV-127), susceptible (SqMV-22) or recovery (SqMV-3) phenotypes. Post-transcriptional gene silencing (PTGS) was the underlying mechanism for resistance of SqMV-127. Here, experiments conducted to determine the mechanism of the recovery phenotype and whether enhanced resistance could be obtained by combining transgenes from susceptible and recovery plants are reported.

Comparative Virus Resistance and Fruit Yield of Transgenic Squash with Single and Multiple Coat Protein Genes.

Five transgenic squash lines expressing coat protein (CP) genes from cucumber mosaic cucumovirus (CMV), zucchini yellow mosaic potyvirus (ZYMV), and watermelon mosaic virus 2 potyvirus (WMV 2) were analyzed in the field for their reaction to mixed infections by these three viruses and for fruit production. Test plants were exposed to natural inoculations via aphids in trials simulating the introduction of viruses by secondary spread from mechanically infected susceptible border row plants. Plants of transgenic line CZW-3 expressing the CP genes from CMV, ZYMV, and WMV 2 displayed the highest level of resistance with no systemic infection, although 64% exhibited localized chlorotic dots which were mainly confined to older leaves.