Editing plant genomes without introducing foreign DNA into cells may alleviate regulatory concerns related to genetically modified plants. We transfected preassembled complexes of purified Cas9 protein and guide RNA into plant protoplasts of Arabidopsis thaliana, tobacco, lettuce and rice and achieved targeted mutagenesis in regenerated plants at frequencies of up to 46%. The targeted sites contained germline-transmissible small insertions or deletions that are indistinguishable from naturally occurring genetic variation.

The recent development of adenine base editors (ABEs) has enabled efficient and precise A-to-G base conversions in higher eukaryotic cells. Here, we show that plant-compatible ABE systems can be successfully applied to protoplasts of Arabidopsis thaliana and Brassica napus through transient transfection, and to individual plants through Agrobacterium-mediated transformation to obtain organisms with desired phenotypes. Targeted, precise A-to-G substitutions generated a single amino acid change in the FT protein or mis-splicing of the PDS3 RNA transcript, and we could thereby obtain transgenic plants with late-flowering and albino phenotypes, respectively. Our results provide 'proof of concept' for in planta ABE applications that can lead to induced neo-functionalization or altered mRNA splicing, opening up new avenues for plant genome engineering and biotechnology.

To develop a controlled-release fertilizer (CRF) suitable for nutrient absorption characteristics of Phalaenopsis, four kinds of new controlled-release fertilizer (NCRF 1-4) with different dissolution rates were developed and studied to determine the concentration and amount suitable for growth of Phalaenopsis. To make NCRF, new acryl-based polymers were developed and used as fertilizer coating solutions. In addition, a fluidized bed coater for coating fertilizer was developed and used in this study. To test the growth of Phalaenopsis, 10-monthold Phalaenopsis seedlings were planted in plastic pots (diameter 10 cm) filled with 100% Sphagnum moss and cultivated for approximately 100 days from May 29, 2015, to September 11, 2015. NCRF 1, NCRF 2, and Osmocote, an imported fertilizer, consistently exhibited release patterns of fertilizer nutrients in a directly proportional form; however, NCRF 3 and NCRF 4 displayed a sigmoid-like tendency of fertilizer nutrient release with a slower initial dissolution rate. Furthermore, leaf length, leaf width, fresh weigh, and root weight of Phalaenopsis were the highest when growing in 1.5 g/pot of NCRF 3 fertilizer, and the pH and electrical conductivity (EC) of the soil were stable at this concentration of NCRF 3. Based on our results, we suggest that 1.5 g/pot of NCRF 3 fertilizer is the ideal concentration and fertilizer for growing Phalaenopsis.

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