CRISPR technology towards genome editing of the perennial and semi-perennial crops citrus, coffee and sugarcane.

Gene editing technologies have opened up the possibility of manipulating the genome of any organism in a predicted way. CRISPR technology is the most used genome editing tool and, in agriculture, it has allowed the expansion of possibilities in plant biotechnology, such as gene knockout or knock-in, transcriptional regulation, epigenetic modification, base editing, RNA editing, prime editing, and nucleic acid probing or detection. This technology mostly depends on in vitro tissue culture and genetic transformation/transfection protocols, which sometimes become the major challenges for its application in different crops. Agrobacterium-mediated transformation, biolistics, plasmid or RNP (ribonucleoprotein) transfection of protoplasts are some of the commonly used CRISPR delivery methods, but they depend on the genotype and target gene for efficient editing. The choice of the CRISPR system (Cas9, Cas12), CRISPR mechanism (plasmid or RNP) and transfection technique (Agrobacterium spp., PEG solution, lipofection) directly impacts the transformation efficiency and/or editing rate. Besides, CRISPR/Cas technology has made countries rethink regulatory frameworks concerning genetically modified organisms and flexibilize regulatory obstacles for edited plants. Here we present an overview of the state-of-the-art of CRISPR technology applied to three important crops worldwide (citrus, coffee and sugarcane), considering the biological, methodological, and regulatory aspects of its application. In addition, we provide perspectives on recently developed CRISPR tools and promising applications for each of these crops, thus highlighting the usefulness of gene editing to develop novel cultivars.

Atributos fenológicos e agronômicos em cultivares de cafeeiro arábica / Phenological and agronomic attributes in arabica cultivars of coffee tree

O objetivo deste trabalho foi avaliar o efeito do ano agrícola sobre os atributos fenológicos e agronômicos em diferentes cultivares de cafeeiro arábica. Os atributos fenológicos determinados foram estádios de frutificação, de gema dormente até fruto seco e duração do ciclo. Como atributos agronômicos, foram avaliados o rendimento, a produtividade, os tipos e o tamanho de grãos. O período entre antese até fase chumbinho não variou entre as cultivares para o ano agrícola 2004/2005, já para o ano agrícola seguinte a cultivar 'Icatu Precoce' apresentou antecipação da fase chumbinho, e a cultivar 'Obatã' atrasou o início do ciclo fenológico em relação às demais cultivares estudadas. A duração do ciclo variou com o ano agrícola, o que possibilitou a confirmação da influência dos efeitos ambientais nessa característica. Os atributos agronômicos (produtividade, rendimento, tipo e tamanho de grãos), independentemente das cultivares, também variaram com o ano agrícola.

The aim of this study was to evaluate cultivars of Coffea arabica on the basic of phenology and agronomic attributes a function of the year of production. The phenological attributes were evaluated regarding phenology and percentage of cherry fruits at harvesting time. Agronomic traits evaluated included productivity and outturn, type of seeds and grain size. The period between the blooms till the fruits at the beginning of growth did not vary among the coffee trees in the year of production 2004/2005. While in the following year the Icatu Precoce cultivar presented anticipation of the fruits at the beginning of growth and the 'Obatã' cultivar delayed the beginning of the phenological cycle due to the other cultivars studied. The duration of each cycle varied with the year of production and then all the coffee trees in this study could be differentiated on the timing of maturation (early, middle and late). The agronomic attributes (productivity, outturn, type and size of grains), regardless the different cultivars of coffee also varied with the year of production.

Altered expression of the caffeine synthase gene in a naturally caffeine-free mutant of Coffea arabica.

In this work, we studied the biosynthesis of caffeine by examining the expression of genes involved in this biosynthetic pathway in coffee fruits containing normal or low levels of this substance. The amplification of gene-specific transcripts during fruit development revealed that low-caffeine fruits had a lower expression of the theobromine synthase and caffeine synthase genes and also contained an extra transcript of the caffeine synthase gene. This extra transcript contained only part of exon 1 and all of exon 3. The sequence of the mutant caffeine synthase gene revealed the substitution of isoleucine for valine in the enzyme active site that probably interfered with enzymatic activity. These findings indicate that the absence of caffeine in these mutants probably resulted from a combination of transcriptional regulation and the presence of mutations in the caffeine synthase amino acid sequence.

Altered expression of the caffeine synthase gene in a naturally caffeine-free mutant of Coffea arabica

In this work, we studied the biosynthesis of caffeine by examining the expression of genes involved in this biosynthetic pathway in coffee fruits containing normal or low levels of this substance. The amplification of gene-specific transcripts during fruit development revealed that low-caffeine fruits had a lower expression of the theobromine synthase and caffeine synthase genes and also contained an extra transcript of the caffeine synthase gene. This extra transcript contained only part of exon 1 and all of exon 3. The sequence of the mutant caffeine synthase gene revealed the substitution of isoleucine for valine in the enzyme active site that probably interfered with enzymatic activity. These findings indicate that the absence of caffeine in these mutants probably resulted from a combination of transcriptional regulation and the presence of mutations in the caffeine synthase amino acid sequence.