Improvement Of Crop Traits Research Articles

Engineering plant architecture via CRISPR/Cas9-mediated alteration of strigolactone biosynthesis

BackgroundPrecision plant genome engineering holds much promise for targeted improvement of crop traits via unprecedented single-base level control over the genetic material. Strigolactones (SLs) are a key determinant of plant architecture, known for their role in inhibiting shoot branching (tillering).ResultsWe used CRISPR/Cas9 in rice (Oryza sativa) for targeted disruption of CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), which controls a key step in SL biosynthesis. The ccd7 mutants exhibited a striking increase in tillering, combined with a reduced height, which could be rescued by application of the synthetic SL analog GR24. Striga germination assays and liquid chromatography–mass spectrometry analysis showed that root exudates of ccd7 mutants were also SL deficient.ConclusionsTaken together, our results show the potential and feasibility of the use of the CRISPR/Cas9 system for targeted engineering of plant architecture and for elucidating the molecular underpinnings of architecture-related traits.

REGENERATION AND TRANSFORMATION SYSTEM IN EGYPTIAN SWEET POTATO (Ipomoea batatas Lam.) CULTIVARS

Genetic transformation is considered as one of the most favorable options for improvement of crop traits. In this study the regeneration frequency and transformation system were established on the Egyptian sweet potato (Ipomoea batatas (L.) Lam.) cv. Abees and Mabruka. The effect of different hormone combinations and type of explant on shoot regeneration was evaluated. The regeneration percentages from Abees and Mabruka cv. 26.3 and 13.3%, respectively were obtained on Murashige and Skoog MS basal salt mixture + 1.0 mg/l BA + 30.0 g/l sucrose + 2.2 g/l Phytagel with Abees cv. and the same media was used for cv. Mabruka with only cytokinin type different as 5.0 Kin and shoots were rooted on MS medium + 30 g/l sucrose and 2.2 g/l Phytagel. The Agrobacterium-mediated and microprojectile bombardement transformation system were successfully introducing the reporter gus and selectable bar marker genes in the sweet potato explants under pressure of 900 and 1100 psi and microcarrier travel distance (6 and 9 cm). Incorporation and expression of the gus and bar genes into sweet potato plants were confirmed using polymerase chain reaction (PCR) and GUS histochemical assay. Several factors were found to be important for regeneration and transformation in sweet potato. The most effective factors were plant genotype and the type of explants. Co-cultivation time and optical density of the Agrobacterium suspension were also critical for sweet potato transformation. This work is an attempt to open the door for further genetic improvement of sweet potato using important agronomic traits.

Efficient CRISPR/Cas9-Mediated Genome Editing Using a Chimeric Single-Guide RNA Molecule.

The CRISPR/Cas9 system has been applied in diverse eukaryotic organisms for targeted mutagenesis. However, targeted gene editing is inefficient and requires the simultaneous delivery of a DNA template for homology-directed repair (HDR). Here, we used CRISPR/Cas9 to generate targeted double-strand breaks and to deliver an RNA repair template for HDR in rice (Oryza sativa). We used chimeric single-guide RNA (cgRNA) molecules carrying both sequences for target site specificity (to generate the double-strand breaks) and repair template sequences (to direct HDR), flanked by regions of homology to the target. Gene editing was more efficient in rice protoplasts using repair templates complementary to the non-target DNA strand, rather than the target strand. We applied this cgRNA repair method to generate herbicide resistance in rice, which showed that this cgRNA repair method can be used for targeted gene editing in plants. Our findings will facilitate applications in functional genomics and targeted improvement of crop traits.

Heritability of targeted gene modifications induced by plant-optimized CRISPR systems.

The Streptococcus-derived CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated protein 9) system has emerged as a very powerful tool for targeted gene modifications in many living organisms including plants. Since the first application of this system for plant gene modification in 2013, this RNA-guided DNA endonuclease system has been extensively engineered to meet the requirements of functional genomics and crop trait improvement in a number of plant species. Given its short history, the emphasis of many studies has been the optimization of the technology to improve its reliability and efficiency to generate heritable gene modifications in plants. Here we review and analyze the features of customized CRISPR/Cas9 systems developed for plant genetic studies and crop breeding. We focus on two essential aspects: the heritability of gene modifications induced by CRISPR/Cas9 and the factors affecting its efficiency, and we provide strategies for future design of systems with improved activity and heritability in plants.

English

  Genetic transformation is considered as one of the most promising options for improvement of crop traits. Current transformation methods for sweetpotato depend on plant regeneration through organogenesis or somatic embryogenesis. Somatic embryogenesis and plant regeneration at a high frequency has been restricted to a few sweetpotato varieties. Three auxins namely:  2,4-dichlorophenoxyacetic acid (2,4-D), 4-fluoroamphetamine (4-FA) and 4,5-trichlorophenoxyacetic acid (2,4,5-T)were investigated in this study for enhancing somatic embryogenesis from various plant organs of recalcitrant African sweetpotato cultivars. 2,4-D was found to be the best (p ≤ 0.05) for induction of embryogenic callus. Cultivar Bwanjule had the highest (20.2%) embryogenic callus frequency among the five African cultivars tested. The highest number of plants in this study was regenerated from the non-African cultivar variety Jonathan on media supplemented with 0.2 mg Zeatin. The emergence of roots from callus of recalcitrant Ugandan cultivars and the comparable high embryogenic responses in this work demonstrate the potential for regenerating plants from African cultivars that have not been regenerated before. The regeneration of roots in this work could be useful for the initiation of root cultures. The most important application of this work is in genetic transformation of sweet potato, particularly for improvement of resistance to weevils.   Key words: Embryogenesis, plant growth regulators, plant regeneration, Ipomoea batatas

Individual fitness versus whole-crop photosynthesis:solar tracking tradeoffs in alfalfa.

Despite the optimism of some molecular biologists, natural selection among the wild ancestors of crops is unlikely to have missed simple genetic improvements that would consistently have enhanced individual fitness. Tradeoff-free opportunities for further improvement of crop traits like photosynthetic efficiency or drought tolerance may therefore be elusive. Opportunities linked to acceptable tradeoffs may be abundant, however. Tradeoffs between individual competitiveness and the collective productivity of plant communities (e.g. those linked to height) have been key to past increases in yield potential. Solar tracking by leaves could involve such tradeoffs, if photosynthetic benefits to tracking leaves are outweighed by increased shading of leaves lower in the canopy. This hypothesis was tested using rotation in the horizontal plane to disrupt solar tracking in alfalfa. In sparse canopies, solar tracking increased net canopy photosynthesis, but rarely by more than 3%. As leaf area increased, solar tracking tended to decrease net canopy photosynthesis, despite edge effects in our 1-m2 artificial communities, which probably exaggerated net photosynthetic benefits of tracking. Computer modeling suggested that the season-long effects of solar tracking on community productivity can be negative. Solar tracking may have persisted, nonetheless, because individuals whose leaves track the sun increase shading of competitors.

Genetic transformation of Prunus domestica L. using the hpt gene coding for hygromycin resistance as the selectable marker

The study and development of transformation technology with new selection schemes is important for various fundamental studies and for crop trait improvement via genetic engineering. Here we have shown that hygromycin resistance is an effective system for plum genetic transformation. Embryonic axes of mature seeds were co-cultivated with Agrobacterium strain LBA4404 containing the pC1381 plasmid carrying the hygromycin phosphotranferase gene ( hpt) and β-glucuronidase (GUS) gene or with strain EHA105 containing the plasmid pC1301 carrying the same marker and reporter genes. The latter strain containing a pC2301 plasmid carrying the neomycin phosphotransferase gene ( nptII) gene was used as a control. Infected explants were placed on shoot induction medium containing either 5 mg L −1 hygromycin or 75 mg L −1 kanamycin for selection. Green shoots developed from the explants under hygromycin pressure. These shoots showed continued and vigorous growth and development upon transfer onto fresh hygromycin medium. PCR using hpt sequence primers, and Southern blot analysis using a probe from the hpt gene, confirmed the presence of the transgenes and their stable integration in regenerated plants. Full transgenic plants were obtained in a greenhouse. Hygromycin selection was very effective and no escapes were observed. The study demonstrated that hygromycin resistance can be used as an effective selectable marker for plum transformation. The new system developed here is important and useful for multiple gene transformation in plum.