LaCl3 Treatment Improves Agrobacterium-mediated Immature Embryo Genetic Transformation Frequency of Maize

Agrobacterium-mediated genetic transformation of immature embryos is important for gene-function studies and molecular breeding of maize. However, the relatively low genetic transformation frequency remains a bottleneck for applicability of this method, especially on commercial scale. We report that pretreatment of immature embryos with LaCl3 (a Ca2+ channel blocker) improves the infection frequency of Agrobacterium tumefaciens, increases the proportion of positive calluses, yields more positive regenerated plantlets, and increases the transformation frequency from 8.40% to 17.60% for maize. This optimization is a novel method for improving the frequency of plant genetic transformations mediated by Agrobacterium tumefaciens.

LaCl3 Treatment Improves the Agrobacterium-mediated Immature Embryo Genetic Transformation Efficiency in Maize

Agrobacterium mediated genetic transformation of immature embryo plays an important auxiliary role in the study of gene function and molecular breeding in maize. However, the relatively low genetic transformation efficiency is still the bottleneck of the application of this method, especially in commercial scale production application. In this study, we found that pretreatment of immature embryos with LaCl 3 , a Ca 2+ channel blocker, could improve the infection efficiency of Agrobacterium tumefaciens , increase the proportion of resistant calluses, obtain more positive regenerated plantlets, and finally improve the transformation efficiency in maize. This optimization provides a new direction for improving the efficiency of plant genetic transformation mediated by Agrobacterium tumefaciens .

Genome-Wide Identification and Expression Profiling Analysis of WOX Family Protein-Encoded Genes in Triticeae Species

The WOX family is a group of plant-specific transcription factors which regulate plant growth and development, cell division and differentiation. From the available genome sequence databases of nine Triticeae species, 199 putative WOX genes were identified. Most of the identified WOX genes were distributed on the chromosomes of homeologous groups 1 to 5 and originated via the orthologous evolution approach. Parts of WOX genes in Triticum aestivum were confirmed by the specific PCR markers using a set of Triticum. durum-T. aestivum genome D substitution lines. All of these identified WOX proteins could be grouped into three clades, similar to those in rice and Arabidopsis. WOX family members were conserved among these Triticeae plants; all of them contained the HOX DNA-binding homeodomain, and WUS clade members contained the characteristic WUS-box motif, while only WUS and WOX9 contained the EAR motif. The RNA-seq and qPCR analysis revealed that the TaWOX genes had tissue-specific expression feature. From the expression patterns of TaWOX genes during immature embryo callus production, TaWOX9 is likely closely related with the regulation of regeneration process in T. aestivum. The findings in this study could provide a basis for evolution and functional investigation and practical application of the WOX family genes in Triticeae species.

CRISPR/Cas9-mediated genome editing in rice cv. IPB3S results in a semi-dwarf phenotypic mutant

Nurhayati, Ardie SW, Santoso TJ, Sudarsono. 2021. CRISPR/Cas9-mediated genome editing in rice cv. IPB3S results in a semi-dwarf phenotypic mutant. Biodiversitas 22: 3792-3800. IPB3S is Indonesian lowland rice and high-yielding cultivar. However, plant height posture makes it prone to lodging which could reduce the yield. This study aimed to edit the GA20Ox2 gene by introducing CRISPR/Cas9 GA20Ox2 construct into IPB3S and developing the semidwarf rice mutants. Immature embryo explants of IPB3S were used for the transformation process mediated by EHA105 strain of Agrobacterium tumefaciens carrying pC1300-Cas9/GA20Ox2 and by changing the regeneration medium composition. PCR analysis showed that rice cv. IPB3S genetic transformation gained putative mutant T0 lines carrying the hpt gene (growth efficiency was 47.9%, while transformation efficiency was 19.3%). Using the developed regeneration medium, we have obtained 24 putative rice cv. IPB3S T0 mutant lines carrying hpt. The best medium for regenerating IPB3S was the A medium (regeneration efficiency 73.3%). IPB 8 and IPB 14 were potential to evaluate in the next generation. The shortest plant height for the T1 generation was observed in the IPB 8-3 mutant.

Improved Transformation and Regeneration of Indica Rice: Disruption of SUB1A as a Test Case via CRISPR-Cas9

Gene editing by use of clustered regularly interspaced short palindromic repeats (CRISPR) has become a powerful tool for crop improvement. However, a common bottleneck in the application of this approach to grain crops, including rice (Oryza sativa), is efficient vector delivery and calli regeneration, which can be hampered by genotype-dependent requirements for plant regeneration. Here, methods for Agrobacterium-mediated and biolistic transformation and regeneration of indica rice were optimized using CRISPR-Cas9 gene-editing of the submergence tolerance regulator SUBMERGENCE 1A-1 gene of the cultivar Ciherang-Sub1. Callus induction and plantlet regeneration methods were optimized for embryogenic calli derived from immature embryos and mature seed-derived calli. Optimized regeneration (95%) and maximal editing efficiency (100%) were obtained from the immature embryo-derived calli. Phenotyping of T1 seeds derived from the edited T0 plants under submergence stress demonstrated inferior phenotype compared to their controls, which phenotypically validates the disruption of SUB1A-1 function. The methods pave the way for rapid CRISPR-Cas9 gene editing of recalcitrant indica rice cultivars.

The gene TaCB1 overcomes genotype dependency in wheat genetic transformation

Genotype dependency is the most important factor in wheat genetic transformation, which further limits wheat improvement by transgenic integration and genome editing approaches. The application of regeneration related genes during in vitro culture could potentially contribute to enhancement of plant transformation efficiency. In the present study, a wheat gene TaCB1 in the WUSCHEL family was identified to dramatically increase the transformation efficiencies of many wheat varieties without genotype dependency after its over-expression. The expression of TaCB1 in wheat calli did not prohibit shoot differentiation and root development. The application of TaCB1 can lighten the requirement to wheat immature embryo for plant regeneration. Transgenic wheat plants can be clearly recognized by the visible phenotype of wide flag leaves. The promise function of TaCB1 on improving transformation efficiency was also tested in T. monococcum, triticale, rye, barley, and maize.

MicroRNA Zma-miR528 Versatile Regulation on Target mRNAs during Maize Somatic Embryogenesis

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the accumulation and translation of their target mRNAs through sequence complementarity. miRNAs have emerged as crucial regulators during maize somatic embryogenesis (SE) and plant regeneration. A monocot-specific miRNA, mainly accumulated during maize SE, is zma-miR528. While several targets have been described for this miRNA, the regulation has not been experimentally confirmed for the SE process. Here, we explored the accumulation of zma-miR528 and several predicted targets during embryogenic callus induction, proliferation, and plantlet regeneration using the maize cultivar VS-535. We confirmed the cleavage site for all tested zma-miR528 targets; however, PLC1 showed very low levels of processing. The abundance of zma-miR528 slightly decreased in one month-induced callus compared to the immature embryo (IE) explant tissue. However, it displayed a significant increase in four-month sub-cultured callus, coincident with proliferation establishment. In callus-regenerated plantlets, zma-miR528 greatly decreased to levels below those observed in the initial explant. Three of the target transcripts (MATE, bHLH, and SOD1a) showed an inverse correlation with the miRNA abundance in total RNA samples at all stages. Using polysome fractionation, zma-miR528 was detected in the polysome fraction and exhibited an inverse distribution with the PLC1 target, which was not observed at total RNA. Accordingly, we conclude that zma-miR528 regulates multiple target mRNAs during the SE process by promoting their degradation, translation inhibition or both.

Callus culture in vitro in the experimental evaluation of drought resistance in cereals (review)

One of the directions in plant biotechnological research is the use of callus cultures in vitro. The callus is an integrated system that is formed both exogenously (as a result of the proliferation of surface cells of various tissues of the plant organism) and endogenously (in the depth of these tissues). Initially, callus consists of homogeneous cells gradually transformed into a system of groups of heterogeneous cells with specific morphogenetic potencies. These potencies are realized by various ways of morphogenesis in vitro, including those leading to the formation of full-fledged plants-regenerants. In this review, using cereals as an example, we analyze the literature and our data on the use of calli in vitro as experimental systems for studying the plant stress-resistance to abiotic factors, especially to the drought factor. The advantages and limitations of using callus cultures in vitro are considered. The prospects of studying the mechanisms of action of abiotic stressors and resistance to them at the cellular and tissue levels under model conditions of callus culture in vitro are shown. Attention is paid to the assessment of the drought resistance of cereals under selective conditions simulating drought in vitro by such a parameter as the growth activity of calli. The issue of studying anti-stress effects in callus cultures in vitro is analyzed on the example of brassinosteroids. The prospects of using the model system “immature embryo in vivo–embryonic morphogenic callus in vitro” for rapid assessment of the effect of anti-stress plant growth regulators are considered. It is emphasized that the basis for using calli as the model system is both the significant role of the cell in all morphogenetic events of the plant organism in vivo and in vitro and the similarity of the responses of plants in vivo, calli in vitro, regenerants of callus origin in vitro and ex vitro.