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.

Efficiency of selection–biotechnological system of selection for creation of breeding source material of sunflower resistant to herbicides and broomrape

The sunflower is a strategically important oil crop. Every year the area under this crop grows, and the rapid returning of sunflowers back to the fields provokes the formation of new more aggressive races of broomrape (Orobanche cumana Wallr.). Broomrape is a parasite that interferes with the normal development of sunflower and can lead to significant crop losses. For creating a sunflower hybrid (F1) it is needed to cross the parental components, which have a complex of important traits, among which there is a resistance to the herbicides and a broomrape. Considering that the creation of each of the components of the hybrid involves many years of painstaking work in the breeding process, modern approaches and methods are used to accelerate the creation of a new source material. Thus, using the technology of cultivating immature embryos in vitro culture, it is possible to reduce the time to create lines resistant to herbicides, for example. And during selection for resistance to pathogenic organisms, testing is most often used against an artificial infectious background, both in the field and in laboratory conditions, in order to differentiate the material on this basis. The aim of this work was to establish the effectiveness system when creating an initial breeding material resistant to herbicides and broomrape. As a result of testing the lines on an artificial infectious background, was identified plants which have high resistance to the G-race broomrape and were isolated from hybrid combinations resistant to tribenuron-methyl and imidazolinones. Thus, among the analyzed plants which are resistant to tribenuron‐methyl, four lines were isolated, which are highly resistant to the G-race broomrape from a hybrid combinations BH0118/SURES–2 (101/1, 101/4, 101/6, 101/7), and BH0318/SURES–2 (101/21, 101/24, 101/28, 101/30), and five lines (101/11, 101/12, 101/16, 101/17, 101/18) from a hybrid combination BH0218/SURES–2. Among imidazolinone-resistant sunflower lines – line 3 was isolated as highly resistant to the G-race broomrape.

An efficient sorghum transformation system using embryogenic calli derived from mature seeds

Significant progress has been made on sorghum transformation in the last decades; however, the transformation process has been constrained by the availability of immature embryos because most of the researchers have utilized immature embryos as favorable explants. Although immature embryos have been proven to be optimal for tissue culture and transformation, isolation of immature embryos is time-consuming, labor-intensive, and limited by warm weather. In this study, we developed an efficient genetic transformation system using mature seeds as explants. The nptII and gus gene, used as the selective marker and report gene respectively, have been co-transformed by particle bombardment. After optimization of tissue culture, the G418 concentration, and transgenic, the average transformation frequency at 13.33% was achieved routinely. The transgenic events and transgene copy numbers were determined by PCR and RT-PCR, respectively. The geneticin selection and GUS staining on T1 seedlings confirmed that the transgenic plants were heritable. Our results demonstrated that the efficient sorghum transformation system has been established using mature seeds as explants. This transformation system will promote sorghum research on genetic engineering and genome editing without seasonal weather conditions restriction and explant resources restriction.

CALLUS FORMATION AND PLANT REGENERATION FROM IMMATURE EMBRYOS OF PIGMENTED UPLAND RICE (Oryza sativa cv. Tadong)

The application of biotechnology in upland rice improvement programs depends on the availability of efficient regeneration protocols. Although protocols for shoot regeneration of upland rice are available, none has been reported for pigmented cultivars. This study reports on a protocol for callus induction and regeneration of Tadong, a pigmented upland rice cultivar from Sabah. For callus induction, immature embryos were cultured on media containing 2,4-Dichlorophenoxyacetic (2,4-D) at various concentrations (0 – 2.5 mg/L) and on different types of media (MS; MSB5; N6B5; N6). To induce shoot regeneration, callus explants were cultured on MS medium supplemented with combinations of 6-Benzylaminopurine (BAP) at various concentrations (0 – 3.0 mg/L) and 1-Naphthaleneacetic acid (NAA) at 1.0 mg/L. To induce shoot development, callus explants were pre-treated with Thidiazuron (TDZ) at various concentrations (0-1.0 mg/l) and exposed to different desiccation periods (0 – 72 hours). 2,4-Dichlorophenoxyacetic at 2.5 mg/L and N6B5 medium resulted in the highest percentages of explant forming callus which were 60.3 ± 17.0 % and 58.7 ± 9.8 % respectively. The regeneration media failed to induce shoot on callus explants, instead, green spots were formed on the surface of the callus. The green spots were stimulated to develop into shoots when the callus explants were pre-treated with 0.5 mg/L TDZ or exposed to partial desiccation for 24 h, the percentages of explant forming shoot were 35.7 ± 4.8 % and 47.7 ± 6.8 % respectively. Shoots developed into complete plants on hormone-free MS medium and acclimatized.

Whole-genome sequence analysis of mutations in rice plants regenerated from zygotes, mature embryos, and immature embryos

Somaclonal variation was studied by whole-genome sequencing in rice plants (Oryza sativa L., ‘Nipponbare’) regenerated from the zygotes, mature embryos, and immature embryos of a single mother plant. The mother plant and its seed-propagated progeny were also sequenced. A total of 338 variants of the mother plant sequence were detected in the progeny, and mean values ranged from 9.0 of the seed-propagated plants to 37.4 of regenerants from mature embryos. The ratio of single nucleotide variants among the variants was 74.3%, and the natural mutation rate calculated using the variants in the seed-propagated plants was 1.2 × 10−8. The percentage and the mutation rate were consistent with the values reported previously. Plants regenerated from mature embryos had significantly more variants than different progeny types. Therefore, using zygotes and immature embryos can reduce somaclonal variation during the genetic manipulation of rice.

Development of a Transformable Fast-Flowering Mini-Maize as a Tool for Maize Gene Editing

Maize (Zea mays ssp. mays) is a popular genetic model due to its ease of crossing, well-established toolkits, and its status as a major global food crop. Recent technology developments for precise manipulation of the genome are further impacting both basic biological research and biotechnological application in agriculture. Crop gene editing often requires a process of genetic transformation in which the editing reagents are introduced into plant cells. In maize, this procedure is well-established for a limited number of public lines that are amenable for genetic transformation. Fast-Flowering Mini-Maize (FFMM) lines A and B were recently developed as an open-source tool for maize research by reducing the space requirements and the generation time. Neither line of FFMM were competent for genetic transformation using traditional protocols, a necessity to its status as a complete toolkit for public maize genetic research. Here we report the development of new lines of FFMM that have been bred for amenability to genetic transformation. By hybridizing a transformable maize genotype high Type-II callus parent A (Hi-II A) with line A of FFMM, we introgressed the ability to form embryogenic callus from Hi-II A into the FFMM-A genetic background. Through multiple generations of iterative self-hybridization or doubled-haploid method, we established maize lines that have a strong ability to produce embryogenic callus from immature embryos and maintain resemblance to FFMM-A in flowering time and stature. Using an Agrobacterium-mediated standard transformation method, we successfully introduced the CRISPR-Cas9 reagents into immature embryos and generated transgenic and mutant lines displaying the expected mutant phenotypes and genotypes. The transformation frequencies of the tested genotypes, defined as the numbers of transgenic event producing T1 seeds per 100 infected embryos, ranged from 0 to 17.1%. Approximately 80% of transgenic plants analyzed in this study showed various mutation patterns at the target site. The transformable FFMM line, FFMM-AT, can serve as a useful genetic and genomic resource for the maize community.

The “embryo in planta – callus in vitro” system: cytophysiological aspects (by wheat example)

Plant regeneration from calli in vitro is an integral part of a number of biotechnologies. Immature embryos (IE) are particularly promising as explants for obtaining morphogenic calli (MC) in cereals.