Regenerated Rice Plants Research Articles

Vascular system of rice leaves depending on ploidy level

The current paper has provided data on the study of the vascular system of flag leaves of rice plants grown in a greenhouse in the period 2022–2023. The objects of the study were 24 rice samples developed at the FSBSI “ARC “Donskoy” using the method of androgenesis and cell culture. The purpose of the work was to determine the degree of development of the vascular system of flag leaves of regenerated rice plants with different ploidy levels (1n, 2n, 4n). When studying the vascular system of plant leaves (the number and area of bundles), it was found that haploid plants had smaller vascular bundles compared to diand tetraploid samples. The average beam diameter had dimensions of 59.5, 69.3 and 75.3 µm, the area of one beam was 2815.6, 3827.2 and 4540.5 µm², respectively. In the leaves of rice samples, a larger number of small and large vascular bundles have been formed with increasing ploidy level. Their number was 36–44 in haploids, 40–52in diploids, 52–60 in tetraploids. The average number of bundles was 40.3, 46.6 and 55.2, respectively. The venation pattern was individual for each sample. Between single large bundles there are from one to seven small veins, most often 4–6 pieces. Thus, samples with different ploidy levels differ in the anatomical structure of the leaves, which ultimately affects their morphology and productivity.

A piggyBac-mediated transgenesis system for the temporary expression of CRISPR/Cas9 in rice.

SummaryTargeted mutagenesis via CRISPR/Cas9 is now widely used, not only in model plants but also in agriculturally important crops. However, in vegetative crop propagation, CRISPR/Cas9 expression cassettes cannot be segregated out in the resulting progenies, but must nevertheless be eliminated without leaving unnecessary sequences in the genome. To this end, we designed a piggyBac‐mediated transgenesis system for the temporary expression of CRISPR/Cas9 in plants. This system allows integration into the host genome of piggyBac carrying both CRISPR/Cas9 and positive selection marker expression cassettes from an extrachromosomal double‐stranded transfer DNA (dsT‐DNA), with subsequent excision of the transgenes by the re‐transposition of piggyBac from the host genome after successful induction of targeted mutagenesis via CRISPR/Cas9. Here, we demonstrate that the transgenesis system via piggyBac transposition from T‐DNA works to deliver transgenes in rice. Following positive–negative selection to exclude transgenic cells randomly transformed with T‐DNA, piggyBac‐mediated transgenesis from the extrachromosomal dsT‐DNA was successful in ca. 1% of transgenic callus lines. After temporary expression of CRISPR/Cas9 within piggyBac, we confirmed, in a proof‐of‐concept experiment, that piggyBac could be excised precisely from the genome via the stably transformed transposase PBase. Even after excision of piggyBac, CRISPR/Cas9‐induced targeted mutations could be detected in the endogenous gene in regenerated rice plants. These results suggest that our piggyBac‐mediated transgenesis system will be a valuable tool in establishing efficient CRISPR/Cas9‐mediated targeted mutagenesis in vegetatively propagated crops.

Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity

Cytosine base editors (CBEs) generate C-to-T nucleotide substitutions in genomic target sites without inducing double-strand breaks. However, CBEs such as BE3 can cause genome-wide off-target changes via sgRNA-independent DNA deamination. By leveraging the orthogonal R-loops generated by SaCas9 nickase to mimic actively transcribed genomic loci that are more susceptible to cytidine deaminase, we set up a high-throughput assay for assessing sgRNA-independent off-target effects of CBEs in rice protoplasts. The reliability of this assay was confirmed by the whole-genome sequencing (WGS) of 10 base editors in regenerated rice plants. The R-loop assay was used to screen a series of rationally designed A3Bctd-BE3 variants for improved specificity. We obtained 2 efficient CBE variants, A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3, and the WGS analysis revealed that these new CBEs eliminated sgRNA-independent DNA off-target edits in rice plants. Moreover, these 2 base editor variants were more precise at their target sites by producing fewer multiple C edits.

Increasing fidelity and efficiency by modifying cytidine base-editing systems in rice

The efficiency of plant cytidine base-editing systems is limited, and unwanted mutations frequently occur in transgenic plants. We increased the cytidine editing frequency and fidelity of the plant base editor 3 (BE3) and targeted activation-induced cytidine deaminase (CDA) (target-AID) systems by coexpressing three copies of free uracil–DNA glycosylase (UDG) inhibitor (UGI). The editing efficiency of the improved BE3 and CDA systems reached as high as 88.9% and 85.7%, respectively, in regenerated rice plants, with a very low frequency of unwanted mutations. The low editing frequency of the BE3 system in the GC context could be overcome by the modified CDA system. These results provide a high-fidelity and high-efficiency solution for rice genomic base editing.

Diverse chromosomal rearrangements induced by Ac/Ds at the OsRLG5 locus in regenerated rice plants

Closely-located transposable elements (TEs) can induce chromosomal breakage and rearrangements via alternative transposition (AT) reactions. Our previous study showed that diverse chromosomal rearrangements were induced by two inversely-oriented Ds elements at the OsRLG5 (Oryza sativa receptor-like kinase gene 5) locus in regenerated rice plants. Here, several novel chromosomal rearrangements were identified in a population of tissue culture regenerants, and their proposed Ac/Ds-transposition mechanisms were discussed. In the first type of event, AT was induced by the same Ds element on each of two sister chromatids and resulted in the inversion of genomic DNA flanking the Ds element. In the second type of event, DNA segments of the inter-transposon segment (ITS) region were deleted or inverted. This novel local rearrangement likely resulted from an AT reaction of two directly-oriented Ds elements located on the same chromatid. The 5' and 3' termini of two Ds elements were re-inserted into two different sites inside the ITS, which generated the subsequent deletion and inversion. In the third type of event, two inversely-oriented Ds elements induced homologous recombination and chromosomal translocation. Together, these results showed the diversity of Ac/Ds-mediated chromosomal rearrangements and demonstrated the utility of the experimental system for examining the mechanisms underlying Ac/Ds AT reactions.

Variation in Spikelet-Related Traits of Rice Plants Regenerated from Mature Seed-Derived Callus Culture

Callus is An Excellent Source For in Vitro Plant Regeneration, But Plants Regenerated from Callus Sometimes Show Phenotypic And Genotypic Variation from The initial Plants. in This Study, The Variation in Spikelet-Related Traits of The Rice Plants Regenerated from Calluses And Their Performance in The Paddy Field Were Examined. The Phenotypic Variation in Spikelet-Related Traits of The Regenerated Plants Was Not Always in A Reduction in Their Mean Value. For instance, Panicle Length, Spikelet Number And Fertile Spikelet Number of indonesian Rice Genotypes Ciapus And Bp-140 in The Regenerated Plants Were Significantly Greater Than Those of The initial Plants (Developed from The Seeds). The Spikelet Fertility of The Regenerated Rice Plants Was Not Significantly Lower Than That of The initial Plants Except in Ciapus And Bp-140. The Occurrence of Somaclonal Variants Varied With The Genotype. Ciapus And Bp-140, Which induce Many Somaclonal Variants, Are Suggested To Be Valuable For Genetic, Breeding Or Functional Genomic Studies, While Fatmawati, Which is Stable, Could Be Used For Genetic Transformation Study.

Effectiveness of genotype-based selection in the production of marker-free and genetically fixed transgenic lineages: ectopic expression of a pistil chitinase gene increases leaf-chitinase activity in transgenic rice plants without hygromycin-resistance gene

An extra copy of a rice pistil chitinase gene, termed chip, which is expressed predominantly in floral organs just before anthesis, was introduced into an economically important cultivar of rice (Oryza sativa L. cv. Koshihikari) together with the hygromycin-resistance gene (hph) as a marker gene by co-transformation. Over 100 regenerated rice plants (R0) and their self-pollinated first and second generations (R1 and R2) were genotypically characterized by PCR-based genomic DNA analysis of the two transgenes to obtain stable transgenic lines, in which chip was inserted into a single locus separately from hph. We obtained homozygous chip-transgenic R1 lines free of hph that stably expressed the chip mRNA and protein in rice leaf tissue. Chitin-hydrolytic activity in the leaves of these transgenic lines was higher than in those of non-transgenic controls. The resistance of the transgenic lines to rice blast disease fungus (Magnaporthe grisea) was examined by smearing water agar mixed with overcrowded conidia (race 007.0) on mechanically injured leaves, showing tendency to blast-disease resistance. Thus, the genotype-based selection was demonstrated to be effective at obtaining genetically stable transgenic plant lines without marker genes.

Characterization of Some Chromosomal Aberrations in Regenerated Rice Plants (Oryza sativa)

Somaclonal variation was studied in two Iranian land races of O. sativa spp. japonica var Hassani and O. sativa spp. indica var Sadridomsiah and 2000 plants of each cultivar were cytogenetically examined in two steps. In the first step, chromosome counts of root tips was used to detect ploidy levels and aneuploids of regenerated plants. In the second step, chromosomal aberrations were characterized by pachytene analysis of PMC’s. Ploidy levels were seen between n and 4n (haploids to tetraploids) in both cultivars with diploid resource (2n = 2x = 24). The total rate of variation for Hassani (japonica) was 13.7% including 10.8% for changes in chromosome number (ploidy levels and aneuploids) and 2.9% for chromosomal aberrations such as deficiency. A total rate of variation for Sadridomsiah (indica) was 15.6% including 12.4% for change in numbers and 3.2% for aberrations in construction. Most of important cytological mutations were observed in various chromosomes among regenerated plants of cultivars. Neither nullisomics nor inversions were distinguished in any samples.

벼에서 캘러스 유기원와 계대배양 기간에 따른 재분화 계통의 주요 농업형질 변이

Variations of agronomic traits were evaluated on the progenies of regenerated rice plants of different callus origin and subculture period. From eighty-eight percent to ninety percents of ovary culture-derived plants (OCP) and anther culture-derived plants (ACP) were not segregated within the lines in major agronomic characters. Compare to ACP and OCP lines, the seed culture derived lines (SCP) showed more segregation (17％) in major agronomic traits among the lines. The most frequent segregating traits were grain fertility (6.1％) in ACP, leaf color (4.2％) in OCP and grain fertility (4.9％) and leaf color (4.9％) in SCP lines. The SCP line showed more variation in culm length, panicle length and heading date than those of OCP and ACP lines. The variation of agronomic traits in SCP lines was tended to increase with prolonged subculture. Culm and panicle length were shorter than those of original cultivar in all three types of tissue culture-derived lines (OCP, ACP and SCP).

DNA Polymorphism among Rice Somaclones

Molecular markers were used to detect the influence of high concentrations of 2,4 dichlorophenoxyacetic acid (2,4-D) in the callusing media on DNA variations in regenerated rice plants. Restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and polymerase chain reaction (PCR) based RFLP analysis were carried out on 12 somaclones of Oryza sativa L. cv. B-370. In vitro culture induced DNA variations were detected in the regenerated plants but the effect of high auxin concentration in the medium could not be revealed. In a second study, fingerprinting of 15 semi-dwarf, high yielding somaclones of B-370 was carried out using RAPD technique. Amplification using 20 random primers produced a total of 167 DNA bands out of which 97 bands were polymorphic. A total of 32 unique DNA bands were detected across all the somaclones and they could be grouped based on their similarity to B-370. RAPD analysis helped to reveal similarity or differences among the somaclones while fingerprinting using additional RAPD markers was not successful.

Recovery of transgenic rice plants expressing the rice dwarf virus outer coat protein gene (S8)

The coding region of the eighth largest segment (S8) of the rice dwarf virus (RDV) was obtained from a RDV Fujian isolate. It was then cloned into pTrcHisA for expression in E. coli and into vector pE3 for plant transformation. By using callus derived from mature rice embryos as the target tissue, we obtained regenerated rice plants after bombardment of the former with plasmid pE3R8 containing the RDV S8 gene and the marker gene neomycin phosphotransferase (NPT II). Southern blotting confirmed the integration of the RDV S8 gene into the rice genome. The expression of the outer coat protein in both E. coli and rice plants was confirmed by western blotting. The recovery of transgenic rice plants expressing S8 gene is an important step towards studying the function of the RDV genes and obtaining RDV-resistant rice plants.

Somatic cell culture of rice cultivars with different grain types: Somaclonal variation in some grain and quality characters

Variations in some grain and quality characters in progenies of regenerated rice plants were studied. Grain length and weight decreased significantly, yet gel consistency increased. Variations in these quantitative characters of all cultivars studied were consistent, showing the tendencies of the variations. Grain protein contents of the somaclones were higher in one cultivar. Variability of most traits was increased by combining low-dosage radiation and tissue culture.