Functional Validation of cas9/guideRNA Constructs for Site-Directed Mutagenesis of Triticale ABA8′OH1 loci

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale ABA 8′-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticale can be increased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes.

CORRECTION OF THE TEMPERATURE DRIFT IN THE GEOPHYSICAL INSTRUMENT OF GROUND-BASED INDUCTION FREQUENCY SOUNDING

A technique for correcting the temperature drift in the measurements of the geophysical instrument of ground-based induction frequency sounding has been developed. The device is considered as a measure of the apparent electrical resistivity of the conducting half-space. In the process of manufacturing the equipment, the temperature coefficient of resistance (TCR) of the device is experimentally measured for each frequency separately. The TCR of the device is used to bring the measured resistance values to a certain normalized temperature, for example, 20 °C. The results of the tests performed were processed and systematized using Microsoft Excel. The graphs of the dependence of resistance on temperature are built. The developed methods are intended to improve the accuracy of data processing obtained with the help of equipment operating under conditions of changing ambient temperatures.

Functional Validation of cas9/guideRNA Constructs for Site-directed Mutagenesis of Triticale ABA8’OH1 loci

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like Triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in Triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the Triticale ABA 8’-hydroxylase 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in Triticale can be increased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in Triticale genes.

Improved Anther Culture Media for Enhanced Callus Formation and Plant Regeneration in Rice (Oryza sativa L.)

Anther culture technique is the most viable and efficient method of producing homozygous doubled haploid plants within a short period. However, the practical application of this technology in rice improvement is still limited by various factors that influence culture efficiency. The present study was conducted to determine the effects of two improved anther culture media, Ali-1 (A1) and Ali-2 (A2), a modified N6 medium, to enhance the callus formation and plant regeneration of japonica, indica, and hybrids of indica and japonica cross. The current study demonstrated that genotype and media had a significant impact (p < 0.001) on both callus induction frequency and green plantlet regeneration efficiency. The use of the A1 and A2 medium significantly enhanced callus induction frequency of japonica rice type, Nipponbare, and the hybrids of indica × japonica cross (CXY6, CXY24, and Y2) but not the indica rice type, NSIC Rc480. However, the A1 medium is found superior to the N6 medium as it significantly improved the green plantlet regeneration efficiency of CXY6, CXY24, and Y2 by almost 36%, 118%, and 277%, respectively. Furthermore, it substantially reduced the albino plantlet regeneration of the induced callus in two hybrids (CXY6 and Y2). Therefore, the improved anther culture medium A1 can produce doubled haploid rice plants for indica × japonica, which can be useful in different breeding programs that will enable the speedy development of rice varieties for resource-poor farmers.

Development and Characterization of Wheat-Agropyron cristatum Introgression Lines Induced by Gametocidal Genes and Wheat ph1b Mutant

The P genome of Agropyron cristatum Gaertn. contains many desirable genes that can be utilized as genetic resources to improve wheat. In this research, we used both the gametocidal chromosome 2Cc and the pairing homologous gene (Ph1b) mutant to induce structural aberrations and translocations between wheat and the 4P, 5P, and 6P genome chromosomes. By using the two approaches, a total of 19 wheat-A. cristatum translocations have been identified, in which 13 were induced by the Triticum aestivum cv. Chinese Spring (CS) ph1b mutant (CS ph1b) and six were induced by gametocidal chromosome 2Cc from Aegilops cylindrica Host. The wheat-4P, -5P and -6P A. cristatum translocations were characterized by in situ hybridization and by a set of conserved orthologous set (COS) molecular markers. The aberrations included centromeric translocations, terminal translocations, dicentric translocations, and deletions. The average induction frequency of chromosome structural aberrations was 10.9% using gametocidal 2Cc chromosome and 8.8% using ph1b mutant. The highest frequency obtained was for chromosome 4P using both approaches. All the wheat-A. cristatum translocation lines obtained were valuable for identifying A. cristatum chromosome 4P, 5P, and 6P related genes. In addition, these lines provided genetic resources and new germplasm accessions for the genetic improvement of wheat.

Efficient protoplast isolation from ovule-derived embryogenic callus inCitrusvolkameriana

The present study reports on the isolation of viable protoplast from ovule-derived embryogenic calli of Volkameriana (Citrus volkameriana L.), which is a rootstock in high demand for lemon production.Ovules of C. volkameriana isolated at 3 different immature fruit stages, comprising4, 8, and 12 weeks after anthesis (WAA),were cultured on5 different media in order to produce embryogenic callus lines as a source material for protoplast isolation. EME medium (MT basal medium + 0.5 gL–1 malt extract), with the addition of phytohormones [kinetin (KIN), 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP)] at different concentrations, were tested for callogenesis. According to 2-way ANOVA, significant effects were determined as a result of the immature fruit stage and type of culture media (P ≤ 0.01) on the callogenesis and embryogenic callus induction frequency. First, callus formation was recorded after 4 WAA on medium comprising EME + 2,4-D (1.0 mg L–1) + BAP (0.5 mg L–1). Callus induction frequency was the highest (90.00%) in the same culture medium when the ovules wereculturedat8 WAA. In addition, culturing the ovules isolated from 12 WAA immature fruits of C. volkameriana resulted in the highest indirect somatic embryogenesis (24%). Embryogenic callus initiation was the highest (25.56%) using EME + KIN (1.0 mg L–1) and ovules cultured at 8 WAA (14%) resulted in the highest embryogenic callus formation. Effects of different enzyme concentrations on the efficiency of protoplast isolation were calculated using the hemocytometer cell counting method. The combination of 2% cellulase and 0.2% pectinase gave the highest numbers of protoplasts, at 12.33 × 105protoplast/mL. Embryogenic callus lines obtained by culturing ovules of C. volkameriana yielded high-quality protoplasts after isolation and could be useful as a protoplast source for further somatic hybridization studies.

Genetics of anther culture capability traits and its association with seed yield traits in sesame

Successful development of heterotic hybrids in the autogamous sesame crop depends largely on the general combining ability of the parents as well as the specific combining ability of the hybrids produced from them. Homogeneity of Vr -Wr values and non-significance of regression coefficient (b) in a 6 parents half diallal cross were found in respect of capsule length, seeds per capsule, plant height, callus induction frequency, days to callus induction and response to somatic embryogenesis indicating validity of Hayman’s assumptions. The estimates of D, H1 and H2 components indicated predominance of non-additive gene action in the inheritance of these six traits, and the ADD estimates indicated overdominance for all these characters. The estimates of F and KD/KR indicated presence of more of dominant alleles in the parental population except for response to somatic embryogenesis. Strong positive association was recorded between seed yield and in vitro traits viz., callus induction frequency, callus fresh weight and somatic embryogenesis. This indicates that anther culture technique can assist in early screening for combining ability of parents as well as direct selection for heterotic crosses at an earlier stage of hybrid breeding programme.

Callus induction and axillary shoot formation in Asparagus racemosus Willd.

An experiment was performed to establish a regeneration protocol for an important medicinal plant, Asparagus racemosus. In the present investigation, nodal and internodal explants were employed for callus induction and axillary shoot formation. Maximum callus induction frequency was found on MS medium fortified with 2,4-D (1.0 mg/L) along with NAA (1.0 mg/L) and BAP (0.5 mg/L). However, individual effects of 2,4-D or NAA with BAP showed least callus induction. The higher concentrations of 2,4-D and BAP decreased the response of explants. However, maximum axillary shoot formation was observed on MS medium adjuvanted with BAP (2.0 mg/L) and NAA (0.5 mg/L).

Response to Callus Induction and Regeneration of Newly Released BRRI Rice Varieties

A study was carried out for developing an efficient callus induction and regeneration system for three newly developed BRRI varieties namely BRRI dhan86, BRRI dhan87 and BRRI dhan89. Dehusked seeds were plated onto MS and N6 media with two hormone combinations for callus induction. Calli obtained from each callus induction medium were transferred to four different regeneration media. Callus induction frequency and regeneration ability were significantly influenced by rice varieties, and interactions of variety and media. Among the media compositions, the highest callus (59.44%) were obtained from C1 (MS+2mg/l 2,4-D) followed by C2 ( MS+2 mg/l 2,4-D+0.5 mg/l kinetin) , C3 ( N6+2 mg/l 2,4-D) and C4 (N6+2 mg/l 2,4-D+0.5 mg/l kinetin) medium. The highest regeneration (45.74%) was obtained from R2 (MS+4 mg/ml BAP+1.2 mg/ml kinetin+0.5 mg/ml NAA), followed by R3 (1 mg/ml BAP+1 mg/ml Kinetin+1 mg/ml NAA), R4 (2 mg/ml kinetin+1 mg/ml NAA+300 mg casein hydrolysate) and R1 (2 mg/ml BAP+1 mg/ml kinetin+1 mg/ml NAA). BRRI dhan86 showed the highest regeneration ability (53.06%) than the other two varieties. It is observed that all varieties performed better in C1 medium for callus induction and R2 medium for regeneration. This study also revealed that BRRI dhan86 was more responsive to callus induction and regeneration of green plants than the other two varieties. Bangladesh Rice j. 2019, 23(2): 17-25

An efficient protocol for regenerating shoots from paper mulberry (Broussonetia papyrifera) leaf explants

Paper mulberry (Broussonetia papyrifera) is a tree species that has many economic, ecological, and social uses. This study developed an efficient protocol for regenerating shoots from leaf explants using Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth regulators (PGRs), which play vital roles in shoot regeneration. The best result, 86.67% induction frequency and 4.35 shoots per explant, was obtained in the MS medium containing 2.0 mg/L N6-benzyladenine (BA) and 0.05 mg/L indole-3-butyric acid. The effects of explant age, orientation, and genotype were also investigated. Explants from young leaves had a greater regeneration frequency than those from old leaves, and the results were better when the distal end of the leaf explant contacted the medium versus the proximal end. Approximately 70.96% of the shoots rooted well in the MS medium containing 0.4 mg/L α-naphthalene acetic acid (NAA). Although some genotypes achieved poorer results, the regeneration protocol is still applicable for mass multiplication and genetic transformation.