Genetic Variability Studies in M5 Generations of Determinate Early Maturing Cluster Bean [Cyamopsis tetragonoloba (L.) Taub] (MDU-1) Mutants for Yield and Yield Attributing Characters

Background: MDU-1 is a high yielding, indeterminate cluster bean variety. In order to develop determinate mutant with high yield, MDU-1 cluster bean variety was irradiated using gamma rays, electron beam and combination of gamma rays and EMS with different doses and the variability induced in M5 generations were studied. Methods: Twenty one best individual plants from M4 generation were selected based on plant height and higher yield and forwarded to M5 generation where they were evaluated in RBD with three replications. Fourteen non-segregating mutant lines with desirable traits were identified in M5 generation and were evaluated for variability along with the parent MDU1. Result: Five best mutant lines namely ACMC-020-04, ACMC-020-11, ACMC-020-08, ACMC-020-10 and ACMC-020-11 were selected from the 14 accessions based on per se performance and variability analysis. The selected mutants needs to be forwarded for stability testing in different environments.

Generating Novel Male Sterile Tomatoes by Editing Respiratory Burst Oxidase Homolog Genes

Hybrid breeding of tomatoes (Solanum lycopersicum), an important vegetable crop, is an effective way to improve yield and enhance disease and stress resistance. However, the efficiency of tomato hybridization is hindered by self-fertilization, which can be overcome using male sterile lines. It has been reported that reactive oxygen species (ROS) act as a key regulator for anther development, mediated by RBOH (Respiratory Burst Oxidase Homolog) genes. Here, two tomato anther-expressed genes, LeRBOH (Solyc01g099620) and LeRBOHE (Solyc07g042460), were selected to cultivate novel tomato male sterile strains. By using a CRISPR/Cas9 system with a two-sgRNA module, the lerboh, lerbohe, and lerboh lerbohe mutant lines were generated, among which the lerbohe and lerboh lerbohe mutants displayed complete male sterility but could accept wild-type pollens and produce fruits normally. Further analysis uncovered significantly decreased ROS levels and abnormal programmed cell death in lerboh lerbohe anthers, indicating a key role of ROS metabolism in tomato pollen development. Taken together, our work demonstrates a successful application of gene editing via CRISPR/Cas9 in generating male sterile tomatoes and afforded helpful information for understanding how RBOH genes regulating tomato reproduction process.

Mutational Analysis of a Wheat O-methyltransferase Involved in Flavonoid Metabolism

Methylated flavones, and tricin in particular, have been implicated in protecting wheat plants against a variety of biotic and abiotic stresses. Methylated flavones are produced via O-methylation of the hydroxyl groups in flavones, which is catalyzed by O-methyltransferases (OMTs). To examine the role of wheat OMT2 in methylated flavone biosynthesis and facilitate interrogation of tricin functions in wheat-environment interactions, loss-of-function mutants of OMT2 homoeologs, omt-A2 and omt-B2, were identified from a tetraploid wheat Targeting Induced Local Lesions in Genomes (TILLING) mutant population and crossed to generate the omt-A2omt-B2 double mutant. Although tricin and most other soluble phenolics did not differ in leaves and glumes of TILLING control and the omt-A2, omt-B2, and omt-A2 omt-B2 mutants, chlorogenic acid was increased in glumes of omt-A2 omt-B2 relative to TILLING control, suggesting that it might serve as a substrate for OMT2. The omt2 mutant lines showed similar growth phenotypes as well as comparable lignin deposition in cell walls of stems compared to TILLING control. These results collectively suggest that OMT2 and its close homolog OMT1 may possess overlapping activities in tricin production, with OMT1 compensating for the missing OMT2 activities in the omt2 mutant lines.

​Binasoybean-6: A High Yielding Mutant Soybean Variety Developed through Sustainable Mutation Breeding

Background: Soybean is an important source of food, protein and oil and hence more research is essential to increase its yield under different agro-ecological conditions, including stress. In this regard, four popular soybean varieties viz. Shohag, BDS-4, BAU-S/64 and BARI Soybean-5 were irradiated using Co60 gamma rays to create genetic variation for earliness, higher seed yield and other desirable agronomic traits. Methods: The experiments were conducted at Bangladesh Institute of Nuclear Agriculture (BINA) Headquarters farm, Mymensingh during 2006-2009 and 28 elite mutant lines were selected for evaluation. The mutant line, SBM-22 derived from mother variety BARI Soybean-5 irradiated with 300Gy of gamma rays was found to be superior compared to other mutants. Considering the superior performance of mutant SBM-22 including 28 mutants and mother check variety BARI Soybean-5, were evaluated through different trials. The evaluation trials were conducted at different agro-ecological zones of the country during Rabi season (January to April) of 2010-2018. Result: Significant variations were observed both in individual location and over locations for all traits. Reactions to major diseases and insect-pests infestation were also studied. Due to better performance of the mutant SBM-22, Bangladesh Institute of Nuclear Agriculture (BINA) applied to the National Seed Board (NSB) of Bangladesh for registration as an important soybean variety “Binasoybean-6”. Consequently, the NSB of Bangladesh registered SBM-22 as an improved soybean variety in 2019 as Binasoybean-6 for commercial cultivation.

1H NMR-Based Chemometrics to Gain Insights Into the Bran of Radiation-Induced Colored Wheat Mutant

Recently, wheat has attracted attention as a functional food, rather than a simple dietary energy source. Accordingly, whole-grain intake increases with an understanding of bioactive phytochemicals in bran. The development of colored wheat has drawn more attention to the value of bran owing to its nutritional quality, as well as the antioxidant properties of the colorant. The present 1H NMR-based chemometric study evaluated the compositional improvement of radiation-induced mutants in purple wheat by focusing on the predominant metabolites with high polarity. A total of 33 metabolites, including three choline derivatives, three sugar alcohols, four sugars, 13 amino acids, eight organic acids, and two nucleosides, were identified throughout the 1H NMR spectra, and quantification data were obtained for the identified metabolites via peak shape-based quantification. Principal component and hierarchical cluster analyses were conducted for performing multivariate analyses. The colored original wheat was found to exhibit improvements compared to yellow wheat in terms of the contents of primary metabolites, thus highlighting the importance of conducting investigations of polar metabolites. The chemometrics studies further revealed mutant lines with a compositional enhancement for metabolites, including lysine, proline, acetate, and glycerol.

Targeted Deletion of the First Intron of the Wxb Allele via CRISPR/Cas9 Significantly Increases Grain Amylose Content in Rice

Background The rice Waxy (Wx) gene plays a major role in seed amylose synthesis and consequently controls grain amylose content. Wx gene expression is highly regulated at the post-transcriptional level. In particular, the GT/TT polymorphism at the 5′splicing site of its 1st intron greatly affects this intron’s splicing efficiency and defines two predominant Wx alleles, Wxa and Wxb. Wxa rice often harbours intermediate to high amylose contents, whereas Wxb rice exhibits low to intermediate amylose contents. By deleting the Wx 1st intron using CRISPR/Cas9 technology, we generate a completely novel Wx allele and further investigate how intron removal affects Wx gene expression and rice grain amylose content. Results CRISPR/Cas9-mediated targeted deletion of the Wx 1st intron was performed on 4 rice inbred lines: KY131 (Wxb), X32 (Wxb), X35 (Wxa) and X55 (Wxlv). Deletion of the 1st intron occurred in 8.6–11.8% of the primary transformants of these 4 inbred lines. Compared to wild-type plants, amylose content was significantly increased from 13.0% to approximately 24.0% in KY131 and X32 mutant lines, which both carried the Wxb allele. However, no significant difference in amylose content was observed between wild-type plants and X35 and X55 mutant lines, which carried the Wxa and Wxlv alleles, respectively. Wx gene expression analysis of wild-type plants and mutants yielded results that were highly consistent with amylose content results. KY131 and X32 mutants accumulated increased levels of steady mRNA transcripts compared with wild-type plants, whereas steady mRNA levels were not altered in X35 and X55 mutants compared with wild-type plants. Grain quality, including appearance quality and eating and cooking quality, which are tightly associated with amylose content, was also assessed in wild-type and mutant plants, and data were presented and analysed. Conclusions This study presents a novel and rapid strategy to increase amylose content in inbred rice carrying a Wxb allele. Our data strongly suggest that the 1st intron of the Wx gene regulates Wx gene expression mainly at the post-transcriptional level in rice. This finding is in contrast to a previous hypothesis suggesting that it influences Wx gene transcription. In addition, removal of the first intron generates a completely novel Wx allele. Further studies on this new Wx allele will provide invaluable insights into the regulation of Wx gene expression, which will help researchers engineer new Wx alleles to facilitate the breeding of rice cultivars with better eating and cooking quality.

Integrated transcriptome and proteome analysis reveals brassinosteroid-mediated regulation of cambium initiation and patterning in woody stem.

Wood formation involves sequential developmental events requiring the coordination of multiple hormones. Brassinosteroids (BRs) play a key role in wood development, but little is known about the cellular and molecular processes that underlie wood formation in tree species. Here, we generated transgenic poplar lines with edited PdBRI1 genes, which are orthologs of Arabidopsis vascular-enriched BR receptors, and showed how inhibition of BR signaling influences wood development at the mRNA and/or proteome level. Six Populus PdBRI1 genes formed three gene pairs, each of which was highly expressed in basal stems. Simultaneous mutation of PdBRI1–1, −2, −3 and − 6, which are orthologs of the Arabidopsis vascular-enriched BR receptors BRI1, BRL1 and BRL3, resulted in severe growth defects. In particular, the stems of these mutant lines displayed a discontinuous cambial ring and patterning defects in derived secondary vascular tissues. Abnormal cambial formation within the cortical parenchyma was also observed in the stems of pdbri1–1;2;3;6. Transgenic poplar plants expressing edited versions of PdBRI1–1 or PdBRI1–1;2;6 exhibited phenotypic alterations in stem development at 4.5 months of growth, indicating that there is functional redundancy among these PdBRI1 genes. Integrated analysis of the transcriptome and proteome of pdbri1–1;2;3;6 stems revealed differential expression of a number of genes/proteins associated with wood development and hormones. Concordant (16%) and discordant (84%) regulation of mRNA and protein expression, including wood-associated mRNA/protein expression, was found in pdbri1–1;2;3;6 stems. This study found a dual role of BRs in procambial cell division and xylem differentiation and provides insights into the multiple layers of gene regulation that contribute to wood formation in Populus.

Yield components of the M7 Kipas Putih mutant soybean

This study aimed to obtain the homogeny of M8 soybean lines in an effort to release new high yielding varieties. The research was conducted in Lamsidaya village, and the observation of yield components was carried out at the Plant Breeding Laboratory, Faculty of Agriculture, Universitas Syiah Kuala. This study did not use an experimental design, so that the quantitative characters were observed individually from each plant, then analyzed by estimating the heritability broad sense value and the progress of selection. The material used were var. Kipas Putih and var. Dega1 as control, eight of 7th generation Kipas Putih mutant lines (B4, B7, B10, B12, B13, B15, B18 and B22). All genotypes were planted in rectangular plot 10 m x 1 m, drainage 50 cm, and planting distance 50 cm x 30 cm. Plants were maintained in accordance with the recommendation for soybean cultivation from Balitkabi, and fertilizer NPK (16:16:16) was given 200 g/plot (200 kgha-1). Parameters observed included plant height, flowering time, harvest time, number of productive branches, total number of pods per plant, number of filled pods per plant, percentage of filled pods per plant, seed weight per plant, weight of 100 seeds, seed weight per plot, and yield potential. All quantitative characters indicated increase on selection except for the character of plant height, percentage of filled pods and weight of 100 seeds because the heritability value was low. The B18 line has the potential to become a new high yielding variety because it has a high yield potential (3.02 tons ha-1) with a large seed size compared to the other seven mutant lines and is also higher than the parent Kipas Putih and var Dega 1 varieties.

Aklimatisasi dan Respon Pertumbuhan Mutan Leucaena Leucocephala Varietas Tarramba Teradaptasi Asam

Acclimatization is the final stage of plant propagation in tissue culture techniques that can determine the success of the nursery process. This study aimed to observe the growth response during the acclimatization stage of the acid-adapted of Leucaena leucocephala Tarramba variety, which developed from tissue culture techniques. The design used in this study was a completely randomized design (CRD) with the cultivation of 11 mutant lines from tissue culture, namely M1-M11 (an acid-adapted mutant from tissue culture addition of 1 ppm IBA) and 2 controls (lamtoro broodstock without gamma irradiation) namely K0 (lamtoro broodstock resulting from tissue culture addition of 0 ppm IBA), K1 (breeding lamtoro from tissue culture addition of 1 ppm IBA). The variables observed were the level of plant viability, plant height, and number of leaves. The results showed that the acclimatization of the plant Leucaena leucocephala to tissue culture production on the M3 and M9 mutant lines gave the best response to plant morphological growth up to 5 WAP (weeks after planting). Key words: acclimatization, IBA hormone, Leucaena leucocephala, tissue culture

A novel zebrafish model of SPEG-related centronuclear myopathy (CNM): characterization and comparison with other CNM model zebrafish

Centronuclear myopathy (CNM) is a congenital neuromuscular disorder caused by pathogenic variation in genes associated with membrane trafficking and excitation-contraction coupling (ECC). Bi-allelic autosomal recessive mutations in striated muscle enriched protein kinase (SPEG) account for a subset of CNM patients. Previous research has been limited by the perinatal lethality of Speg knockout mice. Thus, the precise biological role of SPEG in skeletal muscle remains unknown. To address this issue, we generated zebrafish spega, spegb, and spega/spegb (speg-DKO) mutant lines. We demonstrate that speg-DKO zebrafish faithfully recapitulate multiple phenotypes associated with human CNM, including disruption of the ECC protein machinery, dysregulation of calcium homeostasis during ECC, and impairment of muscle performance. Taking advantage of the availability of zebrafish models of multiple CNM genetic subtypes, we compared novel and known disease markers in speg-DKO with mtm1-KO and DNM2-S619L transgenic zebrafish. We observed desmin (DES) accumulation common to all CNM subtypes, and Dnm2 upregulation in muscle of both speg-DKO and mtm1-KO zebrafish. In all, we establish a new model of SPEG-related CNM, and identify abnormalities in this model suitable for defining disease pathomechanisms and evaluating potential therapies.