Non-mutant Plants Research Articles

Mutagenesis and Identification of Sugarcane Mutants Using Survival on Polyethylene Glycol and Leaf Damage under Managed Water Stress

Drought causes severe damage to sugarcane, reducing its product yield. Given Thailand’s weather conditions and topography, a breeding program to develop new sugarcane genotypes with a high tolerance for water stress is important to the sugarcane industry. This study created new water stress tolerant sugarcane genotypes using ethyl methanesulfonate (EMS) mutagenesis in the sugarcane cultivar Khon Kaen 3. Using 16 mM of EMS for 4 h induced callus mutagenesis (survival rate, 57.5%). The survival rates of calli treated with 10 mM of EMS for 2 and 4 h in selective media with 15% PEG were higher than that of non-EMS-treated calli. The selected calli survived and grew on selective media with 20% PEG, while non-EMS-treated calli did not grow. The mutant plantlets developed from EMS-treated calli on selective media with 20% PEG for 4 weeks had varying survival rates: 72.25% (10 mM of EMS for 2 h), 75.85% (10 mM of EMS for 4 h), and 60.61% (16 mM of EMS for 4 h). Both healthy mutant sugarcane plants (2,086) and non-mutant plants (234) were cultured on the media with 20% PEG for 16 weeks. Of these, 462 mutant sugarcane plantlets survived and developed on the media, but all the non-mutant sugarcane plantlets died during the selection process. Mutagenesis induced using treatment 4 produced the highest frequency of mutant sugarcane plantlets with water-stress tolerance (45.5%). In total, 136 selected mutant sugarcane plants were transplanted to a greenhouse for evaluation under managed water stress. Fourteen mutant sugarcane plants stayed green after the third cycle of water stress, but the KK3 sugarcane cultivar showed damage on 50% of the leaves. Thus, EMS mutagenesis and evaluation using in vitro and greenhouse methods were successful in developing new sugarcane clones with high water-stress tolerance, which is important for sugarcane breeding programs.

Sensitivity of Seeds to Chemical Mutagens, Detection of DNA Polymorphisms and Agro-Metrical Traits in M1 Generation of Coffee (Coffea arabica L.)

Coffee (Coffea Arabica L.) is threatened by biotic and abiotic stresses. Nevertheless, the breeding of Arabica coffee is restricted due to its low genetic diversity. Crop improvement via mutagenesis represents an alternative for increasing genetic variability and facilitating breeding. In this sense, coffee seeds cv. Catuai were treated for 8 h with a solution of sodium azide (NaN3) (0, 50, 75, 100, and 125 mM) and ethyl methane sulfonate (EMS) (0, 80, 160, 240, 320, and 400 mM). The genetic variability induced in coffee plants after mutagenic treatment with sodium azide was determined by RAPD and AFLP analyses. As the concentration of applied NaN3 and EMS increased, the germination, seedling height, and root length decreased. The LD50 values for NaN3 and EMS were between 50-75 mM and 160-240 mM, respectively. For the 12 RAPD primers evaluated, a total of 46 fragments were obtained of which 34 were polymorphic bands (74%). The amplification with six AFLP selective primer combinations allowed the identification of 36 polymorphisms (17.8%). The analysis revealed that both NaN3 and EMS induced variability within the DNA regions amplified with AFLP and RAPD markers. Finally, under field conditions, significant differences were noticed with respect to plant height, number of nodes in the orthotropic stem, and number of branches of the M1 mutant (NaN3-treated) plants compared to the non-mutant plants. Optimal conditions for NaN3 and EMS mutagenesis using seeds were determined and the optimized conditions have been used to generate a NaN3 mutant M1 coffee var. Catuai population.

Mutations in sorghum SBEIIb and SSIIa affect alkali spreading value, starch composition, thermal properties and flour viscosity

Key messageSeven novel alleles of SBEIIb and one allele of SSIIa co-segregated with the ASV phenotype and contributed to distinct starch quality traits important for food-processing applications.Sorghum is an important food crop for millions of people in Africa and Asia. Whole-genome re-sequencing of sorghum EMS mutants exhibiting an alkali spreading value (ASV) phenotype revealed candidate SNPs in Sobic.004G163700 and Sobic.010G093400. Comparative genomics identified Sobic.010G093400 as a starch synthase IIa and Sobic.004G163700 as a starch branching enzyme IIb. Segregation analyses showed that mutations in Sobic.010G093400 or Sobic.004G163700 co-segregated with the ASV phenotype. Mutants in SSIIa exhibited no change in amylose content but expressed lower final viscosity and lower starch gelatinization temperature (GT) than starches from non-mutant plants. The sbeIIb mutants exhibited significantly higher amylose levels and starch GT and lower viscosity compared to non-mutant starches and ssIIa mutants. Mutations in SBEIIb had a dosage-dependent effect on amylose content. Double mutants of sbeIIb and ssIIa resembled their sbeIIb parent in amylose content, starch thermal properties and viscosity profiles. These variants will provide opportunities to produce sorghum varieties with modified starch end-use qualities important for the beer brewing and baking industries and specialty foods for humans with diabetes.

Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology.

Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology. This powerful tool allows substantial improvement in plant traits in addition to those provided by classical breeding. Here, we demonstrate the development of virus resistance in cucumber (Cucumis sativus L.) using Cas9/subgenomic RNA (sgRNA) technology to disrupt the function of the recessive eIF4E (eukaryotic translation initiation factor 4E) gene. Cas9/sgRNA constructs were targeted to the N' and C' termini of the eIF4E gene. Small deletions and single nucleotide polymorphisms (SNPs) were observed in the eIF4E gene targeted sites of transformed T1 generation cucumber plants, but not in putative off-target sites. Non-transgenic heterozygous eif4e mutant plants were selected for the production of non-transgenic homozygous T3 generation plants. Homozygous T3 progeny following Cas9/sgRNA that had been targeted to both eif4e sites exhibited immunity to Cucumber vein yellowing virus (Ipomovirus) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-W. In contrast, heterozygous mutant and non-mutant plants were highly susceptible to these viruses. For the first time, virus resistance has been developed in cucumber, non-transgenically, not visibly affecting plant development and without long-term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants.

The circadian clock‐associated gene gigantea1 affects maize developmental transitions

The circadian clock is an internal timing mechanism that allows plants to make developmental decisions in accordance with environmental conditions. In model plants, circadian clock-associated gigantea (gi) genes are directly involved in control of growth and developmental transitions. The maize gigantea1 (gi1) gene is the more highly expressed of the two gi homeologs, and its function is uncharacterized. To understand the role of gi1 in the regulatory networks of the maize circadian clock system, gi1 mutants were evaluated for changes in flowering time, phase change and growth control. When grown in long-day (LD) photoperiods, gi1 mutants flowered earlier than non-mutant plants, but this difference was not apparent in short-day (SD) photoperiods. Therefore, gi1 participates in a pathway that suppresses flowering in LD photoperiods, but not in SD. Part of the underlying cause of early flowering was up-regulated expression of the FT-like floral activator gene zea mays centroradialis8 (zcn8) and the CONSTANS-like flowering regulatory gene constans of zea mays1 (conz1). gi1 mutants also underwent vegetative phase change earlier and grew taller than non-mutant plants. These findings indicate gi1 has a repressive function in multiple regulatory pathways that govern maize growth and development.

Towards a reporter system to identify regulators of cross-talk between salicylate and jasmonate signaling pathways in Arabidopsis

The plant signaling hormones salicylic acid (SA) and jasmonic acid (JA) are regulators of inducible defenses that are activated upon pathogen or insect attack. Cross-talk between SA- and JA-dependent signaling pathways allows a plant to finely tune its response to the attacker encountered. In Arabidopsis, pharmacological experiments revealed that SA exerts a strong antagonistic effect on JA-responsive genes, such as PDF1.2, indicating that the SA pathway can be prioritized over the JA pathway. SA-mediated suppression of the JA-responsive PDF1.2 promoter was exploited for setting up a genetic screen aiming at the isolation of signal transduction mutants that are impaired in this cross-talk mechanism. The PDF1.2 promoter was fused to the herbicide resistance gene BAR to allow for life/death screening of a population of mutagenized transgenic plants. Non-mutant plants should survive herbicide treatment when methyl jasmonate (MeJA) is applied, but suppression of the JA response by SA should be lethal in combination with the herbicide. Conversely, crucial SA/JA cross-talk mutants should survive the combination treatment. SA effectively suppressed the expression of the PDF1.2::BAR transgene. However, suppression of the BAR gene did not result in suppression of herbicide resistance. Hence, a screening method based on quantitative differences in the expression of a reporter gene may be better suited to identify SA/JA cross-talk mutants. Here, we demonstrate that the PDF1.2::GUS reporter will be excellently suited in this respect.

Suggested mnemonics for cloned DNA corresponding to enzymes involved in starch metabolism

All known isoforms of ADPglucose synthase are heterotetramers composed of two dissimilar subunits. The deduced primary structures in hand can be divided into five groups based upon fractional identity. The relationship between two proteins in different groups is 50 to 60% identity, while two proteins within a group are 85 to 95% identical. All known enzymes from nonmutant plants contain one subunit from one of the groups of protein and one subunit from one of the other four categories of proteins. We think the database should implicitly indicate this. There are two possibilities for this group.

ABNORMAL GUARD CELL DEVELOPMENT IN AN OLIVE NECROTIC MUTANT OF MAIZE

A study of a mutant variety of Zea mays (ON8147) revealed that the mutant plants, in contrast with normal maize plants, do not exhibit a light‐induced increase in the rate of transpiration, and that the ontogeny of the stomatal complex is abnormal. In later stages of differentiation, the guard cells of mutant plants deteriorate, leaving the mature stomata with only the two subsidiary cells. The subsidiary cells in stomata of mutant leaves are similar to those of normal leaves with respect to their capacity to accumulate K+ in the dark, but they do not lose K+ in the light, as do subsidiary cells of stomata of nonmutant plants. It is suggested that impairment of guard cell function causes death of the mutant plant seedlings primarily by restricting CO2 entry into the leaf.

Abnormal Guard Cell Development in an Olive Necrotic Mutant of Maize

A study of a mutant variety of Zea mays (ON8147) revealed that the mutant plants, in contrast with normal maize plants, do not exhibit a light-induced increase in the rate of transpiration, and that the ontogeny of the stomatal complex is abnormal. In later stages of differentiation, the guard cells of mutant plants deteriorate, leaving the mature stomata with only the two subsidiary cells. The subsidiary cells in stomata of mutant leaves are similar to those of normal leaves with respect to their capacity to accumulate K+ in the dark, but they do not lose K+ in the light, as do subsidiary cells of stomata of nonmutant plants. It is suggested that impairment of guard cell function causes death of the mutant plant seedlings primarily by restricting CO2 entry into the leaf.