In vitro androgenesis: spontaneous vs. artificial genome doubling and characterization of regenerants.

Androgenesis has become the most frequently chosen method of doubled haploid (DH) production in major crops. Theoretically, plantlets derived from in vitro cultured microspore encompass half of the normal chromosome number of donor plants and thus, considered to be haploid. However, depending on species/genotype and the method of haploid production, either via anther or isolated microspore culture, different ratios of spontaneous DHs and diploid (2n) or even polyploid plants originating from somatic tissues or unreduced gametes may also arise in the cultures. Adopting the method of haploid identification, anti-microtubular agent for restoring fertility, and discriminating spontaneous DHs from undesired heterozygote plants will substantially affect the success of androgenesis in breeding programs. The recent advances in the last 2 decades have made it possible to characterize the in vitro regenerants efficiently either prior to genome duplication or using in breeding programs. The herein described approaches and antimicotubular agents are, therefore, expected to improve the efficiency of DH-based breeding pipeline through the in vitro androgenesis.

Microspore embryogenesis in Brassica: calcium signaling, epigenetic modification, and programmed cell death.

MAIN CONCLUSION: Stress induction followed by excessive calcium influx causes multiple changes in microspores resulting in chromatin remodeling, epigenetic modifications, and removal of unwanted gametophytic components via autophagy, switching microspores towards ME. In Brassica, isolated microspores that are placed under specific external stresses can switch their default developmental pathway towards an embryogenic state. Microspore embryogenesis is a unique system that speeds up breeding programs and, in the context of developmental biology, provides an excellent tool for embryogenesis to be investigated in greater detail. The last few years have provided ample evidence that has allowed Brassica researchers to markedly increase their understanding of the molecular and sub-cellular changes underlying this process. We review recent advances in this field, focusing mainly on the perception to inductive stresses, signal transduction, molecular and structural alterations, and the involvement of programmed cell death at the onset of embryogenic induction.

Molecular characterization and expression analysis of SERK1 and SERK2 in Brassica napus L.: implication for microspore embryogenesis and plant regeneration.

KEY MESSAGE: The BnSERK1 and BnSERK2 are involved in the process of microspore embryogenesis induction, development, and plantlet regeneration. Little is known about regulatory role of somatic embryogenesis-related kinase (SERK) genes family in the induction of microspore embryogenesis, development and plant regeneration. In this study, the expression of two SERK genes (SERK1 and SERK2) was assessed during the microspore embryogenesis and plantlet regeneration in Brassica napus L. The BnSERK1 was severely up-regulated 1-5 days following microspore culture and its expression drastically decreased in the globular-heart and also torpedo staged microspore-derived embryos (MDEs). In addition, high levels of BnSERK1 transcript were detected in the MDE maturation phase and in the roots and shoots of the regenerated plantlets which indicates a broader role(s) of BnSERK1 in the organ formation, rather than being specific to the embryogenesis. Results of partial sequencing indicated that the BnSERK1 shares a conserved serine-threonine kinase catalytic domain and exhibited 95 % similarity with AtSERK1, CsSERK1, BrSERK1, NaSERK1, and NbSERK1. A steady increase in the expression of BnSERK2 was observed during the MDE initiation and development so that, the highest expression was noted in the MDE maturation phase i.e., late cotyledonary MDEs. Our results also indicated low amounts of BnSERK2 transcript at the onset of rhyzogenesis but significantly higher expression in the developing roots. In contrast, the BnSERK2 strongly up-regulated during the both initially and developed shoots. The BnSERK2 shares highly conserved LRR-RLK domain when compared with different species tested so that, high homology (100 %) was noticed with BrSERK2. Based on our findings, MDE formation and plantlet regeneration seem to be correlated with both BnSERK1 and BnSERK2 expression.

Methods for Chromosome Doubling.

The completely homozygous genetic background of doubled haploids (DHs) has many applications in breeding programs and research studies. Haploid induction and chromosome doubling of induced haploids are the two main steps of doubled haploid creation. Both steps have their own complexities. Chromosome doubling of induced haploids may happen spontaneously, although usually at a low rate. Therefore, artificial/induced chromosome doubling of haploid cells/plantlets is necessary to produce DHs at an acceptable level. The most common method is using some mitotic spindle poisons that target the organization of the microtubule system. Colchicine is a well-known and widely used antimitotic. However, there are substances alternative to colchicine in terms of efficiency, toxicity, safety, and genetic stability, which can be applied in in vitro and in vivo pathways. Both pathways have their own advantages and disadvantages. However, in vitro-induced chromosome doubling has been much preferred in recent years, maybe because of the dual effect of antimitotic agents (haploid induction and chromosome doubling) in just one step, and the reduced generation of chimeras. Plant genotype, the developmental stage of initial haploids, and type-concentration-duration of application of antimitotic agents, are top influential parameters on chromosome doubling efficiency. In this review, we highlight different aspects related to antimitotic agents and to plant parameters for successful chromosome doubling and high DH yield.

Hydrological drought persistence and recovery over the CONUS: A multi-stage framework considering water quantity and quality.

Hydrological droughts have considerable negative impacts on water quantity and quality, and understanding their regional characteristics is of crucial importance. This study presents a multi-stage framework to detect and characterize hydrological droughts considering both streamflow and water quality changes. Hydrological droughts are categorized into three stages of growth, persistence, retreat, and water quality variables (i.e., water temperature, dissolved oxygen concentration, and turbidity) are utilized to further investigate drought recovery. The framework is applied to 400 streamflow gauges across the Contiguous United States (CONUS) over the study period of 1950-2016. The method is illustrated for the 2012 US drought, which affected most of the nation. Results reveal the duration, frequency, and severity of historical droughts in various regions as well as their spatial consistencies and heterogeneities. Furthermore, duration of each stage of drought (i.e., growth, persistence, and retreat) is also assessed and the spatial patterns are diagnosed across the CONUS. Considering the water quality variables, increased water temperature (4 °C on average) and reduced dissolved oxygen concentration (2.5 mg/L on average) were observed during drought episodes, both of which impose severe consequences on ecology of natural habitats. On the contrary, turbidity was found to decrease during droughts, and indicate a sudden increase when drought terminates, due to increase in runoff. Varied drought recovery durations are perceived for different water quality variables, and in general, it takes about two more months for water quality variables to recover from a drought, following the hydrological drought termination.

Evaluation of Root Canal Anatomy in Mandibular Incisors Using CBCT Imaging Technique in an Iranian Population.

STATEMENT OF THE PROBLEM: To perform a successful endodontic treatment, sufficient knowledge about the number of root canals and their morphology is essential. Missed normal variations may burden this treatment. PURPOSE: This study aimed to evaluate the presence of the second canal in the roots of mandibular central and lateral incisors in an Iranian population by employing CBCT images. MATERIALS AND METHOD: This cross-sectional study recruited 180 CBCT image of mandible to evaluate the number of roots as well as the number and types of root canals. Data for each sample were collected in a data collection form set and analyzed by chi-square test using SPSS17 software. RESULTS: A total of 681 permanent mandibular incisors were assessed. All samples had one root. Most of the samples (70.3%) had only one canal (type 1 Vertucci classification). The frequency of dual-canal in samples was 29.7%; the prevalence of dual-canal in mandibular lateral teeth (35%) was more than the mandibular central teeth (23.9%, p< 0.05). Following type 1 canal, type 3 (15.7%), type 5 (12.9%), type 4 (0.7%), and type 2 (0.3%) canals had the highest frequencies respectively. CONCLUSION: Based on this study, presence of a second canal in mandibular lateral teeth (35%) is more common than in mandibular central teeth (23.9 %). The most common canal type observed was type 1 (according to Vertucci classification) followed by type 3.

Effect of Variation in Viscosity Grade of Ethycellulose on Theophylline Microcapsule Properties Prepared by Emulsion Solvent Evaporation.

BACKGROUND: There are conflicting reports regarding the effect of polymer viscosity grade on microcapsule properties. The aim of the present study was to investigate the effect of just viscosity grade of ethylcellulose (EC) (not polymeric solution) on properties of theophylline microcapsules prepared by emulsion solvent evaporation. METHODS: The effect of EC viscosity grade and drug:polymer ratio was investigated on microcapsule properties (yield, particle size, morphology, surface characteristics and drug release). Differential scanning calorimetry (DSC) and X-ray powder diffraction (XRD) were implemented to study the interaction and solid state of drug. The microcapsules were compressed in the presence of excipients and drug release was evaluated. RESULTS: The yield of microencapsulation and encapsulation efficiency at 1:1 drug:polymer ratio was dependent on EC viscosity. Microcapsules were spherical with some pores on their surfaces. The number of pores was more and their size was bigger for EC 100 cP microcapsules. Theophylline remained in crystalline form after encapsulation. DSC studies confirmed lack of interaction between drug and polymer. The drug release was rapid at 2:1 drug:polymer whilst it was slowed down at 1:1 drug:polymer ratio. Microcapsules obtained from EC 100 cP showed slightly faster drug release at latter ratio. Marginal changes in release rate were observed after compression of microcapsules. CONCLUSIONS: All viscosity grades of EC were able to sustain the release of the drug from microcapsules. Considering the similar release profiles for microcapsules prepared from different viscosities of EC, the use of lower viscosity grade of EC is recommended due to the ease of production and also less processing time.

Effects of Heat Shock and 2,4-D Treatment on Morphological and Physiological Characteristics of Microspores and Microspore-Derived Doubled Haploid Plants in Brassica napus L.

BACKGROUND: Stresses such as heat shock, starvation, or osmotic is essential to lead isolated microspores towards embryogenesis. Despite the effectiveness of stresses in embryogenesis, they exert adverse effects on metabolism and growth of the regenerated plants. OBJECTIVES: The effects of heat shock and 2,4-D treatment on total protein content of treated microspores, morphological and physiological characteristics of the doubled haploid (DH) plants were assessed. MATERIALS AND METHODS: Buds containing mid- to late- uninucleate microspores were used for microspore culture. Microspores were isolated and cultured in NLN-13 medium and incubated at 30ºC for 14 days or treated with 2,4-D (35 mg.L-1) for 30 min to induce embryogenesis. Microspore-derived embryos were transferred onto B5 medium for plantlet regeneration. Ploidy level of the regenerated plantlets was determined using Partec flow cytometry. Spectrophotometric readings were carried out at 490, 663 and 645 nm to determine Chl a-b and carotenoids contents. TRIzol and cetyl-threeethyl-ammonium bromide (CTAB) were used for protein extraction from microspores and leaves. Length and width of stomata and pollen grains were also photographed using light microscope (Olympus). RESULTS: Applied stressors significantly reduced total protein content of treated microspores however, protein content and concentration of chlorophyll a and b of the DH plants were only increased by heat shock treatment when compared with the donor plant 'Hyola 420'. In contrast, carotenoids were not affected by applied stressors. Longer and wider stomata were observed by 2,4-D treatment but, the length of pollen grains was significantly decreased following heat shock and 2,4-D treatment. CONCLUSIONS: Total protein content of cultured microspores, concentration of chlorophyll a and b, length and width of stomata of microspore-derived doubled haploid plants were significantly affected by the type of inductive stresses. However, carotenoids were more stable and not affected by applied stressors.