Spatio-Temporal Distribution of Cell Wall Components in the Placentas,Ovules and Female Gametophytes of Utricularia during Pollination

In most angiosperms, the female gametophyte is hidden in the mother tissues and the pollen tube enters the ovule via a micropylar canal. The mother tissues play an essential role in the pollen tube guidance. However, in Utricularia, the female gametophyte surpasses the entire micropylar canal and extends beyond the limit of the integument. The female gametophyte then invades the placenta and a part of the central cell has direct contact with the ovary chamber. To date, information about the role of the placenta and integument in pollen tube guidance in Utricularia, which have extra-ovular female gametophytes, has been lacking. The aim of this study was to evaluate the role of the placenta, central cell and integument in pollen tube pollen tube guidance in Utricularia nelumbifolia Gardner and Utricularia humboldtii R.H. Schomb. by studying the production of arabinogalactan proteins. It was also determined whether the production of the arabinogalactan proteins is dependent on pollination in Utricularia. In both of the examined species, arabinogalactan proteins (AGPs) were observed in the placenta (epidermis and nutritive tissue), ovule (integument, chalaza), and female gametophyte of both pollinated and unpollinated flowers, which means that the production of AGPs is independent of pollination; however, the production of some AGPs was lower after fertilization. There were some differences in the production of AGPs between the examined species. The occurrence of AGPs in the placental epidermis and nutritive tissue suggests that they function as an obturator. The production of some AGPs in the ovular tissues (nucellus, integument) was independent of the presence of a mature embryo sac.     

Function Analysis of the PR55/B Gene Related to Self-Incompatibility in Chinese Cabbage Using CRISPR/Cas9

Chinese cabbage, a major crop in Korea, shows self-incompatibility (SI). SI is controlled by the type 2A serine/threonine protein phosphatases (PP2As). The PP2A gene is controlled by regulatory subunits that comprise a 36 kDa catalyst C subunit, a 65 kDa regulatory A subunit, and a variety of regulatory B subunits (50–70 kDa). Among them, the PP2A 55 kDa B regulatory subunit (PR55/B) gene located in the A05 chromosome has 13 exons spanning 2.9 kb, and two homologous genes, Bra018924 and Bra014296, were found to be present on the A06 and A08 chromosome, respectively. In this study, we performed a functional analysis of the PR55/B gene using clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9 (CRISPR/Cas9)-mediated gene mutagenesis. CRISPR/Cas9 technology can be used to easily introduce mutations in the target gene. Tentative gene-edited lines were generated by the Agrobacterium-mediated transfer and were selected by PCR and Southern hybridization analysis. Furthermore, pods were confirmed to be formed in flower pollination (FP) as well as bud pollination (BP) in some gene-edited lines. Seed fertility of gene-edited lines indicated that the PR55/B gene plays a key role in SI. Finally, self-compatible T-DNA-free T₂ gene-edited plants and edited sequences of target genes were secured. The self-compatible Chinese cabbage developed in this study is expected to contribute to Chinese cabbage breeding.     

Species Phylogeny versus Gene Trees: A Case Study of an Incongruent Data Matrix Based on Paphiopedilum Pfitz. (Orchidaceae)

The phylogeny of the genus Paphiopedilum based on the plastome is consistent with morphological analysis. However, to date, none of the analyzed nuclear markers has confirmed this. Topology incongruence among the trees of different nuclear markers concerns entire sections of the subgenus Paphiopedilum. The low-copy nuclear protein-coding gene PHYC was obtained for 22 species representing all sections and subgenera of Paphiopedilum. The nuclear-based phylogeny is supported by morphological characteristics and plastid data analysis. We assumed that an incongruence in nuclear gene trees is caused by ancestral homoploid hybridization. We present a model for inferring the phylogeny of the species despite the incongruence of the different tree topologies. Our analysis, based on six low-copy nuclear genes, is congruent with plastome phylogeny and has been confirmed by phylogenetic network analysis.     

An Efficient Agrobacterium-Mediated Transformation System for Poplar

Poplar is a model system for the regeneration and genetic transformation of woody plants. To shorten the time required for studies of transgenic poplar, efforts have been made to optimize transformation methods that use Agrobacterium tumefaciens. In this study, an Agrobacterium infective suspension was treated at 4 °C for at least 10 h before infecting explants. By transforming the Populus hybrid clone “Nanlin895” (Populus deltoides × P. euramericana) with Agrobacterium harboring the PBI121:CarNAC6 binary vector, we showed that the transformation efficiency was improved significantly by multiple independent factors, including an Agrobacterium infective suspension with an OD₆₀₀ of 0.7, an Agrobacterium infection for 120 min, an Agrobacterium infective suspension at a pH of 5.0, an acetosyringone concentration of 200 µM, a cocultivation at 28 °C, a cocultivation for 72 h and a sucrose concentration of 30 g/L in the cocultivation medium. We also showed that preculture of wounded leaf explants for two days increased the regeneration rate. The integration of the desired gene into transgenic poplars was detected using selective medium containing kanamycin, followed by southern blot analysis. The expression of the transgene in the transgenic lines was confirmed by northern blot analysis.     

An Improved Protocol for Agrobacterium-Mediated Transformation in Subterranean Clover (Trifolium subterraneum L.)

Subterranean clover (Trifolium subterraneum) is the most widely grown annual pasture legume in southern Australia. With the advent of advanced sequencing and genome editing technologies, a simple and efficient gene transfer protocol mediated by Agrobacterium tumefaciens was developed to overcome the hurdle of genetic manipulation in subterranean clover. In vitro tissue culture and Agrobacterium transformation play a central role in testing the link between specific genes and agronomic traits. In this paper, we investigate a variety of factors affecting the transformation in subterranean clover to increase the transformation efficiency. In vitro culture was optimised by including cefotaxime during seed sterilisation and testing the best antibiotic concentration to select recombinant explants. The concentrations for the combination of antibiotics obtained were as follows: 40 mg L⁻¹ hygromycin, 100 mg L⁻¹ kanamycin and 200 mg L⁻¹ cefotaxime. Additionally, 200 mg L⁻¹ cefotaxime increased shoot regeneration by two-fold. Different plant hormone combinations were tested to analyse the best rooting media. Roots were obtained in a medium supplemented with 1.2 µM IAA. Plasmid pH35 containing a hygromycin-resistant gene and GUS gene was inoculated into the explants with Agrobacterium tumefaciens strain AGL0 for transformation. Overall, the transformation efficiency was improved from the 1% previously reported to 5.2%, tested at explant level with Cefotaxime showing a positive effect on shooting regeneration. Other variables in addition to antibiotic and hormone combinations such as bacterial OD, time of infection and incubation temperature may be further tested to enhance the transformation even more. This improved transformation study presents an opportunity to increase the feeding value, persistence, and nutritive value of the key Australian pasture.     

Mono- and diammonium phosphates and triple superphosphate as sources of P in a calcareous soil

Recovery of phosphorus from monoammonium phosphate (MAP), diammonium phosphate (DAP) and triple superphosphate (TSP), at rates of 0, 15, 30, or 45 mg P kg^–1 was determined in a pot experiment on a Calcaric Lithosol soil (21% CaCO₃). At the 15 mg P kg^–1 rate DAP was as effective as MAP and more effective than TSP in supplying P, but it was less effective than MAP and TSP at the higher rates of 30 and 45 mg P kg^–1. At the two higher P rates residual bicarbonate extractable P was also significantly lower with DAP. Yield dry matter was not affected by the source of P.     

Developmental changes in fatty acid composition of oil in kernel fractions of corn (Zea mays L.)

Gas liquid chromatography was used to determine the change in fatty acid composition of oil from three kernel fractions (pericarp, endosperm and germ) during kernel maturation of four inbred lines of corn. Inbred lines were sibpollinated, and sampling of ears began six days after pollination (DAP) and continued at three day intervals until 33 DAP and then at weekly intervals until 54 DAP. Proportion of palmitic acid in the pericarp oil rapidly decreased between 6 and 12 DAP while oleic and linoleic acids increased during the same period. Changes in fatty acid composition of oil from the endosperm during kernel maturation were erratic and no consistent trends were evident. In the germ oil, palmitic and linolenic acid proportions decreased during kernel maturation, while oleic acid decreased and linoleic acid increased during kernel maturation for three of the four inbred lines. By about 24 to 27 DAP, the fatty acid composition of oil in the mature kernel was established. Since kernel fractions are of different genetic origin, a study of developmental changes in lipid classes or in fatty acid composition of oil should be limited within kernel fractions that have a similar genetic constitution. Approved as Journal Series Paper No. 723, University of Georgia, College of Agriculture Experiment Stations.     

Plant Regeneration and Somatic Embryogenesis from Immature Embryos Derived through Interspecific Hybridization among Different Carica Species

Plant regeneration and somatic embryogenesis through interspecific hybridization among different Carica species were studied for the development of a papaya ringspot virus-resistant variety. The maximum fruit sets were recorded from the cross of the native variety C. papaya cv. Shahi with the wild species C. cauliflora. The highest hybrid embryos were recorded at 90 days after pollination and the embryos were aborted at 150 days after pollination. The immature hybrid embryos were used for plant regeneration and somatic embryogenesis. The 90-day-old hybrid embryos from the cross of C. papaya cv. Shahi × C. cauliflora showed the highest percentage of germination, as well as plant regeneration on growth regulators free culture medium after 7 days pre-incubation on half-strength MS medium supplemented with 0.2 mg/L BAP, 0.5 mg/L NAA and 60 g/L sucrose. The 90-day-old hybrid embryos from the cross of C. papaya cv. Shahi × C. cauliflora produced maximum callus, as well as somatic embryos when cultured on half-strength MS medium containing 5 mg/L 2,4-D, 100 mg/L glutamine, 100 mg/L casein hydrolysate and 60 g/L sucrose. The somatic embryos were transferred into half-strength MS medium containing 0.5 mg/L BAP and 0.2 mg/L NAA and 60 g/L sucrose for maturation. The highest number of regenerated plants per hybrid embryo (10.33) was recorded from the cross of C. papaya cv. Shahi × C. cauliflora. Isoenzyme and dendrogram cluster analysis using UPGMA of the regenerated F₁ plantlets confirmed the presence of the hybrid plantlets.     

Clade-Specific Plastid Inheritance Patterns Including Frequent Biparental Inheritance in Passiflora Interspecific Crosses

Plastid inheritance in angiosperms is presumed to be largely maternal, with the potential to inherit plastids biparentally estimated for about 20% of species. In Passiflora, maternal, paternal and biparental inheritance has been reported; however, these studies were limited in the number of crosses and progeny examined. To improve the understanding of plastid transmission in Passiflora, the progeny of 45 interspecific crosses were analyzed in the three subgenera: Passiflora, Decaloba and Astrophea. Plastid types were assessed following restriction digestion of PCR amplified plastid DNA in hybrid embryos, cotyledons and leaves at different developmental stages. Clade-specific patterns of inheritance were detected such that hybrid progeny from subgenera Passiflora and Astrophea predominantly inherited paternal plastids with occasional incidences of maternal inheritance, whereas subgenus Decaloba showed predominantly maternal and biparental inheritance. Biparental plastid inheritance was also detected in some hybrids from subgenus Passiflora. Heteroplasmy due to biparental inheritance was restricted to hybrid cotyledons and first leaves with a single parental plastid type detectable in mature plants. This indicates that in Passiflora, plastid retention at later stages of plant development may not reflect the plastid inheritance patterns in embryos. Passiflora exhibits diverse patterns of plastid inheritance, providing an excellent system to investigate underlying mechanisms in angiosperms.     

Improved Transformation and Regeneration of Indica Rice: Disruption of SUB1A as a Test Case via CRISPR-Cas9

Gene editing by use of clustered regularly interspaced short palindromic repeats (CRISPR) has become a powerful tool for crop improvement. However, a common bottleneck in the application of this approach to grain crops, including rice (Oryza sativa), is efficient vector delivery and calli regeneration, which can be hampered by genotype-dependent requirements for plant regeneration. Here, methods for Agrobacterium-mediated and biolistic transformation and regeneration of indica rice were optimized using CRISPR-Cas9 gene-editing of the submergence tolerance regulator SUBMERGENCE 1A-1 gene of the cultivar Ciherang-Sub1. Callus induction and plantlet regeneration methods were optimized for embryogenic calli derived from immature embryos and mature seed-derived calli. Optimized regeneration (95%) and maximal editing efficiency (100%) were obtained from the immature embryo-derived calli. Phenotyping of T₁ seeds derived from the edited T₀ plants under submergence stress demonstrated inferior phenotype compared to their controls, which phenotypically validates the disruption of SUB1A-1 function. The methods pave the way for rapid CRISPR-Cas9 gene editing of recalcitrant indica rice cultivars.     

Protective Roles of Gadd45 and MDM2 in Blueberry Anthocyanins Mediated DNA Repair of Fragmented and Non-Fragmented DNA Damage in UV-Irradiated HepG2 Cells

Growth Arrest and DNA Damage-inducible 45 (Gadd45) and MDM2 proteins, together with p21 and p53, play important roles in cell cycle checkpoints, DNA repair, and genome integrity maintenance. Gadd45 and MDM2 were activated and transcribed instantly by UV irradiation, whereas blueberry anthocyanins (BA) decreased the gene and protein expression levels in HepG2 cells for up to 24 h, and gradually restored the UV-induced fragmented and non-fragmented DNA damage of the nucleus at a time point of 12 h. Nevertheless, UV-irradiated HepG2 cell arrests occurred mainly in the G1 phase, which indicated G1 as a checkpoint. The proteins, p21 and p53, retain cellular integrity, suppressing the oncogenic transformation by interruption of the G1 phase of the cellular cycle, giving time for repairing the damage to DNA, or apoptosis induction if the damage is too severe to be repaired, while MDM2 and Gadd45 concomitantly ensure the presence of p53 and p21. Thus, we conclude that repair, together with Gadd45 and MDM2 genes, were involved in light and dark reaction mechanisms, however, BA could interfere and assist the repair through restoration, although further studies of the complex of the gene cascades triggered and responded to in BA-assisted DNA repair are needed.     

Influences of diallyl phthalate as chain extender on the properties of high molecular weight poly(vinyl chloride) resin

A higher porosity with better thermostability is desirable for poly(vinyl chloride) (PVC) resin. In this study, high molecular weight PVC resins are prepared by vinyl chloride monomer (VCM)‐diallyl phthalate (DAP) suspension copolymerization in a 20‐L reactor at 50 °C using DAP as chain extender. SEM, BET, and analyses of plasticizer absorption results show the high molecular weight poly(vinyl chloride) (HPVC) by DAP‐VCM copolymerization is loose and porous. With increasing DAP content when the mass ratio of DAP/VCM (ω) is below the gel point, the porosity and the degree of polymerization increase. Nevertheless, the bulk density and particle size decrease. When more than the gel point, these relationships are reversed. Thermogravimetric analysis revealed that the HPVC had better thermostability than that of commercial PVC, and its thermostability increases with increasing ω before it reaches the gel point. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 45093.     

Synthesis of novel bisphenol containing phthalazinone and azomethine moieties and thermal properties of cured diamine/bisphenol/DGEBA polymers

A novel bisphenol(1,2-dihydro-2-(4-((4-hydroxy)phenyliminomethylidene)phenyl)-4-(4-((4-(4-hydroxy)phenyliminomethylidene)phenoxy)phenyl)(2H)phthalazin-1-one, DPP) and a diamine(1,2-dihydro-2-(4-aminophenyl)-4-(4-(4-aminophenoxy)phenyl)(2H)phthalazin-1-one, DAP) were synthesized and characterized. The novel epoxy polymers containing phthalazinone and/or azomethine moieties were prepared by binary polymerization of DAP (or DPP) with diglycidyl ether of biphenyl A (DGEBA) and ternary polymerization of hybrid curing agents, DAP/DPP (DAP and DPP under different molar ratios) with DGEBA. The cure behaviors of these new epoxy systems were studied by dynamic differential scanning calorimeter (DSC) and Infrared (IR) scans. Especially, the activation energy of DAP/DGEBA calculated by Kissinger and Ozawa methods were 73.8 and 77.4 kJ/mol, respectively. For ternary epoxy system, it was found that hybrid curing agents of DAP/DPP exhibited significant associated effect on their reactivity towards the oxirane group. Glass transition temperatures (T_g's) of these new epoxy polymers were all above 150 °C from the results of DSC, and the initial thermal decomposition temperatures (T_d,5%'s) and integral procedure decomposition temperatures (IPDT's) of these new epoxy polymers are above 350 and 850 °C, respectively from results of thermogravimetric analyses (TGA). These results show that new epoxy polymers containing phthalazinone and/or azomethine moieties exhibited excellent thermal properties. Especially, thermal properties of the ternary epoxy polymers could be modified by changing the content of DAP and DPP. The linear relationships between char yield (Y_c,wt%) and the structural compositions of these new polymers (weight percentage of phthalazinone, azomethine and nitrogen, C/H weight ratio) were built.