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Abnormal physiological responses of plant cultures such as shoot tip necrosis, callus, and hyperhydricity are some of the most difficult challenges in shoot micropropagation, and their causes are not well understood. Five Murashige and Skoog mineral salt factors, which influence the growth of pear shoot cultures, were tested in a five-dimensional surface response experimental design. Pyrus communis ‘Old Home × Farmingdale 87,’ ‘Horner 51,’ and ‘Winter Nelis’; Pyrus dimorphophylla; and Pyrus ussuriensis ‘Hang Pa Li’ shoot cultures were grown on 43 computer-designed treatments to represent the design space of all possible treatment combinations. Analysis of shoot response to these treatments identified the factors that both contributed to physiological disorders and remedied them. Undesirable callus formation was common for pear shoots cultured on standard medium and decreased on formulations with increased NH 4NO 3, Fe, and mesos (CaCl 2, KH 2PO 4, and MgSO 4) for most genotypes. Shoot tip necrosis varied with the genotype, but low mesos or low nitrogen concentrations contributed to the necrosis. Hyperhydricity was more prominent with low mesos or low NH 4NO 3. Hooked and upwardly curled new leaves were seen in most genotypes and resulted from use of low mesos in P. communis and low nitrogen for ‘Hang Pa Li’ and P. dimorphophylla. Fasciation and hypertrophy were seen infrequently and resulted from wide imbalances in several nutrients simultaneously. In general, standard concentrations of Murashige and Skoog iron and micros combined with high mesos and moderate nitrogen compounds produced normal shoots without physiological disorders. [ABSTRACT FROM AUTHOR]

We document high rates of triploidy in aspen (Populus tremuloides) across the western USA (up to 69% of genets), and ask whether the incidence of triploidy across the species range corresponds with latitude, glacial history (as has been documented in other species), climate, or regional variance in clone size. Using a combination of microsatellite genotyping, flow cytometry, and cytology, we demonstrate that triploidy is highest in unglaciated, drought-prone regions of North America, where the largest clone sizes have been reported for this species. While we cannot completely rule out a low incidence of undetected aneuploidy, tetraploidy or duplicated loci, our evidence suggests that these phenomena are unlikely to be significant contributors to our observed patterns. We suggest that the distribution of triploid aspen is due to a positive synergy between triploidy and ecological factors driving clonality. Although triploids are expected to have low fertility, they are hypothesized to be an evolutionary link to sexual tetraploidy. Thus, interactions between clonality and polyploidy may be a broadly important component of geographic speciation patterns in perennial plants. Further, cytotypes are expected to show physiological and structural differences which may influence susceptibility to ecological factors such as drought, and we suggest that cytotype may be a significant and previously overlooked factor in recent patterns of high aspen mortality in the southwestern portion of the species range. Finally, triploidy should be carefully considered as a source of variance in genomic and ecological studies of aspen, particularly in western U.S. landscapes. [ABSTRACT FROM AUTHOR]

Background: With the objective of combining multiple resistant traits from wild relative species in florist's chrysanthemums, trigeneric hybridization was conducted by crossing two intergeneric F1 hybrids Chrysanthemum grandiflorum x Artemisia vulgaris and Chrysanthemum crassum x Crossostephium chinense. Methodology/Principal Findings: To assess post-pollination phenomena, we investigated pollen germination on the stigma and embryo development, using fluorescence and scanning electron microscopy and paraffin-embedded sections, respectively. We selected eight putative trigeneric hybrid lines that showed the greatest morphological differences from the parents from among the progeny derived via embryo rescue. The hybridity of one trigeneric hybrid was further confirmed by fluorescent genomic in situ hybridization; in addition, the aphid resistance and salt tolerance of this hybrid were higher than those of the chrysanthemum parent and the C. grandiflorum x A. vulgaris F1 hybrid, respectively. Conclusions/Significance: The enhanced aphid resistance of the hybrid line reflects the inheritance of chromosomes from A. vulgaris, which carries genes that encode bioactive components. The enhanced salt tolerance of the trigeneric hybrid is attributable to inheritance of genetic materials from Chrysanthemum crassum and Crossostephium chinense, which act to maintain the compartmentation of Na+ and K+ ions and their selective transportation among different organs to avert deleterious effects and protect the photosynthetic apparatus. The results indicate that trigeneric hybridization between different bigeneric hybrids is a promising method for combination of multiple stress-resistance traits for improvement of chrysanthemum. [ABSTRACT FROM AUTHOR]

High resolution melting curve analysis (HRM) has been used as an efficient, accurate and cost-effective tool to detect single nucleotide polymorphisms (SNPs) or insertions or deletions (INDELs). However, its efficiency, accuracy and applicability to discriminate microsatellite polymorphism have not been extensively assessed. The traditional protocols used for SSR genotyping include PCR amplification of the DNA fragment and the separation of the fragments on electrophoresis-based platform. However, post-PCR handling processes are laborious and costly. Furthermore, SNPs present in the sequences flanking repeat motif cannot be detected by polyacrylamide-gel- electrophoresis based methods. In the present study, we compared the discriminating power of HRM with the traditional electrophoresis-based methods and provided a panel of primers for HRM genotyping in Citrus. The results showed that sixteen SSR markers produced distinct polymorphic melting curves among the Citrus spp investigated through HRM analysis. Among those, 10 showed more genotypes by HRM analysis than capillary electrophoresis owing to the presence of SNPs in the amplicons. For the SSR markers without SNPs present in the flanking region, HRM also gave distinct melting curves which detected same genotypes as were shown in capillary electrophoresis (CE) analysis. Moreover, HRM analysis allowed the discrimination of most of the 15 citrus genotypes and the resulting genetic distance analysis clustered them into three main branches. In conclusion, it has been approved that HRM is not only an efficient and cost- effective alternative of electrophoresis-based method for SSR markers, but also a method to uncover more polymorphisms contributed by SNPs present in SSRs. It was therefore suggested that the panel of SSR markers could be used in a variety of applications in the citrus biodiversity and breeding programs using HRM analysis. Furthermore, we speculate that the HRM analysis can be employed to analyse SSR markers in a wide range of applications in all other species. [ABSTRACT FROM AUTHOR]

The success of germination, growth and final yield of every crop depends to a large extent on the quality of the seeds used to grow the crop. Seed quality is defined as the viability and vigor attribute of a seed that enables the emergence and establishment of normal seedlings under a wide range of environments. We attempt to dissect the mechanisms involved in the acquisition of seed quality, through a combined approach of physiology and genetics. To achieve this goal we explored the genetic variation found in a RIL population of Solanum lycopersicum (cv. Moneymaker) x Solanum pimpinellifolium through extensive phenotyping of seed and seedling traits under both normal and nutrient stress conditions and root system architecture (RSA) traits under optimal conditions. We have identified 62 major QTLs on 21 different positions for seed, seedling and RSA traits in this population. We identified QTLs that were common across both conditions, as well as specific to stress conditions. Most of the QTLs identified for seedling traits co-located with seed size and seed weight QTLs and the positive alleles were mostly contributed by the S. lycopersicum parent. Co-location of QTLs for different traits might suggest that the same locus has pleiotropic effects on multiple traits due to a common mechanistic basis. We show that seed weight has a strong effect on seedling vigor and these results are of great importance for the isolation of the corresponding genes and elucidation of the underlying mechanisms. [ABSTRACT FROM AUTHOR]

Biological invasions can cause major ecological and economic impacts. During the early stages of invasions, eradication is desirable but tactics are lacking that are both effective and have minimal non-target effects. Mating disruption, which may meet these criteria, was initially chosen to respond to the incursion of light brown apple moth, Epiphyas postvittana (LBAM; Lepidoptera: Tortricidae), in California. The large size and limited accessibility of the infested area favored aerial application. Moth sex pheromone formulations for potential use in California or elsewhere were tested in a pine forest in New Zealand where LBAM is abundant. Formulations were applied by helicopter at a target rate of 40 g pheromone per ha. Trap catch before and after application was used to assess the efficacy and longevity of formulations, in comparison with plots treated with ground-applied pheromone dispensers and untreated control plots. Traps placed at different heights showed LBAM was abundant in the upper canopy of tall trees, which complicates control attempts. A wax formulation and polyethylene dispensers were most effective and provided trap shut-down near ground level for 10 weeks. Only the wax formulation was effective in the upper canopy. As the pheromone blend contained a behavioral antagonist for LBAM, 'false trail following' could be ruled out as a mechanism explaining trap shutdown. Therefore, 'sensory impairment' and 'masking of females' are the main modes of operation. Mating disruption enhances Allee effects which contribute to negative growth of small populations and, therefore, it is highly suitable for area-wide control and eradication of biological invaders. [ABSTRACT FROM AUTHOR]

Male sterile and seedless characters are highly desired for citrus cultivar improvement. In our breeding program, a male sterile cybrid pummelo, which could be considered as a variant of male fertile pummelo, was produced by protoplast fusion. Herein, ecotopic stamen primordia initiation and development were detected in this male sterile cybrid pummelo. Histological studies revealed that the cybrid showed reduced petal development in size and width, and retarded stamen primordia development. Additionally, disorganized cell proliferation was also detected in stamen-like structures (fused to petals and/or carpel). To gain new insight into the underlying mechanism, we compared, by RNA-Seq analysis, the nuclear gene expression profiles of floral buds of the cybrid with that of fertile pummelo. Gene expression profiles which identified a large number of differentially expressed genes (DEGs) between the two lines were captured at both petal primordia and stamen primordia distinguishable stages. For example, nuclear genes involved in nucleic acid binding and response to hormone synthesis and metabolism, genes required for floral bud identification and expressed in particular floral whorls. Furthermore, in accordance with flower morphology of the cybrid, expression of PISTILLATA (PI) was reduced in stamen-like structures, even though it was restricted to correct floral whorls. Down-regulated expression of APETALA3 (AP3) coincided with that of PI. These finding indicated that, due to their whorl specific effects in flower development, citrus class-B MADS- box genes likely constituted 'perfect targets' for CMS retrograde signaling, and that dysfunctional mitochondria seemed to cause male sterile phenotype in the cybrid pummelo. [ABSTRACT FROM AUTHOR]

The fundamental niche defined by five ions, NO3 -, PO4 3-, K+ , was mapped for Chlorella vulgaris (Trebouxiophyceae) and Peridinium cinctum (Dinophyceae) growth rates and maximum cell densities in batch cultures. A five dimensional ion-mixture experimental design was projected across a total ion concentration gradient of 1 to 30mM to delineate the ion-based, "potential" niche space, defined as the entire n-dimensional hypervolume demarcated by the feasible ranges of the independent factors under consideration. The growth rate-based, fundamental niche volumes overlapped for ca. 94% of the ion mixtures, although the regions of maximal growth rates and cell densities were different for each alga. Both C. vulgaris and P. cinctum exhibited similar positive responses to cations and negative responses to anions. It was determined that total ion concentration for these five ions, from 1 to 30 mM, did not directly affect either growth rate or maximal cell density for either alga, although it did play an interactive role with several ions. This study is the first that we are aware of to attempt the mapping of a multivariate, ion-based, fundamental niche volume. The implications of the experimental design utilized and the potential utility of this type of approach are discussed. [ABSTRACT FROM AUTHOR]+, and Cl- , was mapped for Chlorella vulgaris (Trebouxiophyceae) and Peridinium cinctum (Dinophyceae) growth rates and maximum cell densities in batch cultures. A five dimensional ion-mixture experimental design was projected across a total ion concentration gradient of 1 to 30mM to delineate the ion-based, "potential" niche space, defined as the entire n-dimensional hypervolume demarcated by the feasible ranges of the independent factors under consideration. The growth rate-based, fundamental niche volumes overlapped for ca. 94% of the ion mixtures, although the regions of maximal growth rates and cell densities were different for each alga. Both C. vulgaris and P. cinctum exhibited similar positive responses to cations and negative responses to anions. It was determined that total ion concentration for these five ions, from 1 to 30 mM, did not directly affect either growth rate or maximal cell density for either alga, although it did play an interactive role with several ions. This study is the first that we are aware of to attempt the mapping of a multivariate, ion-based, fundamental niche volume. The implications of the experimental design utilized and the potential utility of this type of approach are discussed. [ABSTRACT FROM AUTHOR]

The root weevil Diaprepes abbreviatus L. has been continuously reared since 1992 on an artificial diet (F1675) first reported in 1982. The weight of weevils reared on F1675 was more than twice as great as field-collected individuals. Recently, several ingredients included in the original F1675 diet were shown to have little or no effect on insect performance. We examined the effects of varying three ingredients of a simplified diet on the weight of adult D. abbreviatus. We used a geometric design combined with response surface models to evaluate the effects of the amount and proportion of diet ingredients to identify optimal diet recipes for normative weight gain defined in relation to field-collected D. abbreviatus from locations in Florida. Our results showed that it is possible to produce adults of normative weight or, for that matter, any mean weight within a wide range by varying the proportions of cottonseed meal, wheat germ and cellulose, a non-nutritive filler. Although wheat germ contributed to greater weight gain, survival of larvae to adult was lower on diets containing only wheat germ compared with diets containing only cottonseed meal as the principal nutritive components. The analyses of all the variables measured indicate that cottonseed meal is the only major nutritive component, in addition to standard vitamin and salt mixes, required to produce adult D. abbreviatus of normative weight. [ABSTRACT FROM AUTHOR]

Abnormal physiological responses of plant cultures such as shoot tip necrosis, callus, and hyperhydricity are some of the most difficult challenges in shoot micropropagation, and their causes are not well understood. Five Murashige and Skoog mineral salt factors, which influence the growth of pear shoot cultures, were tested in a five-dimensional surface response experimental design. Pyrus communis ‘Old Home × Farmingdale 87,’ ‘Horner 51,’ and ‘Winter Nelis’; Pyrus dimorphophylla; and Pyrus ussuriensis ‘Hang Pa Li’ shoot cultures were grown on 43 computer-designed treatments to represent the design space of all possible treatment combinations. Analysis of shoot response to these treatments identified the factors that both contributed to physiological disorders and remedied them. Undesirable callus formation was common for pear shoots cultured on standard medium and decreased on formulations with increased NH 4NO 3, Fe, and mesos (CaCl 2, KH 2PO 4, and MgSO 4) for most genotypes. Shoot tip necrosis varied with the genotype, but low mesos or low nitrogen concentrations contributed to the necrosis. Hyperhydricity was more prominent with low mesos or low NH 4NO 3. Hooked and upwardly curled new leaves were seen in most genotypes and resulted from use of low mesos in P. communis and low nitrogen for ‘Hang Pa Li’ and P. dimorphophylla. Fasciation and hypertrophy were seen infrequently and resulted from wide imbalances in several nutrients simultaneously. In general, standard concentrations of Murashige and Skoog iron and micros combined with high mesos and moderate nitrogen compounds produced normal shoots without physiological disorders. [ABSTRACT FROM AUTHOR]

Seven putative protease inhibitor (PPI) cDNAs, representing four protein families, were isolated from a grapefruit (Citrus paradisi Macf. Cv. Marsh) immature fruit flavedo cDNA library. Cloned open reading frames encoded proteins with similarity to, and protein signatures for: legume Kuntiz inhibitors (lkiL-1, lkiL-2, lkiL-3), potato trypsin inhibitor I (ptiIL-1), serpins (serpL-1), cystatins (cystL-1), and gamma thionins (gthL-1). Response of transcript abundance to fruit development and leaf wounding was determined for all but lkiL-1 using real-time RT-PCR. Immature leaves had the highest transcript levels for all PPIs. The gthL-1 transcript in immature leaves was the most abundant transcript but was absent from healthy mature leaves. In fruit flavedo, transcripts for all PPIs were most abundant in youngest fruit (<15 mm dia. fruit), and declined during development, but displayed different patterns of developmental change. Mechanical or Diaprepes root weevil (DRW) feeding damage to leaves caused a <10-fold reduction or had no effect on transcript level with the exception of gthL-1 which, as a result of damage, increased >50-fold in mature leaves and decreased >1400-fold in immature leaves. This developmental control of transcript response to wounding in a woody perennial is opposite of what has been observed for defensive proteinase inhibitors (PIs) in other plants (typically herbaceous and/or annual plants), where younger leaves typically invoke a higher defensive proteinase inhibitor transcript accumulation than older tissues. Except for gthL-1, the PPI transcripts were minimally responsive or unresponsive to wounding. Changes in PPI transcript levels suggest diverse roles for the products of these genes in citrus, with only gthL-1 responding in a defense-like manner. [Copyright &y& Elsevier]

Testing the function of therapeutic compounds in plants is an important component of agricultural research. Foliar and soil-drench methods are routine but have drawbacks, including variable uptake and the environmental breakdown of tested molecules. Trunk injection of trees is well-established, but most methods for this require expensive, proprietary equipment. To screen various treatments for Huanglongbing, a simple, low-cost method to deliver these compounds to the vascular tissue of small greenhouse-grown citrus trees infected with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) or infested with the phloem-feeding CLas insect vector Diaphorina citri Kuwayama (D. citri) is needed. To meet these screening requirements, a direct plant infusion (DPI) device was designed that connects to the plant's trunk. The device is made using a nylon-based 3D-printing system and easily obtainable auxiliary components. The compound uptake efficacy of this device was tested in citrus plants using the fluorescent marker 5,6-carboxyfluorescein-diacetate. Uniform compound distribution of the marker throughout the plants was routinely observed. Furthermore, this device was used to deliver antimicrobial and insecticidal molecules to determine their effects on CLas and D. citri respectively. The aminoglycoside antibiotic streptomycin was delivered into CLas-infected citrus plants using the device, which resulted in a reduction in the CLas titer from 2 weeks to 4 weeks post treatment. Delivering the neonicotinoid insecticide imidacloprid into D. citri-infested citrus plants resulted in a significant increase in psyllid mortality after 7 days. These results suggest that this DPI device represents a useful system for delivering molecules into plants for testing and facilitate research and screening purposes.

Clustered regularly interspaced short palindromic repeats (CRISPR) technology is a powerful genome editing tool. Recently developed CRISPR-Cas12a system confers several advantages over CRISPR-Cas9, making it ideal for use in plant genome editing and crop improvement. While traditional transformation methods based on plasmid delivery pose concerns associated with transgene integration and off-target effects, CRISPR-Cas12a delivered as ribonucleoproteins (RNPs) can effectively alleviate these potential issues. Here we present a detailed protocol for LbCas12a-mediated genome editing using RNP delivery in Citrus protoplasts. This protocol provides a comprehensive guideline for RNP component preparation, RNP complex assembly and delivery, and editing efficiency assessment., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Anther and microspore culture for producing haploid plants are very complex systems and include general effects where the specific effects must be identified and optimized to develop culture systems capable of producing the large numbers of haploids required by breeding programs. These general effects include genotype, physiological state of the source plant, age of the anthers and microspores, preculture treatments, culture conditions, and culture media. Design of experiments (DoE) is an experimental approach specifically designed to identify and optimize the multiple factors that make up complex systems, and is ideally suited for developing in vitro systems to produce haploids. The basic DoE strategy starts by screening multiple factors thought to affect the responses being measured. Screening identifies factors with large and small effects. Factors with large effects are used to manipulate the system, and are moved to the DoE optimization phase such as response surface methodology. Factors with small or trivial effects are eliminated from further consideration, and this simplifies the system. The basic concepts of fractional factorial designs and how to use them are explained. Fractional factorials are the most important DoE screening tool and are the first experiments run before DoE optimization experiments. To illustrate the unique properties of fractional factorials, a detailed example is provided that includes all of the calculations so that no statistical software is required., (© 2021. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)