Noemi Tel-Zur

Modified CTAB Procedure for DNA Isolation from Epiphytic Cacti of the Genera Hylocereus and Selenicereus (Cactaceae)

We present a simple protocol for DNA isolation from climbing cacti, genera Hylocereus and Selenicereus. The abundant polysaccharides present in Hylocereus and Selenicereus species interfere with DNA isolation, and DNA extracts, rich in polysaccharides, are poor templates for amplification using polymerase chain reaction (PCR). We used roots as the source tissue due to the lower viscosity of the extracts relative to that of other tissues. The extraction and isolation procedure we devised consists of the following steps: (1) three washes of ground tissue with the extraction buffer to remove the polysaccharides; (2) extraction with high-salt (4 M NaCl) cetyltrimethylammonium bromide (CTAB) buffer to remove the remaining polysaccharides; (3) removal of RNA by RNase; (4) phenol:chloroform extraction to remove proteins; (5) chloroform extraction to remove remaining phenols. The yields ranged from 10 to 20 μg DNA/g fresh roots. DNA samples prepared by our method were consistently amplifiable in the RAPD reaction and gave reproducible profiles.

Cytology and mating systems in the climbing cacti Hylocereus and Selenicereus

Chromosome numbers and meiotic behavior are reported for the climbing cacti species Hylocereus undatus, Hylocereus polyrhizus, and Selenicereus megalanthus. The Hylocereus spp. are diploid (2n = 22), while S. megalanthus is a tetraploid (2n = 44). Irregular chromosome disjunction at anaphase I in pollen mother cells of S. megalanthus is probably the major cause of its reduced pollen viability and may contribute to low seed set, low number of viable seeds and, consequently, low fruit mass. A pollination study confirmed self-incompatibility in H. polyrhizus and a weakened incompatibility reaction in H. undatus and S. megalanthus. Major crossability barriers do not exist between the Hylocereus spp. investigated. Reciprocal intergeneric crosses were successful between Hylocereus spp. and S. megalanthus, suggesting that an Hylocereus sp. might be one of the diploid progenitors of the tetraploid S. megalanthus. The implications of the results on cacti nomenclature and systematics are briefly discussed.

Morphological, cytological and metabolic consequences of autopolyploidization in Hylocereus (Cactaceae) species.

BackgroundGenome doubling may have multi-level effects on the morphology, viability and physiology of polyploids compared to diploids. We studied the changes associated with autopolyploidization in two systems of somatic newly induced polyploids, diploid-autotetraploid and triploid-autohexaploid, belonging to the genus Hylocereus (Cactaceae). Stomata, fruits, seeds, embryos, and pollen were studied. Fruit pulp and seeds were subjected to metabolite profiling using established gas chromatography-mass spectrometry (GC-MS) and ultra-performance liquid chromatography (UPLC) Q-TOF-MS/MS (time of flight)-protocols.ResultsAutopolyploid lines produced lower numbers of tetrads, larger pollen grains with lower viability, larger stomata with lower density, and smaller fruits with lower seed numbers and decreased seed viability. The abundance of sugars was lower in the fruits and seeds of the two duplicated lines than in their donor lines, accompanied by increased contents of amino acids, tricarboxylic acid (TCA) cycle intermediates, organic acids and flavonoids. Betacyanins, the major fruit pigments in diploid and triploid donors, decreased following genome doubling. Both autopolyploid Hylocereus lines thus exhibited unfavorable changes, with the outcome being more dramatic in the autohexaploid than in the autotetraploid line.ConclusionInduced autotetraploid and autohexaploid lines exhibited morphological and cytological characteristics that differed from those of their donor plants and that were accompanied by significant metabolic alterations. It is suggested that a developmental arrest occurs in the fruits of the autohexaploid line, since their pericarp shows a greater abundance of acids and of reduced sugars. We conclude that genome doubling does not necessarily confer a fitness advantage and that the extent of alterations induced by autopolyploidization depends on the genetic background of the donor genotype.

Growth Platform-Dependent and -Independent Phenotypic and Metabolic Responses of Arabidopsis and Its Halophytic Relative, Eutrema salsugineum , to Salt Stress

Eutrema salsugineum maintains its salt tolerance under very different growth conditions even though its development and metabolism show substantial growth condition-dependent differences. Comparative studies of the stress-tolerant Arabidopsis (Arabidopsis thaliana) halophytic relative, Eutrema salsugineum, have proven a fruitful approach to understanding natural stress tolerance. Here, we performed comparative phenotyping of Arabidopsis and E. salsugineum vegetative development under control and salt-stress conditions, and then compared the metabolic responses of the two species on different growth platforms in a defined leaf developmental stage. Our results reveal both growth platform-dependent and -independent phenotypes and metabolic responses. Leaf emergence was affected in a similar way in both species grown in vitro but the effects observed in Arabidopsis occurred at higher salt concentrations in E. salsugineum. No differences in leaf emergence were observed on soil. A new effect of a salt-mediated reduction in E. salsugineum leaf area was unmasked. On soil, leaf area reduction in E. salsugineum was mainly due to a fall in cell number, whereas both cell number and cell size contributed to the decrease in Arabidopsis leaf area. Common growth platform-independent leaf metabolic signatures such as high raffinose and malate, and low fumarate contents that could reflect core stress tolerance mechanisms, as well as growth platform-dependent metabolic responses were identified. In particular, the in vitro growth platform led to repression of accumulation of many metabolites including sugars, sugar phosphates, and amino acids in E. salsugineum compared with the soil system where these same metabolites accumulated to higher levels in E. salsugineum than in Arabidopsis. The observation that E. salsugineum maintains salt tolerance despite growth platform-specific phenotypes and metabolic responses suggests a considerable degree of phenotypic and metabolic adaptive plasticity in this extremophile.

Floral biology of Ziziphus mauritiana (Rhamnaceae)

Floral development of the synchronous dichogamous species Ziziphus mauritiana, as followed by light and scanning electron microscopy (SEM), was divided into 11 stages using a series of landmark events. Main cellular events happen synchronously in the female and the male structures, such as meiosis in micro- and macrosporocyte cells, tetrad microspore formation and appearance of the functional megaspore cell, and onset of embryo sac differentiation coinciding with mitosis in the microspores. The last stage was characterized by anthesis and continued development of the flower, beginning with anther dehiscence (male phase) and proceeding to the female phase, which was characterized by style elongation. Flowers exhibit synchronous protandrous dichogamy; anthesis takes place in the morning (group A, e.g., clone Q-29) and afternoon (group B, e.g., clone B5/4). Stigma receptivity started after the male phase and occurred synchronously and complementarily with pollen dispersal in the two clones. Pollen viability and production were similar in the two clones, but the pollen diameter of Q-29 was significantly larger than that of B5/4. This study provides the basis for understanding the biological mechanisms regulating floral development, thus expanding the prospects for Z. mauritiana breeding programs and for further molecular and genetic studies of this species.

Testing a Hypothesis of Intergeneric Allopolyploidy in Vine Cacti (Cactaceae: Hylocereeae)

Abstract Allopolyploidy is common in angiosperms, but only rarely involves different genera. One hypothesized case of intergeneric allopolyploidy is Hylocereus megalanthus, a member of Cactaceae tribe Hylocereeae, a group of vine cactus species, some of which are known for their edible fruits (“pitahaya” or “dragon fruit”). This polyploid species has been interpreted as morphologically intermediate between Hylocereus and Selenicereus. Plastid and nuclear ribosomal DNA ITS sequences from all H. megalanthus individuals sampled are either identical (plastid), or form a monophyletic clade despite considerable intraindividual polymorphism (ITS). Plastid and ITS phylogenies both show H. megalanthus nested within a well-supported Hylocereus, which in turn is nested within a paraphyletic Selenicereus. The absence of more than one lineage of ITS in H. megalanthus is consistent with autopolyploidy, but could be due to inter-homoeologue concerted evolution. Numerous low-copy nuclear genes were tested for utility in these vine cacti, and two (phyC and Vatp1) were sampled from H. megalanthus and a subset of Hylocereus and Selenicereus species. In both cases, H. megalanthus haplotypes were more closely related to each other than to other Hylocereus or Selenicereus haplotypes. Thus, we found no evidence for allopolyploidy, let alone intergeneric allopolyploidy, in H. megalanthus.

Morphological changes and self-incompatibility breakdown associated with autopolyploidization in Hylocereus species (Cactaceae)

Chromosome duplication—autopolyploidization—may affect plant morphology and breeding systems, ultimately enabling the production of improved genotypes. In this study, the autotetraploid lines obtained from the self-incompatible diploid Hylocereus monacanthus and the autooctapolyploid lines obtained from the self-compatible tetraploid H. megalanthus were studied and compared with the donor accessions. The resulting H. monacanthus autotetraploids exhibit lower fruit weight, seed number, and pollen viability than the donor plant, but it has larger pollen grains. Although the resulting H. megalanthus autooctaploids had larger pollen grains and lower pollen viability compared with the donor plant, only aborted fruits were obtained from these lines. The most valuable change observed was the breakdown of the self-incompatibility system in the H. monacanthus autotetraploid lines. This research provides important information on the horticultural value of vine cacti autopolyploid lines.

Anastatica hierochuntica, an Arabidopsis Desert Relative, Is Tolerant to Multiple Abiotic Stresses and Exhibits Species-Specific and Common Stress Tolerance Strategies with Its Halophytic Relative, Eutrema (Thellungiella) salsugineum

The search for novel stress tolerance determinants has led to increasing interest in plants native to extreme environments – so called “extremophytes.” One successful strategy has been comparative studies between Arabidopsis thaliana and extremophyte Brassicaceae relatives such as the halophyte Eutrema salsugineum located in areas including cold, salty coastal regions of China. Here, we investigate stress tolerance in the desert species, Anastatica hierochuntica (True Rose of Jericho), a member of the poorly investigated lineage III Brassicaceae. We show that A. hierochuntica has a genome approximately 4.5-fold larger than Arabidopsis, divided into 22 diploid chromosomes, and demonstrate that A. hierochuntica exhibits tolerance to heat, low N and salt stresses that are characteristic of its habitat. Taking salt tolerance as a case study, we show that A. hierochuntica shares common salt tolerance mechanisms with E. salsugineum such as tight control of shoot Na+ accumulation and resilient photochemistry features. Furthermore, metabolic profiling of E. salsugineum and A. hierochuntica shoots demonstrates that the extremophytes exhibit both species-specific and common metabolic strategies to cope with salt stress including constitutive up-regulation (under control and salt stress conditions) of ascorbate and dehydroascorbate, two metabolites involved in ROS scavenging. Accordingly, A. hierochuntica displays tolerance to methyl viologen-induced oxidative stress suggesting that a highly active antioxidant system is essential to cope with multiple abiotic stresses. We suggest that A. hierochuntica presents an excellent extremophyte Arabidopsis relative model system for understanding plant survival in harsh desert conditions.

Public Knowledge and Perceptions of Safety Issues Towards the Use of Genetically Modified Forest Trees: A Cross-Country Pilot Survey

Information on public awareness and acceptance issues regarding the use of Genetically Modified (GM) trees in forestry is lacking, although such information is available for GM organisms in agriculture. This is mainly due to the fact that in Europe there is no authorization for commercial planting of GM forest trees. To address this issue and within the frame of a European COST Action on the Biosafety of Transgenic Forest Trees (FP0905), a KAP (Knowledge Attitude Practice ) cross-country pilot survey was conducted among university students of different disciplines as sampling subjects. In total, 1920 completed questionnaires from 16 European and non-European countries were evaluated. The results provided novel cross-country insights into the level of public knowledge, particularly of young people and their perceptions on safety issues related to the use of GM forest trees , as well as on their attitude towards the acceptance of GM forest trees cultivation. The majority of the respondents, which was more than 60 % in all countries, approved the use of GM forest trees for commercial plantations , excluding natural forests. The majority of respondents also appeared willing to buy products from such plantations, such as wood products, pulp and paper. Over 80 % of the respondents from all countries were in favour of using labelling to identify products of GM origin, while more than 80 % of those would prefer that this labelling be legally mandatory. The top three benefits that were rated as very important in all countries involved the potential lower demand of the GM forest plantations for pesticides, the potential of GM forest trees for restoration of contaminated soils and the potential higher GM forest tree productivity. The top three GM forest tree risks that were perceived as serious hazards in all countries included the potential loss of biodiversity due to gene flow between transgenic and wild trees, the adverse effects of biotrophic processes on host ecosystems and the cultural adaptation to changing biodiversity conditions due to transgene escape. Overall, lack of knowledge regarding the potential benefits and potential risks of the cultivation of GM forest trees was observed in almost all surveyed countries.

Intergeneric hybridization within the tribe Hylocereeae, subfamily Cactoideae (Cactaceae)

Controlled hand-cross-pollinations were performed among the genera Hylocereus, Selenicereus, and Epiphyllum and synthetic intergeneric hybrids were obtained. Parental lines and the intergeneric hybrids were studied for their nuclear DNA (2C) content and for their morphological and phenological characteristics. Hybrids between Hylocereus and Selenicereus species produced viable seeds, a finding that showed the lack of post-zygote barriers. Partial to complete sterility was observed in the intergeneric Epiphyllum x Selenicereus hybrids. The dominant relationship between the alleles controlling morphological and phenological traits was elucidated. Among the traits studied, the presence of spines and/or hairy-spines on the fruit peel, the co-dominance of the ripening time, and the dominance of the high number of shoot ribs were the most significant characteristics proving hybridization. Further development of species-specific molecular markers will be valuable for a suitable taxonomic description of these spe...