Tissue culture-induced DNA methylation polymorphisms in repetitive DNA of tomato calli and regenerated plants

The propagation of plants through tissue culture can induce a variety of genetic and epigenetic changes. Variation in DNA methylation has been proposed as a mechanism that may explain at least a part of these changes. In the present study, the methylation of tomato callus DNA was compared with that of leaf DNA, from control or regenerated plants, at MspI/HpaII sites around five middle-repetitive sequences. Although the methylation of the internal cytosine in the recognition sequence CCGG varied from zero to nearly full methylation, depending on the probe used, no differences were found between callus and leaf DNA. For the external cytosine, small differences were revealed between leaf and callus DNA with two probes, but no polymorphisms were detected among DNA samples of calli or DNA samples of leaves of regenerated plants. When callus DNA cut with HindIII was studied with one of the probes, H9D9, most of the signal was found in high-molecular-weight DNA, as opposed to control leaf DNA where almost all the signal was in a fragment of 530 bp. Also, an extra fragment of 630 bp was found in the callus DNA that was not present in control leaf DNA. Among leaves of plants regenerated from tissue culture, the 630-bp fragment was found in 10 of 68 regenerated plants. This 630-bp fragment was present among progeny of only 4 of these 10 plants after selfing, i.e. it was partly inherited. In these cases, the fragment was not found in all progeny plants, indicating heterozygosity of the regenerated plants. The data are interpreted as indicating that a HindIII site becomes methylated in callus tissue, and that some of this methylation persists in regenerated plants and is partly transmitted to their progeny.     

Genetic and epigenetic stability of cryopreserved and cold-stored hops (Humulus lupulus L.)

Conventional cold storage and cryopreservation methods for hops (Humulus lupulus L.) are available but, to our knowledge, the genetic and epigenetic stability of the recovered plants have not been tested. This study analyzed 51 accessions of hop using the molecular techniques, Random Amplified DNA Polymorphism (RAPD) and Amplified Fragment Length Polymorphism (AFLP), revealing no genetic variation among greenhouse-grown controls and cold stored or cryopreserved plants. Epigenetic stability was evaluated using Methylation Sensitive Amplified Polymorphism (MSAP). Over 36% of the loci were polymorphic when the cold and cryo-treated plants were compared to greenhouse plants. The main changes were demethylation events and they were common to the cryopreserved and cold stored plants indicating the possible effect of the in vitro establishment process, an essential step in both protocols. Protocol-specific methylation patterns were also detected indicating that both methods produced epigenetic changes in plants following cold storage and cryopreservation.     

Assessment of somaclonal variation in <Emphasis Type="Italic">Dieffenbachia</Emphasis> plants regenerated through indirect shoot organogenesis

The occurrence of somaclonal variation among regenerants derived through indirect shoot organogenesis from leaf explants of three Dieffenbachia cultivars Camouflage, Camille and Star Bright was evaluated. Three types of somaclonal variants (SV1, SV2, and SV3) were identified from regenerated plants of cv. Camouflage, one type from cv. Camille, but none from cv. Star Bright. The three variants had novel and distinct foliar variegation patterns compared to cv. Camouflage parental plants. Additionally, SV1 was taller with a larger canopy and longer leaves than parental plants and SV2. SV2 and SV3 did not produce basal shoots (single stem) but basal shoot numbers between SV1 and parental plants were similar ranging from three to four. The variant type identified from regenerated cv. Camille had lanceolate leaves compared to the oblong leaves of the parent. This variant type also grew taller and had a larger canopy than parental plants. The rates of somaclonal variation were up to 40.4% among regenerated cv. Camouflage plants and 2.6% for regenerated cv. Camille. The duration of callus culture had no effect on somaclonal variation rates of cv. Camouflage as the rates between plants regenerated from 8 months to 16 months of callus culture were similar. The phenotypes of the identified variants were stable as verified by their progenies after cutting propagation. This study demonstrated the potential for new cultivar development by selecting callus-derived somaclonal variants of Dieffenbachia.     

Mitochondrial DMA variation in plants regenerated from embryogenic callus cultures of CMS triticale

The mitochondrial DNA (mtDNA) organization of primary hexaploid cytoplasmic male-sterile (CMS) triticale regenerants containing Triticum timopheevi cytoplasm was analysed by hybridization experiments and compared with the mitochondrial genome organization of the corresponding regenerants with maintainer cytoplasm. Callus cultures had been derived from immature embryos, and 623 triticale plants were regenerated via somatic embryogenesis after three to four subcultures. The chondriome of 159 regenerants was investigated with regard to somaclonal variation. Six different mitochondrial gene probes and four different restriction enzymes were used for Southern blot analyses by the non-radioactive digoxigenin labeling technique. Alloplasmic regenerants showed a gain or loss of hybridization signals up to a high percentage, while euplasmic ones revealed only minor variability with respect to band stoichiometries. In 24 cases rearrangements in the mtDNA were proved. We suppose that recombination processes and selective amplification events are responsible for these findings.     

Assessment of genetic and epigenetic changes following cryopreservation in papaya

A vitrification based cryopreservation technique for storage of in vitro shoot tips of papaya has been tested to ensure applicability across a range of genotypes and to assess the stability of both genotype and phenotype of such clonal material following cryopreservation. Shoot tips of 12 genotypes were cryopreserved, recovery rates were determined and resultant plants were screened for genetic and epigenetic changes. Genomic DNA structure was explored using polymerase chain reaction (PCR) based randomly amplified DNA fingerprinting (RAF), and methylation patterns were monitored using the amplified DNA methylation polymorphism (AMP) PCR technique. Plantlets were recovered following cryopreservation in all but one genotype and recovery rates of 61-73% were obtained from six genotypes. The regenerated plantlets showed varying levels of genomic DNA modifications (0-10.07%), and methylation modifications (0.52-6.62%) of detected markers. These findings have not been reported previously for papaya, and indicate some genotype dependent variability in DNA modifications occur following cryopreservation which may result in somaclonal variation.     

Meiotic and isozymic characterization of plants regenerated from euploid and selfed monosomic tall fescue embryos

Summary Tissue culture of tall fescue (Festuca arundinacea Schreb., 2n=6x=42) would be enhanced by improving the callus induction and plant regeneration efficiency, and evaluating the meiotic and isozymic variation induced by culture. Mature embryos were cultured from four lines of Kenhy tall fescue and from the progeny of three selfed monosomics. Evaluation of six media-auxin combinations showed callus initiation was greatest on SH medium with 2.5 mg/l 2,4,5-T or 7.4 mg/l pCPA, while plant regeneration was greatest on SH medium with 0.5 mg/l 2,4-D. Cytological analyses of 27 plants derived from euploid parents showed a high frequency of aneuploidy (15/27). Chromosome numbers of aneuploids ranged from 36 to 41, with one plant having 80 chromosomes and two plants being asynaptic. Two of ten monosomic-derived plants were euploid, five were monosomic, one was monosomic with a fragment and two were double monosomic. Zymograms of the parents and regenerants were obtained for the enzymes ACPH, ADH, GOT, 6-PGD and PGI. Isozyme variation was observed for two groups of plants derived from the same Kenhy embryos. One group of four monosomic-derived plants differed for the enzymes GOT and ACPH, and all four plants had a PGI pattern. different from that of the parental monosomic plant. This indicated loss of a PGI allele, probably as a result of callus culture.Contribution No. 89-3-141 of the Kentucky Agricultural Experiment Station in cooperation with the USDA-ARS. Part of thesis research for senior author's M. S. degree     

Phenotypic and karyotypic status of Beta vulgaris plants regenerated from direct organogenesis in petiole culture

Summary A method for high frequency in vitro regeneration from petiole explants was tested on nine breeding lines of Beta vulgaris L. from the haploid, diploid and tetraploid levels. Regenerants could be obtained without a callus step, from excised petioles derived either from axillary buds sprouted in vitro or from field grown plants, by plating the explants on MS medium supplemented with TIBA (2,3,5-triiodobenzoïc acid) and BAP (6-Benzylaminopurine). The multiple shoots obtained were then rooted in vitro and transferred to soil. In some cases, these adventitious shoots were also used as a petiole explant source for further petiole culture cycles, and the phenotypic characteristics and ploidy status of the regenerants were investigated after one or three petiole culture cycles. Conventional shoot apex culture was used as an in vitro control. Phenotypic variations such as differences in morphology and changes in in vitro growth behaviour, were noticed. Chloroplast and chromosome counts indicated that the alterations in morphogenetic pathway could not be explained by the occurrence of gross cytogenetic abnormalities such as aneuploidy or myxoploidy. Our results suggest that the altered morphology is caused by the presence of the exogenous antiauxin (TIBA) during the in vitro phase. Following transfer to the greenhouse, none of these variations persisted and cytogenetic analyses revealed karyotypic stability in all the plants studied, even after three petiole culture cycles. An assessment of the in vitro petiole culture method as a true-to-type multiplication method for Beta vulgaris is made.     

Structure and inheritance of ribosomal DNA variants in cultivated and wild hop, Humulus lupulus L.

Genetic variation was assessed among cultivated and wild hop, Humulus lupulus, by restriction fragment length polymorphisms (RFLPs) of the ribosomal RNA genes (rDNA). Two rDNA length variants of 10.3 and 9.3 kbp represented by three phenotypes designated A, B and C were detected with XhoI. Restriction-site mapping showed that hop rDNA is structurally similar to those of most higher plants. A high level of homogeneity existed in rDNA repeat lengths among the diverse hop genotypes. Generally, phenotype A was predominant in wild and cultivated European and Asian genotypes; phenotype B in North American cultivars; while phenotype C was present only in native North American hop, providing a potential molecular marker for the identification of this germ plasm. The rDNA data provided genetic evidence for the separation of native and cultivated American genotypes and supports the hypothesis that North American hop cultivars are of hybrid origin from European and native American genotypes. The segregation of rDNA phenotypes in four F₁ families suggests that a single locus with two co-dominant alleles controls genetic variability for rDNA variants in hop.     

NaCl-tolerant plants of Poncirus trifoliata regenerated from tolerant cell lines

Summary Salt-tolerant cell lines of citrus rootstock (Poncirus trifoliata cv Pomeroy) were selected by subculturing embryo-derived calli on media containing sublethal concentrations of NaCl (5 and 10 g/l). Selected lines showed a normal growth in the presence of salt at the concentrations used for selection, and salt tolerance persisted after a passage on a salt-free media. Their K⁺ and Ca²⁺ content remained higher than in control cells for increasing NaCl concentration in the medium, suggesting a modification of cell membrane permeability as the main cause of NaCl tolerance. Shoots and plants regenerated from selected cell lines showed improved growth and salt tolerance. Calli induced from these plants tolerated a salt concentration of 10 g/l, indicating the persistance of the selected trait.     

Somaclonal variation of the mitochondrial ATPase subunit 6 gene region in regenerated triticale shoots and full-grown plants

Comparative hybridization analyses of total DNA from fertile and cytoplasmic male-sterile (CMS) triticale plants which had been regenerated from embryogenic callus cultures revealed the organization and variation of the mitochondrial atp6 gene region. In order to compare different developmental phases, we analysed mitochondrial DNA (mtDNA) from both the shoots and full-grown regenerants. Somaclonal variants were identified on the basis of differences in the mtDNA from fertile and CMS triticale. Several shoots as well as all of the full-grown plants analysed showed somaclonal variation. This phenomenon could be traced back to having primarily orginated from the influence of the nuclear background, which give rise to a stoichiometric increase in a rye-specific orf25 gene copy, and a tissue culture-induced combination of fertile and CMS-specific mtDNA organization of the atp6 gene area. The latter event is probably caused by the homologous recombination of repetitive sequences that may be accompanied by selective amplifications.     

Field performance of transgenic potato plants compared with controls regenerated from tuber discs and shoot cuttings

Summary The objective of this study was to separate and determine effects on the field performance of transgenic potatoes that originate from the tissue culture process of transformation and from the genes inserted. The constructs introduced contained the reporter gene for betaglucuronidase (GUS) under the control of the patatin promoter (four different constructs) and the neomycin phosphotransferase gene under the control of the nopaline synthase promoter. Both genes might be expected to have a neutral effect on plant phenotype. The field performance of transgenic plants (70 independent transformants) was compared with non-transgenic plants regenerated from tuber discs by adventitious shoot formation and from shoot cultures established from tuber nodal cuttings. Plants from all three treatments were grown in a field trial from previously field-grown tubers, and plant performance was measured in terms of plant height at flowering, weight of tubers, number of tubers, weight of large tubers and number of large tubers. There was evidence of somaclonal variation among the transgenic plants; mean values for all characters were significantly lower and variances generally higher than from plants derived from nodal shoot cultures. A similar change in means and variances was observed for the non-transgenic tuber-disc regenerants when compared with shoot culture plants. Plant height, tuber weight and tuber number were, however, significantly lower in transgenic plants than in tuber-disc regenerants, suggesting an effect on plant performance either of the tissue culture process used for transformation or of the genes inserted. There were significant differences between constructs for all five plant characters. The construct with the smallest segment of patatin promoter and the lowest level of tuber specificity for GUS expression had the lowest values for all five characters. It is proposed that the nature of GUS expression is influencing plant performance. There was no indication that the NPTII gene, used widely in plant transformation, has any substantial effect on plant performance in the field.     

Initiation and growth characterization of some hop cell suspension cultures

Cell suspension cultures of some hop (Humulus lupulus L.) cultivars were initiated from corresponding callus cultures induced on different media. Dissimilation curves were determined to characterize the growth of the suspension cultures maintained in Gamborg's B5 and in Murashige and Skoog's medium. Both media proved to be suitable; a comparison of the curves obtained for suspension cultures of the hop cultivar Wye Northdown grown in both media did not reveal striking differences. For four hop cell lines, the concentration of nitrate and sugar in the culture medium was analysed by HPLC during the growth of their suspension cultures. The cell suspension cultures of the various hop cultivars were also screened for the presence of bitter acids by HPLC and of volatile compounds by capillary GC. However, neither bitter acids nor volatile compounds could be detected; the addition of a non-toxic lipophylic phase (XAD-2, XAD-1180 or Miglyol 812) to the culture media did not help to induce the formation of volatile compounds.This paper will also appear as a chapter of the Ph.D. thesis of the first author; this thesis will be distributed among colleagues only.     

Development of a shoot multiplication system for hop (Humulus lupulus L.)

Summary Nodal explants from hop were exposed to plant growth regulators to determine suitable media for initiation from axillary buds and subsequent micropropagation. Efficient culture establishment (96.6% of explants) was achieved on Murashige and Skoog (MS) medium (modified to contain 1 mg l⁻¹ thiamine hydrochloride) supplemented with 0.57 μM indoleacetic acid (IAA) and 2.22 μM 6-benzylaminopurine (BA). Subsequent transfer of explants to treatments containing an auxin ([1-naphthaleneacetic acid], NAA or IAA) and BA, 6-[γ,γ-dimethylallylamino]purine (2iP), kinetin (KIN) or thidiazuron (N-phenyl-N′-1,2,3-thidiazol-5-ylurea [TDZ]) resulted in significantly different amounts of multiplication. Optimal TDZ-supplemented media elicited a greater than threefold increase in the number of shoots and nodes generated per explant compared to optimal media containing BA, 2iP and KIN. Shoots were successfully rooted using half-strength MS supplemented with 5.71 μM IAA and 4.9 μM indolebutyric acid (IBA), and regenerated plants were successfully transferred to soi. An overall micropropagation schedule, which can be implemented into hop commercialization programs, includes: (i) establishment in MS with 0.57 μM IAA and 2.22 μM BA; (ii) multiplication in MS with 0.57 μM IAA and 2.27 μM TDZ; (iii) elongation in MS; and (iv) rooting in half-strength MS with 5.71 μM IAA and 4.9 μM IBA.     

Trinucleotide microsatellite repeat is tightly linked to male sex in hop (<Emphasis Type="Italic">Humulus lupulus</Emphasis> L.)

Hop is a dioecious perennial, the female plants of which are cultivated for production of resin, used mainly in the brewing industry. Sex determination of hop plants is phenotypically determined in the second year of growth, so early sex determination at seedling stage, before planting, is important for breeding and cultivation purposes. We analyzed a microsatellite locus to determine sex specific differences in hop. Alleles of the locus showed tight linkage to male character, since no cross-over event was detected in two analyzed families with 181 progenies. The complete heterozygosity in 50 analyzed diverse males indicates a potential use of this marker for any desired parental pair. The marker is amplified in homologous chromosomes, which rules out incorrect determination of non-amplified samples, as can be the case with the dominant (presence/absence) type of markers. The described microsatellite locus is also highly variable, with 35 sized alleles, the distribution of which in different hop germplasms and their sequence variability are discussed. The sex specific marker determined in our study can contribute to studies of sex determination mechanisms and can be readily used in hop breeding and cultivation.     

Somaclonal variation in rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.) analyzed using polymorphic and sequenced AFLP markers

Amplified fragment length polymorphism (AFLP) analysis of 24 in vitro regenerated rye plants was performed in order to evaluate the somaclonal variation rate in this species and to identify rye genomic regions where mutations are preferentially promoted by in vitro culture processes. Regenerated plants were obtained from cell lines derived from immature embryos and plants were regenerated by somatic embryogenesis. Twenty-three regenerants showed variation when compared against sibling plants obtained from the same cell line. A total number of 887 AFLP markers were scored, and 8.8% identified the same polymorphism in plants obtained independently from different cell lines, revealing putative mutational hot spots. Using controlled crossings and analysis of the corresponding progenies, we were able to verify the genetic stability in the next generation for only five of these polymorphisms. The nucleotide sequence of the AFLP amplicon of four of the polymorphic markers was obtained, but only the sequence of two markers was clearly identified in the databases. The sequence of marker A1-303 was identified as part of a tandemly repeated sequence, the 120-bp family, which is located at telomeric regions and is widely distributed among rye chromosomes. The marker A5-375 showed high similarity with regions of Angela retrotransposons.     

Culture-induced variation in plants of Coffea arabica cv. caturra rojo,regenerated by direct and indirect somatic embryogenesis

Amplified fragment-length polymorphism (AFLP) was used to evaluate the stability of DNA in regenerated plantlets of Coffea arabica obtained by direct (DSE) and indirect somatic embryogenesis (ISE). Cluster analysis using the unweighted pair-group method (UPGMA), showed no specific grouping pattern related to the type of embryogenesis. These results suggest that the somatic embryogenesis (SE) process has a mechanism for the selection of normal and competent cells. Bulked DNA from regenerated plants obtained by DSE and ISE, and from the mother plants, was used to characterize specific AFLP fragments associated with each SE process. Twenty-three primer combinations were tested. A total of 1446 bands were analyzed, with 11.4% being polymorphic and 84% being specific for regenerated plants. Furthermore, specific bands were detected for DSE, ISE, and the mother plants. These results indicate that the SE process induces rearrangements at the DNA level and demonstrates discrepancies between the mechanisms involved in each SE process. Coffea arabica breeding programs that involve DSE and ISE can use AFLP as an additional tool for assessing DNA stability.     

Determination of genetic stability using isozymes and RFLPs in beet plants regenerated in vitro

Summary Sugar, fodder and garden beet (Beta vulgaris L.) plants have been regenerated in culture from a range of expiant material. Of the regenerants 764 were subjected to isozyme analysis using eight enzyme-specific stains, and 60 were subjected to RFLP analysis using three cDNA probes. Both molecular techniques allowed the identification of somaclonal variant plants. Assessment of the numbers of variant isozymes and restriction fragments has allowed the calculation of the approximate percentage of variant alleles occurring in any one somaclonal regenerant, namely between 0.05% and 0.1%.     

Susceptibility of hop to Phorodon humuli

A portion of the USDA World Hop (Humulus lupulus L.) Germ Plasm Collection was screened for antibiosis to the hop aphid, Phorodon humuli (Shrank). Hop aphid development and reproduction on the retained accessions 58016, 21090M, 21211, 21240, and 60038 were then compared to hop aphids reared on the commercial hop cultivars (cvs) L-8, Perle, Chinook, Galena, Eroica, Willamette, and Cascade. In general, hop aphids matured faster and reproduced more on the commercial cvs than on 21090M, 58016, 21211, or 21240. Accession 60038, of note because it supported unusually high numbers of aphids during screening, was found to be the most susceptible of any hop tested. Mortality and natality schedules were then combined to construct matrix models of aphid population growth on 60038, 58016, Cascade, and Perle (the most and least susceptible noncommercial and commercial hops, respectively). Beginning with one one-day-old nymph per leaf, the models predicted that P. humuli would require 24 days to surpass an action threshold of 100 aphids per leaf on 58016. This was seven days longer than on the susceptible Cascade and five days longer than on Perle. A commercial hop with the antibiosis of 58016 would probably require fewer insecticide applications during the growing season, thus retarding the development of insecticide resistance in P. humuli and enhancing the effectiveness of integrated management programs by protecting beneficial insects.

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Zusammenfassung Ein Teil der Genbank des USDA von Hopfen (Humulus lupulus L.) wurde auf Resistenz gegen Phorodon humuli (Shrank) getestet. Dabei wurden die besonders widerstands fähigen Klone 58016, 21090M, 21211, 21240 sowie Klon 60038, der ungewöhnlich viele Blattläuse ertrug, ausgewählt. Anschließend wurde die Entwicklung und Vermehrung der Blattläuse auf den ausgewählten Klonen und auf den Handelssorten (cvs) L-8, Perle, Chinook, Galena, Eroica, Willamette, Cascade untersucht. Im Allgemeinen entwickelten sich die Blattläuse auf den Handelssorten reascher und vermehrten sich stärker als auf den resistenten Klonen. Klon 60038 war dagegen die anfälligste Sorte. Mortalitäts- und Natlitätsdaten wurden kombiniert um Matrixmodelle des Populationswachstums der Blattläuse auf 60038, 58016, Cascade und Perle (die am meisten und am wenigsten anfälligen kommerziellen und nichtkommerziellen Sorten) zu konstruieren. Die Modelle begannen bei 1 Larve pro Blatt und sagten voraus, dass die Aktionsgrenze von 100 Tieren pro Blatt auf 58016 nach 24 Tagen erreicht wird. Das ist 7 Tage später als auf den anfälligen Sorten Cascade und 5 Tage später als auf Perle. Eine kommerzielle Sorte mit der Anfälligkeit von 58016 würde vermutlich während der Vegetationszeit weniger Insektizidspritzungen benötigen. Damit würde die Resistenzentwicklung bei Phorodon humuli verlangsamt und die Wirkung von IPM Programmen durch Schutz der natürlichen Feinde verbessert.

     

Detection of epigenetic variation in tissue-culture-derived plants of <Emphasis Type="Italic">Doritaenopsis</Emphasis> by methylation-sensitive amplification polymorphism (MSAP) analysis

Somaclones exhibiting variations with flower characteristics were recovered from the tissue-culture-derived plants of Doritaenopsis. Two molecular techniques, random amplified polymorphic DNA (RAPD) and methylation-sensitive amplification polymorphism (MSAP) analyses, were used to characterize the somaclones. RAPD analysis, using 100 randomly selected primers, failed to differentiate variants and normal plants, even though some primers (six out of 100 primers) exhibited 6–10 distinct banding patterns. However, MSAP analysis revealed the differences in the DNA methylation patterns in the normal and variant plants which were correlated with phenotypic variation. In all, 311, 337, 366, and 343 fragments were obtained with normal and V1, V2, and V3 variant plants, respectively; each representing recognition site cleaved by either or both of the isoshizomers were amplified using 12 combination of primers. A total of 36 (11.6%), 77 (22.9%), 73 (19.9%), and 47 (13.7%) sites were found to be methylated at cytosine in the genomes of normal and V1, V2, and V3 variant Doritaenopsis plants. This study demonstrates usefulness of MSAP to detect DNA methylation events in tissue cultured Doritaenopsis plants.