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.     

B-Chromosomes and E-1 isozyme activity in mosaic bulbs of Scilla autumnalis (Liliaceae)

The electrophoretic patterns of esterase (E-1), alcohol dehydrogenase (ADH), and glutamate oxaloacetate transaminase (GOT) isozymes were studied in two Spanish populations of the lily Scilla autumnalis with B-chromosome carrying individuals. The E-1 isozyme activity appears only in those individuals with B-chromosomes. None of the bulbs free of B's show it. Five bulbs, mosaic for B-content, were identified. Electrophoretic analysis shows that these bulbs are characterised by mosaicism for E-1 isozyme activity. An analysis of individual roots by both electrophoretic and cytological methods shows that tissue mosaicism for B-content correlates with tissue mosaicism for E-1 isozyme activity. The electrophoretic analysis of different roots from bulbs heterozygous for the Est-1 locus indicates that the structural gene for E-1 is not located on the B-chromosome itself. Rather there is a derepressor effect of Bs on E-1 isozyme activity. Since ADH and GOT patterns are unaffected by the presence of B-chromosomes it is clear that they do not exhibit a generalised derepressor effect.     

Detection of Somaclonal Variations in Tissue Culture-Derived Date Palm Plants Using Isoenzyme Analysis and RAPD Fingerprints

Isoenzyme analysis and activities of peroxidase (PER), polyphenol oxidase (POD) and glutamate oxaloacetate (GOT) and randomly amplified polymorphic DNA (RAPD) fingerprints were used to analyze somaclonal variations in tissue culture-derived date palm plants. The frequency of somaclonal variations was found to be age dependent. Similar isoenzyme patterns for PER and GOT were detected in all analyzed plants. However, variations in activities of the three enzymes studied and in POD isoenzymes were detected. RAPD analysis showed genetic variations in approximately 4 % of the analyzed plants (70 regenerants). The genetic variations were only detected in 6- and 12-months-old cultures. It was observed also that all morphologically abnormal shoots showed genetic variations at the molecular level.     

RAPD polymorphisms among variant and phenotypically normal rice (Oryza sativa var.indica) somaclonal progenies

Summary RAPD analysis was performed among eight rice somaclonal families known to vary for specific characters and four somaclonal families which were phenotypically normal. The parental cultivar,indica rice cv. FR13A, was found to be homogeneous and homozygous at all but one of the 45 RAPD loci. Polymorphisms were found at 28 of the 45 bands among the somaclonal families, including both loss of parental bands, and the appearance of novel non-parental bands. Segregation data revealed both heterozygous and homozygous mutation events, with recessive mutations more prevalent than dominant. All somaclonal families differed significantly from the parental material, indicating that genomic alterations occurred in all families regardless of phenotype. None of the variant families could be regarded as isogenic lines of FR13A at the DNA level. However, some of the DNA level variation may be in highly repeated sequences with no phenotypic effects. The implications for somaclonal breeding and genetic engineering programs are discussed.     

Effects of genetically modified amylopectin-accumulating potato plants on the abundance of beneficial and pathogenic microorganisms in the rhizosphere

In this study, the potential effects of a genetically modified (GM) amylopectin-accumulating potato line (Solanum tuberosum L.) on plant beneficial bacteria and fungi as well as on phytopathogens in the rhizosphere were investigated in a greenhouse experiment and a field trial. For comparison, the non-transgenic parental cultivar of the GM line and a second non-transgenic cultivar were included in the study. Rhizospheres were sampled during young leaf development (EC30) and at florescence (EC60). The microbial community composition was analysed by real-time PCR to quantify the abundances of Pseudomonas spp., Clavibacter michiganensis, Trichoderma spp. and Phytophthora infestans. Additionally, total bacterial and fungal abundances were measured. None of the examined gene abundance patterns were affected by the genetic modification when wild type and GM line were compared. However, significant differences were observed between the two natural potato cultivars, especially during the early leaf development of the plants. Furthermore, gene abundance patterns were also influenced by the plant developmental stage. Interestingly, the impact of the cultivar and the plant vegetation stage on the microbial community structure was more pronounced in field than in greenhouse. Overall, field-grown plants showed a higher abundance of microorganisms in the rhizosphere than plants grown under greenhouse conditions.     

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     

Assessing the potential for unintended effects in genetically modified potatoes perturbed in metabolic and developmental processes. Targeted analysis of key nutrients and anti-nutrients

Targeted compositional analysis was carried out on transgenic potato tubers of either cultivar (cv.) Record or cv. Desirée to assess the potential for unintended effects caused by the genetic modification process. The range of transgenic lines analysed included those modified in primary carbohydrate metabolism, polyamine biosynthesis and glycoprotein processing. Controls included wildtype tubers, tubers produced from plants regenerated through tissue culture (including a callus phase) and tubers derived from transformation with the ‘empty vector’ i.e. no specific target gene included (with the exception of the kanamycin resistance gene as a selectable marker). Metabolite analysis included soluble carbohydrates, glycoalkaloids, vitamin C, total nitrogen and fatty acids. Trypsin inhibitor activity was also assayed. These cover the major compounds recommended by the OECD in their Consensus Document on Compositional Considerations for New Varieties of Potatoes: Key Food and Feed Nutrients, Anti-Nutrients and Toxicants (2002). Data was statistically analysed using analysis of variance (ANOVA) for individual compounds and, where applicable, principal component analysis (PCA). In general, targeted compositional analysis revealed no consistent differences between GM lines and respective controls. No construct specifically induced unintended effects. Statistically significant differences between wildtype controls and specific GM lines did occur but appeared to be random and not associated with any specific construct. Indeed such significant differences were also found between wildtypes and both tissue culture derived tubers and tubers derived from transformation with the empty vector. This raises the possibility that somaclonal variation (known to occur significantly in potato, depending on genotype) may be responsible for an unknown proportion of any differences observed between specific GM lines and the wildtype. The most obvious differences seen in GC-MS profiles were between the two potato varieties used in the study.     

Genetic control of alcohol dehydrogenase isozymes in Triticum dicoccum

By means of the zymogram technique, two types of electrophoretic patterns were found for the enzyme alcohol dehydrogenase (ADH) in Triticum dicoccum, a tetraploid species of wheat. Each strain examined showed three bands of ADH activity. The two types of patterns found differed with respect to the relative electrophoretic mobilities and staining intensities of the bands. Evidence that the variation between the patterns is controlled at a single gene locus by two codominant alleles was obtained from appropriate genetic crosses. Heterozygotes for the allelic difference have five bands of ADH activity. These probably represent six different forms of the enzyme, two of which have coincident electrophoretic mobilities. These observations support the hypothesis that ADH exists as a dimer in T. dicoccum. It is probable that the enzyme subunits are coded for by duplicated ADH gene loci, each of which was contributed to the original tetraploid wheats by one of the two diploid progenitors of that group.This paper is Technical Article No. 7983, Texas Agricultural Experiment Station.     

Epigenetic aspects of somaclonal variation in plants

Somaclonal variation is manifested as cytological abnormalities, frequent qualitative and quantitative phenotypic mutation, sequence change, and gene activation and silencing. Activation of quiescent transposable elements and retrotransposons indicate that epigenetic changes occur through the culture process. Epigenetic activation of DNA elements further suggests that epigenetic changes may also be involved in cytogenetic instability through modification of heterochromatin, and as a basis of phenotypic variation through the modulation of gene function. The observation that DNA methylation patterns are highly variable among regenerated plants and their progeny provides evidence that DNA modifications are less stable in culture than in seed-grown plants. Future research will determine the relative importance of epigenetic versus sequence or chromosome variation in conditioning somaclonal variation in plants.     

Rapid production of transgenic sugarcane with the introduction of simple loci following biolistic transfer of a minimal expression cassette and direct embryogenesis

A protocol is described that supports the production of transgenic sugarcane plants ready for transfer to soil within 3 mo from culture initiation. Biolistic gene transfer into cross-sections of immature leaf whorl explants followed by direct somatic embryogenesis resulted in the stable genetic transformation of the commercially important sugarcane cultivar CP 88-1762. Accelerating the production of transgenic sugarcane plants not only saves time and effort but will likely also minimize somaclonal variation. Southern blot analysis revealed simple transgene integration patterns ranging from one to five hybridization products. NPTII-ELISA confirmed that most of the transgenic plants expressed the transgene stably in vegetative progeny. Using a minimal, linear expression cassette (MC) without vector backbone sequences for the biolistic gene transfer and reducing the amount of MC to 10 ng per shot may have led to simple transgene integration and stable transgene expression. Therefore, this protocol has great potential for the generation of commercial transgenic sugarcane events.     

植物人工异源多倍体的遗传及后遗传变化

据估计,70%以上的显花植物在其生活史上至少发生过一次以上的多倍化。传统的有关多倍性的观点认为,多倍体基因组应是其双亲基因组的积加。但是,有些合成异源多倍体的基因组发生了广泛的遗传及后遗传变化。这些变化包括亲本DNA序列丢失、核仁显性、DNA甲基化模式改变、基因沉默、反转座子激活等。亲本序列丢失可能与部分同源序列间重组有关,而亲本基因沉默可能与同源性依赖的基因沉默及RNA干涉等有关。     

Somaclonal variation and resistance to Verticillium wilt in lucerne,Medicago sativa L., plants regenerated from callus

Somaclonal variation in disease reaction type to infection by the vascular wilt pathogen Verticillium albo-atrum Reinke & Berth was assessed in a population of lucerne plants regenerated from callus lines obtained from a susceptible cultivar. Disease severity in the regenerant population was reduced by comparison with parental controls. Seed progeny and plants recovered via a second tissue culture cycle reverted to mainly susceptible reaction types. In a further experiment a low molecular weight toxic fraction from culture filtrates of the fungus was incorporated into the callus medium prior to regeneration. Toxin treatment reduced the regenerative capacity of callus, and there was little evidence for a higher frequency of wilt resistant plants in populations selected at low toxin concentrations. The results suggest that somaclonal variation as an alternative breeding strategy for disease resistance in lucerne offers no advantages over conventional recurrent selection.     

Field assessment of dihaploid Solatium tuberosum and S. brevidens somatic hybrids

Summary Following both chemical and electrical fusion of protoplasts of a dihaploid line of potato (Solanum tuberosum), (PDH40), with those of the wild species, Solanum brevidens, 11 and 40 somatic hybrid plants, respectively were obtained. Fifteen of these somatic hybrid genotypes and the two parents were studied further in a small field trial to assess field performance and phenotypic variability. In the UK, somatic hybrid plants are classified as genetically engineered organisms, and the UK Advisory Committee on Genetic Manipulation have imposed various restrictions on field experiments. Examination of the somatic hybrids in the field showed extensive phenotypic variability, and no two genotypes were identical. Some of the variation reflected changes in chromosome numbers, but other factors were also involved. Half the somatic hybrid genotypes produced tubers in the field, although the tubers were smaller and differed morphologically from those of PDH40. The results of the study suggest that the extent of somaclonal variation manifested in somatic hybrids is greater than that found in protoplast-derived plants of potato. The implications of this observation and the current regulations concerning field experiments of somatic hybrid plants in the UK are discussed.     

转基因白桦的遗传变异分析

应用细胞学方法分析了由农杆菌介导法获得的转基因白桦的细胞学变异情况,结果表明转基因白桦的染色体变异频率为78.5％,远远高于非转基因白桦的变异频率(15.3％),且变异以非整倍体占多数。同时用RAPD标记方法研究了转基因白桦在DNA水平的变异情况,结果显示DNA多态性指数为31.67,并与其它转基因植物的变异情况作了比较研究。最后分析、讨论了产生变异的原因:(1)组织培养过程中产生突变;(2)外源基因的整合及重排时宿主基因组的插入位点及相邻基因转录表达的干扰;(3)应用抗生素和除草剂等筛选转基因植株时促进了转基因植株的变异程度。并提出减少转基因植物体细胞克隆变异的建议。     

小麦与多年生黑麦草原生质体的电融合及杂种愈伤组织的形成

多年生黑麦草(Lolium perenne L.)悬浮培养细胞来源的原生质体和小麦(Triticumaestivum L.)含叶绿体的悬浮培养细胞来源的原生质体间,用直流方波脉冲进行电融合,获得了体细胞杂种愈伤组织。小麦的原生质体经过碘乙酰胺失活。愈伤组织的形态和颜色被用作识别预期杂种的标记。为了对杂种愈伤组织进行同工酶分析,观察了亲本的9种同工酶谱,其中3种在亲本间表现出差异(ADH、GOT 和 SDH)。酒精脱氢酶(ADH)的分析结果表明,有6个细胞系表现出杂种带。这些细胞系经过其他两种同工酶分析和 rDNA 探针杂交试验表明,一个细胞系表现出基本完全的亲本基因组间的组合,其余5个细胞系是部分杂种。     

An assessment of somaclonal variation in linseed (Linum usitatissimum)

Somaclonal lines of linseed from the parent cultivar Norlin were produced from a callus-based in oitro regeneration system (the R₀ generation). In field trials conducted over two seasons, 47 R₁ (plants produced from the R₀ generation) and 20 R₂ somaclonal lines (plants produced from the R₁ generation) were compared to the parent cultivar Norlin for quantitative characters. Irrespective of the genotype, traits in R₁'s and R₂'s were assessed on the basis of regression analysis as showing heritabilities of between 28% and 64%. Generally, the somaclonal variation assessed during these early generations revealed some detrimental traits, e.g. lower seed yield than the parent (control) cultivar and reduced 1000 seed weights, but a few lines were identified which had early or late flowering dates, improved seed yield and increased 1000 seed weights. It is concluded that somaclonal variation could be of value as an adjunct to classical breeding.     

Linkage relationships among five enzyme-coding gene loci in the copepod Tigriopus californicus: A genetic confirmation of achiasmatic meiosis

Linkage relationships among five polymorphic enzyme-coding gene loci in the marine copepod Tigriopus californicus have been determined using electrophoretic analysis of progeny from laboratory matings. Phosphoglucose isomerase (PGI; EC 5.3.1.9) was found to be tightly linked to glutamate-pyruvate transaminase (GPT; EC 2.6.1.2), with only one recombinant observed in 364 progeny; glutamate-oxaloacetate transaminase (GOT; EC 2.6.1.1) is linked to the PGI-GPT pair, with a recombination fraction of approximately 0.20 in male double heterozygotes. Phosphoglucomutase (PGM; EC 2.7.5.1) and an esterase (EST; EC 3.1.1.1) are not linked to the PGI, GPT, GOT grouping, which has been designated linkage group I. Reciprocal crosses have revealed that no recombination occurs in female T. californicus; this observation confirms a previous report that meiosis in female Tigriopus is achiasmatic.     

Comparative electrophoretic studies of proteins and enzymes of some Brassica species

Summary A considerable amount of variation with respect to soluble proteins and esterase isoenzyme pattern was observed between different species of Brassica. Naturally occurring amphidiploids had comparable proteins and isoenzyme patterns to either one or both of the parental species. The species relationship based on percentage homology of protein and esterase bands revealed that B. nigra and B. campestris are the parental species of B. carinata and not B. nigra and B. oleracea, as suggested on the basis of cytological studies. Elimination of a pair of chromosomes might have resulted into 2n=34 in the case of B. carinata. Further studies are needed to confirm this view. The peroxidase and catalase isoenzyme patterns did not show much variation in different species and amphidiploids.     

Fermentation and malate metabolism in response to elevated CO2 concentrations in two strawberry cultivars

Concentrations of acetaldehyde, ethanol, ethyl acetate (EA), organic acids and activities and gene expression of alcohol dehydrogenase (ADH; EC 1.1.1.1), pyruvate decarboxylase (PDC; EC 4.1.1.1), alcohol acyltransferase (AAT; EC 1.4.1.14), malate dehydrogenase (MDH; EC 1.1.1.37), malic enzyme (ME; EC 1.1.1.40) and glutamate dehydrogenase (EC 1.4.1.14) were investigated in two strawberry ( Fragaria × ananassa Duch) cultivars with different responses to CO₂ during storage. 'Jewel' fruit treated with CO₂ accumulated acetaldehyde and ethanol but little EA, while 'Cavendish' accumulated little acetaldehyde or ethanol but accumulated EA. In CO₂-treated fruit, PDC activity was positively correlated with EA accumulation in 'Jewel' but not in 'Cavendish', while no differential effect of atmosphere was observed on its gene expression. ADH activity and gene expression show a correlation with ethanol accumulation in 'Cavendish'. In 'Jewel', there was a positive correlation between ADH gene expression and enzyme activity; however, this correlation does not explain ethanol accumulation in this cultivar. EA accumulation did not show any correlation with AAT activity and gene expression in any of the cultivars. Succinate concentrations were highest and those of malate lowest in CO₂-treated fruit of both cultivars, but MDH and ME activities were not affected by CO₂. Gene expression of MDH and ME were not affected by atmosphere in 'Cavendish', although in 'Jewel' the MDH expression was slightly lower in CO₂- than air-treated fruit. The results of this study show that differences in fermentation products and malate accumulation in CO₂-treated strawberry fruit are not consistently correlated with enzyme activities and gene expression.