Stable gene expression in transgenic apple tree tissues and segregation of transgenes in the progeny — Preliminary evidence

Summary We report here, for the first time, the stable expression and Mendelian segregation of transgenes in a tree species. So far we have evidence for a 1 : 1 segregation of thenos gene in the R1 of transgenic apple progeny. In addition we present evidence for stable gene expression of bothnos and the co-transferred genenptII in the fruit flesh of the apple fruit some 7 years after the initial transformations.     

The Introduction of Transgenes to Control Blackheart in Pineapple (Ananas Comosus L.) cv. Smooth Cayenne by Microprojectile Bombardment

Summary A transformation technique for the introduction of transgenes to control blackheart by particle bombardment has been developed for pineapple cv. Smooth Cayenne. Leaf callus cultures capable of high frequency organogenesis with a short regeneration time were used as explant material. Gus and gfp reporter genes were used to observe and determine transient and stable expression. The ppo gene, isolated from pineapple, was introduced to control blackheart. Co-transformation occurred with constructs containing the nptII gene conferring geneticin resistance. We have recovered 15 independent transgenic gus and gfp lines each from 8 separate experiments and 22 ppo lines from 11 experiments. Gus, gfp, ppo and nptII positive plants have been regenerated, which have been shown by Southern blot analysis to be stable transgenics containing multiple copies of the introduced genes. These results show that biolistic gene delivery in pineapple can be successfully achieved at an acceptable efficiency of 0.21–1.5% for genetic improvement of ’Smooth Cayenne’, the industry standard throughout the world.     

Transge ne inheritance, segregation and expression in bread wheat

Transgene integration, inheritance and expression were studied in six transgenic wheat (Triticum aestivum) lines produced by co-bombardment with two plasmids containing marker genes and genes encoding HMW subunits of wheat glutenin, respectively. Transgene insertion number ranged from 1 to approximately 15. Within a transgenic locus the majority of plasmid copies were found at dispersed genomic sites separated by intervening DNA. However, evidence was obtained for the arrangement of introduced plasmid copies as concatamers and for plasmid truncation and rearrangement. Transgenes were frequently located in genetically unlinked chromosome sites, resulting in independent segregation of loci among progeny. In two lines this gave rise to progeny containing only the gene of interest. Transgenes were inherited in the T₁ generation as a dominant trait although Mendelian segregation ratios were not always observed. No evidence of co-suppression of endogenous HMW subunits was observed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of fertile transgenic Brassica napus by Agrobacterium-mediated transformation of protoplasts

A protocol for Agrobacterium tumefaciens‐mediated transformation of Brassica napus mesophyll protoplasts is described. A strain with a neomycin phosphotransferase (nptII) gene and a KCS gene under control of a napin promoter was used at co‐cultivation. Transformed protoplasts were regenerated to fertile and morphologically normal transgenic plants. Transformants were confirmed by PCR of the nptII gene and NAP/KCS expression cassette, and Southern blot analysis. Seeds of the transformants showed a changed fatty acid profile: two transformants had a higher erucic acid level and differed significantly from that of B. napus. Genetic analysis of the progeny revealed that the kanamycin resistance introduced was inherited in a Mendelian fashion.     

Transgenic barley by particle bombardment. Inheritance of the transferred gene and characteristics of transgenic barley plants

Summary Transgenic barley plants (Hordeum vulgare L. cv. Kymppi) were obtained by particle bombardment of various tissues. Immature embryos and microspore-derived cultures were bombarded with gold particles coated with plasmid DNA carrying the gene coding for neomycin phosphotransferase II (NPTII), together with plasmid DNA containing the gene for -glucuronidase (GUS).Bombarded immature embryos were grown to plants without selection and NPTII activity was screened in small plantlets. One plant proved to be transgenic (T₀). This chimeric plant passed the transferred nptII gene to its T₁ progeny. The presence of the nptII gene was demonstrated by the PCR technique and enzyme activity was analyzed by an NPTII gel assay. Four T₀ spikes and 15 T₁ offspring were transgenic. The integration and inheritance was confirmed by Southern blot hybridization. Transgenic T₂ and T₃ plants were produced by isolating embryos from green grains of transgenic T₁ and T₂ plants, respectively and growing them to plants. After selfing, the ratio of transgenic to non-transgenic T₂ offspring was shown to follow the rule of Mendelian inheritance. The general performance of transgenic plants was normal and no reduction in fertility was observed.Microspore-derived cultures were bombarded one and four weeks after microspore isolation. After bombardment, cultures were grown either with or without antibiotic selection (geneticin ^R or kanamycin). When cultures were grown without selection and regenerated plants were transferred to kanamycin selection in rooting phase, one out of a total of about 1500 plants survived. This plant both carried and expressed the transferred nptII gene. The integration was confirmed by Southern blot hybridization. This plant was not fertile.     

Correlated inheritance of sex expression and fruit shape in Cucumis

Genetic control of sex expression in particular strains of Cucumis melo and C. sativus is under the control of a single locus. A dominant allele, A, is responsible for monoecious sex expression; the recessive allele, a, is responsible for andromonoecious sex expression. The evidence suggests that in C. melo fruit shape is determined by a single gene with incomplete dominance plus minor modifying genes, linked in coupling phase with the gene for determination of sex expression. In C. sativus the evidence does not completely exclude the possibility that the alleles for sex determination are pleiotropic and, therefore, are also the major determiners of fruit shape. However, evidence in favor of close linkage between genes determining sex and fruit shape is given and the author favors an interpretation of close linkage between two subunits of a complex gene. Evolutionary significance is seen in the presence of correlated inheritance of sex expression and fruit shape in three species of two genera of the Cucurbitaceae.     

Inheritance of resistance to angular leaf spot in Nicotiana tabacum L. cultivars burley 21 and kentucky 14

Summary The genetics of resistance to angular leaf spot caused by Pseudomonas syringae pv. tabaci in Nicotiana tabacum cultivars Burley 21 and Kentucky 14 was investigated by studying disease reactions to three isolates of parental, F₁, F₂ and backcross generations derived from crosses between the resistant cultivars and the susceptible cultivar Judy's Pride. Studies were conducted in the greenhouse and in field plant beds. Chi-square values were computed to determine whether the observed ratios for disease reactions deviated from expected Mendelian ratios for a single, dominant gene controlling resistance to angular leaf spot in tobacco. Based on the resistance of the F₁ and the backcross generation to the resistant parent (BC-R), a 3 resistant: 1 susceptible segregation ratio in the F₂, and a 1 resistant: 1 susceptible segregation ratio in the backcross generation to the susceptible parent (BC-S), it was concluded that resistance to three isolates of Pseudomonas syringae pv. tabaci is governed by a single, dominant gene.     

Segregation Analysis of Isozyme Markers on Isolated Microspore-Derived Embryos in Brassica napus L.

The segregation of isozyme genetic markers was studied on embryos arising from isolated microspore cultures in live rapeseed F1 hybrid genotypes. Out of the ten isozyme genes considered, live (Aco-1, Aco-3, Lp-1, Pgm-3, Tpi-1) did not segregate according to expected Mendelian ratios in at least two F1 crosses. F2 plants, generated from the same hybrids, included in the analysis, were tree of segregation distortions. Linkage analysis between each segregating enzyme locus in each progeny revealed independance between these markers. In this paper, the results of the linkage analysis as well as the origins of the distortions are discussed. The existence of androgenic embryogenesis genes may be responsible for the abnormal segregations of the five isozyme genes.     

Optimal sampling strategies for evaluating fruit softening after harvest in apple breeding

Environmental variance components associated with year, tree, and harvest date were estimated for fruit softening after harvest in apple (Malus × domestica Borkh.) to determine their relative importance and design optimum sampling strategies to discriminate genotypes in apple breeding. Fruit were stored after harvest under 20± 2 ^∘C and 80± 5%RH. Softening was evaluated by adapting the change in firmness during storage to a linear regression and defining the regression coefficient as the softening rate. Environmental variances associated with genotype × year interaction, among trees, year × tree interaction, and among harvest dates were all very small, namely, 2.7, 0.1, 5.2, and 5.7%, respectively, to the total variance obtained from the analysis of variance for the softening rate. The variance associated with genotype, at 57.3%, was very large. On the basis of the number of fruit necessary for firmness measurements, two times harvest is an efficient strategy to determine a genotype mean for the softening.     

The potential role of PR-8 gene of apple fruit in the mode of action of the yeast antagonist,Candida oleophila,in postharvest biocontrol of Botrytis cinerea

Several pathogenesis-related (PR) genes of apple have been cloned and their response to different pathogens has been studied. Different PR genes, however, may have a variable response depending on the specific organ or tissue as well as microbe. The objective of the current study was to characterize the expression of specific apple PR genes in fruit tissues in response to the antagonistic yeast, Candida oleophila (a common postharvest biocontrol agent), and the fungal pathogen, Botrytis cinerea (a major postharvest pathogen). Apple PR-5 and PR-8 gene expression was characterized in fruit in response to C. oleophila and B. cinerea. Results indicated that PR-8 expression was significantly elevated in response to both fungi. In contrast, neither C. oleophila nor B. cinerea treatment markedly affected PR-5 expression. The PR-8 gene was then synthesized and cloned into a Pichia pastoris expression system to study the antifungal activity of the PR-8 protein against B. cinerea both in vitro and in vivo. Collectively, the PR-8 gene of apple is associated with the response to B. cinerea infection, and may play a role in the mechanism by which C. oleophila effectively inhibits B. cinerea disease in apple fruit, namely by the induction of this specific host PR gene.     

The V f gene for scab resistance in apple is linked to sub-lethal genes

Summary V _f is the most widely used resistance gene in the breeding for scab resistant apple cultivars. Distorted segregation ratios for V _f -resistance have frequently been reported. Here we revealed that sub-lethal genes caused the distorted segregation. The inheritance of V _f was examined in six progenies by testing linked molecular markers. Three progenies showed distorted segregations that could be explained by three sub-lethal genes (sl1, sl2 and sl3), of which sl1, sl2 were closely linked to V _f . The s11 gene was located at about 14 cM from V _f and expressed itself only in the presence of another independently segregating sub-lethal gene sl3. Only the double homozygous recessive genotypes (sl1sl1 sl3sl3) were lethal, which occurred at first as dwarf and poor vigour plants during the first three months after germination. The sl2 gene was also linked to V _f and its lethality was expressed prior to seed germination and also required the homozygous recessive presence of sl3. The map position of sl3 has not yet been identified. The linkage of V _f to sub-lethal genes usually results in a shortage of V _f -resistant progenies. But in some exceptional crosses, it will lead to abundance of resistant seedling.An erratum to this article can be found at     

Correlation of isoenzyme polymorphism and Bayoud-disease resistance in date palm cultivars and progeny

Summary Seedlings of date palm (Phoenix dactylifera L.), obtained from seven cultivars crossed with two males, were analyzed by polyacrylamide gel electrophoresis for esterase (EST), glutamate oxaloacetate transaminase (GOT), endopeptidase (ENP) and alcohol dehydrogenase (ADH) polymorphisms. Eleven, eight, five and two phenotypes were revealed for the enzymes tested, respectively. Seedlings of F₁ populations derived from Bayoud (Fusarium)-resistant and low fruit quality cultivars were characterized by a high electrophoretic polymorphism, when compared with progenies of Bayoud-susceptible and high fruit quality cultivars. In almost all cases, the most frequent electrophoretic phenotypes scored for each enzyme in different F₁ populations, were similar to those of the corresponding parent cultivars. Heterozygous phenotypes have been found for, at least, 3 loci; Got-2, Est-1 and Enp, indicating that such loci could be used to screen for hybrid seedlings. The expected Mendelian segregation of allozymes has been observed for these 3 loci, in many F₁ populations. It seems that progenies of Bayoud-resistant cultivars are characterized by a high level of electrophoretic polymorphism. The estimation of this index and the search for genetic linkage with segregating allozymes, may be biochemical criteria useful as an aid in distinguishing date palm seedling populations derived from Bayoud-resistant cultivars and suitable for breeding programs.     

Role of the genes Md-ACO1 and Md-ACS1 in ethylene production and shelf life of apple (Malus domestica Borkh)

Shelf life determines the economic life time of mature apples, which can be either freshly harvested or stored. Good shelf life is highly associated with a slow decrease of fruit firmness at room temperature. Apple is a climacteric fruit, in which loss of firmness seems to be physiologically related to ethylene. Ethylenes biosynthetic pathway is controlled by two large gene families coding for 1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxydase (ACO).In this study, one ACS and one ACO gene were examined for their effect on ethylene production and shelf life in apple using gene specific molecular marker, and have also been positioned on a molecular marker linkage map. The ACO marker was developed in this research and mapped on linkage group (LG) 10 of the crosses Prima × Fiesta and Fuji × Mondial Gala, within the 5% border of a previously identified fruit firmness QTL [Theor Appl Genet 100 (2000) 1074]. We denoted this locus as Md-ACO1. In addition, we mapped the previously developed Md-ACS1 marker [Theor Appl Genet 101 (2000) 742] on LG15.Studies on the cross Fuji × Braeburn revealed that Md-ACS1 and Md-ACO1 independently affect the internal ethylene concentration (IEC) as well as shelf life of apple, Md-ACS1 having the strongest effect. Descendants homozygous for Md-ACS1-2 and Md-ACO1-1 showed to have the lowest ethylene production as well as superior shelf-life. These two genes are candidates to be included in marker assisted breeding.     

Genetic control of fertility in the oil palm (Elaeis guineensis Jacq.)

Summary A study was conducted on the segregating populations from tenera × tenera, tenera x fertile Pisifera crosses and tenera selfings. Pisifera palms were categorised as (1) fertile Pisifera palms if producing mature ripe bunches regularly under natural conditions, and (2) partially female sterile pisifera palms if producing a few or no bunches in several years.Based on observations on the segregation patterns of the fruit forms (dura, tenera and pisifera) and bunch production patterns, a genetic model was formulated to explain the genetic basis of fertility in the oil palm. Fertility in the oil palm is shown to be controlled by a single gene which is linked to the gene controlling fruit form. Chi-square tests confirmed that the model agreed with the segregating ratios of fruit form and fertility observed.The implications of this finding with regard to oil palm breeding and improvement, and the potential of the fertile pisifera palm for increasing palm oil yields in plantations are discussed.     

An allelic series at the Co-1 locus conditioning resistance to anthracnose in common bean of Andean origin

In this study, we characterized the genetic resistance of the Andean bean cultivars Kaboon and Perry Marrow and their relation to other sources of anthracnose resistance in common bean. Based on the segregation ratio (3R:1S) observed in two F₂ populations we demonstrated that Kaboon carries one major dominant gene conferring resistance to races 7 and 73 of Colletotrichum lindemuthianum. This gene in Kaboon is independent from the Co-2 gene and is an allele of the Co-1 gene present in Michigan Dark Red Kidney (MDRK) cultivar. Therefore, we propose the symbol CO-1 ² for the major dominant gene in Kaboon. The Co-1 is the only gene of Andean origin among the Co anthracnose resistance genes characterized in common bean. When inoculated with the less virulent Andean race 5, the segregation ratio in the F₂ progeny of Cardinal and Kaboon was 57R:7S (p = 0.38). These data indicate that Kaboon must possess other weaker dominant resistance genes with a complementary mode of action, since Cardinal is not known to possess genes for anthracnose resistance. Perry Marrow, a second Andean cultivar with resistance to a different group of races, was shown to possess another resistant allele at the Co-1 locus and the gene symbol Co-1 ³ was assigned. In R × R crosses between Perry Marrow and MDRK or Kaboon, no susceptible F₂ plants were found when inoculated with race 73. These findings support the presence of a multiple allelic series at the Andean Co-1 locus, and have major implications in breeding for durable anthracnose resistance in common bean. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evaluation of the role of the endo-β-(1,4)-glucanase gene FaEG3 in strawberry fruit softening

In strawberry, Fragaria × ananassa Duch., fruit, two different endo-β-1,4-glucanase genes, FaEG1 and FaEG3, also named Cel1 and Cel2, are expressed during the softening process that occurs during fruit ripening. It has also been suggested that FaEG3, which contains a putative cellulose-binding domain, could play a key role in fruit development, since previous attempts to down-regulate this gene through transgenesis have been unsuccessful. In this investigation, we obtained transgenic strawberry plants containing an antisense sequence of the FaEG3 gene under the control of the 35S promoter. Ripened fruit from transgenic lines (Acel lines) showed large variation in FaEG3 silencing, but fruit firmness was similar to control fruit in all the lines. Two Acel lines showing almost 95% reductions in FaEG3 mRNA levels were selected for further study. In these lines, FaEG3 down-regulation was high, from 78 to 95%, at all fruit developmental stages, whereas FaEG1 was only slightly suppressed. In spite of the high FaEG3 silencing achieved, EGase activity was not modified in ripe fruit. At the cell wall level, walls from transgenic ripe fruit contained a significantly higher amount of the 4 M KOH fraction, which is enriched in hemicellulosic polymers. The analysis of this fraction by size exclusion chromatography showed that transgenic cell walls contained a smaller amount of higher molecular mass polymers than controls. Altogether, these results indicate that FaEG3 does not play a key role either in fruit development or fruit softening. However, its silencing affects the amount and, in a minor way, the size of hemicellulosic polymers.