Genetic control of albinism in pickerelweed (Pontederia cordata L.)

Pickerelweed (Pontederia cordata L.) is a diploid (2n = 2x = 16) perennial aquaphyte. Preliminary studies revealed that a group of nonalbino pickerelweed plants maintained for breeding and inheritance studies regularly produced albino seedlings. The objective of this experiment was to determine the number of loci, number of alleles, and gene action controlling albinism in pickerelweed. Five nonalbino parental lines were used in this experiment to create S(1) and F(1) populations. F(2) populations were produced through self-pollination of F(1) plants. Evaluation of S(1), F(1), and F(2) generations allowed us to identify a single diallelic locus controlling albinism in these populations of pickerelweed, with albinism completely recessive to normal green leaf production. We propose that this locus be named albino with alleles A and a.     

Antioxidative response of Golden Agave leaves with different degrees of variegation under high light exposure

Variegated mutants are spotted rare in wild, and especially in the high light (HL) conditions. In addition, these plants are cultivated by a growing number of horticulturists due to their high economic value. In this experiment, the antioxidative response of both albino and green tissues from a mutant plant Golden Agave, which possesses leaves with different degrees of variegation, was investigated under normal or HL exposure, respectively. Severe oxidative stress was observed in green tissues of high albinism leaves (30?C40?% albino tissues, muy) over low albinism counterparts (less than 10?% albino tissues, mug). Compared with green tissues, low oxidative damage (relative electrolyte leakage and protein damage) and antioxidant enzyme (SOD, CAT, APX, and GR) activities were observed in the albino than in the adjacent green tissues. Photoinactivation of the CAT and the APX enzyme activities were observed in the albino tissues to a significantly level under HL exposure. In addition, lower redox values (ASC/DHA and GSH/GSSG) and higher levels of reactive oxygen species (ROS) were monitored in the green tissues of the muy over mug mutant even under normal light conditions. This work could help us to understand the HL sensitivity of variegated mutant plants better and allow us to improve variegated mutant plant cultivation skills. At last, the ??oxidative stress hypothesis?? was introduced to illustrate the reduced evolutionary advantage of plant variegation phenomenon in the discussion.     

Organelle antioxidants improve microspore embryogenesis in wheat and triticale

Low frequency of green plant production and albinism limits the use of isolated microspore culture (IMC) in cereal breeding programs. The present study was conducted in triticale and bread wheat IMC to increase the production of green plants and minimize albinism. NPB-99?+?10% Ficoll induction medium was supplemented with mitochondrial or plastid antioxidants, in a completely random design, to evaluate their contribution to successful microspore embryogenesis and green plant production. Each group of antioxidants was tested independently: first in triticale and then validated in various spring wheat genotypes. While the response differed by wheat genotype, induction medium supplemented with proline (10 mM) yielded a greater number of embryos/embryo-like structures and green plants in both triticale and wheat. No differences were found with respect to albinism in triticale or wheat except for the cv. Sadash. Among plastid antioxidants tested, glutathione (2 μM) proved to be the best antioxidant to increase embryo and green plant production. Salicylic acid also helped to reduce the number of albino plants in triticale and the wheat genotype SWS366. Overall, induction medium supplemented with proline or glutathione enhanced microspore embryogenesis in both triticale and wheat and increased the number of green plants in the recalcitrant genotypes.     

Establishment of homozygous transgenic rice lines after elimination of unlinked albino traits from transgenic events through segregation

In an analysis of 339 independent T ₀ transgenic rice lines generated by Agrobacterium-mediated transformation, albino plants appeared in the T ₁ generation in two single-copy transgenic lines, O54 and O36 and in one double-copy transgenic line, C18. While the T ₀ plants of these three lines were green, albino and green plants emerged in a 1:3 ratio in the T ₁ generation. The albino phenotype segregated as a monogenic recessive trait. Southern blot analysis of the green and albino plants in the T ₁ generation confirmed that the albino trait and the T-DNA insertion events were unlinked. Segregation of the albino trait from the transgenic trait in the lines O54 and O36 was confirmed in T ₂ and T ₃ generations, respectively. Homozygous transgenic plants free from the albino trait were also identified. In the double-copy transgenic line C18, we genetically separated the two transgenic loci, out-segregated the albino locus from both transgene loci, and identified homozygous plants for each of the transgenic events by Southern blot analysis in the T ₁ generation itself. Thus, we demonstrate that when an albino trait appears in the T ₁ generation and is unlinked to a transgene locus, the albino locus can be segregated from the transgene locus and homozygous transgenic lines free from albinos can be established.     

A transgenic mouse model with inducible Tyrosinase gene expression using the tetracycline (Tet-on) system allows regulated rescue of abnormal chiasmatic projections found in albinism

Congenital defects in retinal pigmentation, as in oculocutaneous albinism Type I (OCA1), where tyrosinase is defective, result in visual abnormalities affecting the retina and pathways into the brain. Transgenic animals expressing a functional tyrosinase gene on an albino genetic background display a correction of all these abnormalities, implicating a functional role for tyrosinase in normal retinal development. To address the function of tyrosinase in the development of the mammalian visual system, we have generated a transgenic mouse model with inducible expression of the tyrosinase gene using the tetracycline (TET-ON) system. We have produced two types of transgenic mice: first, mice expressing the transactivator rtTA chimeric protein under the control of mouse tyrosinase promoter and its locus control region (LCR), and; second, transgenic mice expressing a mouse tyrosinase cDNA construct driven by a minimal promoter inducible by rtTA in the presence of doxycycline. Inducible experiments have been carried out with selected double transgenic mouse lines. Tyrosinase expression has been induced from early embryo development and its impact assessed with histological and biochemical methods in heterozygous and homozygous double transgenic individuals. We have found an increase of tyrosinase activity in the eyes of induced animals, compared with littermate controls. However, there was significant variability in the activation of this gene, as reported in analogous experiments. In spite of this, we could observe corrected uncrossed chiasmatic pathways, decreased in albinism, in animals induced from their first gestational week. These mice could be instrumental in revealing the role of tyrosinase in mammalian visual development.     

Plant tubulin intronics

Introns of plant tubulin genes are useful molecular tools to study IME (Intron Mediated Enhancement of gene expression) and to define plant genetic and evolutionary relationships through ILP (Intron Length Polymorphism). Here we show that the intron present within the 5'UTR sequence of some rice beta-tubulin genes can sustain IME in rice transgenic plants and that degenerated oligonucleotide mixtures designed to amplify the first and the second intron present within the coding sequence of plant beta-tubulin genes can successfully detect ILPs among different bean varieties.     

Novel and recurrent mutations in the tyrosinase gene and the P gene in the German albino population

Albinism is a heterogeneous group of genetic disorders resulting from deficiencies in pigmentation. Clinically, it is divided into ocular (OA) and oculocutaneous albinism (OCA). OCA involves lack of pigment in the skin, hair, and eyes and results from mutations in the tyrosinase gene or in the P gene. OA mainly affects pigmentation in the visual system and may be a mild form of OCA or may be caused by other genetic defects. Clinical diagnosis of albinism type is difficult, because of the observed range of phenotypic variation. Thus, genetic analysis may be helpful with respect to a more accurate diagnosis. Here, we report the mutational profile, determined by genetic analysis of the tyrosinase and P genes, of a large German albino population. We have revealed a total of 42 distinct mutations, 19 of which are novel. Of the 74 unrelated patients screened, 32 (43%) had mutations in the tyrosinase gene, 16 (22%) had P gene mutations, and 26 (35%) patients had no detectable genetic abnormalities. This defines a population of albino patients who are tyrosinase-gene- and P-gene-negative and who thus may represent a good study group for searching for additional genes associated with albinism.     

Transgenic plants expressing geminivirus movement proteins: abnormal phenotypes and delayed infection by Tomato mottle virus in transgenic tomatoes expressing the Bean dwarf mosaic virus BV1 or BC1 proteins

Transgenic tomato plants expressing wild-type or mutated BV1 or BC1 movement proteins from Bean dwarf mosaic virus (BDMV) were generated and examined for phenotypic effects and resistance to Tomato mottle virus (ToMoV). Fewer transgenic plants were recovered with the wild-type or mutated BC1 genes, compared with the wild-type or mutated BV1 genes. Transgenic tomato plants expressing the wild-type or mutated BV1 proteins appeared normal. Interestingly, although BDMV induces only a symptomless infection in tomato (i.e., BDMV is not well adapted to tomato), transgenic tomato plants expressing the BDMV BC1 protein showed a viral disease-like phenotype (i.e., stunted growth, and leaf mottling, curling, and distortion). This suggests that the symptomless phenotype of BDMV in tomato is not due to a host-specific defect in the BC1 protein. One transgenic line expressing the BC1 gene did not show the viral disease-like phenotype. This was associated with a deletion in the 3' region of the gene, which resulted in expression of a truncated BC1 protein. Several R0 plants, expressing either wild-type or mutated BV1 or BC1 proteins, showed a significant delay in ToMoV infection, compared with non-transformed plants. R1 progeny plants also showed a significant delay in ToMoV infection, but this delay was less than that in the R0 parents. These results also demonstrate that expression of viral movement proteins, in transgenic plants, can have deleterious effects on various aspects of plant development.     

Protoplast transformation and regeneration of transgenic Valencia sweet orange plants containing a juice quality-related pectin methylesterase gene

Valencia orange [Citrus sinensis (L.) Osbeck] is the leading commercial citrus species in the world for processed juice products; however, the presence of thermostable pectin methylesterase (TSPME) reduces its juice quality. A long-term strategy of this work is to eliminate or greatly reduce TSPME activity in Valencia orange. Previous work resulted in the isolation of a putative TSPME gene, CsPME4, associated with a thermostable protein fraction of Valencia orange juice. To begin research designed to overexpress CsPME4 to verify the thermostability of the protein product and/or to downregulate the gene, a sense gene cassette containing a gene-specific sequence from a putative TSPME cDNA and the enhanced green fluorescent protein (GFP) as a selectable marker was constructed (M2.1). In the work reported here, M2.1 plasmid DNA was transformed (polyethylene glycol-mediated) into protoplasts isolated from an embryogenic suspension culture of Valencia somaclone line B6-68, in an effort to obtain transgenic Valencia lines. A vigorous transformed line was identified via GFP expression, physically separated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. One transgenic proembryo expressing GFP was recovered and multiple shoots were regenerated. The recovery of multiple transgenic plants was expedited by in vitro grafting. Polymerase chain reaction analysis revealed the presence of the PME gene in transgenic plants, and subsequent Southern blot analysis confirmed the presence of the eGFP gene. These transgenic plants show normal growth and minor morphological variation. The thermostability of PME in these plants will be assessed after flowering and fruit set. This is the first successful transfer of a target fruit-quality gene by protoplast transformation with recovery of transgenic plants in citrus. This method of transformation has the advantage over Agrobacterium-mediated transformation in that it requires no antibiotic-resistance genes.     

Green, herbicide-resistant plants by particle inflow gun-mediated gene transfer to diploid bahiagrass (Paspalum notatum)

We have established an efficient particle-bombardment transformation protocol for the diploid non-apomictic genotype of the warm season forage crop Paspalum notatum (bahiagrass). A vector containing a herbicide resistance gene (bar) together with the GUS reporter gene was used in transformation experiments. The bar gene confers resistance to the herbicide bialaphos. An improved culture system, highly regenerative callus, dense in compact polyembryogenic clusters, was produced on medium with a high CuSO4 content at elevated temperature. Target tissue (360 calli) produced under these conditions yielded 52 rooted plants on herbicide-containing medium, and 22 of these plants were PCR-positive. DNA gel blot analysis revealed a copy number of 1-5 for the GUS gene in different independent transformants. There was no correlation between copy number and GUS activity. While conventional cultures yielded exclusively albino plants on herbicide-containing medium, improved culture conditions for the target tissue resulted in the recovery of 100% green transgenic plants. All green herbicide-resistant regenerants were morphological normal and fertile.     

Self-fertile cybrids Nicotiana tabacum (+ Hyoscyamus aureus) with a nucleo-plastome incompatibility

Cytoplasmic hybrids (cybrids) in a novel inter-generic combination, Nicotiana tabacum (+ Hyoscyamus aureus), were generated by fusion of protoplasts from a plastome tobacco albino mutant (line R100a1) and gamma-irradiated green protoplasts of H. aureus. Cybrids possessed a plastome of H. aureus and a rearranged mitochondrial DNA. The cybrids displayed a syndrome of nucleo-plastome incompatibility expressed as a partial chlorophyll-deficiency of cotyledonary and true leaves at the early stage of vegetative development of plants grown from seeds in soil. During later development, the plants restored a normal green coloration. This character is phenotypically indistinguishable from the same syndrome in previously generated cybrids N. tabacum (+ H. nigrum). In contrast to the cybrids N. tabacum (+ H. nigrum), cybrids N. tabacum (+ H. aureus) were self-fertile, and did not manifest other features that were interpreted as nucleo-mitochondrial incompatibilities in N. tabacum (+ H. nigrum) plants. Therefore, the cybrids N. tabacum (+ H. aureus) present a self-propagating system of Nicotiana (+ Hyoscyamus) nucleo-plastome incompatibility in its pure form.     

Rice oxalate oxidase gene driven by green tissue‐specific promoter increases tolerance to sheath blight pathogen (Rhizoctonia solani) in transgenic rice

Rice sheath blight, caused by the necrotrophic fungus Rhizoctonia solani, is one of the most devastating and intractable diseases of rice, leading to a significant reduction in rice productivity worldwide. In this article, in order to examine sheath blight resistance, we report the generation of transgenic rice lines overexpressing the rice oxalate oxidase 4 (Osoxo4) gene in a green tissue‐specific manner which breaks down oxalic acid (OA), the pathogenesis factor secreted by R. solani. Transgenic plants showed higher enzyme activity of oxalate oxidase (OxO) than nontransgenic control plants, which was visualized by histochemical assays and sodium dodecylsulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE). Transgenic rice leaves were more tolerant than control rice leaves to exogenous OA. Transgenic plants showed a higher level of expression of other defence‐related genes in response to pathogen infection. More importantly, transgenic plants exhibited significantly enhanced durable resistance to R. solani. The overexpression of Osoxo4 in rice did not show any detrimental phenotypic or agronomic effect. Our findings indicate that rice OxO can be utilized effectively in plant genetic manipulation for sheath blight resistance, and possibly for resistance to other diseases caused by necrotrophic fungi, especially those that secrete OA. This is the first report of the expression of defence genes in rice in a green tissue‐specific manner for sheath blight resistance.     

Transgenic indica rice resistant to sap-sucking insects

Agrobacterium-mediated genetic transformation has been optimized in indica rice susceptible to sap-sucking insects, viz., brown planthopper (BPH) and green leafhopper (GLH). Snowdrop lectin gene (gna) from Galanthus nivalis, driven by phloem-specific rice-sucrose-synthase promoter, along with herbicide resistance gene (bar) driven by CaMV 35S promoter, was employed for genetic transformation. Embryogenic calli--after co-cultivation with Agrobacterium strain LBA4404 harbouring Ti plasmid pSB111-bar-gna--were selected on the medium containing phosphinothricin. PCR and Southern blot analyses confirmed the stable integration of both the genes into genomes of transgenic (T0) rice plants. Northern and Western blot analyses revealed the expression of gna in the transgenic plants. In the T1 and T2 generations, the gna and bar transgenes showed co-segregation at a ratio of 3 : 1. Plant progenies expressing gna, in T1 and T2, exhibited substantial resistance against BPH and GLH pests. This is the first report dealing with transgenic indica rice exhibiting high resistance to both insects.     

一个新的小麦黄化突变体的遗传研究

摘要: 通过对冬小麦“西农1718” 自然黄化突变体多年连续自交、与突变亲本回交以及与其他基因型进行正反交, 研究突变性状的遗传规律。观察统计发现, 突变体中金黄株发育至抽穗－开花期前后死亡, 黄-绿株自交后代表现为1金黄株︰2黄-绿株︰1绿株, 绿株自交后代不再分离; 黄-绿株与突变亲本回交及与其他基因型正反交, 后代均表现为1黄-绿株︰1绿株。遗传分析表明, 该小麦黄化突变体是由一对核基因控制的不完全显性遗传, 其中, 金黄株是纯合体(au/au), 表现为致死, 黄-绿株为杂合体(Au/au), 绿株为纯合体(Au/Au)。     

Transgenic rice plants: Characterization of two generations of seed progeny

Transgenic rice plants have been regenerated from kanamycin-resistant callus of Oryza sativa (cv. Taipei 309) derived from protoplasts electroporated with pCaMVNEO carrying the neomycin phosphotransferase II ( nptII ) gene. Of 6 randomly selected plants, all contained the nptll gene, but only 2 plants expressed NPTII activity. The transgenic plants were significantly shorter, produced fewer tillers, took longer to flower and had reduced fertility compared to non-transformed protoplastderived plants. Fifty-six seeds collected from one transgenic plant expressing NPTII activity germinated on medium containing kanamycin sulphate to give 16 green, first seed generation (R₁) plants. The latter could be divided into 3 groups: (i) Plants which set seed, had normal floret morphology and produced a total of 76 seeds; (ii) Plants which flowered, but which failed to set seed; (iii) Plants which failed to flower, were shorter and had significantly fewer tillers than plants of groups (i) and (ii). The nptII gene was present in all transgenic R₁ plants, but only 8 plants expressed the gene. Phenotypic characteristics, observed in transgenic R₁ plants were also seen in the transforned R₂ plants. These included reduced stature, a longer vegetative phase and reduced fertility compared to non-transformed plants.     

Transgenic Rice: Characterization of Protoplastderived Plants and their Seed Progeny

Thirty eight green and 2 albino plants were regenerated from400 kanamycin-resistant colonies derived from protoplasts isolatedfrom cell suspensions of Oryza sativa variety Taipei 309 andelectroporated with pCaMVNEO carrying the neomycin phosphotransferaseII (nptII) gene. Twenty of the green transgenic R_o plants weretransferred to the glasshouse, where 3 flowered after 7 months.Of 15 plants analysed by DNA hybridization, all carried thenptll gene, but only 2 of 11 plants assayed for NPTII activityexpressed the nptll gene. One transgenic R_o plant produced 59seeds following self-pollination. The seeds, when germinatedon medium containing kanamycin sulphate, gave 16 green transgenicR, plants. Five transgenic R₁ plants flowered and set seed,7 flowered but failed to produce seeds, while 4 did not producepanicles. Transgenic R_o and R₁ plants were shorter, requiredlonger to flower, and had reduced pollen viability comparedto non-transformed R₁ protoplast-derived plants. The nptII genewas present in all 16 transgenic R₁ plants, but NPTII activitywas detected in only 8 of these plants. Key words: Oryza sativa variety Taipei 309, rice, protoplasts, direct DNA uptake, kanamycin-resistant tissues, transgenic plants, DNA hybridization, neomycin phosphotransferase II (NPTII), gene expression and inheritance     

Relationships between brood size and offspring body size in an ovoviviparous fish: maternal effects and genetic trade-off

Maternal effects can form an important source of variation in offspring fitness and have important evolutionary and ecological consequences. To explore genetic control for body size of newborn offspring, the present study examined maternal brood size and offspring body size in 14 strains and performed cross experiments in an ovoviviparous fish, guppy. Correlation analyses among the strains indicated that the strains with larger brood size have smaller offspring body size. Diallel and reciprocal crosses among four strains revealed a large maternal, but no paternal, contribution to offspring body size. To examine whether offspring body size is determined by maternal genotypes and whether offspring body size correlates with albinism, backcrosses were performed between a wild-type strain with large offspring body size and an albino strain. Offspring body size differed from both the parental strains in one generation of backcrossing but was not significantly different from the paternal strains after three generations. In the backcross generations, the offspring yielded by albino individuals showed significantly smaller body size than those yielded by normal individuals. These results indicated that offspring body size is determined by maternal genotypes, suggesting that offspring body size is influenced by the maternal albinism gene or the maternal locus linked with the albinism locus. The significant negative correlation between maternal brood size and offspring body size detected through our experiments showed that the maternal genetic character of brood size strongly correlated with offspring body size, indicating genetic trade-off between maternal brood size and offspring body size in the guppy.