Transgenic Beet Research Articles

Over-expression of the Hybrid Aspen Homeobox <i>PttKN1</i> Gene in Red Leaf Beet Induced Altered Coloration of Leaves

PttKN1 (Populus tremula × tremuloides KNOTTED1) gene belongs to the KNOXI gene family. It plays an important role in plant development, typically in meristem initiation, maintenance and organogenesis, and potentially in plant coloration. To investigate the gene functions further, it was introduced into red leaf beet by the floral dip method mediated via Agrobacterium tumefaciens. The transformants demonstrated typical phenotypes as with other PttKN1 transformants. These alterations were very different from the morphology of the wild type. Among them, morphological modification of changed color throughout the entire plant from claret of wild type to yellowish green was the highlight in those transgenic PttKN1-beet plants. The result of spraying selection showed that the PttKN1-beet plants had kanamycin resistance. PCR assay of the 35S-Promoter, NPTII and PttKN1 gene, PCR-Southern analysis of the NPTII and PttKN1 gene showed that the foreign PttKN1 gene had successfully integrated into the genome of beet plant. Furthermore, the results of RT-PCR analysis showed that the gene was ectopic expressed in transgenic plants. These data suggested that there is a correlation between the ectopic expression of PttKN1 gene and morphological alterations of beet plants. Pigment content assay showed that betaxanthins concentrations shared little difference between wild type and transgenic lines, while betacyanins content in transgenic plants was sharply decreased, indicating that the altered plant coloration of the transgenic beet plants may be caused by the changed betacyanins content. The tyrosinase study suggested that the sharply decreased of betacyanins content in transgenic plants was caused via the decreased tyrosinase level. Therefore, the reason for the altered plant coloration may be due to partial inhibition of betacyanin biosynthesis that was induced via the pleiotropic roles of PttKN1 gene.

Over-expression of the Hybrid Aspen Homeobox <i>PttKN1</i> Gene in Red Leaf Beet Induced Altered Coloration of Leaves

PttKN1 (Populus tremula × tremuloides KNOTTED1) gene belongs to the KNOXI gene family. It plays an important role in plant development, typically in meristem initiation, maintenance and organogenesis, and potentially in plant coloration. To investigate the gene functions further, it was introduced into red leaf beet by the floral dip method mediated via Agrobacterium tumefaciens. The transformants demonstrated typical phenotypes as with other PttKN1 transformants. These alterations were very different from the morphology of the wild type. Among them, morphological modification of changed color throughout the entire plant from claret of wild type to yellowish green was the highlight in those transgenic PttKN1-beet plants. The result of spraying selection showed that the PttKN1-beet plants had kanamycin resistance. PCR assay of the 35S-Promoter, NPTII and PttKN1 gene, PCR-Southern analysis of the NPTII and PttKN1 gene showed that the foreign PttKN1 gene had successfully integrated into the genome of beet plant. Furthermore, the results of RT-PCR analysis showed that the gene was ectopic expressed in transgenic plants. These data suggested that there is a correlation between the ectopic expression of PttKN1 gene and morphological alterations of beet plants. Pigment content assay showed that betaxanthins concentrations shared little difference between wild type and transgenic lines, while betacyanins content in transgenic plants was sharply decreased, indicating that the altered plant coloration of the transgenic beet plants may be caused by the changed betacyanins content. The tyrosinase study suggested that the sharply decreased of betacyanins content in transgenic plants was caused via the decreased tyrosinase level. Therefore, the reason for the altered plant coloration may be due to partial inhibition of betacyanin biosynthesis that was induced via the pleiotropic roles of PttKN1 gene.

The promoter of the nematode resistance gene Hs1pro-1 activates a nematode-responsive and feeding site-specific gene expression in sugar beet (Beta vulgaris L.) and Arabidopsis thaliana.

The Hs1pro-1 gene confers resistance to the beet cyst nematode Heterodera schachtii in sugar beet (Beta vulgaris L.) on the basis of a gene-for-gene relationship. RNA-gel blot analysis revealed that the transcript of Hs1pro-1 was present in uninfected roots of resistant beet at low levels but increased by about fourfold one day after nematode infection. Treatments of plants with external stimuli including salicylic acid, jasmonic acid, gibberellic acid and abscisic acid as well as wounding or salt stress did not result in changes in the gene transcription, indicating de novo transcription of Hs1pro-1 upon nematode infection specifically. To study transcriptional regulation of Hs1pro-1 expression at the cellular level, a 3082 bp genomic fragment representing the Hs1pro-1 promoter, isolated from the YAC-DNA housing the Hs1pro-1 gene, was fused to the beta-glucuronidase reporter gene (1832prm1::GUS) and transformed into susceptible beet roots and Arabidopsis plants, respectively. Fluorometric and histochemical GUS assays on transgenic beet roots and Arabidopsis plants carrying the 1832prm1::GUS construct demonstrated that the Hs1pro-1 promoter is functional in both species and drives a nematode responsive and feeding site-specific GUS-expression. GUS activity was detected as early as at initiation of the nematode feeding sites and GUS staining was restricted to the nematode feeding sites. To delineate the regulatory domains of the Hs1pro-1 promoter, fusion genes with various 5' deletions of the Hs1pro-1 promoter and the GUS gene were constructed and analysed in transgenic beet roots as well. Cis elements responsible for feeding site-specific gene expression reside between -355 and +247 from the transcriptional initiation site of Hs1pro-1 whereas an enhancer region necessary for higher gene expression is located between -1199 and -705 of the promoter. The Hs1pro-1 promoter drives a nematode feeding site-specific GUS expression in both sugar beet and Arabidopsis suggesting a conserved mechanism of regulation of Hs1pro-1 expression in these two species.

BIOSAFETY OF HYBRIDS BETWEEN TRANSGENIC VIRUS-RESISTANT SUGAR BEET AND SWISS CHARD

One important issue of biosafety research is whether gene flow from trans- genic crops to nontransgenic relatives causes unwanted effects. We carried out field trials with hybrids between transgenic sugar beets, and a close cultivated relative, Swiss chard. This hybrid also acts as a model for hybrids between sugar beet and wild/ weed beet (Beta vulgaris ssp. maritima). Transgenic beets with beet necrotic yellow vein virus (BNYVV) coat protein (cp), phosphinothricin-acetyl-transferase (bar), and neomycin- phospho-transferase (nptll) genes were hand-crossed to Swiss chard. The resulting F1 plants and controls were grown at two different BNYVV infestation levels and three different competitive conditions with Chenopodium album. Transgenic hybrids had consistently high- er biomass than controls under high background BNYVV infestation, and consistently lower biomass than controls under low background infestation. The transgenic hybrids had a significantly lower rate of bolting than controls at all sites. Competition with Ch. album always had a strong negative influence on the performance of all genotypes. We conclude that ecological implications due to the introduction and spread of virus-resistant transgenic hybrids will be observed only in those feral Swiss chard and wild beet populations where fitness is significantly influenced by high infestations of BNYVV.

Biosafety of Hybrids between Transgenic Virus-Resistant Sugar Beet and Swiss Chard

One important issue of biosafety research is whether gene flow from transgenic crops to nontransgenic relatives causes unwanted effects. We carried out field trials with hybrids between transgenic sugar beets, and a close cultivated relative, Swiss chard. This hybrid also acts as a model for "weed beet" hybrids between sugar beet and wild/weed beet (Beta vulgaris ssp. maritima). Transgenic beets with beet necrotic yellow vein virus (BNYVV) coat protein (cp), phosphinothricin-acetyl-transferase (bar), and neomycin-phospho-transferase (nptII) genes were hand-crossed to Swiss chard. The resulting F1 plants and controls were grown at two different BNYVV infestation levels and three different competitive conditions with Chenopodium album. Transgenic hybrids had consistently higher biomass than controls under high background BNYVV infestation, and consistently lower biomass than controls under low background infestation. The transgenic hybrids had a significantly lower rate of bolting than controls at all sites. Competition with Ch. album always had a strong negative influence on the performance of all genotypes. We conclude that ecological implications due to the introduction and spread of virus-resistant transgenic hybrids will be observed only in those feral Swiss chard and wild beet populations where fitness is significantly influenced by high infestations of BNYVV.

Genetic diversity and gene flow between wild, cultivated and weedy forms of Beta vulgaris L. (Chenopodiaceae), assessed by RFLP and microsatellite markers

Beets belonging to the species Beta vulgaris L. can be found in crop, wild and weedy forms, all of which are interfertile. We studied the intra-specific genetic relationships of about 300 individuals from 54 populations of various French geographic origins using nuclear molecular markers (five single-copy RFLP loci and one microsatellite locus). The patterns of diversity were congruent for both types of markers. Genetic diversity in wild beets appeared to be high, both in term of allele number and observed heterozygosity, whereas the narrowness of the cultivated-beet gene pool was confirmed. Genetic distances between all forms showed that weed beets in northern France are intermediates between sugar beet and inland wild beets in south-western France. This analysis allowed us to infer the paternal origin of weed beets and furthermore, is in agreement with a previous study which focused on their maternal origin: weed beet infesting sugar-beet fields originated from accidental and recurrent hybridization between cultivated lines and ruderal inland wild beets during the production of commercial seeds in south-western France. Inland wild beets are genetically close to Mediterranean coastal wild beets, but differ from other coastal forms (from Biscay, Brittany and northern France). The study of gene flow in the beet complex contributes to the risk assessment of transgenic beets.

Nucleic acid and protein elimination during the sugar manufacturing process of conventional and transgenic sugar beets

The fate of cellular DNA during the standard purification steps of the sugar manufacturing process from conventional and transgenic sugar beets was determined. Indigenous nucleases of sugar beet cells were found to be active during the first extraction step (raw juice production) which was carried out at 70°C. This and the consecutive steps of the manufacturing process were validated in terms of DNA degradation by competitive PCR of added external DNA. Each step of the process proved to be very efficient in the removal of nucleic acids. Taken together, the purification steps have the potential to reduce the amount of DNA by a factor of >10 14, exceeding by far the total amount of DNA present in sugar beets. Furthermore, the gene products of the transgenes neomycin phosphotransferase and BNYVV (rhizomania virus) coat protein CP21 were shown to be removed during the purification steps, so that they could not be detected in the resulting white sugar. Thus, sugar obtained from conventional and transgenic beets is indistinguishable or substantially equivalent with respect to purity.

Influence of sugar beet breeding on populations of Beta vulgaris ssp. maritima in Italy

Abstract. It is highly probable that transgenic cultivars of sugar beet may influence wild beets in the seed‐production‐area of northern Italy. For this reason a survey of the local wild beet populations and their habitat characteristics was conducted in 1994/1995, i.e. before transgenic beets and their off spring could have become established. Wild beets (Beta vulgaris ssp. maritima) were found at 21 locations between Trieste and Cesenatico, as part of the natural littoral vegetation classified as Atriplicetum tatarici (Cakiletea maritimae) and Crithmetum (Crithmo‐Staticetea). The analysis of phenotypic attributes leads to a division into three different sub‐populations. Greenhouse studies on the morphology and life‐cycle attributes demonstrated actual gene flow between conventional seed beet and the examined wild beet population.

Competitiveness of transgenic sugar beet resistant to beet necrotic yellow vein virus and potential impact on wild beet populations

Beets are a crop of particular concern regarding invasiveness questions because they commonly become feral due to unintentional hybridization with annual forms of wild beets. In this study the performance of transgenic beets resistant to Beet Necrotic Yellow Vein Virus (BNYVV) was compared to the performance of unmodified material from the same breeding line. Both transgenic and control genotypes were also compared to a conventionally bred variety carrying a similar phenotypic trait. Field tests were developed in a step by step fashion in order to study seed emergence and competitiveness in early life stages. The tests quantified the potential ecological advantage of virus resistance under virus and non-virus infestation conditions. In experimental field releases in 1993 and 1994 in Germany, a small but increasingly clear ‘additive’ ecological advantage of the genetically engineered trait was detected. In both years and all competition treatments, the conventional tolerant variety performed best. An impact of naturalization on natural, non-agricultural habitats may appear in wild beet populations in Italian seed beet production areas. However, a survey of coastal areas of North-Eastern Italy found no virus infestation in 1994, suggesting that an increase in wild beet fitness is unlikely to occur.

Gene Transfer for Herbicide Resistance

Since 1990 Maribo Seed has conducted field trials with transgenic sugarbeet. Glyphosate tolerance has been the main objective. Trials are performed in Denmark, France, England, and Belgium, and in 1993 for the first time in USA. As sugarbeet varieties are hybrids, the transformation can be made on either multigerm, diploid fatherJines or monogerm, diploid motherJines (CMS-lines). Our development work has involved a range of different genetical constructs from Monsanto. The Agrobacterium vector transfers the insert into random chromosomal positions in single or multiple copies. Transformed plants are cloned to about 10 copies each and tested. If the GUS­ gene is present, GUS analysis on pollen can distinguish between plants which are homozygous and heterozygous for the introduced traits. Transgenic beets, but also progenies of transgenic beets, show considerable variation in the expression of the genes inserted (position effects). We have seen indications of interactions between transgenes and native genes. In most cases transgenes segregate according to Mendel's laws. The occurrence of meiotic irregularities, chimerics and multi-copy inserts can add to the complexity of developing transgenic lines, but classical breeding techniques are able to select Jines which are identical to the parental line except for the introduced trait.