Bret Morris

Selectable marker-free transgenic plants without sexual crossing: transient expression of cre recombinase and use of a conditional lethal dominant gene

Transgenic tobacco plants were produced that contained single-copy pART54 T-DNA, with a 35S-uidA gene linked to loxP-flanked kanamycin resistance (nptII) and cytosine deaminase (codA) genes. Retransformation of these plants with pCre1 (containing 35S transcribed cre recombinase and hygromycin (hpt) resistance genes) resulted in excision of the loxP-flanked genes from the genome. Phenotypes of progeny from selfed-retransformed plants confirmed nptII and codA excision and integration of the cre-linked hpt gene. To avoid integration of the hpt gene, and thereby generate plants totally free of marker genes, we attempted to transiently express the cre recombinase. Agrobacterium tumefaciens (pCre1) was cocultivated with leaf discs of two pART54-transformed lines and shoots were regenerated in the absence of hygromycin selection. Nineteen of 773 (0.25%) shoots showed tolerance to 5-fluorocytosine (5-fc) which is converted to the toxic 5-fluorouracil by cytosine deaminase. 5-fc tolerance in six shoots was found to be due to excision of the loxP-flanked region of the pART54 T-DNA. In four of these shoots excision could be attributed to cre expression from integrated pCre1 T-DNA, whereas in two shoots excision appeared to be a consequence of transient cre expression from pCre1 T-DNA molecules which had been transferred to the plant cells but not integrated into the genome. The absence of selectable marker genes was confirmed by the phenotype of the T1 progeny. Therefore, through transient cre expression, marker-free transgenic plants were produced without sexual crossing. This approach could be applicable to the elimination of marker genes from transgenic crops which must be vegetatively propagated to maintain their elite genotype.

Down-Regulation of TM29, a Tomato SEPALLATA Homolog, Causes Parthenocarpic Fruit Development and Floral Reversion

We have characterized the tomato (Lycopersicon esculentum Mill.) MADS box gene TM29 that shared a high amino acid sequence homology to the ArabidopsisSEP1, 2, and 3(SEPALLATA1, 2, and 3) genes. TM29 showed similar expression profiles toSEP1, with accumulation of mRNA in the primordia of all four whorls of floral organs. In addition, TM29 mRNA was detected in inflorescence and vegetative meristems. To understandTM29 function, we produced transgenic tomato plants in which TM29 expression was down-regulated by either cosuppression or antisense techniques. These transgenic plants produced aberrant flowers with morphogenetic alterations in the organs of the inner three whorls. Petals and stamens were green rather than yellow, suggesting a partial conversion to a sepalloid identity. Stamens and ovaries were infertile, with the later developing into parthenocarpic fruit. Ectopic shoots with partially developed leaves and secondary flowers emerged from the fruit. These shoots resembled the primary transgenic flowers and continued to produce parthenocarpic fruit and additional ectopic shoots. Based on the temporal and spatial expression pattern and transgenic phenotypes, we propose that TM29functions in floral organ development, fruit development, and maintenance of floral meristem identity in tomato.

Regeneration of transgenic plants from the commercial apple cultivar Royal Gala.

A transformation system was developed for the commercial apple (Malus X domestica Borkh.) cultivar Royal Gala. Leaf pieces from in vitro-grown shoots were cocultivated for 2 days with Agrobacterium tumefaciens strain LBA4404 containing the binary vectors pKIWI105 or pKIWI110. Shoots were produced on a shooting medium containing kanamycin (50 mg·L−1). A 2-day incubation period on kanamycin-free medium prior to antibiotic selection enhanced the regeneration of kanamycin-resistant shoots. The majority of the kanamycin-resistant shoots also expressed GUS (β-glucuronidase) activity. The putatively transformed shoots were rooted on a medium containing kanamycin (50 mg·L−1). Rooted plants were established in a greenhouse, and plants transformed with pKIWI110, which contains a mutant Arabidopsis acetolactate synthase gene, were shown to be resistant to the herbicide Glean™. Integration of T-DNA into the apple genome was confirmed by PCR and Southern hybridization analyses.

The nucleotide sequence of the infectious cloned dna component of tobacco yellow dwarf virus reveals features of geminiviruses infecting monocotyledonous plants

An infectious clone of the Australian geminivirus tobacco yellow dwarf virus (TobYDV) was constructed from virus-specific double-stranded DNA isolated from infected tobacco and used to demonstrate a single-component genome. The nucleotide sequence of TobYDV DNA comprises 2580 nucleotides. TobYDV DNA has three coding regions, two in the virion sense and one in the complementary sense, homologous to those identified for other geminiviruses, particularly those infecting monocotyledonous (monocot) plants. The complementary sense coding region is comprised of two overlapping reading frames, with an intron of 86 nucleotides. Efficient splicing of the mRNA for this coding region was observed in the infected dicotyledonous (dicot) hosts bean and tobacco despite the intron having an A + U content (57%) more typical of geminiviruses of monocot plants. TobYDV encapsidates a small oligonucleotide able to prime synthesis of the complementary DNA strand in vitro. The TobYDV genome organization, low A + U intron, and encapsidated oligonucleotide primer resemble those of the monocot-infecting geminiviruses. These results strongly suggest that TobYDV is a monocot geminivirus which has become adapted to dicot hosts.

Regulation of the activities of African cassava mosaic virus promoters by the AC1, AC2, and AC3 gene products

DNA fragments comprising each of the promoter regions from the geminivirus African cassava mosaic virus (ACMV) were cloned into the pUC18-based vector, pG1, producing transcriptional fusions with the beta-glucuronidase gene (GUS) and nopaline synthase terminator sequence. The relative activity of each promoter construct was analyzed by a GUS expression assay of extracts from Nicotiana clevelandii protoplasts coelectroporated with the GUS reporter constructs and constructs in which individual ACMV open reading frames (ORFs) were placed under control of a cauliflower mosaic virus 35 S promoter. Results suggest repression of the AC1 gene by its gene product, which is required for ACMV DNA synthesis. The promoter activity observed for the single promoter for the DNA A genes encoding functions of spread and the regulation of replication (AC2 and AC3 ORFs) was unaffected by coelectroporation with any of the ACMV ORF constructs. Promoters for the AV1 (coat protein) gene and the two DNA B genes (BV1 and BC1) were activated by electroporation of the AC2 ORF construct. To a lesser extent promoters for the AV1 and BV1 genes were activated with the AC3 ORF construct. The same pattern of promoter repression and activation was observed when transgenic N. benthamiana plants expressing the GUS reporter constructions were inoculated with ACMV DNA A.

Post-transcriptional silencing of chalcone synthase in petunia using a geminivirus-based episomal vector

A vector that produces DNA replicons (multicopy plant episomes) was constructed using elements of the geminivirus tobacco yellow dwarf virus (TYDV). All plant cells contain an integrated chromosomal T-DNA copy of the TYDV elements that provides a template for the production of episomes in the cell nucleus. Transgenic Petunia hybrida plants containing a CaMV 35S promoter-driven chalcone synthase A (ChsA) gene cloned into the episomal vector produced flowers with a white-spotted phenotype at high frequency. The spots were found at random locations in the petals and occurred in corresponding positions in both the upper and lower epidermis, indicating that the spots were non-clonal. The spotted phenotype was somatically stable and was inherited through meiosis. In white-spotted flower tissue, steady-state ChsA mRNA levels were downregulated but rates of RNA transcription were unaffected, suggesting that the phenotype resulted from post-transcriptional gene silencing of the endogenous and episomal ChsA genes. Increases in both the frequency and extent of gene silencing in flowers correlated with increases in episome copy number in mature flowers, flower buds and young and fully expanded leaves. Relatively small increases in episome copy number (less than threefold) appeared sufficient to trigger the gene-silenced phenotype.

Transformation of citrus embryogenic cells using particle bombardment and production of transgenic embryos

Abstract A transformation system was developed for the citrus variety tangelo ( Citrus reticulata Blanco × C. paradisi Macf.) cultivar ‘Page’. Embryogenic cells from suspension cultures were bombarded with tungsten particles coated with plasmid DNA which contained the uid A gene encoding β-glucuronidase (GUS) and the npt II gene conferring kanamycin resistance. More than 600 transient and 15 stable transformants were obtained per bombardment based on the number of GUS expression foci at 1 day and 8 weeks after bombardment. Pretreatment of cells with high osmotic potential (0.3 M sorbitol + 0.3 M mannitol) was found to enhance both transient and stable transformation efficiency. Between 2 and 3 months following bombardment, calli were formed on a medium containing kanamycin (100 mg·l −1 ). The majority of the kanamycin-resistant calli also expressed GUS activity. From these calli, embryos and plantlets were produced. Integration of the uid A and npt II genes into the tangelo genome was confirmed by polymerase chain reaction (PCR) and Southern analyses.

Ethylene inhibitors enhance in vitro root formation from apple shoot cultures

Abstract Effects of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and three ethylene inhibitors, AgNO3, aminoethoxyvinyglycine (AVG) and CoCl2, on root formation were tested in vitro using shoot cultures of the apple (Malus×domestica Borkh.) cultivar Royal Gala. ACC inhibited root formation by delaying root emergence and increasing callus formation at the bases of shoots. In contrast, ethylene inhibitors promoted root formation. Both AgNO3 and AVG at the appropriate concentrations increased the percentage of shoots producing roots and reduced callus formation at the base of these shoots. AgNO3 stimulated root emergence and enhanced root growth, while AVG increased the number of roots per shoot. CoCl2 slightly increased root number and rooting efficiency. These promotive effects may result from a reduction in ethylene concentration or inhibition of ethylene action. The results found in this study may be used to improve the rooting efficiency of other apple cultivars and rootstocks, and possibly of other plant species.

MDH1: an apple homeobox gene belonging to the BEL1 family.

Differential display was used to isolate genes differentially expressed early in fruit development of apple (Malus domestica Borkh.). This approach resulted in the isolation of MDH1, a homeobox gene with a homeodomain similar to that of BELL1 (BEL1), which is involved in regulation of ovule development in Arabidopsis. However, outside the homeodomain MDH1 is quite different from BEL1. In apple, MDH1 mRNA was predominantly found in flowers, expanding leaves and expanding fruit. In pre-anthesis flowers, in situ hybridization showed that MDH1 mRNA accumulated in ovules. To further investigate the function of this new homeobox gene, MDH1 was transformed into Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. The transgenic Arabidopsis plants showed dwarfing, reduced fertility and changes in carpel and fruit (silique) shape. The size and shape of the cells in the transgenic fruit was irregular. Both the transgenic phenotypes in Arabidopsis and the expression pattern of this gene in apple are consistent with the idea that MDH1 is likely to play an important role in control of plant fertility.

Analysis of the potential promoter sequences of African cassava mosaic virus by transient expression of the beta-glucuronidase gene.

DNA fragments from promoter regions of the geminivirus, African cassava mosaic virus, were cloned into pG1, a vector based on pUC18, producing transcriptional fusions with the beta-glucuronidase (GUS) gene and nopaline synthase termination sequence. The activity of each promoter construct was assessed by analysing the transient expression of GUS in Nicotiana clevelandii protoplasts. The results demonstrated that constructs containing the common region of DNA A showed much stronger promoter activity in the complementary sense than in the viral sense. These results were supported by the analysis of promoter activity in transgenic N. benthamiana plants. In comparison, in protoplasts a region upstream of the AC2 open reading frame was shown to have moderate promoter activity. Unlike DNA A, the complementary sense DNA B promoter constructs had weak activity; the viral sense DNA B promoter constructs appeared to be regulated by host factors. The implications of these results for the regulation of early and late genes are discussed.