In Planta Transformation of Tomato

In this study, in planta transformation of tomato (Solanum lycopersicum L.), using fruit injection and floral dip, is reported. Agrobacterium tumefaciens strain EHA 105 containing one of three constructs, i.e., pROKIIAP1GUSint (carrying the Apetala 1 [AP1] gene), pROKIILFYGUSint (carrying the LEAFY [LFY] gene), or p35SGUSint (carrying the β-glucuronidase [GUS] gene), was used for plant transformation. For fruit injection transformation, no significant effects (p > 0.05) of the construct used were observed. The highest frequency of transformation was obtained following 48-h incubation of tomato fruit with bacterial cells harboring either one of the three constructs; transformation frequencies of 17%, 19%, and 21% for AP1, LFY, and GUS gene constructs, respectively, were obtained. When fruit maturity was evaluated in fruit injection experiments, mature red fruit resulted in higher frequency of transformants than immature green fruit with 40%, 35%, and 42% for AP1, LFY, and GUS gene constructs, respectively. For floral dip transformation, a higher number of transformants was obtained when the GUS gene construct was used instead of either the AP1 or LFY gene construct, thus suggesting a possible inhibitory effect of the flowering genes used. When flowers were transformed prior to rather than following pollination, they yielded a higher transformation frequency, 12% for the LFY construct and 23% for the GUS construct (p < 0.05), although no transformant was obtained with the AP1 gene construct. All putative GUS-positive transformants were analyzed using polymerase chain reaction and confirmed for the presence of the transgene. Compared to control plants, transgenic plants carrying either the AP1 or LFY transgene flowered earlier and showed several different morphological characters.     

The common bean growth habit gene PvTFL1y is a functional homolog of Arabidopsis TFL1

In a common bean plant exhibiting determinate growth, the terminal shoot meristem switches from a vegetative to reproductive state, resulting in a terminal inflorescence. Contrary to this, indeterminate growth habit results in a terminal meristem that remains vegetative where it further regulates the production of lateral vegetative and reproductive growth. In the last century, breeders have selected determinate growth habit, in combination with photoperiod insensitivity, to obtain varieties with a shorter flowering period, earlier maturation and ease of mechanized harvest. Previous work has identified TFL1 as a gene controlling determinate growth habit in Arabidopsis thaliana. In this work, we have validated that the Phaseolus vulgaris candidate gene, PvTFL1y, is the functional homolog of TFL1 using three independent lines of evidence. First, in a population of ~1,500 plants, PvTFL1y was found to co-segregate with the phenotypic locus for determinate growth habit (fin) on chromosome 01. Second, using quantitative PCR, we found that two unique haplotypes associated with determinacy at the PvTFL1y locus, a 4.1-kb retrotransposon and a splice-site mutation, cause mRNA abundance to decrease 20–133 fold, consistent with the recessive nature of fin. Finally, using a functional complementation approach, through Agrobacterium-mediated transformation of determinate Arabidopsis, we rescued tfl1-1 mutants with the wild-type PvTFL1y gene. Together, these three lines of evidence lead to the conclusion that PvTFL1y is the functional homolog of the Arabidopsis gene, TFL1, and is the gene responsible for naturally occurring variation for determinacy in common bean. Further work exploring the different haplotypes at the PvTFL1y locus may lead to improved plant architecture and phenology of common bean cultivars.     

In-vitro flower formation in leaf explants of tomato: effect of NaCl

Leaf explants of 24 cultivars and 2 F1 hybrids of the common tomato (Lycopersicon esculentum Mill.) and ofL. pimpinellifolium Brezh. were cultured on Murashige-Skoog medium containing different concentrations of NaCl. The cultures of 11 genotypes formed flower buds when cultured on medium containing 0.5% NaCl. Flower formation occurred either by direct differentiation from the leaf cultures or by transition of the apices of regenerated shoots from the vegetative state to floral buds. No flower formation occurred on medium without NaCl or media with 1.0% NaCl or more. There existed great differences in the capacity of in-vitro flower formation in the tomato leaf explants among the genotypes tested. The genotypes whose explants did form flowers were all of determinate growth habit.     

Dt2 Is a Gain-of-Function MADS-Domain Factor Gene That Specifies Semideterminacy in Soybean

Similar to Arabidopsis thaliana, the wild soybeans (Glycine soja) and many cultivars exhibit indeterminate stem growth specified by the shoot identity gene Dt1, the functional counterpart of Arabidopsis TERMINAL FLOWER1 (TFL1). Mutations in TFL1 and Dt1 both result in the shoot apical meristem (SAM) switching from vegetative to reproductive state to initiate terminal flowering and thus produce determinate stems. A second soybean gene (Dt2) regulating stem growth was identified, which, in the presence of Dt1, produces semideterminate plants with terminal racemes similar to those observed in determinate plants. Here, we report positional cloning and characterization of Dt2, a dominant MADS domain factor gene classified into the APETALA1/SQUAMOSA (AP1/SQUA) subfamily that includes floral meristem (FM) identity genes AP1, FUL, and CAL in Arabidopsis. Unlike AP1, whose expression is limited to FMs in which the expression of TFL1 is repressed, Dt2 appears to repress the expression of Dt1 in the SAMs to promote early conversion of the SAMs into reproductive inflorescences. Given that Dt2 is not the gene most closely related to AP1 and that semideterminacy is rarely seen in wild soybeans, Dt2 appears to be a recent gain-of-function mutation, which has modified the genetic pathways determining the stem growth habit in soybean.     

Effect of an enhanced CaMV 35S promoter and a fruit-specific promoter on uida gene expression in transgenic tomato plants

Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T₀ and T₁ plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T₁ plants segregated in a 3∶1 Mendelian ratio.     

Allelic variation at the linked AP1 and PhyC loci in hexaploid wheat is associated but not perfectly correlated with vernalization response

Vernalization requirement is an important trait in temperate crop plants such as wheat and must be considered when selecting varieties for cultivation under different climatic conditions. To determine the growth habit of wheat varieties, plants need to be grown under different vernalization regimes, a lengthy but necessary process for breeders involved in crossing winter with spring germplasm. If haplotypes can be associated with growth habit, then molecular marker assays that are reliable, cheap, and quick can be developed to assist in the selection of plants with the desired phenotype. We have analyzed 81 accessions that have different vernalization requirements and putative different origins of spring habit for sequence variation at the Apetala1 (AP1) locus, which underlies Vrn-1, and at the linked Phytochrome C (PhyC) locus. Good correspondence was found between the AP1 genotype and the PhyC haplotype for 77 of the 81 accessions. Two varieties displayed a recombination event between the AP1 and PhyC loci, and one variety carried a recombinant PhyC gene. In addition, one variety carried an apparent AP1 winter allele, but displayed the Vrn-A1 spring habit. The PhyC haplotype for this variety also indicated the presence of a Vrn-A1 spring allele. Our data suggest that both the AP1 promoter region and PhyC SNPs can be used as diagnostic markers for vernalization response at the vrn-A1 locus, but that neither are perfect tags.     

Plant growth and development influenced by transgenic insertion of bacterial chitinolytic enzymes

The role of the chitinolytic enzymes in plants is not necessarilyrestricted to plant defense. Tomato plants transformed with an endochitinaseand a chitobiosidase gene from Streptomyces albidoflavus andgrowth under greenhouse conditions showed a significant reduction in plantheight, and reduced time to flowering compared with the control(non-transformed) plants. The levels of chitobiosidase and endochitinaseactivity in the transgenic tomato plants were positively correlated with earlyflowering, and negatively correlated with plant height. We have not determinedwhether these effects are exclusively due to the expression of the transgenesof endochitinase and chitobiosidase from S. albidoflavus orthe additive effect of these 2 enzymes combined with the endogenouschitinolytic enzymes produced by the plants. However, when control plants were trimmed,early flowering was observed compared with the controls that were not trimmed, whichindicates that wound induced proteins such as chitinolytic enzymes affect thetime of flowering. In addition, the expression of the endochitinase andchitobiosidase genes significantly increased the number of flowers and fruit onthe plants, resulting in an increase in yield of fruit. One of the primarygoals of crop breeding programs is to increase the productivity of plants. These twogenes were directly associated with plant productivity, and should be studied further.     

FLORAL INDUCTION DOES NOT ALTER THE GROWTH PARAMETERS OF FUCHSIA

Floral induction by night interruption of Fuchsia hybrida cv. Lord Byron, a quantitative long-day plant with decussate phyllotaxis and an indeterminate flowering habit, altered neither the rate of leaf initiation nor the rate of leaf expansion; nor did flower initiation and development change the vegetative growth of the plants. This was diagnosed using plastochron duration and plastochron ratio measurements before, during, and after a 10-day induction period. A comparison between indeterminate and determinate flowering is made using these two parameters.     

Effects of simulated herbivory in three old field Compositae with different inflorescence architectures

The effects of simulated herbivory (early or late defoliation and cutting of the flowering shoot) on the growth and reproduction of three species of monocarpic composite forbs (Crepis pulchra, Picris hieracioides and C. foetida) with different inflorescence architectures were studied in experimental plots. For the three species studied, early defoliation had no significant effect on subsequent growth. In contrast, late defoliation, occurring at the start of the season of drought, had a negative effect on growth and reproduction in the two Crepis species, particularly C. foetida, but had less effect on P. hieracioides. Sexual biomass was more clearly affected by late defoliation than was vegetative biomass, although the effects differed markedly among species possibly as a result of differences in phenology. Clipping the flowering shoot removed about 3 times less biomass than late defoliation and had little effect on vegetative biomass. It had much greater effects on the sexual biomass in P. hieracioides and C. pulchra, and resulted in the production of many shoots sprouting from the rosette, allowing the treated plants to regain a vegetative biomass close to that of control plants. Clipping did however lead to the production of shorter shoots and a reduction in the number of capitula formed. In C. foetida, much branching occurred even when the main shoot was not cut; the architecture of individual plants was therefore only slightly changed by clipping the apical bud and the sexual biomass of this species was not affected by ablation of the flowering shoot. Overcompensation was found in only two families of C. pulchra for vegetative biomass. No over-compensation was found for sexual biomass, despite an increase in the number of flowering shoots in C. pulchra and P. hieracioides following clipping. However situations close to compensation for the vegetative biomass in the three species and in P. hieracioides for the sexual biomass were recorded. The response of the three study species to simulated herbivory were related to their architecture and to the time of defoliation.     

Development of a Simple PCR-based Marker for the Determination of Growth Habit in Vicia faba L. using a Candidate Gene Approach

We have developed the first molecular marker suitable for the selection of determinate growth habit in faba bean using the candidate gene approach. We obtained the sequences of TFL1/CEN like genes from public databases and designed primers on conserved domains. We used three cultivars with determinate growth habit and four accessions (two cultivars and two lines) with indeterminate growth habit. All these genotypes are used in our faba bean breeding program. A single monomorphic PCR fragment was obtained. A set of restriction enzymes was assayed. The enzyme Hind1II produced a clear polymorphism between determinate and indeterminate genotypes. This new cleaved amplified polymorphism (CAP) marker was tested using an F₂ population contrasting for growth habit derived from the cross ‘Verde Bonita’ × 2N52. This marker showed 100% efficiency in discriminating both types of genotypes. Moreover, the codominancy of this marker allows the detection of heterozygous individuals facilitating the breeding process when pyramiding different genes. The perfect cosegregation of the marker with the trait indicates that an orthologue of TFL1/CEN controls the growth habit in faba bean. This marker has been tested in all the genotypes used in our faba bean breeding program as donors of the determinate growth habit. Therefore, it is expected to work well in all the crosses performed with these parental lines as happens in the F₂ tested. The CAPS marker developed in this work will be useful for Marker Assisted Selection programs. In addition, this marker is useful for quality control to determinate the percentage of outsider seeds in commercial seed lots. Moreover, it is a valuable tool to breeders when submitting new faba bean varieties for registration since the method allows guaranteeing that outsider plants remains under the requested limit for registration.     

Ectopic expression of a poplar APETALA3-like gene in tobacco causes early flowering and fast growth

A MADS-box gene, designated PtAP3, was isolated from a floral bud cDNA library derived from Populus tomentosa. Analysis by multiple alignments of both nucleotide and amino acid sequences, together with phylogenetic analysis, revealed that PtAP3 is an ortholog of Arabidopsis AP3. Analysis of RNA extracts from vegetative and reproductive tissues of P. tomentosa by RT-PCR indicated that PtAP3 is expressed in roots, stems, leaves and vegetative and floral buds. Notably, the expression of PtAP3 fluctuated during floral bud development between September and February with differences between male and female buds. In the former, a gradual down-regulation during this period, interrupted by a slight up-regulation in December, was followed by a sharper up-regulation on February. In developing female floral buds, expression was stable from September to November, sharply up-regulated in December, and then gradually down-regulated until February. The functional role of PtAP3 was investigated in transgenic tobacco plants. Of 25 transformants, nine displayed an earlier flowering phenotype compared with the wild type plants. Furthermore, transgenic tobacco had faster growth and more leaves than untransformed controls. The traits proved to be heritable between the T0 and T1 generations. Our results demonstrate a regulatory role of the PtAP3 gene during plant flowering and growth and suggest that the gene may be an interesting target for genetic modification to induce early flowering in plants.     

Gibberellic acid causes earlier flowering and synchronizes fruit ripening of coffee

The effect of 100 mgl^–1 gibberellic acid (GA₃) on flowering and fruit ripening synchrony, fruit set, fruit fresh weight, and vegetative growth were studied for different size classes of coffee (Coffea arabica L. cv. Guatemalan) flower buds. Flower buds that were > 4 mm, but not developed to the candle stage at the time of GA₃ treatment, reached anthesis 20 days earlier than the controls, and their development was independent of precipitation, unlike the controls. Fruit from buds that were treated with GA₃ at the candle stage showed earlier and more synchronous ripening than the control, although no differences in flowering were found during anthesis. Buds that were smaller than 4 mm at the time of treatment did not respond to GA₃ applications. Treatment with GA₃ did not affect fruit set, fresh weight of fruits, or vegetative shoot growth.     

Increased Growth and Yield of Hydroponically Grown Greenhouse Tomato Plants Inoculated with Arbuscular Mycorrhizal Fungi and Fusarium oxysporum f. sp. radicis-lycopersici

The arbuscular mycorrhizal fungi Glomus monosporum, G. vesiculiferum, G. deserticola, G. intraradices, G. mosseae, and two unidentified species were tested to determine their effect on plant growth and fruit production of tomato (Lycopersicon esculentum Mill.) cv. Trust inoculated with Fusarium oxysporum f. sp. radicis-lycopersici (FORL) under near-commercial greenhouse conditions. Inoculation with G. monosporum and G. mosseae significantly increased fruit yield and fruit number of tomato plants grown hydroponically in sawdust. Plant height and plant dry weight increased significantly when inoculated with G. monosporum and G. mosseae. Further, plants inoculated with G. monosporum and G. mosseae showed significantly lower FORL root infection than the untreated control plants.     

Over-expression of a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene improves salt tolerance in an upland rice

To develop a salt-tolerant upland rice cultivar (Oryza sativa L.), OsNHX1, a vacuolar-type Na⁺/H⁺ antiporter gene from rice was transferred into the genome of an upland rice cultivar (IRAT109), using an Agrobacterium-mediated method. Seven independent transgenic calli lines were identified by polymerase chain reaction (PCR) analysis. These 35S::OsNHX1 transgenic plants displayed a little accelerated growth during seedling stage but showed delayed flowering time and a slight growth retardation phenotype during late vegetative stage, suggesting that the OsNHX1 has a novel function in plant development. Northern and western blot analyses showed that the expression levels of OsNHX1 mRNA and protein in the leaves of three independent transgenic plant lines were significantly higher than in the leaves of wild type (WT) plants. T₂ generation plants exhibited increased salt tolerance, showing delayed appearance and development of damage or death caused by salt stress, as well as improved recovery upon removal from this condition. Several physiological traits, such as increased Na⁺ content, and decreased osmotic potential in transgenic plants grown in high saline concentrations, further indicated that the transgenic plants had enhanced salt tolerance. Our results suggest the potential use of these transgenic plants for further agricultural applications in saline soil.     

Wheat SOC1 functions independently of WAP1/VRN1, an integrator of vernalization and photoperiod flowering promotion pathways

Heading time in bread wheat ( Triticum aestivum L.) is determined by three characters – vernalization requirement, photoperiodic sensitivity and narrow-sense earliness (earliness per se) – which are involved in the phase transition from vegetative to reproductive growth. The wheat APETALA1 ( AP1 )-like MADS-box gene, wheat AP1 ( WAP1 , identical with VRN1 ), has been identified as an integrator of vernalization and photoperiod flowering promotion pathways. A MADS-box gene, SUPPRESSOR OF OVEREXPRESSION OF CO 1 ( SOC1 ) is an integrator of flowering pathways in Arabidopsis . In this study, we isolated a wheat ortholog of SOC1 , wheat SOC1 ( WSOC1 ), and investigated its relationship to WAP1 in the flowering pathway. WSOC1 is expressed in young spikes but preferentially expressed in leaves. Expression starts before the phase transition and is maintained during the reproductive growth phase. Overexpression of WSOC1 in transgenic Arabidopsis plants caused early flowering under short-day conditions, suggesting that WSOC1 functions as a flowering activator in Arabidopsis . WSOC1 expression is affected neither by vernalization nor photoperiod, whereas it is induced by gibberellin at the seedling stage. Furthermore, WSOC1 is expressed in transgenic wheat plants in which WAP1 expression is cosuppressed. These findings indicate that WSOC1 acts in a pathway different from the WAP1 -related vernalization and photoperiod pathways.