Tomato POLLEN DEFICIENT 2 encodes a G-type lectin receptor kinase required for viable pollen grain formation.

Pollen development is a crucial biological process indispensable for seed set in flowering plants and for successful crop breeding. However, little is known about the molecular mechanisms regulating pollen development in crop species. This study reports a novel male-sterile tomato mutant, pollen deficient 2 (pod2), characterized by the production of non-viable pollen grains and resulting in the development of small parthenocarpic fruits. A combined strategy of mapping-by-sequencing and RNA interference-mediated gene silencing was used to prove that the pod2 phenotype is caused by the loss of Solanum lycopersicum G-type lectin receptor kinase II.9 (SlG-LecRK-II.9) activity. In situ hybridization of floral buds showed that POD2/SlG-LecRK-II.9 is specifically expressed in tapetal cells and microspores at the late tetrad stage. Accordingly, abnormalities in meiosis and tapetum programmed cell death in pod2 occurred during microsporogenesis, resulting in the formation of four dysfunctional microspores leading to an aberrant microgametogenesis process. RNA-seq analyses supported the existence of alterations at the final stage of microsporogenesis, since we found tomato deregulated genes whose counterparts in Arabidopsis are essential for the normal progression of male meiosis and cytokinesis. Collectively, our results revealed the essential role of POD2/SlG-LecRK-II.9 in regulating tomato pollen development.

Tomato CRABS CLAW paralogues interact with chromatin remodelling factors to mediate carpel development and floral determinacy.

CRABS CLAW (CRC) orthologues play a crucial role in floral meristem (FM) determinacy and gynoecium formation across angiosperms, the key developmental processes for ensuring successful plant reproduction and crop production. However, the mechanisms behind CRC mediated FM termination are far from fully understood. Here, we addressed the functional characterization of tomato (Solanum lycopersicum) paralogous CRC genes. Using mapping-by-sequencing, RNA interference and CRISPR/Cas9 techniques, expression analyses, protein-protein interaction assays and Arabidopsis complementation experiments, we examined their potential roles in FM determinacy and carpel formation. We revealed that the incomplete penetrance and variable expressivity of the indeterminate carpel-inside-carpel phenotype observed in fruit iterative growth (fig) mutant plants are due to the lack of function of the S. lycopersicum CRC homologue SlCRCa. Furthermore, a detailed functional analysis of tomato CRC paralogues, SlCRCa and SlCRCb, allowed us to propose that they operate as positive regulators of FM determinacy by acting in a compensatory and partially redundant manner to safeguard the proper formation of flowers and fruits. Our results uncover for the first time the physical interaction of putative CRC orthologues with members of the chromatin remodelling complex that epigenetically represses WUSCHEL expression through histone deacetylation to ensure the proper termination of floral stem cell activity.

Approaching the genetic dissection of indirect adventitious organogenesis process in tomato explants.

The screening of 862 T-DNA lines was carried out to approach the genetic dissection of indirect adventitious organogenesis in tomato. Several mutants defective in different phases of adventitious organogenesis, namely callus growth (tdc-1), bud differentiation (tdb-1, -2, -3) and shoot-bud development (tds-1) were identified and characterized. The alteration of the TDC-1 gene blocked callus proliferation depending on the composition of growth regulators in the culture medium. Calli from tds-1 explants differentiated buds but did not develop normal shoots. Histological analysis showed that their abnormal development is due to failure in the organization of normal adventitious shoot meristems. Interestingly, tdc-1 and tds-1 mutant plants were indistinguishable from WT ones, indicating that the respective altered genes play specific roles in cell proliferation from explant cut zones (TDC-1 gene) or in the organization of adventitious shoot meristems (TDS-1 gene). Unlike the previous, plants of the three mutants defective in the differentiation of adventitious shoot-buds (tdb-1, -2, -3) showed multiple changes in vegetative and reproductive traits. Cosegregation analyses revealed the existence of an association between the phenotype of the tdb-3 mutant and a T-DNA insert, which led to the discovery that the SlMAPKKK17 gene is involved in the shoot-bud differentiation process.

The Tomato SlVIPP1 Gene Is Required for Plant Survival Through the Proper Development of Chloroplast Thylakoid Membrane.

Since membranes play essential roles in all living beings, all cells have developed mechanisms for efficient and fast repair of membrane damage. In Escherichia coli, the Phage shock stress A (PspA) protein is involved in the maintenance of the integrity of its inner membrane in response to the damage produced by exposure to stress conditions. A role in thylakoid membrane maintenance and reorganization has been proposed for Vesicle Inducing Protein in Plastid 1 (VIPP1), the putative PspA ortholog in Arabidopsis thaliana. While some membranes of plant cells have been extensively studied, the biosynthesis and maintenance of chloroplast thylakoid membrane remains poorly known. Here, we report the cloning and functional characterization of the tomato (Solanum lycopersicum L.) ortholog of Escherichia coli PspA and Arabidopsis thaliana VIPP1, which we dubbed SlVIPP1. Our genetic and molecular characterization of slvipp1, an insertional mutant, allowed us to conclude that the tomato SlVIPP1 gene is needed for development, as Arabidopsis VIPP1, but not Escherichia coli PspA. Homozygous slvipp1 tomato plants are albino and exhibit early lethality and highly aberrant chloroplast development with almost complete absence of thylakoids. The phenotype of tomato RNAi lines and that of additional slvipp1 alleles generated by CRISPR/Cas9 gene editing technology confirmed that the morphological and histological aberrations shown by slvipp1 homozygotes are caused by VIPP1 lack of function. We also found that tomato SlVIPP1 overexpression does not cause any visible effect on plant morphology and viability. Our work with slvipp1 plants evidences that SlVIPP1 is an essential gene required for tomato survival, since its function is crucial for the proper formation and/or maintenance of thylakoid membranes.

Na+ transporter HKT1;2 reduces flower Na+ content and considerably mitigates the decline in tomato fruit yields under saline conditions.

Genes encoding HKT1-like Na+ transporters play a key role in the salinity tolerance mechanism in Arabidopsis and other plant species by retrieving Na+ from the xylem of different organs and tissues. In this study, we investigated the role of two HKT1;2 allelic variants in tomato salt tolerance in relation to vegetative growth and fruit yield in plants subjected to salt treatment in a commercial greenhouse under real production conditions. We used two near-isogenic lines (NILs), homozygous for either the Solanum lycopersicum (NIL17) or S. cheesmaniae (NIL14) allele, at HKT1;2 loci and their respective RNAi-Sl/ScHKT1;2 lines. The results obtained show that both ScHKT1;2- and SlHKT1;2-silenced lines display hypersensitivity to salinity associated with an altered leaf Na+/K+ ratio, thus confirming that HKT1;2 plays an important role in Na+ homeostasis and salinity tolerance in tomato. Both silenced lines also showed Na+ over-accumulation and a slight, but significant, reduction in K+ content in the flower tissues of salt-treated plants and consequently a higher Na+/K+ ratio as compared to the respective unsilenced lines. This altered Na+/K+ ratio in flower tissues is associated with a sharp reduction in fruit yield, measured as total fresh weight and number of fruits, in both silenced lines under salinity conditions. Our findings demonstrate that Na+ transporter HKT1;2 protects the flower against Na+ toxicity and mitigates the reduction in tomato fruit yield under salinity conditions.

The res (restored cell structure by salinity) tomato mutant reveals the role of the DEAD-box RNA helicase SlDEAD39 in plant development and salt response.

Increasing evidences highlight the importance of DEAD-box RNA helicases in plant development and stress responses. In a previous study, we characterized the tomato res mutant (restored cell structure by salinity), showing chlorosis and development alterations that reverted under salt-stress conditions. Map-based cloning demonstrates that RES gene encodes SlDEAD39, a chloroplast-targeted DEAD-box RNA helicase. Constitutive expression of SlDEAD39 complements the res mutation, while the silencing lines had a similar phenotype than res mutant, which is also reverted under salinity. Functional analysis of res mutant proved SlDEAD39 is involved in the in vivo processing of the chloroplast, 23S rRNA, at the hidden break-B site, a feature also supported by in vitro binding experiments of the protein. In addition, our results show that other genes coding for chloroplast-targeted DEAD-box proteins are induced by salt-stress, which might explain the rescue of the res mutant phenotype. Interestingly, salinity restored the phenotype of res adult plants by increasing their sugar content and fruit yield. Together, these results propose an unprecedented role of a DEAD-box RNA helicase in regulating plant development and stress response through the proper ribosome and chloroplast functioning, which, in turn, represents a potential target to improve salt tolerance in tomato crops.

ENO regulates tomato fruit size through the floral meristem development network.

A dramatic evolution of fruit size has accompanied the domestication and improvement of fruit-bearing crop species. In tomato (Solanum lycopersicum), naturally occurring cis-regulatory mutations in the genes of the CLAVATA-WUSCHEL signaling pathway have led to a significant increase in fruit size generating enlarged meristems that lead to flowers with extra organs and bigger fruits. In this work, by combining mapping-by-sequencing and CRISPR/Cas9 genome editing methods, we isolated EXCESSIVE NUMBER OF FLORAL ORGANS (ENO), an AP2/ERF transcription factor which regulates floral meristem activity. Thus, the ENO gene mutation gives rise to plants that yield larger multilocular fruits due to an increased size of the floral meristem. Genetic analyses indicate that eno exhibits synergistic effects with mutations at the LOCULE NUMBER (encoding SlWUS) and FASCIATED (encoding SlCLV3) loci, two central players in the evolution of fruit size in the domestication of cultivated tomatoes. Our findings reveal that an eno mutation causes a substantial expansion of SlWUS expression domains in a flower-specific manner. In vitro binding results show that ENO is able to interact with the GGC-box cis-regulatory element within the SlWUS promoter region, suggesting that ENO directly regulates SlWUS expression domains to maintain floral stem-cell homeostasis. Furthermore, the study of natural allelic variation of the ENO locus proved that a cis-regulatory mutation in the promoter of ENO had been targeted by positive selection during the domestication process, setting up the background for significant increases in fruit locule number and fruit size in modern tomatoes.

Alq mutation increases fruit set rate and allows the maintenance of fruit yield under moderate saline conditions.

Arlequin (Alq) is a gain-of-function mutant whose most relevant feature is that sepals are able to become fruit-like organs due to the ectopic expression of the ALQ-TAGL1 gene. The role of this gene in tomato fruit ripening was previously demonstrated. To discover new functional roles for ALQ-TAGL1, and most particularly its involvement in the fruit set process, a detailed characterization of Alq yield-related traits was performed. Under standard conditions, the Alq mutant showed a much higher fruit set rate than the wild type. A significant percentage of Alq fruits were seedless. The results showed that pollination-independent fruit set in Alq is due to early transition from flower to fruit. Analysis of endogenous hormones in Alq suggests that increased content of cytokinins and decreased level of abscisic acid may account for precocious fruit set. Comparative expression analysis showed relevant changes of several genes involved in cell division, gibberellin metabolism, and the auxin signalling pathway. Since pollination-independent fruit set may be a very useful strategy for maintaining fruit production under adverse conditions, fruit set and yield in Alq plants under moderate salinity were assessed. Interestingly, Alq mutant plants showed a high yield under saline conditions, similar to that of Alq and the wild type under unstressed conditions.

Phenotypic and genetic characterization of tomato mutants provides new insights into leaf development and its relationship to agronomic traits.

BACKGROUND: Tomato mutants altered in leaf morphology are usually identified in the greenhouse, which demands considerable time and space and can only be performed in adequate periods. For a faster but equally reliable scrutiny method we addressed the screening in vitro of 971 T-DNA lines. Leaf development was evaluated in vitro in seedlings and shoot-derived axenic plants. New mutants were characterized in the greenhouse to establish the relationship between in vitro and in vivo leaf morphology, and to shed light on possible links between leaf development and agronomic traits, a promising field in which much remains to be discovered. RESULTS: Following the screening in vitro of tomato T-DNA lines, putative mutants altered in leaf morphology were evaluated in the greenhouse. The comparison of results in both conditions indicated a general phenotypic correspondence, showing that in vitro culture is a reliable system for finding mutants altered in leaf development. Apart from providing homogeneous conditions, the main advantage of screening in vitro lies in the enormous time and space saving. Studies on the association between phenotype and nptII gene expression showed co-segregation in two lines (P > 99%). The use of an enhancer trap also allowed identifying gain-of-function mutants through reporter expression analysis. These studies suggested that genes altered in three other mutants were T-DNA tagged. New mutants putatively altered in brassinosteroid synthesis or perception, mutations determining multiple pleiotropic effects, lines affected in organ curvature, and the first tomato mutant with helical growth were discovered. Results also revealed new possible links between leaf development and agronomic traits, such as axillary branching, flower abscission, fruit development and fruit cracking. Furthermore, we found that the gene tagged in mutant 2635-MM encodes a Sterol 3-beta-glucosyltransferase. Expression analysis suggested that abnormal leaf development might be due to the lack-off-function of this gene. CONCLUSION: In vitro culture is a quick, efficient and reliable tool for identifying tomato mutants altered in leaf morphology. The characterization of new mutants in vivo revealed new links between leaf development and some agronomic traits. Moreover, the possible implication of a gene encoding a Sterol 3-beta-glucosyltransferase in tomato leaf development is reported.

Developmental role of the tomato Mediator complex subunit MED18 in pollen ontogeny.

Pollen development is a crucial step in higher plants, which not only makes possible plant fertilization and seed formation, but also determines fruit quality and yield in crop species. Here, we reported a tomato T-DNA mutant, pollen deficient1 (pod1), characterized by an abnormal anther development and the lack of viable pollen formation, which led to the production of parthenocarpic fruits. Genomic analyses and the characterization of silencing lines proved that pod1 mutant phenotype relies on the tomato SlMED18 gene encoding the subunit 18 of Mediator multi-protein complex involved in RNA polymerase II transcription machinery. The loss of SlMED18 function delayed tapetum degeneration, which resulted in deficient microspore development and scarce production of viable pollen. A detailed histological characterization of anther development proved that changes during microgametogenesis and a significant delay in tapetum degeneration are associated with a high proportion of degenerated cells and, hence, should be responsible for the low production of functional pollen grains. Expression of pollen marker genes indicated that SlMED18 is essential for the proper transcription of a subset of genes specifically required to pollen formation and fruit development, revealing a key role of SlMED18 in male gametogenesis of tomato. Additionally, SlMED18 is able to rescue developmental abnormalities of the Arabidopsis med18 mutant, indicating that most biological functions have been conserved in both species.

The SlCBL10 Calcineurin B-Like Protein Ensures Plant Growth under Salt Stress by Regulating Na+ and Ca2+ Homeostasis.

Characterization of a new tomato (Solanum lycopersicum) T-DNA mutant allowed for the isolation of the CALCINEURIN B-LIKE PROTEIN 10 (SlCBL10) gene whose lack of function was responsible for the severe alterations observed in the shoot apex and reproductive organs under salinity conditions. Physiological studies proved that SlCBL10 gene is required to maintain a proper low Na+/Ca2+ ratio in growing tissues allowing tomato growth under salt stress. Expression analysis of the main responsible genes for Na+ compartmentalization (i.e. Na+/H+ EXCHANGERs, SALT OVERLY SENSITIVE, HIGH-AFFINITY K+ TRANSPORTER 1;2, H+-pyrophosphatase AVP1 [SlAVP1] and V-ATPase [SlVHA-A1]) supported a reduced capacity to accumulate Na+ in Slcbl10 mutant leaves, which resulted in a lower uploading of Na+ from xylem, allowing the toxic ion to reach apex and flowers. Likewise, the tomato CATION EXCHANGER 1 and TWO-PORE CHANNEL 1 (SlTPC1), key genes for Ca2+ fluxes to the vacuole, showed abnormal expression in Slcbl10 plants indicating an impaired Ca2+ release from vacuole. Additionally, complementation assay revealed that SlCBL10 is a true ortholog of the Arabidopsis (Arabidopsis thaliana) CBL10 gene, supporting that the essential function of CBL10 is conserved in Arabidopsis and tomato. Together, the findings obtained in this study provide new insights into the function of SlCBL10 in salt stress tolerance. Thus, it is proposed that SlCBL10 mediates salt tolerance by regulating Na+ and Ca2+ fluxes in the vacuole, cooperating with the vacuolar cation channel SlTPC1 and the two vacuolar H+-pumps, SlAVP1 and SlVHA-A1, which in turn are revealed as potential targets of SlCBL10.

Albino T-DNA tomato mutant reveals a key function of 1-deoxy-D-xylulose-5-phosphate synthase (DXS1) in plant development and survival.

Photosynthetic activity is indispensable for plant growth and survival and it depends on the synthesis of plastidial isoprenoids as chlorophylls and carotenoids. In the non-mevalonate pathway (MEP), the 1-deoxy-D-xylulose-5-phosphate synthase 1 (DXS1) enzyme has been postulated to catalyze the rate-limiting step in the formation of plastidial isoprenoids. In tomato, the function of DXS1 has only been studied in fruits, and hence its functional relevance during plant development remains unknown. Here we report the characterization of the wls-2297 tomato mutant, whose severe deficiency in chlorophylls and carotenoids promotes an albino phenotype. Additionally, growth of mutant seedlings was arrested without developing vegetative organs, which resulted in premature lethality. Gene cloning and silencing experiments revealed that the phenotype of wls-2297 mutant was caused by 38.6 kb-deletion promoted by a single T-DNA insertion affecting the DXS1 gene. This was corroborated by in vivo and molecular complementation assays, which allowed the rescue of mutant phenotype. Further characterization of tomato plants overexpressing DXS1 and comparative expression analysis indicate that DXS1 may play other important roles besides to that proposed during fruit carotenoid biosynthesis. Taken together, these results demonstrate that DXS1 is essentially required for the development and survival of tomato plants.

A collection of enhancer trap insertional mutants for functional genomics in tomato.

With the completion of genome sequencing projects, the next challenge is to close the gap between gene annotation and gene functional assignment. Genomic tools to identify gene functions are based on the analysis of phenotypic variations between a wild type and its mutant; hence, mutant collections are a valuable resource. In this sense, T-DNA collections allow for an easy and straightforward identification of the tagged gene, serving as the basis of both forward and reverse genetic strategies. This study reports on the phenotypic and molecular characterization of an enhancer trap T-DNA collection in tomato (Solanum lycopersicum L.), which has been produced by Agrobacterium-mediated transformation using a binary vector bearing a minimal promoter fused to the uidA reporter gene. Two genes have been isolated from different T-DNA mutants, one of these genes codes for a UTP-glucose-1-phosphate uridylyltransferase involved in programmed cell death and leaf development, which means a novel gene function reported in tomato. Together, our results support that enhancer trapping is a powerful tool to identify novel genes and regulatory elements in tomato and that this T-DNA mutant collection represents a highly valuable resource for functional analyses in this fleshy-fruited model species.

The sodium transporter encoded by the HKT1;2 gene modulates sodium/potassium homeostasis in tomato shoots under salinity.

Excessive soil salinity diminishes crop yield and quality. In a previous study in tomato, we identified two closely linked genes encoding HKT1-like transporters, HKT1;1 and HKT1;2, as candidate genes for a major quantitative trait locus (kc7.1) related to shoot Na+ /K+ homeostasis - a major salt tolerance trait - using two populations of recombinant inbred lines (RILs). Here, we determine the effectiveness of these genes in conferring improved salt tolerance by using two near-isogenic lines (NILs) that were homozygous for either the Solanum lycopersicum allele (NIL17) or for the Solanum cheesmaniae allele (NIL14) at both HKT1 loci; transgenic lines derived from these NILs in which each HKT1;1 and HKT1;2 had been silenced by stable transformation were also used. Silencing of ScHKT1;2 and SlHKT1;2 altered the leaf Na+ /K+ ratio and caused hypersensitivity to salinity in plants cultivated under transpiring conditions, whereas silencing SlHKT1;1/ScHKT1;1 had a lesser effect. These results indicate that HKT1;2 has the more significant role in Na+ homeostasis and salinity tolerance in tomato.

Heterologous expression of the yeast HAL5 gene in tomato enhances salt tolerance by reducing shoot Na+ accumulation in the long term.

For salt tolerance to be achieved in the long-term plants must regulate Na(+)/K(+) homeostasis over time. In this study, we show that the salt tolerance induced by overexpression of the yeast HAL5 gene in tomato (Solanum lycopersicum) was related to a lower leaf Na(+) accumulation in the long term, by reducing Na(+) transport from root to shoot over time regardless of the severity of salt stress. Furthermore, maintaining Na(+)/K(+) homeostasis over time was associated with changes in the transcript levels of the Na(+) and K(+) transporters such as SlHKT1;2 and SlHAK5. The expression of SlHKT1;2 was upregulated in response to salinity in roots of transgenic plants but downregulated in the roots of wild-type (WT) plants, which seems to be related to the lower Na(+) transport rate from root to shoot in transgenic plants. The expression of the SlHAK5 increased in roots and leaves of both WT and transgenic plants under salinity. However, this increase was much higher in the leaves of transgenic plants than in those of WT plants, which may be associated with the ability of transgenic leaves to maintain Na(+)/K(+) homeostasis over time. Taken together, the results show that the salt tolerance mechanism induced by HAL5 overexpression in tomato is related to the appropriate regulation of ion transport from root to shoot and maintenance of the leaf Na(+)/K(+) homeostasis through modulation of SlHKT1 and SlHAK5 over time.

Production of engineered long-life and male sterile Pelargonium plants.

BACKGROUND: Pelargonium is one of the most popular garden plants in the world. Moreover, it has a considerable economic importance in the ornamental plant market. Conventional cross-breeding strategies have generated a range of cultivars with excellent traits. However, gene transfer via Agrobacterium tumefaciens could be a helpful tool to further improve Pelargonium by enabling the introduction of new genes/traits. We report a simple and reliable protocol for the genetic transformation of Pelargonium spp. and the production of engineered long-life and male sterile Pelargonium zonale plants, using the pSAG12::ipt and PsEND1::barnase chimaeric genes respectively. RESULTS: The pSAG12::ipt transgenic plants showed delayed leaf senescence, increased branching and reduced internodal length, as compared to control plants. Leaves and flowers of the pSAG12::ipt plants were reduced in size and displayed a more intense coloration. In the transgenic lines carrying the PsEND1::barnase construct no pollen grains were observed in the modified anther structures, which developed instead of normal anthers. The locules of sterile anthers collapsed 3-4 days prior to floral anthesis and, in most cases, the undeveloped anther tissues underwent necrosis. CONCLUSION: The chimaeric construct pSAG12::ipt can be useful in Pelargonium spp. to delay the senescence process and to modify plant architecture. In addition, the use of engineered male sterile plants would be especially useful to produce environmentally friendly transgenic plants carrying new traits by preventing gene flow between the genetically modified ornamentals and related plant species. These characteristics could be of interest, from a commercial point of view, both for pelargonium producers and consumers.

Overexpression of dehydrin tas14 gene improves the osmotic stress imposed by drought and salinity in tomato.

One strategy to increase the level of drought and salinity tolerance is the transfer of genes codifying different types of proteins functionally related to macromolecules protection, such as group 2 of late embryogenesis abundant (LEA) proteins or dehydrins. The TAS14 dehydrin was isolated and characterized in tomato and its expression was induced by osmotic stress (NaCl and mannitol) and abscisic acid (ABA) [Godoy et al., Plant Mol Biol 1994;26:1921-1934], yet its function in drought and salinity tolerance of tomato remains elusive. In this study, transgenic tomato plants overexpressing tas14 gene under the control of the 35SCaMV promoter were generated to assess the function of tas14 gene in drought and salinity tolerance. The plants overexpressing tas14 gene achieved improved long-term drought and salinity tolerance without affecting plant growth under non-stress conditions. A mechanism of osmotic stress tolerance via osmotic potential reduction and solutes accumulation, such as sugars and K(+) is operating in tas14 overexpressing plants in drought conditions. A similar mechanism of osmotic stress tolerance was observed under salinity. Moreover, the overexpression of tas14 gene increased Na(+) accumulation only in adult leaves, whereas in young leaves, the accumulated solutes were K(+) and sugars, suggesting that plants overexpressing tas14 gene are able to distribute the Na(+) accumulation between young and adult leaves over a prolonged period in stressful conditions. Measurement of ABA showed that the action mechanism of tas14 gene is associated with an earlier and greater accumulation of ABA in leaves during short-term periods. A good feature for the application of this gene in improving drought and salt stress tolerance is the fact that its constitutive expression does not affect plant growth under non-stress conditions, and tolerance induced by overexpression of tas14 gene was observed at the different stress degrees applied to the long term.

An insertional mutagenesis programme with an enhancer trap for the identification and tagging of genes involved in abiotic stress tolerance in the tomato wild-related species Solanum pennellii.

Salinity and drought have a huge impact on agriculture since there are few areas free of these abiotic stresses and the problem continues to increase. In tomato, the most important horticultural crop worldwide, there are accessions of wild-related species with a high degree of tolerance to salinity and drought. Thus, the finding of insertional mutants with other tolerance levels could lead to the identification and tagging of key genes responsible for abiotic stress tolerance. To this end, we are performing an insertional mutagenesis programme with an enhancer trap in the tomato wild-related species Solanum pennellii. First, we developed an efficient transformation method which has allowed us to generate more than 2,000 T-DNA lines. Next, the collection of S. pennelli T(0) lines has been screened in saline or drought conditions and several presumptive mutants have been selected for their salt and drought sensitivity. Moreover, T-DNA lines with expression of the reporter uidA gene in specific organs, such as vascular bundles, trichomes and stomata, which may play key roles in processes related to abiotic stress tolerance, have been identified. Finally, the growth of T-DNA lines in control conditions allowed us the identification of different development mutants. Taking into account that progenies from the lines are being obtained and that the collection of T-DNA lines is going to enlarge progressively due to the high transformation efficiency achieved, there are great possibilities for identifying key genes involved in different tolerance mechanisms to salinity and drought.

Genetic and physiological characterization of the arlequin insertional mutant reveals a key regulator of reproductive development in tomato.

The genetic and phenotypic characterization of a new tomato (Solanum lycopersicum) insertional mutant, Arlequin (Alq) is reported. Alq mutant plants were affected in reproductive development and their sepals were homeotically converted into fleshy fruit-like organs. Molecular analysis demonstrated that a single copy of T-DNA was present in the mutant genome while genetic analysis confirmed that the mutant phenotype co-segregated with the T-DNA insertion and was inherited as a monogenic semi-dominant trait. The histological and scanning electron microscope analyses revealed cell identity changes in both external and internal tissues of Alq sepals. Flowers developed by Alq homozygous plants showed a severe mutant phenotype, since after fruit set, not only did the sepals become succulent but they also followed a ripening pattern similar to that of normal fruits. From a metabolic viewpoint, Alq sepals also behaved like a fruit, as they acquired the properties of a sink that acted alternatively and independently to the fruit. In fact, expression of regulatory genes controlling tomato fruit ripening was detected in Alq sepals at similar levels to those observed in mature fruits. Furthermore, the Alq mutation inhibited the development of the abscission zone in tomato flowers indicating that the JOINTLESS gene is regulated by ALQ. Results from the genetic and developmental characterization of the Alq tomato mutant suggest that the ALQ gene participates in the regulatory pathway controlling fruit ripening of tomato.

Efficient transformation of Kalanchoe blossfeldiana and production of male-sterile plants by engineered anther ablation.

Engineered male sterility in ornamental plants has many applications such as facilitate hybrid seed production, eliminate pollen allergens, reduce the need for deadheading to extend the flowering period, redirect resources from seeds to vegetative growth, increase flower longevity and prevent gene flow between genetically modified and related native plants. We have developed a reliable and efficient Agrobacterium-mediated protocol for the genetic transformation of different Kalanchoe blossfeldiana commercial cultivars. Transformation efficiency for cv. 'Hillary' was 55.3% whereas that of cv. 'Tenorio' reached 75.8%. Selection was carried out with the nptII gene and increasing the kanamycin concentration from 25 to 100 mg l(-1) allowed to reduced escapes from 50 to 60% to virtually 0%. This method was used to produce male-sterile plants through engineered anther ablation. In our approach, we tested a male sterility chimaeric gene construct (PsEND1::barnase) to evaluate its effectiveness and effect on phenotype. No significant differences were found in the growth patterns between the transgenic lines and the wild-type plants. No viable pollen grains were observed in the ablated anthers of any of the lines carrying the PsEND1::barnase construct, indicating that the male sterility was complete. In addition, seed set was completely abolished in all the transgenic plants obtained. Our engineered male-sterile approach could be used, alone or in combination with a female-sterility system, to reduce the invasive potential of new ornamentals, which has become an important environmental problem in many countries.