Transgenic tomato (Lycopersicon esculentum L.) transformed with a binary vector in Agrobacterium rhizogenes: Non-chimeric origin of callus clone and low copy numbers of integrated vector T-DNA

Summary We transformed tomato (Lycopersicon esculentum L.) by using Agrobacterium rhizogenes containing two independent plasmids: the wild-type Ri-plasmid, and the vector plasmid, pARC8. The T-DNA of the vector plasmid contained a marker gene (Nos/Kan) encoding neomycin phosphotransferase which conferred resistance to kanamycin in transformed plant cells. Transgenic plants (R ₀) with normal phenotype were regenerated from transformed organogenic calli by the punctured cotyledon transformation method. Southern blot analysis of the DNA from these transgenic plants showed that one or two copies of the vector plasmid T-DNA, but none of the Ri-plamid T-DNA, were integrated into the plant genome. Different transgenic plants derived from the same callus clone showed an identical DNA banding pattern, indicating the non-chimeric origin of these plants. We also transformed tomato by using A. tumefaciens strain LBA4404 containing a disarmed Ti-plasmid (pAL4404), and a vector plasmid (pARC8). Transgenic plants derived via A. tumefaciens transformation, like those via A. rhizogenes, contained one to two copies of the integrated vector T-DNA. The kanamycin resistance trait in the progeny (R ₁) of most transgenic plants segregated at a ratio of 3:1, suggesting that the vector T-DNAs were integrated at a single site on a tomato chromosome. In some cases, the expression of the marker gene (Nos/Kan) seemed to be suppressed or lost in the progeny.     

Transformation of cultivated tomato by a binary vector in Agrobacterium rhizogenes: transgenic plants with normal phenotypes harbor binary vector T-DNA,but no Ri-plasmid T-DNA

Summary Cultivated tomato was genetically transformed using two procedures. In the first procedure, punctured cotyledons were infected with disarmed Agrobacterium tumefaciens strain LBA4404 or with A. rhizogenes strain A4, each containing the binary vector pARC8. The chimeric neomycin phosphotransferase (NPT II) gene on pARC8 conferred on transformed plant cells the ability to grow on medium containing kanamycin. Transformation reproducible yielded kanamycin-resistant transformants in different tomato genotypes. NPT II activity was detected in transformed calli and in transgenic plants. All of these plants were phenotypically normal, fertile and set seeds. Using the second procedure, inverted cotyledons, we recovered transformed tomato plants from A. rhizogenes-induced hairy roots. In this case, all of the transgenic plants exhibited phenotypes similar to hairy root-derived plants reported for other species. Southern blot analysis on these plants revealed that the plant DNA hybridized with both probes representing pARC8-T-DNA, and the T-DNAs of the A4 Ri-plasmid. However, southern analysis on those phenotypically normal transgenic plants from the first procedure revealed that only the pARC8-T-DNA was present in the plant genome, thus indicating that the pARC8-T-DNA integrated into the plant genome independently of the pRi A4-T-DNA. Genetic analysis of these phenotypically normal transgenic plants for the kanamycin-resistance trait showed Mendelian ratios, 31 and 11, for selfed (R1) and in crossed progeny, respectively.     

Efficient transformation of potato (Solanum tuberosum L.) using a binary vector in Agrobacterium rhizogenes

Summary We transformed three potato (Solanum tuberosum L.) genotypes by using A. rhizogenes or a mixture of A. rhizogenes and A. tumefaciens. Inoculations of potato stem segments were performed with Agrobacterium rhizogenes AM8703 containing two independent plasmids: the wild-type Ri-plasmid, pRI1855, and the binary vector plasmid, pBI121. In mixed inoculation experiments, Agrobacterium rhizogenes LBA1334 (pRI1855) and Agrobacterium tumefaciens AM8706 containing the disarmed Ti-plasmid (pAL4404) and the binary vector plasmid (pBI121) were mixed in a 11 ratio. The T-DNA of the binary vector plasmid pBI121 contained two marker genes encoding neomycin phosphotransferase, which confers resistance to kanamycin, and -glucuronidase. Both transformation procedures gave rise to hairy roots on potato stem segments within 2 weeks. With both procedures it was possible to obtain transformed hairy roots, able to grow on kanamycin and possessing -glucuronidase activity, without selection pressure. The efficiency of the A. rhizogenes AM8703 transformation, however, was much higher than that of the mixed transformation. Up to 60% of the hairy roots resulting from the former transformation method were kanamycin resistant and possessed -glucuronidase activity. There was no correlation between the height of the kanamycin resistance and that of the -glucuronidase activity in a root clone. Hairy roots obtained from a diploid potato genotype turned out to be diploid in 80% of the cases. Transformed potato plants were recovered from Agrobacterium rhizogenes AM8703-induced hairy roots.     

Somaclonal variation versus chemically induced mutagenesis in tomato (Lycopersicon esculentum L.)

Summary A comparison was made of the type and frequency of mutational events found in the progeny of tomato plants regenerated after one passage in vitro with those induced by chemical mutagenesis with ethyl methane sulphonate. Several mutants were recovered in the progeny of regenerated and mutagenized plants of two cultivars of tomato. They can be grouped into the following categories: seedling lethality, male sterility, resistance to Verticillium, short stature, change in number of lateral shoots or in leaf shape. The results indicate that the two sources of variability differ in their effect, changing the spectrum and frequency of the mutants as well as, at least in some cases, their pattern of segregation.     

Callose accumulation during treatment of tomato (Lycopersicon esculentum L.) cells with biotic elicitors

Time-course of induced accumulation of callose in tomato cells has been studied. Localization of callose in L. esculenthum cells was investigated by fluorescent microscopy technique, and the optimal time for its determination was found. Callose accumulation in tomato cells treated with different biotic elicitors was determined. Nonlinear dependence between callose accumulation and concentration of chitin oligomers (with 3-5 N-acetylglucosamine fragments) was established. Increasing of callose accumulation in tomato cells was proportional to the increase of concentration ofchitin dimer and chitosan in the culture medium.     

Enzymic properties and capacities of developing tomato (Lycopersicon esculentum L.) fruit plastids

To characterize the developmental stage of tomato fruits, chlorophyll content, photosynthetic O2 evolution and CO2 fixation of pericarp slices were determined. During the first developmental stages a higher expression level of the triose phosphate translocator was detected. Transport measurements revealed that both the hexose phosphate and the triose phosphate translocator are very likely to be active at this time. Plastidic and cytosolic fructose-1,6-bisphosphatase are active in green fruit pericarp, whereas in red pericarp only the cytosolic form is present. Tomato fruit chloroplasts are able to synthesize starch from Glc6P. Starch synthesis is strongly dependent on the addition of 3PGA and ATP and on plastid illumination. Fruit chloroplasts exhibit very low CO2 fixation rates and so the capacities of green pericarp slices were investigated. In relation to chlorophyll content, pericarp slices show the same capacity of starch synthesis as spinach or potato leaves. To investigate the presence of further reactions consuming the products of photosynthetic electron transport, the GOGAT activity was measured. In the light, glutamine/2-oxoglutarate-dependent formation of glutamate occurred with a high activity. In the presence of Glc6P only 18% of the light activity was obtained. Since the Glc6P-dependent activity is rather low, the release of ¹⁴CO2 from labelled [1-¹⁴C]-Glc6P was also measured. In the dark, the formation of glutamate and oxidation of Glc6P are very tightly coupled to each other in fruit chloroplasts.     

Agravitropic behaviour of roots of rapeseed (Brassica napus L.) transformed by Agrobacterium rhizogenes

Transgenic hairy roots of Brassica napus (cv. Omega) have been developed, using Agrobacterium rhizogenes strain AR 25, for use as a model system in the investigation of physiological and morphological differences between transgenic and normal roots. The basic parameters of growth and normal or altered gravitropical behaviour of hairy roots are for the first time presented in this paper together with an ultrastructural and morphological analysis of the root statocytes. The results obtained also represented the basis for the TRANSF0RM-experiment on the IML-2 mission performed onboard the Space Shuttle Columbia. Typical hairy root traits such as hormone-autonomous growth high growth rate, lateral branching, and changed/absence of gravitropism were detected. The transformed nature of the roots was confirmed by Southern blot analyses. The gravitropical behaviour of apices from hairy root cultures of this clone has been compared with root tips from normal seedlings. While the wild type roots curved progressively with increasing stimulation angles, the transformed roots showed no curvature when stimulated at 45 degrees, 90 degrees or 135 degrees on the ground. The morphology and ultrastructure of the root tip regions were examined by light microscopy and transmission electron microscopy. At the ultrastructural level no major differences could be detected between the roots studied. There was, however, a slight reduction in the starch content of most of the amyloplasts of the transgenic root tips, and the root cap was more V-shaped in the transgenic roots than in the wild type. Preliminary results from the Shuttle experiment TRANSFORM show a random distribution of amyloplasts in the root cells of both transformed and wild type root caps after 14 h on a 1xg centrifuge followed by 37 h in microgravity.     

Purification and characterization of a glucokinase from young tomato (Lycopersicon esculentum L. Mill.) fruit

In order to clearly establish the properties of the enzymes responsible for hexose phosphorylation we have undertaken the separation and characterization of these enzymes present in tomato fruit (Martinez-Barajas and Randall 1996). This report describes the partial purification and characterization of glucokinase (EC. 2.7.1.1) from young green tomato fruit. The procedure yielded a 360-fold enrichment of glucokinase. Tomato fruit glucokinase is a monomer with a molecular mass of 53 kDa. Glucokinase activity was optimal between pH 7.5 and 8.5, preferred ATP as the phosphate donor (K _m = 0.223 mM) and exhibited low activity with GTP or UTP. The tomato fruit glucokinase showed highest affinity for glucose (K _m = 65 μM). Activity observed with glucose was 4-fold greater than with mannose and 50-fold greater than with fructose. The tomato fruit glucokinase was sensitive to product inhibition by ADP (K _i = 36 μM). Little inhibition was observed with glucose 6-phosphate (up to 15 mM) at pH 8.0; however, at pH 7.0 glucokinase activity was inhibited 30–50% by physiological concentrations of glucose 6-phosphate. Received: 4 October 1997 / Accepted: 10 January 1998     

Transient and stable expression of hepatitis B surface antigen in tomato (Lycopersicon esculentum L.)

Cotyledonary leaves of tomato cv. Megha were transformed with the hepatitis B virus ‘s’ gene, which encodes surface antigen. Six plant expression cassettes (pHBS, pHER, pEFEHBS, pEFEHER, pSHER and pEFESHER) were used to assay the possible expression levels by agroinfiltration. The maximum transient expression level of 489.5 ng/g D.W. was noted in pEFEHER-infiltrated cotyledonary leaves. Transgenic tomato plants with pEFEHBS and pEFEHER expression cassettes were regenerated and characterized by molecular analysis. The expression of the antigen in the fruits was confirmed by RT-PCR and ELISA analysis. This is the first report on the expression of hepatitis B surface antigen in tomato.     

Identification and distribution of profilin in tomato (Lycopersicon esculentum Mill.)

Profilin is a G-actin monomer-binding protein which has been shown to participate in actin-based tipgrowth of animal cells. The abundance of profilin in pollen and its occurrence in several vegetable foods raises the question of the role of profilin in plants. First, its distribution throughout various parts of the plant needs to be determined. This paper describes observations on the presence of profilin in the tomato plant (Lycopersicon esculentum Mill.). The distribution of profilin in flower buds, stems, leaves, roots, and fruits of tomato was determined by immunoblotting and by tissue printing, showing that profilin is present in most if not all parts of the tomato plant.We gratefully acknowledge the help provided by Dr. A.T. Jagendorf and the donation of tomato seeds and maize pollen by N. Eanetta and Dr. M. Smith, respectively. The use of Dr. R. Wayne's SZH ILLD dissecting microscope is gratefully acknowledged. This work was aided by helpful discussions with C.S. Combs, Dr. C.A. Conley, and Dr. J. Andersland. This work was supported by a Hatch grant and NRI Competitive Grants Program/USDA 94-37304-1046 to MVP. This material is based upon work supported under a National Science Foundation Graduate Research Fellowship to DWD.     

Increase in the rate of recombinants in tomato (Lycopersicon esculentum L.) after in vitro regeneration

Summary Modification to the cross-over (C. O.) rate of tomato (Lycopersicon esculentum) was attempted by using in vitro plant regeneration. F1 hybrids with the same genetical homozygous background were compared at two loci: bs-ms32 on chromosome I, and aa-d on chromosome II. For each, the genetic distance separating the two markers was about 20 to 30 map units. One cotyledon of each F2 hybrid seedling was used as in vitro tissue culture material, while the rest of the plantlet was grown as a control. Recombination rates of the selfed progenies from each regenerated and matched control couple were compared. For the first set of markers 59,000 seeds were analysed (5 controls' and 7 regenerated progenies), and for the second, 11,000 (5 controls' and 8 regenerated progenies). There were significant increases in the genetic distance between markers in about half the regenerated individuals. For the first set the increases ranged from 6.07 to 6.91 units out of a control distance of the 19.84 to 25.65, corresponding to lengthenings of 30.59 to 35.29%. For the second set they ranged from 4.92 to 6.04 out of a control distance of 25.05 to 26.57, representing increases of 19.64 to 22.75%. Such a phenomenon can be important either from a fundamental or practical viewpoint, regarding selection efficiency in plants, and potential for gene reassortment.The experiment reported in this paper has been submitted by M. Biglary in partial fulfillment of the requirements for Docteur-Ingénieur degree. (Nov. 1982, Laboratoire d'Amélioration des Plantes, Université Paris-Sud, F-91405 Orsay)     

An immunochemical testing of pathophysiological reactions of several PSTVinfected tomato (Lycopersicon esculentum L.) cultivars

Several tomato cultivars were infected with a severe strain of PSTV and a pathophysiological reaction was characterized by means of enzyme-linked immunosorbent assay with serum containing IgGs to disease-associated host-specific leaf proteins. A strong expression of disease-associated symptoms (stunting, epinasty, leaf blade malformation and rugosity) and strong immunochemical reaction was found for cultivars Bizon, Linia, Revermun and Rutgers. The immunochemical assay revealed appearence of a major antigenic protein having a molecular mass of about 70 kD in these cultivars. The immunochemical reaction with disease-associated proteins reached a maximum four weeks after inoculation of PSTV. A weak immunochemical reaction was observed, if proteins from cvs. Sonato, Harzfeuer and Karlik were analyzed. Cvs. Sonato and Harzfeuer did not show characteristic symptoms such as stunting, leaf blade malformation and rugosity. Except for stunting, the same was true for cv. Karlik. On the other hand, no significant difference in PSTV accumulation was observed among the cultivars analyzed. Reciprocal hybrids obtained by crossing cvs. Revermun and Karlik showed strong symptoms of the disease (Revermun-type) and increased activity of nuclease due to PSTV infection. On the contrary, an immunochemical analysis revealed a low level of the disease-associated antigens in these hybrid tomatoes, suggesting rather recessive genetic background determining their expression during PSTV-caused pathogenesis.     

Transformation of eggplant (Solanum melongena L.) using a binary Agrobacterium tumefaciens vector

Summary Kanamycin resistant plants of Solarium melongena L. (eggplant) cv. Picentia were obtained following the cocultivation of leaf explants with Agrobacterium tumefaciens. A disarmed binary vector system containing the neomycin phosphotransferase (NPTII) gene as the selectable marker and chloramphenicol acetyltransferase (CAT) as a reporter gene was utilized. In vitro grown plants were used as sources of explants to produce transgenic plants on selective medium containing 100 mg/l kanamycin. The transformation and expression of the foreign genes was confirmed by DNA hybridizations, leaf disc assays, and by measuring NPTII and CAT enzyme activities. This technique is simple, rapid, efficient, and transgenic eggplants of this commercial cultivar have been transferred to soil where they have flowered and set seed.Abbreviations CAT chloramphenicol acetyltransferase - MS Murashige and Skoog - NPTII neomycin phosphotransferase - NOS nopaline synthase - ZEA zeatin     

Allotriploid somatic hybrids of diploid tomato (Lycopersicon esculentum Mill.) and monoploid potato (Solanum tuberosum L.)

Allotriploid somatic hybrids were obtained from fusions between protoplasts of diploid tomato and monohaploid potato. The selection of fusion products was carried out in two different ways: (1) The fusion of nitrate reductase-deficient tomato with potato gave rise only to hybrid calli if selection was performed on media lacking ammonium. Parental microcalli were rarely obtained and did not regenerate. (2) The fusion of cytoplasmic albino tomato with potato gave rise to albino and green hybrid calli and plants. Allotriploids were identified from the two somatic hybrid populations by counting chloroplast numbers in leaf guard cells and by flow cytometry of leaf tissue. Although some pollen fertility of allotriploids and pollen-tube growth of tomato, potato andLycopersicon pennellii into the allotriploid style were observed, no progeny could be obtained. The relevance of allotriploid somatic hybrids in facilitating limited gene transfer from potato to tomato is discussed.     

Zinc-phosphorus interactions in two cultivars of tomato (Lycopersicon esculentum L.) grown in chelator-buffered nutrient solutions

Zinc-phosphorus interactions have been frequently studied using a diverse number of crop species, but attainment of reproducible Zn deficiencies, especially severe ones, has been hampered by the use of conventional hydroponic solutions wherein contaminating levels of Zn are often near-adequate for normal growth. We utilized novel, chelator-buffered nutrient solutions for precise imposition of Zn deficiencies. Tomato (Lycopersicon esculentum L. cv. Jackpot or Celebrity) seedlings were grown for 15 to 18 d in nutrient solutions containing 200, 600, or 1200 M P, and 0 to 91 M total Zn. Computed free Zn²⁺ activities were buffered at 10^-10.3 M by inclusion of a 100-M excess (above the sum of the micronutrient metal concentrations) of the chelator DTPA. At total added Zn=0, acute Zn deficiency resulted in zero growth after seedling transfer, and plant death prior to termination. Free Zn²⁺ activities 10^-10.6 M resulted in Zn deficiencies ranging from mild to severe, but activities 10^-11.2 were required to cause hyperaccumulation of shoot P to potentially toxic levels. Despite severe Zn deficiency (i.e. ca. 20% of control growth), tissue Zn levels were usually much higher than the widely reported critical value of 20 mg kg^-1, which may be an artifact of the selection of DTPA for buffering free Zn²⁺. Across Zn treatments, increasing solution P depressed growth slightly, especially in Celebrity, but corresponding increases in tissue P (indicative of enhanced P toxicity) or decreases in tissue Zn (P-induced Zn deficiency) were not observed. The depressive effect of P was also not explained by reductions in the water-soluble Zn fraction. Within 40 h, restoration of Zn supply did not ameliorate high leakage rates (as measured by K⁺ efflux) of Zn-deficient roots. Similarly, transfer of Zn-sufficient plants to deficient solutions did not induce leakiness within 40 h. Foliar sprays of ZnSO₄ almost completely corrected both Zn deficiency and membrane leakiness of plants grown in low-Zn solutions. Hence, maintenance of root membrane integrity appears to depend on the overall Zn nutritional status of the plant, and not on the presence of certain free Zn²⁺ levels in the root apoplasm.     

Characterization of the subtilase gene family in tomato (Lycopersicon esculentum Mill.)

The gene family of subtilisin-like serine proteases (subtilases, SBTs) in tomato (Lycopersicon esculentum Mill.) comprises at least 15 members, 12 of which have been characterized in this study. Sequence comparison revealed that tomato subtilases fall into 5 distinct subfamilies. Single genes were shown to exist for LeSBT1, LeSBT2 and tmp, while 5 and 6 genes were found in the LeSBT3/4 and P69 subfamilies, respectively. With the exception of tmp, tomato subtilase genes were found to lack introns. Expression of subtilase genes was confirmed at the mRNA level by northern blot analysis and/or by primer extension experiments. For each of the 5 subtilase subfamilies, a distinctive pattern of expression was observed in tomato organs. At least one of the subtilases was found to be expressed in each organ analysed. Structural features evident from deduced amino acid sequences are discussed with reference to the related mammalian proprotein convertases.