Genetic diversity in cocoa revealed by cDNA probes

The variability of the cocoa (Theobroma cacao) nuclear genome was investigated. A total of 203 cocoa clones was surveyed for restriction fragment length polymorphisms (RFLPs) using four restriction endonuclease and 31 seed cDNA probes. A high level of polymorphism has been found. This study points to a structuring of the species that fits with the distinction between the Criollo and Forastero populations. These results combined with previously obtained nuclear rDNA and mtDNA data allow us to propose new hypotheses on the origin and evolution of the different cocoa populations.     

Genetic transformation of Populus nigra by Agrobacterium tumefaciens

Two clones of Populus nigra L. were tested in vivo and in vitro for their susceptibility to three different Agrobacterium tumefaciens wild-type strains evaluating number and size of resulting calluses. Strain C58 proved to be the most virulent.Various parameters affecting Agrobacterium-mediated transformation of P. nigra clones were further analyzed using ß-glucuronidase gene transient expression. The clone Jean Pourtet proved to be more susceptible than the clone San Giorgio. A. tumefaciens strain A281 pKIWI105 proved to be the most virulent. The optimal procedure involved dipping of leaf discs into a bacterial suspension (7×10⁸ cells/ml) for 20 min, followed by a 48 h co-cultivation period on semi-solid regeneration medium.Leaf explants were co-cultivated with two disarmed A. tumefaciens strains. Plantlets of San Giorgio were regenerated, tested for ß-glucuronidase activity and rooted on selective medium containing kanamycin. Polymerase chain reaction analysis and Southern blot hybridization confirmed the integration of the neomycin phosphotransferase II gene into the poplar genome.Abbreviations BAP 6-benzyl-aminopurine - CaMV Cauliflower Mosaic Virus - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS and gus ß-glucuronidase - hpt hygromycin phosphotransferase - IBA indole-3-butyric acid - KIN kinetin - LB Luria Bertani - MS Murashige and Skoog - NAA ßnaphthaleneacetic acid - NOS Nopaline synthase - NPTII and nptII neomycin phosphotransferase II - PCR Polymerase chain reaction - PVC poly-vinyl-cloride - SDS sodium dodecyl sulfate - SSC sodium cloride-sodium citrate - Tris tris(hydroxymethyl)amino-methane - WPM Woody Plant Medium     

Genotype-independent leaf disc transformation of potato (Solanum tuberosum) using Agrobacterium tumefaciens

Summary Leaves of the in vitro grown potato cultivars Bintje, Berolina, Desiree, and Russet Burbank were wounded and co-cultivated with Agrobacterium strains having chimeric bar and nptII genes on a disarmed T-DNA. Each leaf from these cultivars formed numerous calli on kanamycin-containing medium, and almost all calli regenerated shoots. For Russet Burbank, it was necessary to include AgNO₃ in the medium to obtain efficient shoot regeneration. The transformed plants have one to a few copies of the T-DNA, show NPT-II and PAT activities, and are resistant to high doses of the commercial preparation of phospinotricin (glufosinate). Almost no somaclonal variation was detected in trans-genic plants.     

Abscisic acid metabolism and episodic growth in cocoa

Abscisic acid (ABA) levels and metabolism were investigated in relation to shoot growth in cocoa (Theobroma cacao L.). ABA levels were high (14 nmoles/g fwt) in young flush leaves during shoot growth but gradually declined during the subsequent dormant period. ABA levels were low (1–2 nmoles/g fwt) in mature leaves when the terminal bud re-initiated growth. ABA-glucose ester (ABA-glu) levels were low (3–4 nmoles/g fwt) in the flush leaves during shoot growth and dormancy, however, ABA-glu levels increased more than 7-fold in these same leaves during the next flush cycle. ABA-glu levels then dropped significantly during the dormant period. Radiolabeled-ABA was metabolized to three products in cocoa leaves: ABA-glu, phaseic acid and dihydrophaseic acid. Catabolism of radiolabeled ABA was significantly greater in mature leaves during the dormant period when endogenous levels of ABA were high as compared to the period of active shoot growth when endogenous levels of ABA in mature leaves were low.     

Genetic transformation of prickly-pear cactus (Opuntia ficus-indica) by Agrobacterium tumefaciens

A system for genetic transformation of an elite prickly pear cactus (Opuntia ficus-indica L., cultivar Villa Nueva) by Agrobacterium tumefaciens was developed. Beginning with direct bacterial infection by using a hypodermic syringe to the meristematic tissue termed areoles, transgenic plants were obtained by selection with 100 mg l⁻¹ kanamycin. Transient and stable GUS activities were monitored on kanamycin-resistant shoots and regenerated plants, respectively. Genetic transformation of regenerated plants growing under selection was demonstrated by PCR and Southern blot analysis; transgene copy number in the genome of transgenic plants ranged from two to six, while the transformation frequency obtained by the system reported here was of 3.2%. This method may be useful for routine transformation and introduction of several important genes in prickly pear cactus.     

Transgenic plant production from leaf discs of Moricandia arvensis using Agrobacterium tumefaciens

Summary A high frequency shoot regeneration (80%) was developed from callus of leaf discs and stem internodes of Moricandia arvensis. Leaf discs were shown to be a preferable starting material for transformation experiments. Agrobacterium tumefaciens strain GV3101/pMP90 used in this study contained a binary vector with genes for kanamycin resistance, hygromycin resistance and -glucuronidase (GUS). Maximum transformation efficiency (10.3%) was achieved by using kanamycin at the rate of 200 mg/l as a selection agent. Presence of tobacco suspension culture during co-cultivation and a pre-selection period of seven days after co-cultivation was essential for successful transformation. Transgenic plants grew to maturity and exhibited flowering in a glasshouse. GUS activity was evident in all parts of leaf and the presence of GUS gene in plant gemone was confirmed by PCR analysis.Abbreviations GUS -glucuronidase     

Transformation of Solanum brevidens using Agrobacterium tumefaciens

Leaf pieces of in vitro-cultured plantlets of the wild potato species Solanum brevidens Phil. were cocultivated with Agrobacterium tumefaciens that contained nptII and uidA genes on the disarmed plasmid pBI121. Independent transgenic shoots were regenerated from solidified and liquid medium that contained 50 mg l^–1 kanamycin. Two Agrobacterium strains were investigated for transformation efficiency. GV2260, which contained p35SGUSINT, resulted in a 11% transformation frequency, compared with 1% using LBA4404. Transformation rates were 7% in liquid culture and 3% on solidified medium. All kanamycinresistant, putatively transformed plantlets were confirmed positive by histochemical GUS assays. GUS activity in 22 independently transformed plants was quantified by fluorometric assay. Southern analysis of randomly selected transgenic plants showed that each transgenic plant contained at least one copy of the uidA gene.Abbreviations GUS ß-glucuronidase - MS Murashige-Skoog medium - BA 6-benzylaminopurine - 2ip 6-(, -dimethylallylamino)purine - IAA indole-3-acetic acid - GA₃ gibberellic acid - npt II neomycin phosphotransferase II - NOS nopaline synthase - MUG 4-methyl umbelliferyl glucuronide - MU 7-hydroxy-4-methylcoumarin - X-gluc 5-bromo-4-chloro-3-indolyl ß-D-glucuronic acid     

Factors affecting the rate of T-DNA transfer from Agrobacterium tumefaciens to Nicotiana glauca plant cells

Different factors involved in the early steps of the T-DNA transfer process were studied by using a -glucuronidase gene (gusA) as a reporter in Nicotiana glauca leaf disc transformation experiments. The levels of transient expression of the gusA gene in leaf discs infected with several strains or vir mutants correlated well with their virulence phenotype, except for virC mutants. The rate of T-DNA transfer was shown to be stimulated in the case of non-oncogenic strains by the co-transfer of small amounts of oncogenic genes. It was found that the location of the T-DNA in the Agrobacterium genome affected the T-DNA transfer rate especially in virC mutants. The virC mutants transferred the gusA-containing T-DNA located on a binary vector more efficiently than the oncogenic T-DNA of the Ti plasmid. Although wild-type strains induced high levels of gusA expression early after infection, the gusA expression appeared to be lost late after infection in the infected leaf discs. In contrast, in leaf discs infected by virC mutants the level of gusA expression increased steadily in time. A model explaining these results is presented.     

Haploid transformation in Brassica napus using an octopine-producing strain of Agrobacterium tumefaciens

Summary Microspore-derived embryos of Brassica napus were transformed using the disarmed octopine-producing LBA4404 strain of Agrobacterium tumefaciens containing the binary vector pBin19. Octopine-producing strains have previously been reported to be ineffective in transforming Brassica. Four actively growing yellow/ green sectors were selected from the embryos on 50 mg/l kanamycin and plants regenerated. Analysis for NPT-II activity in these young plants initially indicated no expression of the bacterial NPT-II gene. The plants were nevertheless grown to maturity, selfed and S₁ seed was collected. Three of the S₁ plants produced microspores which were from 4 to 20 times more tolerant to kanamycin than the original parent. Southern analysis revealed that one plant (EC-1) had a single site of insertion and the other two plants (EC-2 and EC-6) had two sites of insertion with sequence homology to the bacterial NPT-II gene. Microspores from the EC-2 and EC-6 transgenics produced embryos on approximately five times the level of kanamycin tolerated by microspores from untransformed plants, while the EC-1 transgenic produced microspores with more than 20 times the tolerance to kanamycin. Analysis of S₁ progeny of the EC-1 transgenic indicated that 100% of the progeny exhibited the trait through both Southern analysis and by expressing tolerance to kanamycin in microspore-derived embryos.     

Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens

A highly efficient gene transfer method mediated by Agrobacterium tumefaciens was developed for Group I indica rice, which had been quite recalcitrant in tissue culture and transformation. Freshly isolated immature embryos from plants grown in a greenhouse were inoculated with A. tumefaciens LBA4404 that harbored super-binary vector pTOK233 or pSB134, which had a hygromycin-resistance gene and a GUS gene in the T-DNA. The efficiency of gene transfer varied with the kinds of gelling agents and the basic compositions of co-cultivation media. The highest activity of GUS after co-cultivation was observed when NB medium solidified with agarose was used. For the subsequent cultures, two types of media (modified NB and CC) were chosen to recover hygromycin-resistant cells efficiently. The transformation protocol thus developed worked very well in all of the varieties tested in this study, and the transformation frequency (number of independent hygromycin-resistant and GUS-positive plants per embryo) reached more than 30% in IR8, IR24, IR26, IR36, IR54, IR64, IR72, Xin Qing Ai 1, Nan Jin 11, and Suewon 258. Most of the transformants (T₀) were normal in morphology and fertile. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the T₀ and T₁ generations. For the recovery of multiple independent transgenic events from a single immature embryo, procedures were developed to section the embryo into as many as 30 pieces after non-selective cultures following co-cultivation. Transformants were then obtained from the pieces cultured on the selective media, and, in the highest case, more than seven independent transgenic plants per original embryo (transformation frequency of 738%) were produced. Thus, the efficiency of transformation was remarkably improved.     

The Agrobacterium tumefaciens T-DNA gene 6b is an onc gene

In this article it is shown that the T-DNA of Agrobacterium tumefaciens contains besides the well-known cyt and aux genes another gene with an oncogenic effect in plants. The gene in question is called 6^b and causes the formation of small tumors in plant species such as Nicotiana glauca and Kalanchoe tubiflora.     

Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system

An efficient genetic transformation protocol has been developed for strawberry cv. Redcoat using Agrobacterium tumefadens. The protocol relies on a high frequency (84%) shoot regeneration system from leaf disks. The leaf disks were inoculated with a non-oncogenic Agrobacterium tumefadens strain MP90 carrying a binary vector plasmid pBI121 which contains a chimeric nopaline synthase (NOS) promoter driven neomycin phosphotransferase (NPT II) gene and a cauliflower mosaic virus 35S (CaMV35S) promoter driven, ß-glucuronidase (GUS) marker gene. The inoculated leaf disks, pre-cultured for 10 days on non-selective shoot regeneration medium, formed light green meristematic regions on selection medium containing 50 g/ml kanamycin. These meristematic regions developed into transformed shoots at a frequency of 6.5% on a second selection medium containing 25 g/ml kanamycin. The selected shoots were multiplied on shoot proliferation medium in the presence of kanamycin. All such shoots were resistant to kanamycin and expressed varying levels of NPT II and GUS enzyme activity. Histochemical assays for GUS activity indicated that the 35S promoter was highly active in meristematic cells of shoot and root apices. Molecular analysis of each transgenic clone confirmed the integration of both marker genes into the strawberry genome. Leaf disks prepared from transformed plants, when put through the second selection cycle on kanamycin, formed callus and exhibited GUS activity. The rooted transformed plants were grown in a greenhouse for further characterization. The protocol may be useful for improvement of strawberry through gene manipulations.NRCC No. 31491During the editorial process, a report has appeared on transformation of strawberry (James et al. 1990 Plant Sci 69:79–94).     

Transfer of non-T-DNA portions of the Agrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of TL-DNA

We introduced a plant selection marker, nptII, to the left of border A in the Agrobacterium Ti plasmid pTiA6. Infection of tobacco leaf discs with the modified Agrobacterium strain gave rise to kanamycin-resistant calli which grew in a hormone-dependent manner. Southern hybridization analysis of DNA isolated from four transformants indicated initiation of DNA transfer at or near border A and absence of T-DNA sequences. These results demonstrate that DNA transfer events starting at a left border on a native Ti plasmid and moving away from the T-DNA region occur and that they can be detected by designing a suitable selection strategy.     

Transformation of forage legumes using Agrobacterium tumefaciens

Summary Galls were induced in six species of forage legumes following inoculation with wild-type strains of A. tumefaciens. The plant species was more influential than the bacterial strain in determining the type of tumour produced. Inoculation of Medicago sativa resulted in small, disorganised tumours. The three Trifolium species, T. repens, T. hybridum and T. pratense, formed galls which tended to produce roots and both Onobrychis viciifolia and Lotus corniculatus produced teratomatous galls. The shoots elongated in the latter species only. In L. corniculatus, tissues that were infected by five bacterial strains were capable of shoot regeneration when cultured on a hormone-free medium. The transformed nature of these shoots was confirmed by their failure to root, the production of callus from leaves cultured on hormone-free medium and the presence of opines.     

Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens

High frequency transformation of Arabidopsis thaliana leaf explants has been obtained using a disarmed Ti plasmid containing the coding region of a neomycin phosphotransferase gene (NPT II) as a selectable marker. The rate of transformation ranged from 55 to 63 percent when acetosyringone (AS), a natural wound response molecule, was added to an Agrobacterium tumefaciens culture prior to incubation with leaf segments. Without acetosyringone, the transformation rate was approximately 2 to 3 percent. Calli resistant to G418 were regenerated into mature flowering plants in the presence of 10 g/ml G418. Southern analysis and neomycin phosphotransferase assays confirmed the insertion and expression of the NPT II gene in regenerated Arabidopsis plants.     

Functional analysis of the Agrobacterium tumefaciens octopine Ti-plasmid left and right T-region border fragments

Summary Border fragments of the octopine Ti-plasmid were tested for their ability to restore tumorigenicity of an avirulent mutant carrying a deleted right border. It was found that neither introduction of left border fragments nor that of small right border fragments at the position of the deletion resulted in a complete restoration of oncogenicity. However, insertion of a larger right border fragment in the deletion mutant gave fully oncogenic strains. In the latter case sequences to the right side of the right border repeat were found to be responsible for a complete restoration of oncogenicity. Also a left border repeat inserted together with this enhancer sequence fully restored the oncogenicity of the deletion mutant. The enhancer-sequence on itself was not able to mediate the transfer of the T-region to the plant cell. Border fragments inserted in inverted orientation in the deletion mutant were able to mediate the transfer of the T-region to the plant cell, but at a reduced frequency.     

Auxin synthesis in Agrobacterium tumefaciens and A. tumefaciens-transformed plant tissue

Problems of IAA biosynthesis, IAA precursors and IAA biosynthetic pathways in Agrobacterium tumefaciens-transformed plant tissue, especially in tobacco tissue culture are reviewed. The knowkedge about levels of IAA and the IAA biosynthetic pathways in Agrobacterium tumefaciens itself is also summarized.Dedical to the late Professor M. Kutáek, a pioneer in auxin biosynthesis research.     

Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

Slow death of Atlantic forest trees in cocoa agroforestry in southeastern Brazil

Cocoa (Theobroma cacao) is cultivated in the states of Bahia and Espírito Santo in eastern Brazil under the so-called cabruca system, where the understorey of native Atlantic forest is cleared and the canopy is thinned out to provide adequate shading for the cocoa trees. Apart from its economic and social role, the cabruca system is said to be important for the conservation of Atlantic forest biodiversity. In this paper we studied tree species richness and forest structure of cabrucas to examine the demographic health of these forests and discuss their long-term survival. Data were collected in 20 farms located alongside a 30km track of the northern margin of the Rio Doce, in northern Espírito Santo. All trees 5cm DBH were identified and their diameter was measured in 80 plots (600m²), totalling 4.8ha of sampled area. Recorded trees were also allocated to four different regeneration phases (pioneers, early secondary, late secondary and climax). The inventory resulted in 507 trees belonging to 105 species in 39 families. This species richness is much lower than in less disturbed forests located in the region. Pioneers and early secondary species dominate the cabruca forest in terms of number of species (56.2%), density (71.0%) and basal area (72.3%). The distribution of diameter frequency showed an imbalance in tree regeneration. Most trees in the range of 5–30cm DBH were pioneers (40.7%), or early secondary species (32.6%), while late secondary and climax trees were less frequent (10.2 and 16.5% of the sampled trees, respectively). The dominance of species of early regeneration phases was also observed for trees >30cm DBH (69.0% of pioneers or early secondary and 31.0% of late secondary or climax species). The results indicated that the cabruca forests are not only less diverse and less dense than secondary or primary forests of the region, but also, and more importantly, their natural succession and gap dynamics are being severely impaired. As a consequence, cabrucas present a structure where tree species of late successional phases are becoming increasingly rare while pioneers and early secondary species are becoming dominant. If current management practices of thinning and clearing of native trees are not improved, the long-term survival of these forests is questionable and their role in maintaining biodiversity in the long run is limited.