Stefano Castiglione

RAPD fingerprints for identification and for taxonomic studies of elite poplar (Populus spp.) clones

RAPD (Random Amplified Polymorphic DNA) fingerprints have recently been used to estimate genetic and taxonomic relationships in plants. In this study RAPD analysis was performed on 32 clones belonging to different species of the genus Populus. Of these, 25 clones are registered in several countries for commercial use and, altogether, cover almost 50% of the worlds cultivated poplars. DNA was prepared from leaves and amplified by PCR using random oligonucleotide primers. Amplification products were separated by agarose-gel electrophoresis to reveal band polymorphisms. Four primers out of the 18 tested, were selected on the basis of the number and frequency of the polymorphisms produced. With these a total of 120 different DNA bands were reproducibly obtained, 92% of which were polymorphic. The polymorphisms were scored and used in band-sharing analyses to identify genetic relationships. With a few but interesting exceptions, these are consistent with the present taxonomy of the genus Populus and with the known predigrees of cultivated poplars. Moreover, the results show that RAPD analysis allows one to discriminate among all tested clones and can, therefore, be recommended as a convenient tool to defend plant breeders rights.

Poplar (Populus nigra L.) plants transformed with aBacillus thuringiensis toxin gene: insecticidal activity and genomic analysis

Insect-resistant poplar (Populus nigra L.) plants have been produced by infecting leaves withAgrobacterium tumefaciens strains carrying a binary vector containing different truncated forms of aBacillus thuringiensis (B.t.) toxin gene under a duplicated CaMV 35S promoter. Putative transgenic plants were propagated by cuttings at two experimental farms (in Beijing and Xinjiang, China). At 2–3 years after transformation, 17 of them were selected on the bases of insect-tolerance and good silvicultural traits, and evaluated for insect resistance, for the presence of theB.t. toxin DNA fragment (Southern blots and PCR) and for the expression of the transgene (western and northern blots). Somaclonal variation, as suggested by the appearance of permanent changes in the shape of the leaves, was also investigated with molecular tools (RFLP (restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA) and microsatellite DNA).Bioassays withApochemia cineraius andLymantria dispar on the leaves of the selected clones showed different and, in some cases, high levels of insecticidal activity. The molecular analysis demonstrated integration and expression of the foreign gene. Somatic changes were correlated to extensive genomic changes and were quantified in dendrograms, in terms of genomic similarity. The analysis of control plants suggested that genomic changes were correlated to thein vitro culture step necessary forA. tumefaciens-mediated gene transfer, rather than to the integration of the foreign genes.Three transgenic clones (12, 153 and 192), selected for insect resistance, reduced morphological changes and promising silvicultural traits, are now under large-scale field evaluation in six different provinces in China.

Nuclear DNA amplification in cultured cells of Oryza sativa L.

SummaryHighly repeated nuclear DNA sequences from suspension cultured cells of Oryza sativa L. cv. ‘Roncarolo’ have been cloned in pBR322. Ten clones with specific digestion patterns have been randomly selected. Nine sequences appear to be organized in a clustered tandem array while one is interpersed in the rice genome. The clones have been used to gather information on: (a) their modulation in cultured cells as compared to whole plant and (b) their distribution in different rice cultivars belonging to the Japonica or Indica subspecies of Oryza sativa L. Hybridization with nuclear DNA isolated either from suspension or from seedlings of the ‘Roncarolo’ cultivar revealed extensive quantitative variations, with most cloned sequences showing amplification (up to 75-fold) in cultured cells. Hybridization with nuclear DNA isolated from seedlings or suspension cultured cells from different cultivars belonging to the Japonica or to the Indica sub-species of O. sativa have shown that (a) amplification also occurs in a similar pattern in the case of DNA from the other tested suspension cultured cell types but not in the case of DNA from seedlings; (b) in some cases the tested sequences show minor but significant variations in different rice accessions.

Hybrid rice (Oryza sativa L.): identification and parentage determination by RAPD fingerprinting

SummaryDNA from three families of rice plants selected in Northern China (each comprising the male sterile, the restorer, the hybrid F1 and the maintainer lines) has been extracted and amplified by PCR with different random DNA primers (RAPD analysis). Then, DNA has been analysed by agarose gel electrophoresis and DNA bands scored as present or absent. The generated matrices are reproducible and amenable for identification of each single plant line. Thus, RAPD fingerprinting of the inbred parental lines and of the resulting hybrid is proposed as a convenient tool for the identification, protection and parentage determination of plant hybrids. Furthermore, by offering a molecular tool to verify the degree of dissimilarity between the parental lines, the RAPD analysis may also be used to search for new parental combinations.

A repeated chromosomal DNA sequence is amplified as a circular extrachromosomal molecule in rice (Oryza sativa L.).

SummaryThe plasmid pE10 is a pBR322-derived plasmid carrying a 4.5 kb rice (Oryza sativa L.) repeated DNA sequence. The cloned sequence has been shown to be amplified in cultured rice cells. The analysis of practically intact chromosomal rice DNA molecules by pulsed field gel electrophoresis has now shown that the amplification is associated with the appearance of extrachromosomal molecules. In fact, pE10 hybridizes exclusively with unfractionated DNA from leaf protoplasts, while it recognizes predominantly an extrachromosomal DNA molecule (ECD) of about 45 kb and its multiples in the case of protoplasts from cultured cells. Insensitivity to the action of the exonuclease Bal31 suggests that the molecule is circular. Analysis of restriction endonuclease products with both standard horizontal and pulsed field gel electrophoresis suggest that the extrachromosomal DNA, and its chromosomal counterpart, is composed of tandemly repeated units of about 7 kb. Thus, the smaller extrachromosomal circle should contain 6–7 repeats, while the sequence cloned in pE10 is a subset of this repeat. The extrachromosomal DNA represents about 1 % of total rice DNA and its level of amplification is not affected by the different phases of growth in culture.

Stability of a foreign gene in transgenic Nicotiana tabacum L. plants during a cycle of dedifferentiation/differentiation

Abstract Protoplasts of Nicotiana tabacum were transformed with the APH(3′)II gene, which confers kanamycin resistance. Plants resistant to kanamycin were differentiated and 3 of them were chosen at random. These were used to study the stability of the foreign gene after a cycle of dedifferentiation, to produce calli, and differentiation, to produce new plants. The effect of the selective pressure was analyzed by performing dedifferentiation and differentiation in the presence or absence of kanamycin. Inbred plants were also produced from the original transformed plants and used as control. Southern blot analysis of DNA extracted from 66 regenerated plants showed in all cases that no detectable alteration occurred both in gene structure and insertion site. Furthermore the specific activity of the APH(3′)II enzyme was shown to be at high level in all regenerated plants regardless of the fact that they were regenerated in the presence or absence of kanamycin. The results described here are experimental evidence that a hybrid forcing gene is rather stable in a heterologous genome even after dedifferentiation of the transformed plants and differentiation in vitro, i.e. in those conditions known to be correlated with extensive somaclonal variation.

Effect of repeated DNA sequences on direct gene transfer in protoplasts of Nicotiana plumbaginifolia

SummaryHighly repeated nuclear DNA sequences from leaves of Nicotiana plumbaginifolia were cloned in pBR322 and tested for their effect on direct gene transfer in protoplasts of the same organism. Protoplasts were prepared from suspension cultures and were incubated in the presence of the plasmid pHP23 carrying the kanamycin resistance gene APH(3′)II and in the presence of the plasmids carrying the cloned sequence. DNA uptake was induced by a polyethyleneglycol (PEG) treatment. Out of the 22 tested clones, 3 significantly stimulated the frequency of appearance of transformed colonies. DNA was extracted from some of the kanamycin-resistant calli obtained by co-transformations. Dot-blots have shown that the stimulatory effect on transformation frequency is often accompanied by a consistent increase in integrated genes sequences.

Direct Gene Transfer in Protoplasts of Nicotiana plumbaginifolia

Different methodologies to transfer foreign genes into higher plants have recently been developed. These include: (1) insertion of the gene into the T-DNA region of the Ti plasmid of Agrobacterium tumefaciens (or of the Ri plasmid of A. rhizogenes) followed by infection of tissues (Gheysen et al. 1985); (2) direct gene transfer from bacterial plasmids to protoplasts (Paszkowski et al. 1984; Shillito et al. 1985); (3) insertion of the gene into a plant virus used to infect plants (Brisson et al. 1984); (4) microinjection of plasmid or nuclear DNA into plant nuclei (Reich et al. 1986); (5) injection of DNA into young floral tillers (De La Pena et al. 1987); (6) delivery of DNA into plant cells using high-velocity microprojectiles (Klein et al. 1987).

State of the foreign gene and of the genome in transgenic rice (Oryza sativa L.).

PCR with random primers (RAPD analysis) performed on the DNA of embryogenic and non-embryogenic suspension cultured rice and of transformed rice plants allows the evaluation of the extent of DNA changes in the different biological materials. This is thus suggested as a convenient approach, in combination with restriction analysis and Southerns blotting, to evaluate the integrity of the foreign gene, the stability of the insertion site and the stability of the whole genome.PCR with random primers (RAPD analysis) performed on the DNA of embryogenic and non-embryogenic suspension cultured rice and of transformed rice plants allows the evaluation of the extent of DNA changes in the different biological materials. This is thus suggested as a convenient approach, in combination with restriction analysis and Southerns blotting, to evaluate the integrity of the foreign gene, the stability of the insertion site and the stability of the whole genome.

Plasticity of the Rice Genome: DNA Amplification in Cultured Cells

A message that geneticists and molecular biologists are increasingly addressing to plant breeders is that the plant genome is remarkably unstable. Plants of a population within a species can show karyotypic variations, with changes in chromosome number or with the appearance of supernumerary or β-chromosomes; but extensive submicroscopic variations, such as movement of transposable elements, chromosome rearrangements and gene amplification may also occur (Durrant 1974; Price et al. 1983; Walbot and Cullis 1983; Marx 1984).