Production of somatic hybrids between frost-tolerant Solanum commersonii and S. tuberosum: characterization of hybrid plants.

Somatic fusion of mesophyll protoplasts was used to produce hybrids between the frost-tolerant species Solanum commersonii (2n=2x=24) and dihaploid S. tuberosum (2n=2x=24). This is a sexually incompatible combination due to the difference in EBN (Endosperm Balance Number, Johnston et al. 1980). Species with different EBNs as a rule are sexually incompatible. Fifty-seven hybrids were analysed for variation in chromosome number, morphological traits, fertility and frost tolerance. About 70% of the hybrids were tetraploid, and 30% hexaploid. Chloroplast counts in stomatal guard cells revealed a low frequency of cytochimeras. The frequency of aneuploids was relatively higher at the hexaploid level (hypohexaploids) than at the tetraploid level (hypotetraploids). The somatic hybrids were much more vigorous than the parents, and showed an intermediate phenotype for several morphological traits and moderate to profuse flowering. Hexaploid hybrid clones were less vigorous and had a lower degree of flowering than the tetraploid hybrid clones. All of the hybrids were female fertile but male sterile except for one, which was fully fertile and self-compatible. Many seeds were produced on the latter clone by selfing and on the male-sterile clones by crossing. The somatic hybrid plants showed an introgression of genes for frost tolerance and an adaptability to cold from S. commersonii. Therefore, the use of these somatic hybrids in breeding for and in genetic esearch on frost tolerance and cold-hardening is suggested.

Nuclear genomic composition of asymmetric fusion products between irradiated transgenic Solanum brevidens and S. tuberosum: limited elimination of donor chromosomes and polyploidization of the recipient genome.

The production of asymmetric somatic hybrid calli after fusion between gamma-irradiated protoplasts from transgenic Solanum brevidens and protoplasts from S. tuberosum are reported. Transgenic (kanamycin-resistant, GUS-positive) S. brevidens plants and hairy root clones were obtained after transformation with Agrobacterium tumefaciens LBA 1060 (pRi1855) (pBI121) and LBA 4404 (pRAL4404) (pBI121), and A. rhizogenes LBA 9402 (pRi1855) (pBI121), respectively. Leaf protoplasts isolated from the transgenic plants or root protoplasts from the hairy root clones were fused with S. tuberosum leaf protoplasts, and several calli were selected on kanamycin-containing medium. The relative nuclear DNA content of the hybrid calli was measured by flow cytometry (FCM), and the percentages of DNA of the S. brevidens and S. tuberosum genomes in the calli were determined by dot blot analysis using species-specific DNA probes. Chromosome-specific restriction fragment length polymorphism (RFLP) markers were used to investigate the elimination of specific S. brevidens chromosomes in the hybrids. The combined data on FCM, dot blot and RFLP analysis revealed that 18-62% of the S. brevidens DNA was eliminated in the hybrid calli and that the RFLP marker for chromosome 7 was absent in seven out of ten calli. The absence of RFLP markers for chromosomes 5 and 11 hardly ever occurred. In most of the hybrids the ploidy level of the S. tuberosum genome had increased considerably.

Tetraploid potato hybrids through protoplast fusions and analysis of their performance in the field.

Data on the production of tetraploid hybrid plants after electrofusion of protoplasts from various diploid (2n = 2x =24) Solarium tuberosum and S. phureja are reported. Ten different fusion combinations were used. Six hybrids with the tetraploid chromosome number 2n=4x=48 were tested under field conditions for their performance in various agronomic traits (tuber type, tuber yield, underwater weight of tubers, numbers of tubers, mean weight per tuber). Tuber yield in five of the six hybrid clones was similar to that of cv Bintje. One hybrid clone (35-4) had three times higher tuber yield than cv Bintje. The mean tuber weight of this hybrid was similar to that of cv Bintje. The results of this study prove that fertile tetraploid somatic hybrids having similar or higher tuber yield than that of cv Bintje can be obtained through somatic hybridization. This technique is now included in commercial potato breeding programs in The Netherlands.

Chromosome elimination and mutation in tetraploid somatic hybrids ofSolanum tuberosum andSolanum phureja.

The hybridity of eleven somatic hybrids between a diploidS. tuberosum and a diploidS. phureja clone could be verified because the parent karyotypes differed in their C-banding patterns. The hybrids were hypotetraploid and some carried structurally rearranged chromosomes and/or minute centric fragments. The nucleolar chromosomes ofS. phureja were eliminated preferentially. The function of the remaining nucleolar organizer regions was not suppressed. Nuclear DNA content was correlated with the hypotetraploid chromosome numbers.

Elimination of Solanum phureja nucleolar chromosomes in S. tuberosum + S. phureja somatic hybrids.

The karyotype of the dihaploid SVP1 line of S. tuberosum (2n=2x=24) showed two nucleolar chromosomes with differently sized satellites. The diploid SVP5 line (2n=2x=24) and tetraploid regenerants of S. phureja had larger but similar satellites. Somatic hybrids between the diploid lines of these potato species with genome combinations 4 tub + 2 ph (plants 1-3), 2 tub + 4 ph (plants 4-7) and 4 tub + 4 ph (plant 8) had lost 2 phureja nucleolar chromosomes if 4 phureja genomes were present. One phureja nucleolar chromosome of plants 1-3 and both of plants 5 and 7 had rearranged satellites. Elimination of the two nucleolar chromosomes occurred preferentially, was under genetic control, and probably took place during early callus development. NOR activity resulting in rear-rangements between NORs may have caused the elimination.

Somatic hybrid potato plants after electrofusion of diploid Solanum tuberosum and Solanum phureja.

Protoplasts from diploid S. tuberosum and diploid S. phureja were electrofused followed by selection of the heterokaryons with a micromanipulator. Visual identification of the heterokaryons was facilitated by fluorescein diacetate staining of the protoplasts from one of the parents, which was grown on herbicide containing medium to induce bleaching of the chlorophyll. In total, 840 heterokaryons showing red (chlorophyll) and yellow-green (fluorescein) fluorescence were selected and cultured at relatively low densities using various feeder systems. Finally, 18 putative hybrid plantlets were obtained and grown to maturity. DNA histograms indicated that the plants were hexaploid, octoploid or mixoploid. With Giemsa C-band pattern analysis of the chromosomes the hybrid character and the combinations of the chromosome sets of all plants investigated could be established.

Electrofusion, a simple and reproducible technique in somatic hybridization of Nicotiana plumbaginifolia mutants.

Electrofusion of protoplasts from two complementary nitrate reductase deficient mutants of Nicotiana plumbaginifolia has resulted in somatic hybrid lines. Mesophyll protoplasts isolated from the cofactor mutant CNX 20 and fluorescein diacetate stained protoplasts derived from a cell suspension culture of the NA 36 line, being defective in the apoenzyme, were used in the fusion experiments. In total, 594 lines were recovered which could proliferate on a selective medium with nitrate as the sole nitrogen source. This is including 141 putative hybrid lines which were obtained after transfer of 1048 heterokaryons with a micromanipulator one day after electrofusion. The hybrid character of some of the selected lines was confirmed by nitrate reductase activity measurements. Plants were grown from hybrid calli.

Lyoluminescence dosimetry part II. State-of-the-art.

This is the second part of a review dealing with the development and current state-of-the-art of lyoluminescence dosimetry. Readout equipment described in the review covers a range of devices from simple manually-operated apparatus to semi-automatic machines suitable for loose powders and for encapsulated dosimeters. The survey of dosimetric properties of mannose and glutamine includes response to X- and gamma-rays, electrons, heavy charged particles and to neutrons. Current applications of lyoluminescence dosimetry include radiotherapy, radiation processing of foods and sterilization of medical products, as well as the use of lyoluminescent phosphors for gamma-ray dose intercomparisons in the high-dose range.

Dosimetry intercomparison for partial body x-irradiation of rats.

An X-ray dosimetry intercomparison for partial body irradiations has been performed among a number of laboratories cooperating within EULEP (European Late Effects Project Group) using mailed acrylic plastic rat phantoms. The main objective of this study was to assess the adequacy of exposure arrangements and dosimetry procedures applied to rat partial body irradiations at the participating institutes. The dose and dose distribution in lung and liver substitute material have been measured with dual thermoluminescent (TL) dosemeters LiF and CaF2:Mn. The doses from scattered radiation in the shielded parts of the phantom have also been assessed. The results of the dose intercomparison show that in the first instance six out of the eight participating institutes obtain dose values which differ by more than 5% from the standard dose. Inconsistencies are observed between the lung and liver doses administered at the same institute. After consultation, four institutes indicated the cause for their deviating dose values and quoted revised results which are closer to the standard value. Large dose gradients are measured, since six out of the eight participants applied unilateral exposures. This project has indicated the need for repeated intercomparisons and for improvements in dosimetry and irradiation procedures for partial body irradiations.

Dosimetry intercomparisons for evaluation of late effects of ionizing radiation.

In 1971, 1973 and 1976 intercomparisons of absorbed dose and dose distribution over a mouse phantom for roentgen irradiation were performed as part of the program of the European Late Effects Project Group (EULEP). Sixteen institutes from 8 countries participated in the sessions of the third intercomparison. In general, progress has been made concerning accuracy and precision of the dosimetry; in 2 cases, discrepancies could be resolved after additional measurements. With regard to the dose distribution over a mouse phantom, the results are not satisfactory; 4 out of 15 participants are still unable to perform uniform irradiations. The necessity of repeated intercomparisons is clearly demonstrated to allow a comparison of biologic results obtained in cooperative research programs.

Lyoluminescence dosimetry in photon and fast neutron beams.

The lyoluminescence (LL) technique using mannose, a monosaccharide, is described. Dose-response curves for 60Co-gamma-rays (5 rad to 120 krad), fission neutrons, 5.3 MeV and 15 MeV neutrons (100 rad to 20 krad) have been measured. The close tissue-equivalence of mannose makes this material well suited for dosimetric use in low energy X-ray fields for radiotherapy and radiobiology. It also provides a cheap, simple and reproducible dosemeter in industrial applications of radiation (sprouting inhibition of onions and potatoes; control of insect infestation). After correction for the gamma contamination of the neutron beam the LL signal per rad in ICRU muscle tissue from the neutron irradiations has been derived and the relative effectiveness of the LL signal for fast neutrons in mannose has been calculated as 0.34 +/- 0.03 (fission neutrons), 0.63 +/- 0.07 (5.3 MeV neutrons) and 0.74 +/- 0.05 (15 MeV neutrons). These results are compared with data from other systems. It is concluded that mannose can be used as a transfer system in neutron dosimetry, if its variation in sensitivity with neutron energy is taken into account.

A thermoluminescence system for the intercomparison of absorbed dose and radiation quality of X-rays with a HVL of 0-1 to 3-0 mm Cu.

A TLD system is described which permits the measurement of absorbed X-ray doses in a water phantom, together with the quality of the incident X-ray beam. The system has been developed for dose and energy intercomparison studies between centres with deep therapy X-ray machines. A distinction has been made between deep therapy treatments and treatments at or near the surface. For beam qualities with a HVL above 1-1 mm Cu, the deep therapy region, measurements have been performed at 5 cm depth in water using CaF2: Mn and LiF (TLD 700). For beam qualities with a HVL of 0-11-1-4 mm Cu, these qualities include the superficial therapy region, measurements with LiF dosemeters at 2 and 10 cm depth in water have been carried out. The uncertainty in the absorbed dose value for X-ray beams with a HVL of 0-11-3-0 mm Cu amounts to +/- 4%; the uncertainty in the determination of the effective energy of the incident beam is +/- 3 to +/- 7%.