Chromosome variation in wheat plants regenerated from cultured immature embryos

Summary A cytological study has been made of plants regenerated from cultured immature embryos of four wheat cultivars (Triticum aestivum, 2n = 6x = 42). In total, 29% of the 192 plants examined were aneuploid with a range in chromosome numbers of 38–45. Evidence of chromosome structural changes was also found. This variation occurred in regenerants of all four cultivars, but there were large differences in the proportions of aneuploids arising from individual cultures which meant that no significant differences could be demonstrated between cultivars. Chromosome abnormalities were present in plants regenerated both from embryogenic cultures and from cultures in which the origin of shoots could not be distinctly defined.     

Shoot Formation from Explant Cultures of Fourteen Potato Cultivars and Studies of the Cytology and Morphology of Regenerated Plants

Plants were regenerated in quantity from cultured excised stem,rachis or leaf pieces of 13 potato (Solarium tuberosum L.) cultivarsusing modifications of a two-step procedure. Pronounced differenceswere observed in the response of different cultivars and explantsources to medium formulations containing different auxins orcytokinins. A few plants were recovered from tuber pieces ofseven cultivars by a different two-step procedure. Morphologicaldifferences from control plants were observed in some of theprimary regenerants. Anthocyanin pigmentation of tubers wasscored and stable changes to red-splashed white or white tuberswere found in some plants regenerated from cv. Desiree. Regenerantsfrom the particoloured cultivars Cara and King Edward oftenlost this trait but it returned to varying extents in subsequentgenerations. Most of the regenerated plants (87 per cent) containedthe euploid number of chromosomes (2n = 4x = 48). The methodsare suitable for production of plants for assessment of somaclonalvariation in a range of genotypes. Potato breeding, somaclonal variation, chromosome variation, skin colour, explant culture, plant regeneration, solanum tuberosum L. potato     

The susceptibility of stems of different potato cultivars to blackleg caused by Erwinia carotovora subsp. atroseptica

The susceptibility of stems of six potato cultivars to Erwinia carotovora subsp. atroseptica was assessed in two years (1981 and 1982) either by direct inoculation in the field or by inoculation of detached stems in the laboratory. These six and a further 22 cultivars were also assessed in three years (1982-84) by inoculating stems of glasshouse-grown plants. Different methods of inoculation and types of inocula were tested. In the field, wooden toothpicks rubbed in bacterial slime were more successful in establishing infection than when dipped in a bacterial suspension, but injection of bacterial suspension with a hypodermic needle was reliable in establishing infection over a range of concentrations. Detached stems were more readily infected and gave more consistent results compared with inoculation in the field. The range of reaction of the six cultivars was similar in both detached stem and glasshouse tests. The early cultivars Pentland Javelin and Ulster Sceptre were most susceptible and of the maincrop cultivars, Maris Piper was intermediate and Desiree and King Edward least susceptible whereas Pentland Crown showed greater resistance in the glasshouse than in the field. Glasshouse tests using hypodermic inoculation indicated a range of susceptibilities; the early cultivars Manna, Maris Bard and Estima were most susceptible and the maincrop Pentland cultivars Crown, Dell, Hawk, Ivory and Squire least susceptible.     

Improvements in regeneration from protoplasts of potato and studies on chromosome stability

Summary Regeneration of plants from protoplasts of potato (Solanum tuberosum) cv. Maris Bard has been achieved from four different initial culture media (ET2, ET3, CLG, VkCLG). These media differed in their hormone, salt and sugar content. Plating efficiencies were highest in the VkCLG medium, but no correlation was found between plating efficiency and regeneration frequency (i.e. the percentage of calli producing shoots). Regeneration frequencies were high on all four media; up to 95% on ET3. Chromosome counts of up to 50 regenerants selected at random from the four treatments showed no significant differences between any of the treatments, in the proportions of plants which were euploid (48), aneuploid at the tetraploid level (48±), and aneuploid with high chromosome numbers (48+ +). Highly significant differences were present, however, between shoots which rooted quickly (predominantly euploid) and those which rooted only after transfer to a rooting medium (predominantly 48+ +). Overall more than 60% of the regenerants were normal (2n=4x=48) and this is a considerable improvement on our earlier work in this cultivar (4% normal). These findings are discussed in relation to factors affecting chromosome stability. Chromosome structural rearrangements are also described.     

Use of resistant and susceptible potato cultivars in the trap cropping of potato cyst nematodes, Globodera pallida and G. rostochiensis

The effects of planting date and growing period of potato cultivars on their efficiency as trap crops for potato cyst nematodes (PCN) were studied. Plots were planted with susceptible or resistant cultivars in April, June and August and these were grown for 5, 6 or 7 wk before removal of the plants by hand lifting. Crops planted in June provided the best overall reductions in PCN population density of up to 95%, with cv. Santé significantly more effective than the other cultivars. Population reductions from the August planting were only slightly less than from planting in June but the tuber yields obtained were much greater: Maris Piper and Maris Bard produced 16.4 and 21.4 t ha^-1 respectively, with 37% and 43% respectively, of a size useful for canning (i.e. between 20 and 40 mm diameter).     

Stable transformation of barley callus using biolistic® particle bombardment and the phosphinothricin acetyltransferase (bar) gene

We have previously studied chromosomal and morphological variation in protoplast cultures of diploid petunia (Petunia hybrida) plants. We found that 85% of the regenerants were tetraploid (2n=4x=28). These plants flowered and set seeds.In the present study, the cytological stability of plants regenerated from leaf mesophyll protoplast cultures derived from the progeny of the self-fertile tetraploid plants was assessed on the basis of mitotic analysis, morphological characters, and protein patterns. When we analyzed the root tip chromosomes of 117 regenerants derived from 39 protoclone calluses, all of the regenerants tested retained the parental chromosome number of 2n=4x=28. One hundred regenerants were further analyzed and displayed normal vegetative morphology and retained the floral characteristics of the seed-derived plants from which they were derived. No significant variations in any character were observed among regenerants. When leaf protein patterns from four regenerated tetraploid protoclones were analyzed by two-dimensional polyacrylamide gel electrophoresis and compared with those of seed-derived plants, the protein patterns exhibited great similarity.The data suggest that tetraploidization of petunia plant increases cytological stability during further in vitro cultures and may play an important role in the genetic stability of regenerant populations.Abbreviations BA benzylaminopurine - IAA indole-3-acetic acid - IEF isoelectric-focusing - PVP polyvinylpyrrolidone - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Detection of somaclonal variation in garlic (Allium sativum L.) using RAPD and cytological analysis

Plants were regenerated by somatic embryogenesis from long-term callus cultures derived from five garlic (Allium sativum L.) cultivars. Thirty-five of these plants were subjected to RAPD analysis. The frequency of variation was found to be cultivar dependent: approximately 1% in the two clones Solent White and California Late and around 0.35% in another three clones, Chinese, Long Keeper and Madena. Certain band changes were found in regenerants of different cultivars, suggesting the existence of a mutation-sensitive part of the garlic genome. The karyotypes of another 75 regenerants derived from the same callus cultures of three parental garlic clones were examined. Of these plants, 9.3% were found to be tetraploids, 4% aneuploid and 2.6% showed a change in the position of the secondary constriction. No association could be shown between the rate of variation for molecular and cytological characters either by comparing cultivars or examining individual regenerants. Received: 30 July 1996 / Revision received: 28 October 1996 / Accepted: 12 November 1996     

Regeneration and characterization of plants obtained from anther cultures in Medicago sativa L

Summary The androgenic ability of four Medicago sativa L. genotype (Boynitza 5, Byala, 494, and 3815) was tested. Callus and organogenesis were induced in all lines studied. The percentage of anthers producing calluses and organogenesis showed wide variation (calluses—from 11% up to 77%; organogenesis—4.8% to 15.2%). It has been established that genotype, nutrient medium composition, and stage of pollen development considerably affected both callus production and organogenesis. Androgenesis in M. sativa could be achieved via callus and direct embryogenesis. About 500 morphologically different regenerants were obtained. Wide variability in chromosome number of regenerated plants was observed by cytological studies. Haploid, dihaploid, as well as mixoploid plants were obtained.     

Molecular cytogenetic analysis of intergeneric chromosomal translocations between wheat (Triticum aestivum L.) and Dasypyrum villosum arising from tissue culture.

Fluorescence in situ hybridization (FISH) was applied with total genomic DNA extracted from Dasypyrum villosum (L.) Candargy as a probe to characterize chromosome translocations arising from tissue culture in hybrids of Triticum aestivum x (T. durum - D. villosum, amphiploid). Chromosome translocations between wheat and D. villosum occurred in callus cells at an average frequency of 1.9%. Translocations existed not only in callus cells but also in regenerants. Three plants with translocation chromosomes were characterized among 66 regenerants of T. aestivum 'Chinese Spring' x 'TH1W' and 'NPFP' x 'TH1'. One of them proved to be a reciprocal translocation with an exchange of about one third of a wheat chromosome arm with about one half of a chromosome arm of D. villosum. The breakpoints of the other two translocations were located at, or near centromeres. The results are similar for both callus cells and regenerants and provide further evidence that translocations take place in tissue culture. Other structural chromosomal changes, for example, fragments, telocentrics, dicentromeres, and deletions, as well as numerical alterations including aneuploidy and polyploidy were recorded both in callus cells and regenerants.     

A method for assessing the field susceptibility of potato cultivars to blackleg (Erwinia carotovora subsp. atroseptica)

In experiments to develop a method for assessing the field susceptibility of potato cultivars to blackleg (Erwinia carotovora subsp. atroseptica) seed tubers were stab-inoculated near the stolon (attachment end), with a suspension of the bacterium, or with water, before planting. Disease symptoms were recorded in three years (1980–1982) and plant growth and yield in 1982. Estima and Maris Bard were the most susceptible cultivars with many plants failing to emerge and most of those that did showing disease symptoms. Pentland Crown was the most resistant: few plants failed to emerge and few showed blackleg. Nevertheless compared with water-inoculated plants bacterial inoculation of the seed tubers of this cultivar caused loss of yield and differences in tuber size distribution. Cara, Wilja and King Edward showed intermediate reactions.     

Lotus corniculatus L.: Morphological and cytological analysis of regenerants from three sources of tissue and selected progeny

Regenerants from three types of tissue, leaf explants (132 plants), leaf protoplasts (68 plants) and cotyledonary protoplasts (119 plants) of L. corniculatus cv Leo differed both morphologically and cytologically from control plants grown from seed. Four categories of chromosome number were found. The frequency and type of variation found in the chromosome numbers of regenerants reflected the method of plant regeneration.Regenerants with both normal and abnormal numbers of chromosomes produced progeny which were cytologically normal and showed only minor morphological changes when compared with control plants.     

Differences in the tolerance of potato cultivars to potato cyst nematodes (Globodera rostochiensis and G. pallida) in field trials with and without nematicides

In field trials Cara, Brio, Maris Piper and Pentland Javelin were consistently more tolerant of damage by Globodera rostochiensis yielding more than Corsair, Pentland Dell, Maris Anchor and Maris Peer, in untreated, heavily infested soil and giving the smallest increases to nematicide treatment. No yield or growth differences were found between plants in untreated and nematicide treated plots at a nematode-free site. The most tolerant cultivars all had a gene (H₁) for resistance to G. rostochiensis derived from Solanum tuberosum ssp. andigena and in soil infested with G. pallida the tolerance of at least one resistant cultivar (Maris Piper) appeared to be lessened. However, some resistant cultivars were comparatively intolerant, even to G. rostochiensis. Early cultivars were generally less tolerant than late maturing cultivars but there were exceptions. Amongst cultivars with resistance derived from Solanum vernei the early cultivar Guardian was more tolerant than the main crop cultivar Corsair. The effect on the yield of several cultivars of a range of densities of G. rostochiensis, produced either by applying different rates of a nematicide or by cropping in the previous year, was examined at two sites. The results indicated that the slope of the regression for yield in relation to nematode density was less for tolerant than intolerant cultivars. At sites infested with G. rostochiensis Maris Piper was found to be consistently more tolerant than Pentland Crown.     

Stem canker (Rhizoctonia solani) on five early and seven maincrop potato cultivars I. Infection of shoots, stolons and tubers

In 1983 and 1984, potato seed tubers of five early and seven maincrop cultivars were inoculated with cultures of Rhizoctonia soluni during planting to simulate severe seed infection. Shoot and stolon infection was assessed in June-August and black scurf on tubers recorded after harvest in October. Almost all shoots of all cultivars had stem canker in both years and disease on shoots, stolons and tubers was more severe in 1984 than in 1983. In 1983 similar amounts of disease developed on all early cultivars and between 11% (Ulster Sceptre) and 32% (Maris Peer) shoots were pruned off. Maris Peer had a stem canker score lower than other cultivars in 1984 but more than half the shoots were pruned off. Shoot pruning on Estima, Ulster Prince and Ulster Sceptre was more common on plants from sprouted than non-sprouted seed. Between 30 and 50% of stolons were pruned off. After harvest in 1985, black scurf was least prevalent on Arran Comet and Maris Peer tubers and in 1984 on Arran Comet and Estima tubers from non-sprouted seed. Of the maincrop cultivars, King Edward plants from sprouted seed had many shoots pruned off in both years. Shoot pruning was also prevalent on Maris Piper and Pentland Squire plants from non-sprouted seed. Record had fewest pruned shoots and stolons and the lowest stem canker score. The disease was more severe on Pentland Crown and Maris Piper plants from non-sprouted than sprouted seed. Black scurf was most common on Cara and King Edward tubers in 1983 and on King Edward and Record tubers in 1984. In both years few shoots but many tubers were infected on plants from non-inoculated seed and the significance of this is discussed.     

Statistical Parameters Reflecting Morphogenetic Capacity of Soft Spring Wheat Calluses

Callus cultures of soft spring wheat were subcultured without separation into explants to follow the line one excised embryo–one callus. This approach revealed the following statistical correlations. Within every cultivar of Triticum aestivum L. and within a row of cultivars arranged in ascending order according to the frequency of embryogenic callus formation, positive correlations (at P = 95) were found between the proliferative activity of callus cells and the frequency of embryogenic callus formation. A reliable intraspecies correlation (significant at P = 95) between multiple regenerations of plants from calluses and the tillering trait (bushiness) of donor plants was also found. We assessed the importance of various statistical parameters of callus cultures for preliminary estimation of morphogenesis efficiency at early stages of culturing. Frequencies of callusogenesis and the growth curves for randomly selected calluses turned out to be noninformative characteristics, unless the morphogenetic activity of calluses was taken into account. The following statistical parameters were found to correlate with the morphogenetic capacity of wheat calluses: gradually increasing coefficients of variation in fresh weight of primary calluses, a larger callus size, and higher fresh weight gain in potentially morphogenetic calluses.     

T-DNA genes to study plant development: precocious tuberisation and enhanced cytokinins in A. tumefaciens transformed potato

Summary Potato Line Mb1501B is a derivative of the cultivar Maris Bard (Solanum tuberosum), transformed with T-DNA from A. tumefaciens strain LBA1501. In culture it grew as frequently branching stunted shoots with a basal callus, lacking roots. These shoots did not form tubers. When grafted, Mb1501B shoots gradually became morphologically more normal and aerial tubers formed readily. Cultured Mb1501B shoots contained 100–200-fold higher concentrations of the biologically-active cytokinins zeatin, zeatin riboside and their corresponding side-chain o-glucosides than untransformed Maris Bard shoots. Cultured Mb1501B shoots contained approximately a 3-fold lower concentration of indole acetic acid (IAA). In grafted Mb1501B plants a 3–10-fold higher concentration of the active cytokinins was found compared with untransformed plants and no difference in IAA concentration.     

Molecular marker analysis of hypoploid regenerants from cultures of barley x Canada wild rye.

Canada wild rye (CWR, Elymus canadensis L., 2n = 4x = 28) is a potential source of genes for disease resistance and environmental tolerance in barley (Hordeum vulgare L., 2n = 2x = 14). Tissue cultures were initiated from immature inflorescences of CWR x 'Betzes' barley hybrids to promote CWR introgression into barley through possible tissue culture induced chromosome breakage and exchange. Among the plants regenerated, some were missing one (2n = 20) or part of one (2n = 20 + telo) chromosome. The objective of this study was to identify the missing chromosome or chromosome arm in these regenerants through the analysis of molecular (RFLP) markers that previously had been mapped in barley. Forty-six hypoploid regenerants that traced to 30 separate explants obtained from 10 interspecific hybrid plants were evaluated. DNA was digested with the restriction enzyme HindIII, Southern blotted, and probed with 39 genomic and cDNA barley clones that identified sequences polymorphic between barley and CWR. Eight of these probes identified band loss patterns that separated the regenerants into two groups. One group, all with barley cytoplasm, were missing a CWR chromosome homoeologous to barley chromosome 3; a second group, all with CWR cytoplasm, were missing a CWR chromosome homoelogous to barley chromosome 7. These results indicated that chromosome elimination in culture was not random. The two cytoplasm groups were further differentiated by probes that identified band shifts. These band shifts were caused by differences in DNA methylation. Key words : Hordeum vulgare, aneuploidy, Elymus canadensis, tissue culture.     

Induced androgenesis in tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> Mill.). III. Characterization of the regenerants

We present data on the morphological, cytological, biochemical and genetic characteristics of tomato regenerants obtained through anther culture. As a result of induced androgenesis, more than 6,000 rooted regenerants were developed that differed both from the donor plants and among each other with respect to habitus and leaf, flower and inflorescence morphology. Cytological analysis revealed a great variability in chromosome number in the cells of the regenerated plants. While most of the regenerants were mixoploid, the majority of the cells had a haploid chromosome number. R₁ and R₂ progenies were tested for their resistance to Clavibacter michiganense subsp. michiganense (Cmm 7). Some of the regenerants were resistant to the pathogen. A biochemical analysis of fruit from R₃ and R₄ plants showed a higher content of dry matter, sugars and vitamin C in the regenerant plants obtained from the hybrids than in those from the cultivars and control plants. The values of the parameters of hybrid regenerants grown in the greenhouse were about 1.5-fold higher than those of the hybrid regenerants grown in the field, and this trend is clearly expressed in all of the hybrid regenerants. The results obtained suggest that induced androgenesis and gametoclonal variation may be used as an additional tool to create a large range of new forms. The application of the latter in breeding programs would accelerate the development of tomato lines and varieties that would be more productive, disease-resistant, highly nutritive and flavour-acceptable.Abbreviations BAP N⁶-Benzylaminopurine - Cmm Clavibacter michiganense subsp. michiganense - cfu Colony-forming units - GA ₃ Gibberellic acid - IBA Indole-3-butyric acid - ms Male sterility - PDA Potato dextrose agar Communicated by H. Lörz     

A comparison of somatic chromosomal instability in tissue culture regenerants from Medicago media Pers

Two cultivars (Heinrichs, Reaver) and two breeding lines (Br1, Le1) from Medicago media were cultured in a media protocol consisting of a high concentration 2,4-D induction step. Regenerants were produced from all four stocks. Representative samples from each regenerant population along with the corresponding control population were cytologically analyzed for chromosomal and pollen abnormalities. While numerical changes in chromosome numbers were found in all regenerant populations, there was considerable variation between the four stock groups. Heteroploidy was observed for both hypo and hyper aneuploid regenerants, but there were no differences in pollen stainability between hypo and hyper aneuploid regenerants and euploid regenerants. Tissue culture regenerants generally produced a lower pollen stainability percent as compared to control populations grown from seeds. Gross and cryptic changes in chromosomes, or hormonal carry over effects or both were considered causes for poor pollen stainability in tissue culture regenerants. Cytological analyses indicate that the cultivar might play an important role in the cytological stability or instability of regenerant populations. Exploitation of this naturally existing situation to produce euploid regenerants for field experiments and to obtain gross cytological stability in somaclones is discussed.     

Variability and correlations in muskmelon in relation to the cultivation method

Summary Six fruit characters have been measured in 23 cultivars of Cucumis melo, representing a wide geographical range. Plants were grown both in the greenhouse and in the field. When the 23 cultivars were analyzed together, the largest component of variance was found between cultivars under both growth conditions, suggesting the existence of large genetic diversity for all the characters studied. Generally, variance between plants within cultivars was less than or equal to variance between fruits within plant. This indicates that environmental variation is the most important part of the variation within cultivars. Correlations between pairs of characters at cultivar, plant and fruit levels were calculated from the variance-covariance components. In the majority of paired traits, the correlation values indicated that genetic and environmental factors may act in the same direction.