Regeneration of fertile green plants from oat isolated microspore culture

Regeneration of fertile green plants from isolated oat microspores is reported for the first time. Factors critical for microspore growth and regeneration include cold pre-treatment, pH of culture medium and the use of conditioned culture medium. It was found that cold pre-treatment at 4°C in the dark for a minimum of 6 weeks was necessary to consistently achieve microspore growth into multicellular structures (MCS). Longer pre-treatments of up to 9 weeks were tested and found to be positively correlated with the number of MCS produced. Microspore culture medium with pH 8.0 produced significantly more MCS larger than eight cells in size than media with pH 5.8. The use of medium conditioned by actively growing barley microspores significantly increased the numbers of MCS larger than eight cells in size compared to non-conditioned media. Plants were regenerated only from cultures using conditioned medium. A total of 2 green plants and 15 albinos were regenerated. Of the green plants, one had the haploid chromosome complement (n = 3x = 21) and the other had the parental hexaploid chromosome complement (2n = 6x = 42) which may be due to spontaneous chromosome doubling. The hexaploid plant set seed naturally and the haploid plant set seed after its chromosome complement was doubled with colchicine.     

Chromosome number and DNA content of tobacco cells adapted to NaCl

Cultured tobacco (Nicotiana tabacum L. cv. Wisconsin 38) cells were found to have altered DNA contents and chromosome numbers after adaptation to NaCl. Cells adapted to 428 mM NaCl were predominately hexaploid compared to the normal tetraploid 2N(2C)=4X=48 chromosome number of unadapted cells. Enrichment of the cell population for hexaploid cells occurred only after exposure to higher NaCl (428 mM), not lower levels of NaCl (171 mM). The majority of adapted cells remain hexaploid for at least 25 cell generations after removal from NaCl exposure. Adapted cell populations were found to have fewer cells with highly polyploid (2N96) nuclei. Salt tolerance of hexaploid cells was not found to be significantly greater than that of tetraploid cells. Cells with higher ploidy levels were less salt tolerant. It is suggested that high levels of NaCl induce polyploidization and that exposure to NaCl selects against cells with very high ploidy levels.     

Ploidy levels of plants regenerated from mixed ploidy maize callus cultures

Summary Haploid and diploid anther-derivedZea mays callus lines were treated with the antimicrotubule herbicide pronamide to produce mixed ploidy callus as determined by flow cytometry. The ploidy levels of the plants regenerated from the callus were determined by counting the leaf epidermal guard cell chloroplast numbers. The proportion of diploid regenerated plants was somewhat lower than the proportion of diploid cells of the callus. The diploid plants regenerated somewhat faster than the haploids. The proportion of tetraploids regenerated from the pronamide treated diploid callus, which originated by spontaneous chromosome doubling, was much lower than the proportion of cells indicating that tetraploid cells survive or regenerate plants at a lower frequency than diploid cells.     

Somatic hybrids of Datura innoxia Mill.+Datura discolor Bernh. and of Datura innoxia Mill.+Datura stramonium L. var tatula L.

Summary Following fusion of protoplasts from a chlorophyll-deficient diploid mutant of Datura innoxia Mill. which can be regenerated to shoots, with green wild-type protoplasts of Datura stramonium L. var. tatula L. which can not, it was possible to isolate 49 green hybrid calli on agar medium. Most of these somatic hybrid calli gave rise to leaves and shoots. The chromosome numbers of the somatic hybrids were determined: 15 were tetraploid (amphidiploid), 24 hexaploid, and the other showed an aneuploid chromosome number.In a similar experiment protoplasts of the Datura innoxia mutant were fused with green wild-type protoplasts of Datura discolor Bernh. which are also not able to be regenerated, four green calli were obtained from which leaves and shoots developed after some transfers on agar medium. Three of them showed the amphidiploid (48) chromosome number, whereas one possessed an aneuploid number of 46 chromosomes.After transfer of rooted shoots to soil flowering plants could be obtained in both combinations. The habits of the somatic hybrids in both combinations were intermediate between the habits of the respective parental plants.Dedicated to my father, Prof. Dr. Theodor Schieder, on the occasion of his 70th birthday.     

Expression of various gametic types in pollen plants regenerated from hybrids between Triticum-Agropyron and wheat

Summary Studies of the chromosomal composition of pollen plants regenerated from the F₁ of hybrids produced from Triticum-Agropyron intermediate type and common wheat demonstrated that various gametic types of the F₁ could be fully expressed at the whole plant level via anther culture. The observed frequency of each of the eight types of pollen plants (based on their chromosome numbers) was in good agreement with the theoretical probabilities as shown by X² analysis. Comparative studies of the chromosome composition of somatic cells and pollen mother cells (PMC's) of selected pollen plants permitted classification of the plants into four distinct classes. The majority of these regenerated pollen plants had identical chromosome numbers in both root tip cells and PMC's. An alien disomic addition line, which was cytologically stable for two generations, was obtained directly from anther culture. Moreover, the addition line exhibits resistance to stripe rust disease, a trait which is conferred by the Agropyron chromosome. We suggest that anther culture techniques provide a unique and expeditious route for the introduction of alien genes or chromosomes into wheat cultivars.     

The origin and meiotic behaviour of hexaploid northern wheatgrass (Agropyron dasystachyum)

The occurrence of hexaploid (2n = 6x = 42) forms in some otherwise natural tetraploid populations of Agropyron dasystachyum (2n = 4x = 28) was cytologically detected and studied. The hexaploid plants are morphologically similar to the tetraploids except for a small reduction in the anther size. The general survey of chromosome numbers of natural Northern Wheatgrass (A. dasystachyum 2n = 4x = 28) populations derived from eight different regions of Alberta indicated that the occurrence of hexaploid variants was not restricted to a single locality. A comparative study of chromosome pairing in the natural and the synthetic hexaploids revealed that the naturally occurring 42-chromosomed plants of A. dasystachyum originated as a result of fertilization between unreduced (SSHH) and the natural (SH) gametes, both coming from the tetraploid form of A. dasystachyum. Based on chromosome pairing, the genomes of the natural hexaploid A. dasystachyum have been designated as SSSHHH. The natural hexaploids appear to intercross among themselves and also with tetraploids producing euploid and aneuploid hybrids. The possible evolutionary significance of these findings is briefly discussed.     

Regulation of karyotype stability in tobacco tissue cultures of normal and tumorous genotypes

Summary Callus cultures of Nicotiana glauca, N. langsdorffii and of their tumor-forming hybrid plants contained a high frequency of cells with irregular chromosome numbers and chromosome aberrations (hypo-, hyper-, polyploid, aneuploid cells; bridges, polytene, broken, fragmented chromosomes, megachromosomes, etc.). Meristematic cells of shoot tips regenerated from the same cultures contained only regular chromosome numbers with normal chromosome structures. Variability in chromosome numbers is a consequence of abnormal mitoses. The data suggest genome segregation in the cultures. Cytological instability appears to be independent of genome segregation composition, genotype, tumorous condition, hormonal requirement and level of ploidy. The karyotype stability of the cultures is only dependent on the degree of organization of tissues and is regulated by factors involved in the control mechanisms of organizational processes.     

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.     

Differential transmission of extra genome chromosomes in pentaploid blueberry

Summary Transmission of extra genome chromosomes by three Vaccinium ashei (2n=6x=72)/V. corymbosum (2n=4x=48) pentaploid hybrids backcrossed to the hexaploid species V. ashei was examined. Chromosome numbers were determined for 36 and 31 progeny representing 5x × 6x and 6x × 5x type crosses, respectively. Chromosome numbers ranged from hypopentaploid (2n=4x+11=59) to hexaploid with means of 2n=66.2 for 5x × 6x progeny and 2n=68.0 for 6x × 5x progeny, representing overall extra genome chromosome gains of 3.3% and 33.3%, respectively. Extra chromosome number distributions for both the 5x × 6x and x × 5x progeny deviated significantly from the theoretical distribution assuming random chromosome transmission and were also found to be heterogeneous. The 2n=5x+9=69 class predominated in 6x × 5x progeny, while a predominate class was lacking in the 5x × 6x progeny. Higher than expected frequencies of plants with chromosome numbers near the pentaploid and hexaploid levels were found in the 5x × 6x progeny, whereas the frequency was only greater at the hexaploid number in 6x × 5x progeny. Present and previous results (Vorsa et al. 1986) indicate that extra genome chromosome transmission in oddploids can be influenced by selection at both gametophytic (pollen) and post-zygotic stages. However, post-zygotic selection may involve two different mechanisms acting concurrently: 1) chromosome imbalance due to aneuploidy and/or 2) endosperm imbalance referring to maternal: paternal genome ratios deviating from 21. Such a mechanism could result in differential transmission rates of extra genome chromosomes in oddploids when crosses are made to differing ploidy levels, and to reciprocal differences as well.     

Intergeneric transfer of cytoplasmic male sterility between Raphanus sativus (cms line) and Brassica napus through cytoplast-protoplast fusion

Summary Cytoplasts isolated from hypocotyl protoplasts of Raphanus sativus cv Kosena (cms line) by ultracentrifugation through Percoll/mannitol discontinuous gradient were fused with iodoacetamide(IOA)-treated protoplasts of Brassica napus cv Westar. Seventeen randomly selected regenerated plants were characterized for morphology and chromosome numbers. All of the regenerated plants had morphology identical to B. napus and 10 of them possessed the diploid chromosome number of B. napus. The remaining plants had chimeric or aneuploid chromosome numbers. The mitochondrial genomes in the 10 fusion products possessing the diploid chromosome numbers of B. napus were examined by Southern hybridization analysis. Four of the 10 plants contained mitochondrial DNA showing novel hybridization patterns. Of these 4 plants, 1 was male sterile, and 3 were male fertile. The remaining plants showed mitochondrial DNA patterns identical to B. napus and were male fertile.     

Selection of Daucus cybrids based on metabolic complementation between X-irradiated D. capillifolius and iodoacetamide-treated D. carota by somatic cell fusion

Summary Protoplasts of Daucus capillifolius isolated from a suspension culture (chromosome number above 60) were X-irradiated over lethal dose (60 krad) just prior to fusion. Protoplasts from D. carota cell line (chromosome number 17) were treated with 15 mM iodoacetamide and fused with the X-irradiated protoplasts. Putative cybrid plants were regenerated on Murashige and Skoog medium (MS) lacking 2,4-D. The regenerated plants possessed chromosome numbers of 17 (2n–1) or 34 (4n–2) and an identical leaf morphology to D. carota. Their mitochondrial DNAs (mtDNAs) were analysed with restriction endonucleases. Novel restriction fragments, not present in mtDNA digests from both parents, were observed in mtDNAs of regenerated plants. These results indicate successful formation of cybrids between D. capillifolius and D. carota by protoplast fusion.     

Polyploidization in leaf callus tissue and in regenerated plants of dihaploid potato

Cytological studies on leaf callus cells and regenerated potato plants suggest that it may be possible to utilize somatic chromosome doubling to obtain tetraploids from outstanding dihaploid breeding clones. The ploidy levels found in callus-derived plants were diploid, tetraploid, and octaploid, but the proportion of these was dependent on the donor genotype. L₁ and L₃ germ layers were studied in more than 300 plants; periclinal ploidy chimerism, an undesirable feature of colchicine doubling, was not found. Leaf callus was more efficiently induced using NAA than 2, 4-D as an auxin source in the Murashige and Skoog medium. A high proportion of dividing cells in young calli were polyploid. The frequency of doubled and octaploid plants regenerated was significantly dependent on donor genotype. The extent of polyploidization was marginally higher after callus growth on a medium containing 2, 4-D than in a medium containing NAA. In some genotypes the chromosome numbers of regenerated plants were variable, being less than tetraploid (mixohypotetraploid). After tuber propagation, the original ploidy level was maintained although mixohypotetraploidy persisted. In a few somatically doubled clones, male fertility was tested and found to be satisfactory with respect to seed-setting.     

Fertile somatic hybrids between Petunia hybrida and a wild species,Petunia variabilis

Somatic hybrid plants were regenerated following electrofusion between leaf mesophyll protoplasts of P. hybrida (2n = 14) and a wild sexually incompatible species, P. variabilis (2n = 18). The selection of hybrids was based on the hybrid vigour, expressed both in the growth of the callus and at the shoot formation stage, resulting from the combination of parental genomes. Calli exhibiting vigorous growth were selected, and upon transfer to regeneration medium gave rise to shoots. Four regenerated plants from three calli had morphological characteristics intermediate between those of the parents. The hybrid nature of these plants was confirmed by chromosome counts as well as isozyme and DNA analyses. They had amphidiploid chromosome numbers (2n = 32) and were fertile. Following self-pollination and backcrossing with P. variabilis, large numbers of F₂ and BC₁ seedlings were obtained.     

Analysis of Chromosome Variation of Plants (R1) Regenerated from Somatic Cell of Wheat (Triticum aestivum)

Abnormal behaviors were observed throughout the whole melosis process of regenerated plants (R0 from somatic cell of Triticum aestivum cv. Ningmai 3. There was variation of chromosome numbers. The type of 2n–2 was frequently found, then the type of 2n–l, while the 2n+l and 2n–4 types were rarely observed. Anomalous ph-nomena such as univalent, multivalent, lagging chromosome, chromosome bridge, fragment and micronuclei were emerged at the meiotic stage of pollen mother cells of the regenerated plants (R1). These variations were different in degree in the 5 tested genotypes.     

毛花猕猴桃原生质体再生植株根尖染色体数目变异与细胞多核现象

对18株毛花猕猴桃(Actinidia eriantha Benth.)原生质体再生植株的体细胞染色体数做了观察,其中12株为整倍体:二倍体(2n=58)和四倍体(2n=116)各6株;另外6株为混倍体,其染色体数目变化在59～203之间。还发现原生质体再生植株有丝分裂间期细胞存在多核现象,有多核细胞的共10株,细胞核数目以双核和三核较常见,最多的有7个核。对照植株为2n=2x=58,未发现多核细胞。     

Somaclonal Variation in Chromosome Number and Nuclei Number of Regenerated Plants from Protoplasts of Actinidia eriantha

By counting the chromosome number of root tip cells in 18 regenerated plants derived from protoplasts of Actinidia eriantha Benth., the authors found 12 euploid plants and 6 mixoploid plants. Of the 12 euploid plants, 6 were diploid (2n = 2x = 58) and the other 6 were tetraploid (2n =4x = 116). The chromosome numbers of the mixoploid plants varied from 59 to 203. Mttltinueleate phenomenon was also observed in the interphase cells of 10 protoplast-derived plants. Cells with binuclei or trinuelei were common and cells having heptanuclei were also seen occassionally. Muhinucleate phenomenon did not occur in the control, i. e., the donor plant, where the chromosome number was 2n = 2x = 58.     

Production of somatic hybrids by electrofusion in Solanum

Summary Conditions are described for large scale electrofusion of mesophyll protoplasts of dihaploid S. tuberosum with those of diploid S. brevidens. Overall fusion frequencies of 20%–30% were achieved, and following fusion, large numbers of protoplast-derived calli were obtained. Putative somatic hybrid plants were selected from the regenerated shoots by examining their morphological characteristics. Twenty-one somatic hybrids were confirmed by isoenzyme analysis and six somatic hybrids were further confirmed by Southern hybridization. Tetraploid hybrids were obtained, but cytogenetic studies indicated that more of the regenerated hybrids were hexaploid than had previously been found following chemical fusion of the same partners. Some advantages of electrofusion over chemical fusion are discussed.     

Karyotypic changes in potato plants regenerated from protoplasts

Over two hundred plants were regenerated from shoot-culture derived proto-plasts of potato (Solanum tuberosum L. cv. Majestic). Some had grossly aberrant phenotypes but the majority were similar to, or indistinguishable from normal control Majestic. Cytological examination showed that on average, 57% of the regenerants had the normal chromosome number (2n=4x=48). The remainder were aneuploids and fell into two classes in approximately equal numbers. The first class was limited at about the euploid level (ie, 2n=44–49). The second class contained plants with higher chromosome numbers ranging from 2n=73 to the octaploid level (2n=8x=96). The overall results represent an improvement over our earlier studies on chromosome variation in protoplast-derived potato plants. In addition, three cases of structural chromosome variation were observed.     

A new cytotype of Jacobaea vulgaris (Asteraceae): frequency,morphology and origin

Jacobaea vulgaris subsp. vulgaris (syn. Senecio jacobaea subsp. jacobaea) constitutes an intricate polyploid complex distributed in Europe. Four cytotypes have been reported in this species, three with euploid (diploid, tetraploid and octoploid; 2n=20, 40 and 80) and one with aneuploid (2n=32) chromosome numbers. Here we report that the diploid chromosome number (2n=20) reported from Bulgaria is due to misidentification with Jacobaea aquatica. On the other hand, we have discovered a new, hexaploid (2n=6x=60) cytotype within J. vulgaris subsp. vulgaris using flow cytometry. The new cytotype occurs within four sympatric populations of otherwise tetraploid and octoploid plants in Pannonia (one locality in the eastern Czech Republic and two localities in southwestern Slovakia) and in Podillya (one locality in western Ukraine). The frequency of hexaploid individuals within 76 studied populations is very low (only 10 of 693 analysed plants), and hexaploids probably represent hybrids between tetraploid and octoploid plants. Three mixed populations with hexaploid plants were subjected to detailed morphological and pollen fertility analyses. Multivariate morphometric analysis reveals partial separation of tetraploid and octoploid plants, whereas hexaploid individuals are similar in morphology to octoploids. In comparison with tetraploids, octoploids and hexaploids exhibit slightly longer ray florets, involucral bracts and tubular florets and more hairy outer achenes. Hexaploid plants display larger pollen grains and lower pollen fertility compared to tetraploids and octoploids.     

Phenotypic and cytological variation among plants derived from anther cultures ofLilium longiflorum

Summary Anther culture of the Easter Lily (Lilium longiflorum; 2n=2x=24) was attempted in order to evaluate its potential in generating haploids for the production of hybrid cultivars. The effects of genotype, temperature (low temperature treatment of buds and high temperature treatment of cultures), sucrose concentration and growth regulators were tested. The most important factors for callus induction were the genotype and the presence of 2,4-dichlorophenoxyacetic acid. Pre-treatments at low or high temperature had no apparent effect, while high sucrose concentration was inhibitory. Callus was derived from 28 of the 108 genotypes tested and plants were regenerated. Phenotypic variations were observed among these regenerants. Somatic chromosome numbers were determined in 42 plants derived from 10 donor genotypes. Thirteen plants were diploid and 29 were mixoploid with chromosome numbers ranging from 11 to 26. Four of the mixoploid plants had a high proportion of cells with haploid chromosome numbers, particularly at early stages of development. Meiosis was examined in plants with flower buds. Most plants had 12 bivalents at Metaphase I, but also aneuploids were observed. Other irregularities included bridges and laggards at Anaphase I. The occurrence of high frequencies of haploid cells (up to 80%) in root tips suggests that some plants may be of gametic origin. Research was supported by the Easter Lily Research Foundation, the Ohio Floriculture Foundation, the Gloeckner Foundation and the Oregon Agricultural Experiment Station (technical paper no. 8398).