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.     

Chromosome analysis by fluorescence in situ hybridization of callus-derived regenerants in Allium cyaneum R.

Investigations were performed to confirm the optimal in vitro culture condition for callus induction and plant regeneration, to observe if somoclonal variation occurs among regenerated plants at the ploidy level and to analyse the chromosomal location of 5S and 18S-26S rRNA gene families using fluorescence in situ hybridization in callus-derived plants of Allium cyaneum. High-est callus initiation was achieved with bulb explants cultured on MS medium supplemented with 2,4-D and BAP at 1 mg l^–1 each. A total of 195 plants was obtained when using MS medium supplemented with 1 mg l^–1 NAA and 5 mg l^–1 BAP; about 92% were diploid having 2n=16; 8% showed a variation in ploidy level. Using digoxigenin-labelled 5S rRNA and biotin-labelled 18S-26S rRNA gene probes, we compared the fluorescence in situ hybridization patterns of autotetraploid plants with the A. cyaneum wild type. The 5S rRNA gene sites were detected on the interstitial region in the short arm of chromosome 4 and on the interstitial region in both arms of chromosome 7. The 18S-26S rRNA gene sites were detected on the terminal region of the short arm, including the satellite of chromosome 5, as well as on a part of chromosome B. The chromosomal location of both rRNA genes in regenerated autotetraploid plants corresponded to those of the wild species. Received: 20 March 1998 / Revision received: 15 June 1998 / Accepted: 8 July 1998     

Flow Cytometry,HPLC-RP,and metAFLP Analyses to Assess Genetic Variability in Somatic Embryo-Derived Plantlets of <Emphasis Type="BoldItalic">Gentiana pannonica</Emphasis> Scop

Cytometric and molecular techniques were used to verify genetic uniformity among somatic embryo-derived plantlets of Gentiana pannonica Scop. Cytometric analysis of regenerants revealed absence of chromosomal changes and alterations in ploidy. However, reverse phase high pressure liquid chromatography detected higher levels of methylation in regenerated plants than those of control plants. These changes were further investigated using a quantitative molecular marker-based approach. This revealed that numerous tissue culture-induced variations, ∼3% (epi)mutations, were observed, including sequence variation and changes in methylation patterns. Moreover, complex patterns of variation, including combinations of genetic and epigenetic changes, were relatively high (ca. 9%). Overall, tissue culture-induced variation reached 16%; while, demethylation was lower than de novo methylation in heterozygotic material and similar in all regenerated plantlets.     

Cytogenetic investigation of geranium plants in tissue culture

Cytogenetic study of six cultivars and six selection lines of geranium (Pelargonium spp.), as well as of 100 plants regenerated from callus cultures has been performed. The majority of cultivars and lines had somatic chromosome numbers 2n = 7x = 56. Among regenerated plants of different cultivars (Rozovaja, Dushistaja, Krunk, Aist, Regar) obtained in vitro from various types of explants (internode, petiole) 61% of diploids and 39% aneuploids were revealed. Chromosome numbers in aneuploids varied from 46 to 82, among them 25.6% regenerated plants had 2n = 72; 10.2% -2n = 68; 5.1% -2n = = 64 and 12.8% -2n = 62. Addition of colhicine to nutrient medium increased the number of aneuploid plants.     

Flow cytometric analysis of somaclonal variation in lineages of Hosta sports detects polyploidy and aneuploidy chimeras

Somaclonal variation of some 124 specially selected cultivars of Hosta Tratt. (Hostaceae) was investigated. Nuclear DNA contents (2C‐value) were measured by flow cytometry of leaves and roots of L1, L2 and L3 layers derived from apical meristems. These values were then converted to inferred ploidies by comparing the measured 2C‐values and ploidy with those of the parent plant. During tissue‐culture propagation, on occasion diploid (L1‐L2‐L3 = 2‐2‐2) hostas give rise to polyploids, such as fully tetraploids (4‐4‐4), and periclinal chimeras, such as partial tetraploids (4‐2‐2). Continual propagation can result in partial tetraploids becoming full tetraploids. Nuclear DNA of some diploids increased with incomplete chromosome sets resulting in fully aneuploids, such as hostas with a DNA ploidy of L1‐L2‐L3 = 2.5‐2.5‐2.5 and 3.7‐3.7‐3.7, and even in aneuploid periclinal chimeras, such as L1‐L2‐L3 = 2.5‐2‐2 and 3.8‐2‐2. The polyploidy of L1, irrespective of the ploidy of L2 and L3, is found to mainly determine the thickness of leaves. Also the higher the ploidy of L1, the wider and more intense in color is the leaf margin. The measurements of Hosta cultivars and their lineages of sports show that chromosome losses or gains are an important source of new cultivars. The complexity of chromosomal distribution in lineages of several Hosta cultivars is discussed.     

Tissue culture in Haworthia

Summary Plants regenerated on two different media (NK and I) from the calluses of simple or cloned subcultures, which were originated from a single stock callus of Haworthia setata derived from its flower bud, were observed for eight characters, i.e., somatic chromosome number in root tips, growth vigor, leaf shape, leaf color, number of stomata per unit leaf area, esterase zymogram, chromosome association at meiotic metaphase I in pollen mother cells, and pollen fertility. From these regenerates plants with different characters from those of the parental plant were obtained. With regards to chromosomal aberrations, tetraploids, aneuploids, plants with a part of the chromosome segment deleted, with reciprocal and non-reciprocal translocations, or with paracentric inversions and those showing sub-chromatid aberrations at meiosis were obtained. The NK medium tended to regenerate more tetraploids and less plants carrying translocation than the I medium.Chromosome variabilities in somatic cells of the regenerates correlated with those of the calluses, from which they regenerated, while they did not correlate with either the meiotic irregularities (chromosome association at MI) or pollen fertility of the regenerates. From these facts, it was concluded that a rather large number of callus cells participate in the regeneration of an individual plant, although, however, only a few limited types of the cells form its germ line.Polyploidy affected growth vigor, leaf shape, stomata number and chromosome association at MI, but its effects were not detected on other characters. Chromosomal aberrations at the diploid level produced no clear changes in the regenerate's phenotype except in meiotic chromosome configuration and pollen fertility.Most chromosomal variants obtained in the present study are already reported in plants collected from wild populations, but plants with the deletion of a whole chromosome (karyotype 7L+6S) or chromosome segment (7L+1M+6S and 14L+2M+12S) have never been reported: this fact suggests that tissue culture is a powerful tool for producing plants with novel karyotypes.Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 436     

Protoclonal variation of plant regeneration in rice

Protoplasts were isolated from callus derived from a single homozygous seed of Oryza sativa L. var. Norin 8. Thirty protoclones were randomly selected and these showed variation in regeneration frequency ranging from 0–87% with an average of 52%. The potential for regeneration of each protoclone as reflected in the regeneration frequency was analyzed five times over a period of 250 days and showed that the protoclones can be classified into three types, namely: protoclones with high regeneration frequency; protoclones with low regeneration frequency, both of which maintained their respective levels of regeneration potential; and protoclones with gradually decreasing regeneration frequency. Secondary protoclones established from protoplasts isolated from some of these protoclones and regenerated 2–3 times for a period of 120 days also showed further reduction in regeneration frequency. The polypeptide composition analyzed by two dimensional gel electrophoresis suggests the presence of specific polypeptides related to regeneration potential. Analysis of ploidy level based on plant morphology and pollen size suggests the predominance of tetraploids among the regenerated plants.     

Characterization of ploidy levels of wheat microspore-derived plants using laser flow cytometry

Summary The purpose of this study was to determine simply and accurately ploidy levels as estimated by changes in nuclear DNA content of wheat (Triticum aestivum L.) plants regenerated from microspore-derived embryos. Using flow cytometry, the nuclear DNA content of green (83) and albino (222) plants derived using anther culture of ‘Bobwhite’ and ‘Pavon 76’, and of their reciprocal F₁ hydrids was estimated. The average DNA concent of the Bobwhite and Pavon 76 standards was 32.46 and 31.28 per nucleus, respectively. Microspore-derived haploid (3X), doubled-haploid (6X), nanoploid (9X), and dodecaploid (12X) plants contained on average 15.44, 30.56, 45.57, and 60.27 pg of DNA, respectively, at a ratio of 1∶1.98∶2.99∶3.90. The frequency of haploids (43.6%) was similar to that of doubled haploids (43.0%), and much larger than the frequency of endopolyploids [nanoploid (1.3%) and dodecaploid (1.0%)] and various aneuploids (11.1%). In terms of genetic stability, green plants had less chromosomal variation than albino plants. The procedure is suitable for rapid determination of the ploidy levels of wheat microspore-derived plants. The knowledge about DNA content or genome size of plants obtained here provides useful information to plant breeders and geneticists interested in using anther culture. Formerly of the Department of Agronomy, University of Nebraska, Lincoln. NE 68583-0915. Formerly of the Center for Biotechnology, University of Nebraska, Lincoln, NE 68588.     

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.     

Population cytology of structural and numerical chromosome variants in theAloïneae (Liliaceae)

The distribution of interchange heterozygotes is analysed in one population of each of three species in the tribeAloïneae (Liliaceae). While one population is shown to be clonal the other two indicate that natural selection eliminates interchange homozygotes and maintains the chromosomal uniformity of the tribe. Analysis of a fourth population containing aneuploids and chromosome deletions shows that mutant plants are capable of interbreeding, but that spread of the aberrations is limited.     

Interspecific hybrids between Medicago sativa L. and annual Medicago containing Alfafa weevil resistance

Non-embryogenic protoplasts of Medicago rugosa and M. scutellata were electro-fused with iodoacetic acid-treated protoplasts of M. sativa (alfalfa). Putative somatic hybrid callus were obtained and some plants regenerated from both combinations. Hybridity of regenerants was confirmed by morphology, molecular means and cytological observations. Parental specific bands were recognized in somatic hybrids by Southern analysis. The somatic hybrids were perennial and their morphology was similar to M. sativa. Cytological observations were carried out on the somatic hybrids, their vegetative clones and self-pollinated offspring. Original somatic hybrids were aneuploids (2n=31–59), but during vegetative proliferation, their chromosome numbers reduced to 32. Those clones of hybrids formed seeds from M. sativa (+) M. rugosa by self-crossing. Chromosomal rearrangements within the parental genomes were observed in vegetative clones of hybrids and their S₁ offspring by Genomic in situ Hybridization (GISH). Some of S₁ offspring from M. sativa (+) M. rugosa showed better spring growth than parental M. sativa and tend to be tolerant to Alfalfa weevil. It was considered that these traits were introduced from the genome transferring M.␣rugosa chromosome to M. sativa. The cell fusion may still have a potential in transferring alien chromosomes in order to increase the genetic variation for crop breeding.     

Chromosome doubling of 2x Solanum species by oryzalin: method development and comparison with spontaneous chromosome doubling in vitro

A potato breeding scheme implies the possibility of ploidy level manipulation either by reducing the chromosome number of cultivars from 48 to 24 to be able to cross them with diploid related species or by doubling diploid material to reach the generally optimal tetraploid level. In vitro spontaneous chromosome doubling is widely used but can lead to somaclonal variation. Since oryzalin has proven to be efficient as a chromosome doubling agent on potato cell suspension cultures, we tried this herbicide on various Solanum species and interspecific diploid hybrids. A 24 h dip in a 28.8 M aqueous oryzalin solution applied on apical buds was the most efficient treatment in terms of tetraploid plant production (mean = 4.1 tetraploid plants for 10 treated buds over 4 genotypes). However 50–100% of the regenerated tetraploid plants acclimatized after in vitro treatment proved to be chimaeric. Consequently, a selection procedure in the progeny was necessary to obtain real and stable doubled clones and final yields were low. This technique is easy to apply and could be a good alternative to chromosome doubling by spontaneous in vitro regeneration in the case of refractory genotypes especially where somaclonal variation is problematic. Percentage of tetraploids among the regenerated plants varied from 6 to 29% with the oryzalin doubling technique while it varied from 20 to 78% by in vitro spontaneous doubling for five diploid genotypes. An observation of the progeny indicated that chimaeras were more frequent using oryzalin (50–100% of the initially supposed tetraploid plants) than when chromosomes doubled spontaneously (4–67% of the initially supposed tetraploid plants).     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated high frequency transformation in dwarf recalcitrant rice cultivars

The Agrobacterium-mediated transformation was done in rice (Oryza sativa L. var. indica) cv. HKR126 and elite cross-bred cv. Pusa Basmati1 (PB1), using strain LBA4404 containing pCAMBIA1300 cloned with gene cassettes; potato proteinase inhibitor and Bacillus thuringiensis endotoxin (plasmid JDW53) or mannitol-1-phosphate dehydrogenase (plasmid RKJ108). Co-cultivation with scutellar-calli derived from mature seeds showed stable and highly efficient transformation. In cvs. HKR126 and PB1, 35 % and 41 % of hygromycin resistant calli were obtained. The transformation efficiency in PB1 (22.0 %) was much higher than in HKR126 (12.5 %). Similarly, PB1 had higher plant regeneration efficiency than HKR126. The shoots regenerated per callus were, 3–4 in HKR126 and 5–6 in PB1. The transformation efficiency with pRKJ108 (18.6 %) was higher than pJDW53 (15.9 %). Polymerase chain reaction (PCR) analysis showed the presence of transgenes in regenerated transgenic plants of both cultivars.     

Somatic embryogenesis from immature zygotic embryos and monitoring the genetic fidelity of regenerated plants in grapevine

Somatic embryogenesis and plant regeneration were successfully established on Nitsch and Nitsch (NN) medium from immature zygotic embryos of six genotypes of grapevine (Vitis vinifera). The optimum hormone combinations were 1.0 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction and 1.0 mg dm⁻³ α-naphthalene acetic acid (NAA) + 0.5 mg dm⁻³ 6-benzyladenine (BA) for embryos production and 0.03 mg dm⁻³ NAA + 0.5 mg dm⁻³ BA for embryos conversion and plant regeneration. The frequency of somatic embryogenesis varied from 10.5 to 37.5 % among six genotypes and 15.5–42.1 % of somatic embryos converted into normal plantlets. The analysis of DNA content determined by flow cytometry and chromosome counting of the regenerated plantlets clearly indicated that no ploidy changes were induced during somatic embryogenesis and plant regeneration, the nuclear DNA content and ploidy levels of the regenerated plants were stable and homogeneous to those of the donor plants. RAPD markers were also used to evaluate the genetic fidelity of plants regenerated from somatic embryos. All RAPD profiles from regenerated plants were monomorphic and similar to those of the field grown donor plants. We conclude that somaclonal variation is almost absent in our grapevine plant regeneration system.     

An efficient mannose selection protocol for tomato that has no adverse effect on the ploidy level of transgenic plants

A protocol for Agrobacterium-mediated transformation with mannose selection was developed for cotyledon petiole, hypocotyl and leaf explants of tomato (Lycopersicon esculentum L. Mill). More than 400 transgenic plants from three tomato varieties were selected with 1% mannose in combination with 0.1–0.5% glucose. Average transformation frequencies ranged from 2.0 to 15.5% depending on the construct, genotype and type of tissue used for transformation. The highest transformation rate was obtained for hypocotyl explants from tomato variety SG048. The ploidy levels of 264 independent transgenic events and 233 non-transgenic plants regenerated from tissue culture were assessed by flow cytometry. The incidence of polyploids within the total population of transgenic plants varied from 10 to 78% and was not significantly different from the non-transgenic population. The greatest variation in the proportion of polyploids was observed in plants derived from different explant types, both in transgenic and non-transgenic regenerants, across three studied genotypes. Transgenic and non-transgenic plants regenerated from leaves included the highest number of normal diploid plants (82–100%), followed by cotyledon petiole-derived plants (63–78%). Transgenic plants produced from hypocotyls contained 22–58% diploids depending on the genotype used in transformation. Results described in this study demonstrate that, although transformation frequencies for leaf tissue are still lower under current protocols, the high percentage of diploids obtained make leaf tissue an attractive transformation target.Abbreviations BAP Benzylaminopurine - MS Murashige-Skoog - MsCHI Medicago sativa chalcone isomerase - PMI Phosphomannose isomerase     

Cytogenetical analysis on aneuploids obtained from pollenclones of rice (Oryza sativa L.)

Summary Rice aneuploids were obtained from 1,715 pollenclones with a mean frequency of 10.2% in anther culture (1983 to 1985). Among the aneuploids obtained, the frequency of primary trisomics ranged from 5.4% to 6,7%, tetrasomics from 1.1% to 1.7% monosomics from 0.9% to 1.3%, nullisomics from 0.5% to 1% and double trisomics from 0.5% to 0.7%. The chromosome complements of those aneuploids were identified by pachytene analysis on the absolute length of the extra chromosomes. Pollen clonal aneuploids showed a different range of variation in agronomic characters from dihaploids of the same origin but the phenotypic variations ressembled those found in aneuploids created by conventional breeding methods. The meiotic chromosome behavior of PMC revealed various chromosomal aberrations of aneuploids: loose pairing, trivalents, univalents, straggling chromosomes, bridges and laggards.     

Ploidy doubling by in vitro culture of excised protocorms or protocorm-like bodies in <Emphasis Type="Italic">Phalaenopsis</Emphasis> species

Endopolyploidy was observed in the protocorms of diploid Phalaenopsis aphrodite subsp. formosana with ploidy doubling achieved by in vitro regeneration of excised protocorms, or protocorm-like bodies (PLBs). Thirty-four per cent of the PLBs regenerated from the first cycle of sectioned protocorms were found to be polyploids with ploidy doubled once or twice as determined by flow-cytometry. The frequency of ploidy doubling increased as the sectioning cycles increased and was highest in diploid followed by the triploid and tetraploid. Regeneration of the endopolyploid cells in the tissue of the protocorms or PLBs is proposed as the source of the development of ploidy doubled plantlets. The frequency of ploidy doubling was similar in seven other Phalaenopsis species, although the rate of increase within cycles was genotype specific. In two species, a comparison of five parameters between 5-month-old diploid and tetraploid potted plants showed only the stomata density differed significantly. The flowers of the tetraploid plant were larger and heavier than those of the diploids. This ploidy doubling method is a simple and effective means to produce large number of polyploid Phalaenopsis species plants as well as their hybrids. The method will be beneficial to orchid breeding programs especially for the interspecific hybridization between varieties having different chromosome sizes and ploidy levels.     

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.     

Comparative cytogenetic analysis of hexaploid <Emphasis Type="Italic">Avena</Emphasis> L. species

Using C-banding method and in situ hybridizatiion with the 45S and 5S rRNA gene probes, six hexaploid species of the genus Avena L. with the ACD genome constitution were studied to reveal evolutionary karyotypic changes. Similarity in the C-banding patterns of chromosomal patterns and in the patterns of distribution of the rRNA gene families suggests a common origin of all hexaploid species. Avena fatua is characterized by the broadest intraspecific variation of the karyotype; this species displays chromosomal variants typical of other hexaploid species of Avena. For instance, a translocation with the involvement of chromosome 5C marking A. occidentalis was discovered in many A. fatua accessions, whereas in other representatives of this species this chromosome is highly similar to the chromosome of A. sterilis. Only A. fatua and A. sativa show slight changes in the morphology and in the C-banding pattern of patterns of chromosome 2C. These results can be explained either by a hybrid origin of A. fatua or by the fact that this species is an intermediate evolutionary form of hexaploid oats. The 7C–17 translocation was identified in all studied accessions of wild and weedy species (A. sterilis, A. fatua, A. ludoviciana, and A. occidentalis) and in most A. sativa cultivars, but it was absent in A. byzantina and in two accessions of A. sativa. The origin and evolution of the Avena hexaploid species are discussed in context of the results.     

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.