Callus Induction,Plant Regeneration and an Assessment of Cytological Variation in Regenerated Plants of Lolium multiflorum L.

Callus cultures were initiated from roots, apical meristem tips and leaf explants of several genotypes of Lolium multiflorum L. (Italian Ryegrass). Genotypes were selected which showed a high frequency of callus initiation and from which plants could be regenerated. Plants could be routinely produced from root-derived callus of only one of the genotypes tested. The selected genotypes were still amenable if the temperature and concentration of 2,4-D in the medium were altered. Increase in temperature caused callus from one genotype to give rise to more albino regenerants. Callus formation and plant regeneration occurred at a higher frequency from diploid than tetraploid explants. All regenerants from the diploid cultures had the 2n = 2x = 14 chromosome number whereas plants regenerated from callus derived from tetraploid cultures lost up to 3 chromosomes.     

Genetic and chromosomal variation inPetunia hybrida plants regenerated from protoplast and callus cultures

Plants regenerated from callus cultures derived from leaf discs and mesophyll protoplasts ofPetunia hybrida cv. Rose of Heaven exhibit a high frequency of genetic and chromosomal variation. Of twelve leaf disc-derived plants examined, only three had the normal diploid chromosome number (2n=14) while seven were tetraploid and two were aneuploid (16 and 27 chromosomes). Of seventeen plants derived from two protoplasts, none had the diploid chromosome number. Most had 28 chromosomes, one 29, two 27, one 26 and one had variable numbers (14–28) in different root tip cells. In all cases aneuploidy was associated with developmental abnormality. In addition, heritable differences in growth, morphology and flower pigmentation were observed in callus-derived tetraploids and diploids, including one diploid which differed from parent plants in at least four characters. These results are discussed in terms of the importance ofPetunia in genetics research and for studies of somaclonal variation.     

甜叶菊同源四倍体离体诱导及鉴定

用不同浓度秋水仙素溶液处理甜叶菊不定芽,诱导同源四倍体,并进行解剖学、染色体鉴定和流式细胞仪鉴定倍性。结果表明:(1)用0.20%的秋水仙素溶液浸泡甜叶菊不定芽12h,同源四倍体诱导率最高,可达32.14%。(2)同源四倍体植株与二倍体(对照)相比,其气孔、叶片等均表现巨大性,且叶片变厚、叶色浓绿、叶片皱缩。(3)对照植株染色体2n=2x=22,四倍体植株染色体2n=4x=44;流式细胞仪倍性鉴定结果显示,对照DNA相对含量为100,四倍体DNA相对含量为200。(4)该研究共鉴定出48株甜叶菊同源四倍体植株,为进行倍性植株的诱导奠定了技术基础,为进一步开展甜叶菊同源四倍体新品种的选育提供了实验材料。     

The Correlation between the Chromosome Variation in Callus and Genotype of Explants of Arabidopsis thaliana

Twelve callus lines of Arabidopsis thaliana were derived from four types of explants excised from diploid plants of two ecotypes (Columbia and Wilna) and autotetraploid plants of the Wilna ecotype. Cytogenetic analysis of the chromosome variation in particular callus lines was carried out for primary culture and callus during 5 months of culture. Ploidy levels of interphase nuclei were estimated by counting the number and size of chromocentres and nuclei of interphase cells. The first polyploid cells in all callus lines were observed during callogenesis. In primary culture the ploidy level ranged between 2 and 15x (10-75 chromosomes). The frequency of polyploid cells was higher in the 5-month old callus culture, but the ploidy level was the same. In the callus lines derived from autotetraploid plants, cells with reduced chromosome number appeared quite frequently along with diploid and polyploid cells.     

Cytogenetic analysis of plants regenerated from colchicine-treated callus cultures of an interspecificHordeum hybrid

Summary Hybrids of Hordeum vulgare (HV) x H. jubatum (HJ) were synthesized for purposes of introgressive breeding, but were sterile and the recovery of pure diploid tillers by colchicine applications in vivo was difficult. Plant regeneration from colchicine-treated callus cultures of the hybrid (HV x HJ) was investigated as a means to produce high numbers of pure diploid, fertile intermediates. 10 of 50 plants regenerated in this manner exhibited variable chromosome numbers with means of approximately 37 (expected diploid number = 42). Cytological examinations of microsporogenesis in all such plants revealed a high incidence of bivalent formation at metaphase I (as compared to nearly complete asynapsis in the F₁), but spindle and chromosome abnormalities in later meiotic stages led to complete sterility. Approximately 40% of HJ plants regenerated from colchicine-treated calli appeared to be pure tetraploids of high fertility. These techniques are hence useful for high frequency production of diploid or polyploid plants.     

In vitro culture ofBellevalia romana (L.) Rchb.

Summary Bellevalia romana (L.) Rchb., a monocotyledonous plant characterized by few (2 n=2 x=8) and very large chromosomes, is a useful subject for studying developmental problemsin vitro. Cytological analysis of callus revealed that the majority of cells were diploid, but the remaining cells had aneuploid nuclei with a wide range of chromosome numbers, tetraploid and haploid nuclei. The frequency of aneuploid and polyploid cells was higher in callus grown in the presence of 2,4-D than in callus grown in NAA plus BAP. These nuclei seemed to increase with the duration of culture. The chromosome number distribution as determined by chromosome counts in calli at different culture times was confirmed by DNA cytophotometry. Chromosome number mosaicism (mixoploidy and aneusomaty) also occurred in all root apices of 9 out of 46 plantlets regenerated from callusvia adventitious shoots.     

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.     

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.     

Chromosomal variation in immature embryo derived calluses of barley (Hordeum vulgare L.)

Summary Chromosome counts of ten morphogenic and seven non-morphogenic immature embryo derived calluses of barley,Hordeum vulgare L. cv. Himalaya, were determined. Morphogenic calluses carried the normal chromosome complement (2n=2x=14) in a majority of the cells. A low frequency of haploid (2n=x=7), triploid (2n=3x=21), tetraploid (2n=4x=28) and octoploid (2n=8x=56) cells were also observed. In contrast, non-regenerability of a callus was attributed to the cells having numerical and structural chromosomal changes. In these calluses, aneuploid cells around diploid, triploid, and tetraploid chromosome numbers predominated. It has been demonstrated that chromosomal changes were induced during the culture and that they did not pre-exist in the cultured barley embryos. Based on this study, it is suggested that chromosome analysis of a non-regenerable callus should be conducted before altering the media composition.     

Cytogenetics of protoplast cultures of Brachycome dichromosomatica and Crepis capillaris and regeneration of plants

Summary Callus derived protoplasts of Brachycome dichromosomatica (2n=2x=4) and Crepis capillaris (2n=2x=6) have been regenerated into karyologically normal plants, i.e. plants without visible alterations of the diploid chromosome set. However, metaphase analysis of protoplast cultures derived from both callus as well as mesophyll cells showed karyological changes in the overwhelming majority of cells in both species leading to multinucleated, polyploid and aneuploid cells. Furthermore, callus derived protoplasts sometimes exhibited changes at the chromosome level as indicated by translocations. The vast majority of aberrant karyotypes arose from failures during mitosis and cytokinesis, pointing to inadequate microtubules as a possible underlying cause. Karyological events of the kind described herein greatly affect the plating efficiency of isolated protoplasts and the viability of protoplast derived calli. Plant regeneration, although demonstrated in this study for the first time in both species, seems to be limited to rarely occurring, protoplast-derived colonies with a relatively stable genome. Our experiments, performed with chromosomal model species, emphasize the need for controlled, non-mutagenic culture conditions.     

Cytological Analysis of Seedling Roots, Transformed Root Cultures and Roots Regenerated from Callus of Swainsona galegifolia (Andr.) R. Br.

The stability of chromosome number was investigated in culturesof roots from Swainsona galegifolia. Roots from germinated seedsor plants grown in vitro when cultured in liquid medium howed90% or more cells with the diploid number of 2n = 32. The remainingcells showed aneuploidy mostly below 32. The stability of chromosomenumbers was not affected by transformation with Agrobacteriumrhizogenes although when roots were transformed with A. rhizogenesLB 9042 the range of chromosome numbers in the few aneuploidcells present was higher than in roots for which strain A4 wasused. In contrast, roots regenerated from callus had only 15%of cells with 2n = 32 and showed a modal number of 18. Six rootcultures established from individual roots regenerated fromcallus showed a wide variation in number (8–83). Fivecultures had a modal number around 18, the sixth, a modal numberof 39 which is above the diploid number. The implication ofthe results for the production of secondary metabolites fromroot culture is discussed. Key words: Agrobacterium rhizogenes, callus cultures, chromosome number, root cultures, Swainsona galegifolia     

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.     

The effect of B chromosomes and T-DNA on chromosomal variation in callus cells and regenerated roots of Crepis capillaris

The chromosome behaviour has been compared in three Crepis capillaris callus culture lines and the roots regenerated from these calli. The calli were obtained from explants derived from plants without and with two B chromosomes and the hairy roots were obtained from plants transformed with Agrobacterium rhizogenes. Cytological studies demonstrated that the presence of additional DNA as B chromosomes or as T-DNA had an influence on the numerical and structural variability of the standard chromosome in long-term callus cultures and in regenerated organs. The callus with two B chromosomes displayed higher levels of polyploidyzation than callus without B chromosomes. The roots regenerated from both these calli were only diploid, while roots regenerated from transformed callus were also polyploid. This revised version was published online in June 2006 with corrections to the Cover Date.     

Peg-mediated fusion of Rubus idaeus (raspberry) and R. fruticosus (blackberry) protoplasts,selection and characterisation of callus lines

ABSTRACT

Cell suspension-derived protoplasts of two cultivated Rubus species, Rubus idaeus-raspberry (subgenus Idaeobatus 2n=2x=14) and R. fruticosus-blackberry (a complex species aggregate within the subgenus Eubatus, 2n=4x=28) were fused using different polyethylene glycol (PEG) fusion treatments. Duration of PEG treatment and choice of culture media influenced the rate of cell divisions and plating efficiency. Colony formation was initiated on solid media for the production of several callus lines. Cytological analyses were performed on selected callus lines with hexaploid chromosome number. Two hexaploid fusion callus lines, selected for their homogeneity in growth and ploidy level, were examined by molecular cytogenetic techniques of fluorescent in situ hybridisation (FISH) and genomic in situ hybridisation (GISH). GISH revealed the presence of the heterokaryon within the fusion callus lines. FISH probed with ribosomal DNA (rDNA) showed variable numbers and sizes of loci. Aberrant distribution and condensation of rDNA were common in interphase cells. FISH results suggest that large karyotype rearrangements occurred, including variation in chromosome number and rDNA loci translocations. Attempts to regenerate plants from the hexaploid callus lines following several applications of plant growth regulator combinations were unsuccessful. This may be attributed to the genomic reorganisation and instability of these long-term fusion callus cultures.     

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).     

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。     

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     

Plant regeneration from hypocotyl and petiole callus of Trifolium pratense L.

Red clover (Trifolium pratense L.) seedlings were screened for the ability to regenerate plantlets from hypocotyl-derived callus tissue. Media sequences described by Beach and Smith (1979) and Collins and Phillips (1982) and a variation using media from both sequences were tested. Plantlets were regenerated from three out of 642 genotypes. In all three cases, callus was initiated on B5C medium and regeneration was accomplished on SPL medium. Attempts to regenerate plants from petiole-derived callus tissue have so far been successful only with regenerants of clone F49. Petiole callus from epicotyl-derived F49 plants proved to be non-regenerative. Pollen viability varied significantly among individuals regenerated from callus cultures of clone F49. Root tip squashes from F49 regenerants revealed the normal diploid chromosome number (2n=14). The frequency of regeneration within progeny from reciprocal crosses between F49 regenerants and several non-regenerative genotypes was 29%.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzylaminopurine - KN kinetin - NAA -naphthaleneacetic acid     

同源四倍体青花菜的核型分析

以四倍体青花菜为材料,采用常规压片法进行核型分析和有丝分裂观察.结果表明:四倍体青花菜核型公式为2n=4x=36=16m+20sm(4 SAT),其中第3、4、7、8对为中着丝粒染色体,第1、2、5、6、9对为近中着丝粒染色体,第6对染色体具随体;核型类型属于2A型,为基本对称型;染色体相对长度组成为2n=36=16 M_2+20 M_1,表明该四倍体青花菜是二倍体加倍得到,为同源四倍体.在部分四倍体根尖中发现非整倍体细胞,其染色体数目变异较大;与二倍体相比,四倍体有丝分裂过程存在双核仁、体细胞配对、染色体桥等异常现象.     

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.