Studies on Initiation of Apple Endosperm Callus and Variations of Chromosomal Ploidy on Callus Cells of Endosperm

The present paper deals with the initiation of endosperm callus of apple and the ploidy on callus cells through subcultures. According to our observations, the low capacity of differentiation has been discussed. The following conclusions are drawn: 1. The callus initiaed at 2–5 layer cells beneath epidermis. Some cells in this region changed into initials of endosperm callus and from initial masses then develop into cellular masses of endosperm callus protruding on the endosperm surface. The differentiation and growth of callus masses through subcultures may carry out in embryonal and transitional cellular masses in the peripheral zone and basal zone. 2. The ploidy in callus cells is very unstable. Through repeated subcultures the ploidy of the callus cells vary greatly. Most cells (about 80%) contain polyploid and a large number of aneuploid neuclei, while the triploid cells only constitute the minority (about 2.5%–3%). Hence the ploidy in callus cells produced by well developed endosperm vary greatly. 3. It is highly probable that the above mentioned variation of ploidy in callus cells of endosperm, particularly within the embryonal and transitional cellular masses is the cause of the low capacity of differentiation.     

Meiotic behavior of pollen mother cells in relation to ploidy level of somatic hybrids between Solanum tuberosum and S. chacoense

Potato somatic hybrids obtained by protoplast fusion between Solanum tuberosum (4x) and Solanum chacoense (2x) were investigated for genome stability and meiotic behavior associated with the pollen viability in order to elucidate the mechanism influencing the fertility of the somatic hybrids. The ploidy level detections conducted in 2004 and 2007 demonstrated that 68 out of 108 somatic hybrids had their ploidy level changed to be uniform and euploidy after successive in vitro subcultures, which mainly occurred in octaploids, aneuploids, and mixoploids, while 74% hexaploids were still stable in their genome dosage in 2007. Different types of abnormal meiotic behavior were observed during the development of pollen mother cells (PMCs) including the formation of univalents, multivalents, laggard chromosomes, and chromosomal bridges, as well as triads and polyads. A higher proportion of abnormal meiosis seemed to be accompanied with a genome dosage higher than the hexaploids expected in this study. A significant positive correlation between defective PMCs and the number of small pollen grains and negative correlation between number of small pollen grains and pollen viability strongly suggested that abnormal meiosis could be a causal factor influencing the fertility of the somatic hybrids. The hexaploids with stable genome dosage and a certain level of fertility will have great potential in a potato breeding program.     

Asymmetric protoplast fusions between wild species and breeding lines of potato – effect of recipients and genome stability

 The objective of this study was to investigate if in asymmetric protoplast fusion experiments the ploidy of the recipient line (di-haploid and tetraploid) has an influence on the extent of the asymmetry of the regenerating fusion products. Nineteen different experiments with the wild species Solanum bulbocastanum and Solanum circaeifolium as donors (irradiated with 210 Gy) and different breeding lines (di-haploid and tetraploid) were carried out. The degree of genome elimination was determined by measuring the relative DNA content using flow cytometry. The data showed that the loss of DNA in hybrid plants was significantly higher for 4x, compared to 2x, plants as recipients. In addition, the stability of asymmetry in the fusion products was studied. For this purpose differences in asymmetry in individual shoots originating from the same callus were analysed. A large variation in the DNA content of individual shoots was detected. Of the 4x to 6x shoots 44% had the same DNA content as another shoot originating from the same callus, 19% had a DNA content between 4x and 6x but different from any other analysed shoot originating from the same callus, 2% were chimeras and 35% had a completely different DNA content (eutetraploid, euhexaploid, eupolyploid or asymmetric with a ploidy level above 6x). RFLP-analysis with single-copy probes of 12 regenerates from six calli (two regenerates per callus) confirmed the assumption that the different regenerates of one callus originate from the same single cell. The analysis of selected regenerates cultivated for a period of more than 1 year demonstrated that the genome of asymmetric regenerates might change during cultivation. Received: 30 April 1998 / Accepted: 24 July 1998     

Cytological Studies on Tissue Culture of Pinus cembra

Callus tissue of Swiss stone pine, Pinus cembra L. var. sibirica Loud., has been grown successfully for 9 months through 7 transfers. Excellent initial growth was obtained from hypocotyl segments of 7-day-old seedlings. A complex agar medium was used supplemented with 2 mg/l 2,4-D, 0.05 mg/l kinetin, 1 g/l Edamin and 10% v/v coconut milk. No deterioration of growth was observed on this medium after numerous transfers. The callus tissue did not produce shoots or roots, but showed an ability for cytodifferentiation. Tracheids or tracheid-like structures were formed in every passage, but the tracheids of the primary callus culture differed markedly from those of the subcultures. The tracheids of the primary culture probably already existed in the hypocotyl, whereas those of the subcultures were formed in vitro. The callus tissue was mainly diploid during the period studied, and more than 86% of the mitoses were diploid. A few mitoses with tetraploidy or a higher ploidy also occurred, but the tissue did not show any tendency to polyploidization.     

The Effect of Growth Conditions and Origin of Tissue 5Phaseolus vulgaris L.)

Callus cultures isolated from various somatic tissues and anthertissue of Phaseolus vulgaris seedlings on a defined growth mediumcontained few diploid cells. The proportion of diploid cellsdid not alter as cultures lost their ability to form vasculartissue. Meristematic cells of roots initiated after transferto induction medium were diploid. All cultures lost their morphogeneticpotential after five to seven subcultures except anther calluswhich formed vascular tissue over a prolonged period of cultureon maintenance medium. After six subcultures anther callus containedmore polyploid cells than somatic cultures. Callus isolated from bean hypocotyl tissue in the presence ofcoconut milk consisted mainly of diploid cells and retainedits morphogenetic potential for a greater number of subculturesthan callus grown on defined medium. Transfer of callus isolatedon the defined medium to medium containing coconut milk increasedthe proportion of diploid cells and prevented further loss ofinorphogenetic potential. An equivalent concentration of cytokininto that in coconut milk prevented the loss of potential butdid not affect the ploidy of the cultures.     

青杄愈伤组织在继代培养中的分化能力及染色体稳定性研究

青杄（Ｐｉｃｅａ ｗ ｉｌｓｏｎｉｉＭａｓｔ．）胚性愈伤组织在改良５９ 附加２, ４－Ｄ及Ｋｔ各１ ｐｐｍ 的培养基上继代３ 年（每月继代１ 次）,仍具有旺盛的增殖能力。在胚性愈伤组织转入１／２改良５９ 并附加ＡＢＡ １ ｐｐｍ 的分化培养基上,约３ 个月左右可分化出大量体细胞胚。体细胞胚分化率达９０％ 以上。经继代３ 年的胚性愈伤组织细胞的染色体倍性十分稳定,其染色体数及核型为２ｎ＝ ２４＝ １６ｍ （６ｓｃ）＋ ８ｓｍ ＋ ２Ｂ。这一结果与由实生苗根尖压片所得结果基本一致     

Shoot-bud regeneration in subcultured callus of engelmann spruce

Summary Callus cultures ofPicea engelmannii (Parry, Engelmann spruce) were initiated and established from mature embryos cultured on von Arnold and Eriksson’s medium (AE) supplemented with N⁶-benzyladenine (10μM) and naphthalene acetic acid (10 μM). Cultures were maintained by subculture at 3-to-4-wk intervals. After three subcultures, callus was transferred to AE medium with only N⁶-benzyladenine (25 μM). Adventitious buds appeared on the surface of the callus after 2-to 4-wk and grew to adventitious shoots on AE medium without growth hormones or on AE medium with kinetin (0.1 μM). Shoot-forming capacity was maintained through 7 further subcultures. This study was supported by the Natural Sciences and Engineering Research Council of Canada grant G1438 to T. A. Thorpe and D. I. Dunstan.     

Somatic chromosome number doubling of selected potato genotypes using callus culture or the colchicine treatment of shoot nodes in vitro

Experiments were carried out to double the somatic cell chromosome numbers of a monoploid and dihaploid of Solanum tuberosum and a genotype of S. circaeifolium subsp. quimense. Colchicine was used in vitro on shoot nodes from which the axillary meristems had been removed. Plants with doubled chromosome numbers were obtained from shoots grown from the tertiary, sub-axillary meristems of all three genotypes. The callus culture of stem and leaf explants was found to produce more shoots with doubled chromosome numbers than the colchicine treatment in the case of the dihaploid and quimense genotypes but no shoots were obtained from callus culture of the monoploid. Fifty-two % of the shoots from the dihaploid and 63% from the quimense clone were ploidy doubled in the case of the best callus culture system. Using a sub-lethal dose of colchicine, the dihaploid yielded 37% ploidy-doubled shoots whereas all the shoots produced from the monoploid were doubled and the quimense clone produced 27% doubled plants. Callus culture was highly dependent upon the type of growth medium and other, unknown, factors. The colchicine treatment, although yielding fewer products, was more reliable for achieving ploidy doubling in selected clones if the number of plants produced is not important.     

Observation on Differentiation Potential and Chromosome Stability of Callus in Subcultures of Picea wilsonii

Embryogenic calli of Picea wilsonii Mast. cultured on modified 59 medium supplemented with 1 ppm 2, 4-D and 1 ppm Kt still remained high reproductive potential after sequential monthly subcultures for 3 years. When the callus were transferred to 1/2 strength modification 59 medium with 1 ppm ABA, a lot of somatic embryos were produced after culturing about 3 monthes. The frequency of callus embryogenesis was above 90 %. Chromosome counting showed that the chromosome ploidy of callus cells of P. wilsonii was stable in subcultures for 3 years. The chromosome number and karyotype formula is 2n= 24= 16m (6sc) +8sm+2B. This result agrees with that form root-tip squash of seedling.     

Stable sodium sulfate tolerance inBrassica napus cv. Westar callus cultures

Summary Sodium-sulfate-tolerant callus ofBrassica napus cv. Westar, selected on medium containing 105 mM Na₂SO₄, was maintained on medium without the salt to test for stability of tolerance. Tolerance to Na₂SO₄ was retained even after 18 subcultures on no salt. This tissue also showed tolerance to K₂SO₄, NaCl, and KCl. However, with the exception of callus grown on KCl fresh weight yields were less than that of tolerant callus maintained continuously on Na₂SO₄. Tolerant callus maintained on no salt had a mixture of the compact morphology of unselected callus and the friable morphology of tolerant callus. Both callus types expressed salt tolerance. Sucrose, reducing sugars and proline concentrations were measured in unselected callus, tolerant callus maintained continuously on Na₂SO₄, and tolerant callus maintained on no salt. Sucrose levels were similar in all cases. Whether maintained on or off Na₂SO₄., tolerant callus had reducing sugars levels three to fou times greater than unselected callus. Tolerant callus maintained on no salt had twice the amount of proline found in the unselected callus. Tolerant callus maintained in the absence of salt had an ash content, sodium concentration, and potassium concentration significantly lower than that of unselected callus. Supported by the Natural Sciences and Engineering Research Council of Canada Strategic Grant nos. G 0949 and G 1642 to T. A. Thorpe, D. M. Reid, and E.C. Yeung.     

SOMATIC EMBRYOGENESIS AND ORGANOGENESIS FROM CALLUS CULTURES OF SOLANUM CAROLINENSE

Culture of stem segments of Solanum carolinense L. on medium supplemented with 10 mg/1 2,4-dichlorophenoxyacetic acid and 1 mg/1 kinetin, induced callus formation. When subcultured on medium lacking 2,4-D but containing a cytokinin, the callus regenerated. The mode of regeneration depended on the type and concentration of cytokinin employed; high concentrations of benzyladenine and all concentrations of kinetin promoted organogenesis, while low concentrations of benzyladenine induced somatic embryogenesis in addition to organogenesis. With age and continued subculture on 2,4-D containing medium, callus progressively lost its ability to regenerate when the auxin was replaced by cytokinin. In conjunction with previous studies on regeneration from anther cultures of S. carolinense, it appears that in both cases, 2,4-D is required for callus initiation and proliferation but must be exchanged for a cytokinin before differentiation will occur. However, since it was not possible to induce embryogenesis in pollen-derived callus, developmental potential may be influenced by the ploidy level of responding cells in culture.     

Synergistic effect of trimethoprim and bavistin for micropropagation of Bacopa monniera

A micropropagation protocol for Bacopa monniera (L.) Wettst., a medicinally important plant, has been developed. Direct organogenesis without callus formation was induced by culturing node, internode and leaf explants on growth regulator free Murashige and Skoog (MS) medium. MS medium supplemented with an antibiotic trimethoprim (TMP) and a fungicide bavistin (BVN) produced axillary shoots from node and adventitious shoot buds on the surface of all explants. The combination of 200 mg dm⁻³ TMP and 200 mg dm⁻³ BVN induced the optimum frequency of shoot formation as well as shoot number. Presence of both TMP and BVN induced multiple axillary shoot formation from the nodal segments and this ability was maintained for four subcultures.     

A tissue culture method for the preservation of Soybean mosaic virus strains

The activity and longevity of Soybean mosaic virus (SMV) in soybean callus culture were investigated with 11 SMV strains which are distinguished by differential reactions on soybean cultivars [Glycine max (L.) Merr.]. Dual cultures (soybean callus and SMV) were initiated by direct culture of SMV-infected leaves from susceptible soybean plants on Msoy and MS agar medium. Established SMV-callus cultures were maintained at 25 °C under light, subcultured to fresh MS medium at 2-month intervals or as necessary, and assayed periodically for virus infectivity. The infected calluses on MS medium grew better and stayed active longer than those on Msoy medium. At 10–15 °C, calluses and SMV were viable and active for 13–15 weeks or longer without subculture. The infectivity of SMV from callus cultures was comparable with that of SMV from infected plants, and remained stable for more than a year through five successive subcultures. Callus tissues of dual cultures were uniformly infected by SMV, thus ensuring infectious subcultures by random transfers. Production of in vitro inoculum can be significantly increased by multiple subcultures. Biological integrity of the SMV cultures was maintained with no change of viral virulence and pathotype. The method is of value for preserving a collection of SMV strains in a highly infectious and readily available form and reduces the chance of contamination or loss in viability.     

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.     

Effect of Some Antibiotics on the In Vitro Morphogenetic Response from Callus Cultures of Coryphantha Elephantidens

The effect of five antibiotics: carbenicillin, chloramphenicol, cefotaxime, kanamycin and hygromycin on the organogenesis from callus cultures of Coryphantha elphantidens (Lem.) Lem. have been studied. Carbenicillin and cefotaxime stimulated shoot regeneration from callus. All antibiotics under study suppressed rooting of in vitro formed shoots. After five sequential subcultures on kanamycin supplemented medium, antibiotic resistant callus was obtained. To study the impact of kanamycin on resistant callus, total protein content was also studied. Selected callus showed a remarkable increase in callus mass. Antibiotic resistant plants have been selected by screening callus pieces on kanamycin supplemented media. Total protein content increased with subsequent subcultures in kanamycin resistant callus. The kanamycin selected shoots withstood the stability test after 2 months on antibiotic free medium. Plants were raised from the callus, which formed roots in 20 mg dm^–3 kanamycin, which was under study.     

In vitro chromosome doubling of nineZantedeschia cultivars

Tetraploid plants have been produced from nineZantedeschia cultivars usingin vitro colchicine treatment. Rapidly-multiplying shoot cultures were treated on a medium containing 0.05% colchicine for 1, 2 or 4 days to induce chromosome doubling. Following the treatment, most shoots were killed but the surviving shoots were multiplied for several subcultures. These shoots were then rootedin vitro and transferred to a greenhouse. Plants were screened 2 months later by measuring stomatal length, and 110 out of 565 plants were selected as putative tetraploids with a stomatal length significantly greater than in diploid control plants. Chromosome counts were carried out on root tips from 44 plants and confirmed that 38 were tetraploids, 2 were chimeras (predominantly tetraploid with a few octoploid cells), and 4 were diploids. Stomatal length has been rechecked in mature tetraploid plants of the cultivar Black Magic, demonstrating that stomatal length is a good indicator of ploidy level inZantedeschia. This study has shown that multiplying colchicine-treated shootsin vitro for several subcultures prior to transfer to soil produced very few chimeras. The stomatal length measurements are non-destructive and allow the rapid screening of a population for tetraploids.     

Multiplication and germination of somatic embryos of American ginseng derived from suspension cultures and biochemical and molecular analyses of plantlets

Summary Seedlings from 11 seed sources (lines) of American ginseng from different geographic regions were evaluated on Murashige and Skoog medium (MS) containing 10 μM α-naphthaleneacetic acid (NAA) and 9 μM 2,4-dichlorophenoxyacetic acid (2,4-D) for callus development and somatic embryo formation. Leaf and stem explants callused at a frequency of 18.2–100%, while somatic embryos were produced from these calluses at a frequency of 25–87.5% after 5 mo. Suspension cultures of nine lines were established by transferring embryogenic callus to MS liquid medium with NAA and 2,4-D at 2.5 and 2.25 μM, respectively, and maintained by subcultures every 8 wk. Globular somatic embryos from these cultures were germinated on half-strength MS containing 1% activated charcoal, and roots >5 mm in length developed within 3 wk. A 7-d exposure to 3 μM gibberellic acid and 5 μM 6-benzylaminopurine significantly enhanced shoot development and promoted further root development. The chromosome number, profiles of the common triterpenoid saponins (ginsenosides), and random amplified polymorphic DNA (RAPD) banding patterns in plantlets derived from suspension culture were compared to those of zygotic seedlings. The chromosome number in root tip cells and suspension cultured cells was 48. Patterns of the six major ginsenosides, determined by thin-layer chromatography, in leaves of tissue culture-derived plantlets were identical to those in seedlings. RAPD patterns among plantlets originating from the same tissue-cultured line were mostly identical; however, altered patterns were observed in some lines that had been maintained in suspension culture for almost 4 yr. The results from this study indicate that propagation of desired ginseng genotypes in suspension culture can be achieved, and that biochemical and molecular markers can be used for authentication of resulting plantlets.     

Haploid callus and regeneration of plants from anthers of Digitalis purpurea L.

Summary Production of callus from anthers of D. purpurea was obtained on several basal media supplemented with various amounts of auxins. Chromosome counts showed that the callus produced was haploid when the anthers 1) were of a dark-brown to black color, and 2) were cultured in the late tetrad stage of microspore development. Subsequent differentiation to plants at high frequencies was possible only 1) when the anthers had been cultured on the medium of Nitsch and Nitsch (Science 163, 85–87; 1969) supplemented with 5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 2) when the callus was transferred to the same medium but without 2,4-D, and 3) when it was cultured under continuous light from fluorescent lamps. Proliferation of the callus and regeneration of plants did not diminish through as many as 20 subcultures. The high frequency of regenerates permits the propagation of a distinct geno-type to a virtually unlimited number of plants. Diploid plants were obtained when the anthers had been cultured in the dark. Tetraploid plants were regenerated by callus from anthers which had been cultured in light. When the time of 2,4-D treatment was shortened a few haploid plants were produced which however did not survive transfer to soil. Cytological observations demonstrated that regeneration started from haploid callus, leading to intermediate degrees of ploidy and finally to diploid plants. Most of the regenerated plants were euploid and flowered and fruited normally under greenhouse and field conditions. If the anther-derived callus was cultured on the medium of Nitsch and Nitsch supplemented with 2.2 mg/l kinetin, plants regenerated only under photoperiodic conditions of 16 h light at 28° and 8 h dark at 20° but the survival was lowered to one third. These plants had a different leaf and flower morphology as compared to the control without kinetin and to the starting material, but their progeny was again essentially normal.     

Nuclear DNA content and chromosome number in somatic hybrid allopolyploids of <Emphasis Type="Italic">Solanum</Emphasis>

An attempt was made to change the proportion of the parental genomes in interspecific hybrids Solanum nigrum + S. tuberosum (ngr + tbr) by means of repeated protoplast fusion. In order to enlarge the potato input into the hybrid genome, the protoplasts of two ngr + tbr hybrids of different ploidy (7x and 8x) were fused with the protoplasts of two different diploid potato clones in three combinations. Protoclonal variability was studied in three populations of new ngr + tbr allopolyploids maintained in vitro. The absolute nuclear DNA content (2C) was measured using flow cytometry to estimate the ploidy of the hybrids. The ploidy level of the selected clones was verified by chromosome counts in root meristems. The newly synthesized allopolyploids (75 clones) showed only a small gain in nuclear DNA content above the mean value determined for the parents, instead of the expected addition of an entire diploid potato genome to the combined parental ngr + tbr genome. An increase in nuclear DNA was observed mostly in the clones having the 7x hybrid as a parent (75% of allopolyploids from two combinations). When the 8x hybrid was used as a parent, only two allopolyploids (5%) exhibited a significantly increased nuclear DNA content. The 8x level of ngr + tbr allopolyploids was shown to be stable and was only occasionally exceeded. Somatic hybrids ngr + tbr offer a model system for studying the molecular mechanism(s) and processes involved in stabilization and establishment of the synthetic Solanum allopolyploids.     

The use of antimicrotubule herbicides for the production of doubled haploid plants from anther-derived maize callus

Summary Four antimicrotubule herbicides, amiprophosmethyl (APM), pronamide, oryzalin, and trifluralin, were evaluated for their ability to induce chromosome doubling in anther-derived, haploid maize callus. Effects of various herbicide treatments on the growth and regenerative capacity of callus along with the ploidy and seed set of regenerated plants were determined. Flow cytometric analysis was also used to measure changes in ploidy levels of callus cells following treatments. More than 50% of the cells were doubled in chromosome number after the haploid callus was treated with 5 or 10 M APM or 10 M pronamide for 3 days. A similar proportion of plants regenerated from the treated callus produced seed upon self-pollination. APM and pronamide did not inhibit callus growth at these concentrations and the treated callus retained a high plant regeneration capacity. Oryzalin very effectively induced chromosome doubling, but severely inhibited the growth of regenerable callus and plant regeneration. Trifluralin induced chromosome doubling in a small proportion of cells at lower concentrations (0.5 and 1 M), however, at a higher concentration (5 M) it inhibited callus growth and plant regeneration. The results indicate that APM and pronamide may be useful agents for inducing chromosome doubling of anther-derived maize haploid callus at very low concentrations.