Ploidy of small individual embryo-like structures from maize anther cultures treated with chromosome doubling agents and calli derived from them

Summary In order to determine the ploidy of individual embryo-like structures (ELSs) following chromosome doubling treatments, a method was developed to determine the DNA content (ploidy level) of nuclei from single ELSs weighing as little as 1 mg using flow cytometry. About half (53%) of the ELSs which formed during anther culture of the maize inbred line used in control medium were haploid, 27% mixoploid and 20% diploid. Gibberellic acid (GA₃) increased the diploid percentage to 52% without affecting the mixoploid frequency (26%). A four day treatment with the chromosome doubling agent colchicine (50M) increased chromosome doubling while oryzalin eliminated the diploidy and mixoploidy. When regenerable callus cultures were initiated from the ELSs none were found to be mixoploid but the haploid and diploid proportions were similar to that of the ELSs analyzed. Regenerable cultures could not be initiated from the colchicine treated ELSs, however. These studies show that with the genotype used here, GA₃ and colchicine increased the amount of chromosome doubling of the ELSs while oryzalin and pronamide did not. The mixoploidy which existed in about 25% of the ELSs was never observed in calli apparently because these structures do not initiate callus or cells of only one ploidy level grew.Abbreviations ELS embryo-like structure - GA₃ gibberellin A₃     

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.     

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.     

Ploidy stability of maize anther culture-derived callus lines

Summary Ploidy levels of 26Zea mays L. anther culture-derived callus lines of the F1 hybrids (H99 × Pa91, Pa91 × FR16, and H99 × FR16) were determined at various times after culture initiation using flow cytometry (for 21 lines) or chromosome counting of callus cells or regenerated plants (for the remaining 5 lines). Twenty of the lines remained haploid, whereas 6 were diploid. The results from flow cytometry, after examining the DNA content of 5000 nuclei of each callus line, show that each callus line consisted of homogenous haploid or diploid cells. Thus for diploid callus lines, spontaneous chromosome doubling must have occurred before or in the early stages of androgenesis, before the initiation of callus cultures. These long-term callus cultures (growing for up to 38 mo.) have stably maintained their ploidy levels so it is unlikely that the culture conditions have caused chromosome doubling. The restriction fragment length polymorphism pattern obtained with 52 to 58 markers for each diploid callus line shows that all the diploid lines are homozygous diploid so each originated from a microspore and not from diploid maternal F1 hybrid tissue.     

Origin of plantlets and callus obtained from chile pepper anther cultures

Summary Androgenesis occurred from chile pepper (Capsicum annuum L.) anthers incubated in a continuous warm environment (29° C) with continuous light. Forty plantes and embryoids were retrieved from anther cultures and anllyzed for isozyme markers. Of these, 35 exhibited a single allele for markers suggesting microspore origin, while 5 were heterozygous indicating somatic tissue origin. Chromosome numbers were confirmed for 21 plantlets, of which 16 were haploid and 5 were diploid. However, two plants exhibited a single allele for an isozyme marker but possessed the diploid chromosome number, suggesting spontaneous doubling. Anther cultures also produced callus. Nearly 92% of the slow-growing calli sampled were heterozygous for the isozyme marker, suggesting somatic tissue origin. More than 46% of the fast-growing calli exhibited only one allele for the marker, indicating microspore origin. Callus did not regenerate plantlets. The occurrence of both heterozygous and homozygous diploid plantlets from pepper anther cultures has important implications for applied breeding programs.     

Nuclear behaviour of colonies and regenerated buds from protoplasts ofNicotiana plumbaginifolia Viviani haploids

The ploidy level variations of protoplast cultures ofNicotiana plumbaginifolla Viviani (n=10) were investigated from protoplast isolation until regenerated buds, using cytophotometric measurements of nuclear DNA content and chromosome counting. An increase in the average nuclear DNA amount has been found to occur in freshly isolated protoplasts after 15 hours of maceration. Cytological abnormalities like nuclear fragmentation, chromatin connections between interphasic nuclei and micronuclei were observed during the following days. Chromosome counting in 15, 30 and 50-day-old calli and in regenerated buds revealed that nuclei are haploid, diploid or aneuploid.Abbreviations p-cells, p-calli or p-colonies protoplast-derived cells, calli or colonies - BAP 6-benzylaminopurine - NAA 1-naphtaleneacetic acid - 2iP ²-isopentyl-aednine     

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.     

Karyotypic changes in callus cultures from haploid and diploid plants of Crepis capillaris (L.) Wallr

Two auxin-heterotrophic callus cultures of Crepis capillaris, one coming from an haploid plant and the other from a diploid one, were studied in regard to karyotypic changes for over a year. The degree of polyploidisation of the originally haploid culture was considerably higher than that of the diploid culture. The frequency of chromosome rearrangements was significantly higher in polyploidised karyotypes than in not polyploidised karyotypes and correspondingly greater in the haploid culture. However, the cytogenetical stability of the cultures cannot be measured only through their degree of polyploidisation: it has been found that new karyotypes also originate through chromosome rearrangements at the same ploidy level as the original explant.     

Spontaneous versus colchicine-induced chromosome doubling in maize anther culture

The range of genetic variation of spontaneous chromosome doubling frequency of maize haploid plantlets derived from in vitro anther culture was evaluated. When regeneration is obtained by direct embryo-genesis, bypassing the callus phase, it appears that the frequency of spontaneous doubling may exceed 40 of the regenerated plantlets. This high frequency may be one consequence of the use of doubled haploid lines derived from anther culture and spontaneous chromosome doubling. We also report an increase, by more than 50, of the productivity of diploid fertile regenerated plantlets produced by colchicine supplemented medium during the cold shock pretreatment of the microspores inside the anthers. Optimization of the treatments and the anther culture procedure are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

The early ontogeny of embryoids and callus from pollen and subsequent organogenesis in anther cultures of Datura metel and rice

Summary Haploidy induction through anther culture has been examined in Datura metel and rice with a view to tracing the precise sequence of development of the pollen, either directly or through an intervening callus, into an embryo and seedling. In D. metel, the vegetative cell of the young pollen grain assumes the major role in formation of embryos whereas the generative cell and its few derivatives degenerate. Embryos and seedlings arising directly from pollen without an intervening callus phase always proved to be haploids, whereas those differentiating from pollen-derived callus gave haploid, diploid and even triploid plants. Cytological analysis of callus tissue showed cells of various ploidy levels ranging from haploid to triploid, and in rare instances even with higher chromosome numbers.In rice anther cultures the embryoids arose from an initial callus phase. Of 15 different rice cultivars tried, only four produced a callus, and in only one, was there differentiation of plants, both haploid and diploid ones. Among other species tried, egg plant has also yielded plantlets through a callus phase whereas only callus production has been achieved in jute, tea and petunia. No response has been obtained in wheat, maize, cotton and coconut.Coconut milk (CM) appears to be the most important component of the medium for the initial induction of embryoids and callus in anther cultures of most of the species tried. However, further growth and differentiation of plants may require a simpler medium; in D. metel, continued culture on CM led to dedifferntiation.Dedicated to the memory of the late Dr. J. P. Nitsch.     

Efficient production of doubled haploid plants through colchicine treatment of anther-derived maize callus

Summary A chromosome doubling technique, involving colchicine treatment of an embryogenic, haploid callus line of maize (Zea mays L., derived through anther culture), was evaluated. Two colchicine levels (0.025% and 0.05%) and three treatment durations (24, 48, and 72 h) were used and compared to untreated controls. Chromosome counts and seed recovery from regenerated plants were determined. No doubled haploid plants were regenerated from calli without colchicine treatment. After treatment with colchicine for 24 h, the callus tissue regenerated about 50% doubled haploid plants. All of the plants regenerated from the calli treated with colchicine for 72 h were doubled haploids, except for a few tetraploid plants. No significant difference in chromosome doubling was observed between the two colchicine levels. Most of the doubled haploid plants produced viable pollen and a total of 107 of 136 doubled haploid plants produced from 1 to 256 seeds. Less extensive studies with two other genotypes gave similar results. These results demonstrate that colchicine treatment of haploid callus tissue can be a very effective and relatively easy method of obtaining a high frequency of doubled haploid plants through anther culture.     

Ploidy-dependent genomic stability in the tissue cultures of ornamental Phlox drummondii Hook

Comparisons of the chromosome numbers, 2C nuclear DNA amounts and karyomorphology were made in explant cultures of diploid (2n = 2x = 14) and autotetraploid (2n = 4x = 28) Phlox drummondii. In 6–36 week old calli derived from diploid internodal segment explants, and in cells of root tips regenerated from such callus, marked differences were observed in chromosome number. The chromosome numbers ranged from 2n = 14 to 2n = 100 and DNA amounts from 8.20 to 63.20 pg in the diploid derived callus, while the extent of variation was much reduced in the regenerated roots. In contrast, the autotetraploid cultures were characterised by the maintenance of the same chromosome number and DNA amounts as the mother plant. Modified chromosome structures were not apparent in any of the cultures. The possible reasons for the chromosomal instability at the diploid level and stability at the tetraploid level are discussed.     

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).     

Ploidy dependence of chromosomal variation in callus cultures ofHyoscyamus muticus L.

Summary Remarkable variation for chromosome number was observed in both diploid (2x) and autotetraploid (4x) callus cultures continuously examined for 12 months at monthly subsculture intervals. Initially, the subcultures exhibited predominantly the genomic level of the starting material which subsequently and gradually developed into heterogenous populations of euploid and aneuploid cells imparting the subcultures a aneusomatic status. The comparison of chromosomal instability recorded in 2x vs. 4x callus cultures revealed that with time both types of calli stabilized at a chromosome number around the 4x level. However, the chromosomal examination of the adventitious roots emerging from the disorganized calli revealed the euploid (both 2x and 4x) chromosome levels suggesting the occurrence of amorphogenetic sieve.This paper is dedicated to Professor A. K.Sharma of Calcutta University on his 65th birthday by his former student U. C. L. and grandstudent S. S. (CIMAP Publication no. 739).     

Ploidy chimeras induced in haploid sporophytes of <Emphasis Type="Italic">Osmunda claytoniana</Emphasis> and <Emphasis Type="Italic">Osmunda japonica</Emphasis>

Haploid sporophytes of Osmunda claytoniana (2n = x = 22) were apogamously produced from calli when cultivated on a hormone-free medium. Flow cytometric analysis showed that ploidy chimeras were spontaneously produced in a haploid sporophyte of O. claytoniana and those of O. japonica that were obtained in the previous study. In the haploid sporophyte of O. claytoniana, a diploid pinnule and a partially diploid terminal segment were produced in a haploid pinna. In O. japonica, a haploid sporophyte yielded a diploid pinna in a haploid frond, and another haploid sporophyte yielded a diploid pinnule in a haploid pinna. Diploid chimeras were large in size and could be readily distinguished from other haploid parts of the fronds. It is likely that the chimeras were produced clonally from a single diploid cell that established chromosome doubling.     

白桦愈伤组织化学诱变

用不同浓度的甲基磺酸乙酯(ethyl methane sulfonate, EMS)对白桦(Betula platyphalla Sak.)愈伤组织进行化学诱变处理。结果表明: EMS诱变剂的浓度和处理时间对愈伤组织的存活率有很大影响。在高浓度EMS短时间处理和低浓度EMS长时间处理条件下得到叶柄、叶片愈伤组织的半致死剂量。通过观察半致死剂量下愈伤组织的染色体发现, 诱变后细胞中单倍体、非整倍体及多倍体比例均高于对照, 这说明EMS的诱变处理引起了愈伤组织细胞中染色体数量的变化。     

Karyological characterization of sugar beet gynogenetic lines cultured in vitro

Flow cytometry was used to screen ploidy levels in 47 cultured in vitro sugar beet gynogenetic lines of various origin and age, obtained after plant regeneration from unfertilized ovules. When donor plants were diploid, the majority of regenerants were found to have cells with 1C, 2C and 4C relative DNA content (mainly haploid and diploid) and there were large differences in the rate of spontaneous in vitro chromosome doubling between individual homozygous lines. Six ovule-derived lines regenerated from fertile and sterile diploid donors of forty-five lines were solid diploids from the very early stages of their in vitro cultivation, and thus could not be classified as doubled haploids. In the case of tetraploid donor plants, the gynogenetic regenerants demonstrated 2x-ploidy level. The results obtained in chimeric plants with both haploid and diploid cells indicated the possibility to overcome mixoploidy by their re-cultivation through generative shoot tip culture. The flow cytometry method confirmed data obtained by conventional microscopic chromosome counting in dividing leaf cells and was found very useful for screening of a large number of regenerants and for characterizing the process of in vitro gynogenetic lines formation in sugar beet.     

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.     

Callus induction,regeneration of haploid plants and chromosome doubling in ovule cultures of pot gerbera (Gerbera jamesonii)

Gynogenetic plants of pot gerbera (Gerbera jamesonii) were successfully produced from cultures of unpollinated ovulesin vitro. Genotypic variations in the number of ovules that formed callus were found among the lines tested. One particularly responsive genotype was found among 17 genotypes tested where the frequency of callus-forming ovules was 17.5%. Four genotypes formed no callus at all. The frequency of shoot formation from the callus varied from 0–19.6% in nine genotypes. Ploidy was determined by flow cytometry, and 37 (80.4%) regenerants were haploid, seven (15.2%) were diploid, and two (4.3%) were mixoploid with both haploid and diploid cells. The doubling of chromosomes was achieved by treatment with 0.05% colchicine for 2–6 din vitro, and 24.2–34.1% of treated haploid plants were found to have been diploidized.Abbreviations BA 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - DAPI 4 ,6-diamidino-2-phenylindole dihydrochloride - MS Murashige and Skoog (1962) basal medium     

Organ-specific and ploidy-dependent somaclonal variation; a new tool in breeding

The organ-specific somaclonal variation means the differences between the variability of somaclones originated from different somatic tissue of plant. Significant differences in some agronomical characters were achieved among somaclones of seed and plumule meristem origin. The ploidy-dependent somaclonal variation means the differences between the variability of somaclones originated from different ploidy-level tissue. Increased variation among regenerated plants was postulated by origin from cultured cells of reduced ploidy level. The comparison of somaclonal variation in the progenies of diploid plants regenerated from callus of haploid and diploid origin supported the ploidy dependent theory. The pollenhaploid somaclone method (PHS-method) was developed and tested for utilization somaclonal variation in rice breeding. The PHS-method comprises the two well-known and widely applied in vitro methods which are the androgenesis (another culture) and genetic instability of cultured haploid somatic cells (callus cultures). Developmental varieties produced by this breeding sheme are under certification in Hungary.