Evaluation of the Effect of Pollen Irradiation on Karyotype Variability in M2 Cotton Plants

The karyotypes of biomorphologically abnormal cotton (Gossypium hirsutum L.) plants obtained in M₂ after pollination with pollen irradiated at dose rates 10, 15, 20, and 25 Gy were studied. Various genomic and chromosomal mutations were detected in 57 M₂ families. The primary monosomics isolated in M₂ were found to be cytologically more stable and more viable, since they had higher meiotic index, pollen fertility, and seed formation. In M₂, a decrease in the number of plants with multiple karyotype aberrations and interchromosomal exchanges with high frequency of multivalent formation was observed. The multivalent configurations had different types and chromosome disjunctions. Their pollen fertility was higher than in translocants found in M₁. Desynapsis often occurred in M₂, including plants with chromosome deficiency or rearrangements. The variation in the number of univalents in various cells was found to result from different expression of synaptic genes. The results indicate stabilization of karyotypes, increase in cytologic stability and viability, and the absence of sterility in aberrant plants.     

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     

Evaluation of the effect of pollen irradiation on karyotype variability in cotton plants

The effect of pollen irradiation at dose rates of 10, 15, 20, and 25 Gy on variability in cotton plants Gossypium hirsutum L. was studied. The modified plants showed a reduced fertility, mainly caused by chromosomal rearragements and genomic mutations during meiosis. The genomic mutations involved primary and tertiary monosomics, monotelodisomics, and a haploid plant. The decrease in meiotic index and pollen fertility in the cotton aneuploids was related not only to aberrations in chromosome pairing but also to genetic features of the original plants. It was found that heterozygosity for interchromosomal exchanges found in M1 plants resulted in the formation of multivalent associations of chromosomes of various forms and types of segregation from translocation complexes. Another result was high variability in pollen fertility. An increase in irradiation dose rate caused an increase in the number of translocants with a high frequency of quadrivalents. The results suggest that the great diversity of forms observed in M1 after pollination with irradiated pollen is determined, first, by elimination of some chromosomes or their arms or the whole paternal genotype and second, by interchromosomal rearrangements. The high variability in pollen fertility of translocants hampers using this trait as a marker of heterozygosity for exchanges in cotton.     

Estimation of efficiency of seed irradiation by thermal neutrons for inducing chromosomal aberration in M2 of cotton Gossipium hirsutum L

Cytogenetic analysis of M2 plants after irradiation of cotton by thermal neutrons was performed in 56 families. In 40 plants of 27 M2 families, different abnormalities of chromosome pairing were found. These abnormalities were caused by primary monosomy, chromosomal interchange, and desynapsis. The presence of chromosome aberrations in some cases decreased meiotic index and pollen fertility. Comparison of the results of cytogenetics analysis, performed in M1 and M2 after irradiation, showed a nearly two-fold decrease in the number of plants with chromosomal aberrations in M2, as well as narrowing of the spectrum of these aberrations. The latter result is explained by the fact that some mutations are impossible to detect in subsequent generations because of complete or partial sterility of aberrant M1 plants. It was established that the most efficient radiation doses for inducing chromosomal aberrations in the present study were 15 and 25 Gy, since they affected survival and fertility of altered plant to a lesser extent.     

Stable progeny production of the amphidiploid resynthesized Brassica napus cv. Hanakkori, a newly bred vegetable

Resynthesized Brassica napus cv. Hanakkori (AACC, 2n?=?38) was produced by cross-hybridization between B. rapa (AA, 2n?=?20) and B. oleracea (CC, 2n?=?18) as a new vegetative crop. Many studies have provided evidences for the instability and close relationship between A and C genome in the resynthesized B. napus cultivars. In fact, seed produced to obtain progeny in Hanakkori had unstable morphological characters and generated many off-type plants. In this study, we investigated the pollen fertility, chromosome number, structure, and behavior linked to various Hanakkori phenotypes to define factors of unstable phenotypic expression in the progeny. Hanakkori phenotypes were categorized into five types. The results of pollen fertility, chromosome number, and fluorescence in situ hybridization analysis for somatic mitosis cells indicated that the off-type plants had lower pollen fertility, aberrant chromosome number, and structures with small chromosome fragments. Observation of chromosomes at meiosis showed that the meiotic division in off-type plants led to appreciably higher abnormalities than in on-type plants. However, polyvalent chromosomes were observed frequently in both on- and off-type plants in diplotene stage of meiosis. We assume that the unstable morphological characters in resynthesized progeny were the result of abnormal division in meiosis. It results as important that the plants of normal phenotype, chromosome structure and minimized abnormal meiosis are selected to stabilize progeny.     

Molecular cytogenetic study on the plants of Elymus nutans with varying fertility on the Qinghai-Tibet Plateau

A molecular cytogenetic investigation was conducted on plants of the allohexaploid species Elymus nutans with varying fertility on the Qinghai-Tibet Plateau. Molecular karyotyping revealed that chromosome variants were distributed unevenly among genomes and among different homologue chromosomes in each genome. The plants with varying fertility exhibited significantly higher numbers of chromosome variants than did the normal fertility samples, although both kinds of plants showed the same pattern of high-to-low polymorphism from the Y to St and H genomes. Heterozygosis and karyotype heterozygosity in the plants with varying fertility were 3- and 13-fold higher than those in normal samples, respectively. Significant negative correlations were found not only between seed setting rates and total genome heterozygosity but also between seed setting rates and heterozygosity of each genome in the plants of varying fertility. Chromosome pairing analysis was performed using genomic in situ hybridization in selected plants of different fertility levels. The pairing of chromosomes at meiotic metaphase I was mostly bivalent, although univalent, trivalent, quadrivalent, and other polyvalents also occurred; in addition, chromosome configuration forms and frequencies varied among the studied samples. ANOVA results showed that the average number of ring bivalents in the Y genome was significantly higher than those in the St and H genomes. Significant positive correlations between pollen grain fertility and ring bivalent number were found in the St and H genomes but not in the Y genome. Furthermore, chromosome configuration parameters (total bivalents, numbers of ring and rod bivalents) were found to be significantly correlated with heterozygosity and seed setting rates in the St and H genomes, respectively, but not in the Y genome. It was inferred that the seed setting rate and pollen grain fertility in E. nutans are strongly influenced by the heterozygosity of each genome, but the Y genome differs from the St and H genomes due to chromosome pair alterations. The St and H genomes may contain more chromosome structural variations than the Y genome in E. nutans.     

加拿大披碱草和圆柱披碱草BC1植株的细胞遗传学鉴定

以加拿大披碱草(2n=4x=28)和圆柱披碱草(2n=6x=42)为材料,对其BC1植株染色体数目、配对构型以及花粉育性和结实性等进行了鉴定。结果显示:BC1代85%以上细胞染色体数目为28条(2n=4x=28);BC1植株的花粉母细胞减数分裂中期Ⅰ染色体配对行为较规则,其平均染色体构型分别为0.04Ⅰ 13.98Ⅱ,且环状二价体多于棒状二价体;BC1植株的花粉可育率和自然结实率分别为84.83%和70.98%,说明BC1植株的育性已得到恢复,为其后代优良株系的选育奠定了基础。     

Evaluation of the Effect of Pollen Irradiation on Karyotype Variability in M1 Cotton Plants

The effect of pollen irradiation at dose rates of 10, 15, 20, and 25 Gy on variability in cotton plants Gossypium hirsutum L. was studied. The modified plants showed a reduced fertility, mainly caused by chromosomal rearrangements and genomic mutations during meiosis. The genomic mutations involved primary and tertiary monosomics, monotelodisomics, and a haploid plant. The decrease in meiotic index and pollen fertility in the cotton aneuploids was related not only to aberrations in chromosome pairing but also to genetic features of the original plants. It was found that heterozygosity for interchromosomal exchanges found in M₁ plants resulted in the formation of multivalent associations of chromosomes of various forms and types of segregation from translocation complexes. Another result was high variability in pollen fertility. An increase in irradiation dose rate caused an increase in the number of translocants with a high frequency of quadrivalents. The results suggest that the great diversity of forms observed in M₁ after pollination with irradiated pollen is determined, first, by elimination of some chromosomes or their arms or the whole paternal genotype and second, by interchromosomal rearrangements. The high variability in pollen fertility of translocants hampers using this trait as a marker of heterozygosity for exchanges in cotton.     

STERILITY BARRIERS OF SOME ARTIFICIAL F1 ORCHID HYBRIDS: MALE STERILITY. I. MICROSPOROGENESIS AND POLLEN GERMINATION

Microsporogenesis, pollen germination and fertility of males gametes were studied in 24 artificial intergeneric and interspecific F₁ hybrids of orchids. Although parental species had the same chromosome number (2n = 40), microsporogenesis of the hybrids was irregular due to the lack of homology of the chromosomes of the parental species. This led to formation of tetrads of microspores without micronuclei, tetrads with 1–8 micronuclei, triads, dyads with and without micronuclei, and monads. Chromosomes numbers found in haploid microsporocytes ranged from 7 to 40; in micronuclei the chromosome number varied between 1 and 5. In terms of pollen germination, three situations were observed: 1) hybrids whose pollen grains did not germinate in the stigma; 2) hybrids in which the pollen tubes grew down in the style, but did not penetrate into the ovary; 3) hybrids in which the pollen tubes grew down normally through the ovary, reaching the ovules. When the pollen tubes did not penetrate the ovary no fruit was formed. Therefore germination tests carried out in vitro may not indicate pollen fertility, because pollen tube growth in the style of the flower may be insufficient to induce fruit formation or to accomplish fertilization.     

Meiotic behaviour and pollen fertility in three species of Zephyranthes (Amaryllidaceae)

Studies on meiotic behaviour and pollen fertility have been carried out in Zephyranthes candida, Z. grandiflora and Z. flava. Maximum meiotic abnormalities in chromosome behaviour were observed in Z. candida and Z. grandiflora. There were variations in the number of bivalent formation, multivalents and anaphasic separation. All types of abnormalities were found to be associated with low percentage of pollen fertility. In Z. flava, chromosomal aberrations were low and pollen fertility was high. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enhanced recovery of doubled haploid lines from parthenogenetic plants of melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.)

Recovery of doubled haploid (DH) progeny from haploid melon plants for use in breeding programs requires efficient chromosome doubling procedures. We describe improved procedures for recovery of fruits and viable seeds from parthenogenetic melon plants. Plant regeneration from nodal explants treated with 500 mg/L colchicine for 12 h was increased from 40 to 88% by transferring the treated explants to medium supplemented with a combination of growth regulators [5 μM IAA; 5 μM BA; 1 μM ABA; 30 μM AgNO₃). Prolonged exposure (2–7 days) to colchicine inhibited regeneration from nodal explants but had less effect on shoot tip explants. Many colchicine-treated plantlets flowered in vitro, allowing early assessment of their male fertility. Production of stained pollen in plants from nodal explants was highest after 0.5–2 days of colchicine treatment and on plants from shoot tips after 1–2 days. In vitro pollen counts correlated well with counts from greenhouse grown plants and with fruit set. The fruit set rate for colchicine-treated plants with a high pollen number was 47%. Appropriate colchicine treatment and culture of nodal explants as well as tip explants can substantially increase the number of fertile plants and DH lines recovered from parthenogenetic melons.     

Occurrence of aneuploid progenies from an asynaptic amphidiploid ofScilla scilloides (lindley) druce II. Mechanism of production of the various aneuploid progenies

The mechanism of production of the various aneuploid progenies was clarified in the asynaptic amphidiploid plants (2n=34+4f+2F, AABB) ofScilla scilloides. Its asynaptic nature and chromosomal stickiness lead to the unequal segregation at anaphase I (AI) in PMC's. The observed values in 18 segregation patterns, 17:17 to 0: 34, were different from the expected values estimated from random segregation of chromosomes. Nevertheless, the preferential transmission of special chromosomes among genomes A (x=8=a₁−a₈) and B (x=9=b₁−b₉) had not occurred. As the result of unequal segregation, the pollen grains with various chromosome numbers were observed. Almost all of the 200 pollen grains contained chromosome numbers more than 17 (range 8 to 34). The observed values of each chromosome number were roughly similar to the expected values of containing the complete set of genome A or B in the random distribution without preferential segregation of chromosomes at AI. The difference between the index of polien mitosis and the pollen fertility was significant in the Wilcoxon matched-pairs signed rank test and suggested the selection for some genomically unbalanced pollen grains during maturation. Consequently, viable pollen grains with various chromosome constitutions are a few (mean pollen fertility of 5.8%) but might produce many aneuploids by self- and cross-pollination.     

Meiotic Analysis of the Regenerated Plants Derived from a Young Spike of Octoploid Triticale ×Secale cereale F1 Hybrid

The regenerated plants had high frequencies of changes in chromosome number and the pairing variation. The chromosome number was more or less variably decreased in different pollen mother cells. Most of regenerated plants were mixoploids. Some had higher frequency of homolgous chromosome pairing because double doses of rye genome had inhibited the effect of Ph gene. But, there was obvious different of chromosome pairing among regenerted plants. The difference was related to numerical chromosome change. Meiosis in a regenerant was analysed with Giemsa-C banding technigue. Partial rye chromosomes did not pair homologously or only very loosely paired. There was moderate level of wheat-wheat homologous partial pairing, however, wheat-rye chromosome pairing also occurred.     

Cytogenetics of a new type of barley with 16 chromosomes

In the progeny of a trisomic type for chromosome 6, Purple, a 16-chromosome type was obtained, which had a pair of new metacentric chromosome 6 in excess. The new metacentric chromosome 6 was shorter than any of the 14 chromosomes of normal barley complement and showed a heteropycnotic nature at late prophase in somatic mitosis. At metaphase I in the plants with 14+one metacentric chromosome 6 (2n=15) the chromosome configuration was exclusively 7_II+1_I indicating that the extra metacentric chromosome 6 could not associate with the normal chromosome 6. At diakinesis and metaphase I in the new 16-chromosome plants most of the sporocytes showed 8_IIor 7_II+2_I. Neither tetravalents nor trivalents were observed at meiosis. The chromosome behaviour at anaphase I and later stages of meiosis was regular in general, resulted in a fairly high pollen fertility of about 61 per cent. Seed fertility however, was very low. The transmission rate of the new metacentric chromosome 6 through the pollen was extremely low in 16-chromosome plants. Possible origin of new basic number and B-chromosome in diploid level through trisomic condition was suggested (Summary see p. 138).Contribution No. 141 of the Department of Plant Science, University of Manitoba.     

Chromosome evolution within theOrnithogalum tenuifolium complex (Hyacinthaceae), with special emphasis on the evolution of bimodal karyotypes

Hypotheses on the evolution of the karyotypes of 8 chromosome races (2n = 4, 6, 8, 10, 12, 16-two forms, 26) within theOrnithogalum tenuifolium complex are discussed. Four of the karyotypes are strictly bimodal: 2n = 8 (6 long and two short chromosomes), 2n = 10 (6 long and 4 short chromosomes), 2n = 12 (6 long and 6 short chromosomes) and 2n = 16 (12 long and 4 short chromosomes). The hypotheses are tested by means of measurements of nuclear DNA content, studies of meiosis and pollen fertility of hybrids, and comparisons of karyotype morphology. The results indicate that the E. African 2n = 12 chromosome race is the most primitive and has given rise to the other chromosome races. The 2n = 6 race is found to have a significantly higher fitness than the 2n = 12 race.     

Introgressive hybridization between a native and an introduced species:Viola lutea subsp.Sudetica versusV. Tricolor

A hybrid swarm betweenViola lutea subsp.sudetica (2n=50, native species) andViola tricolor (2n=26, introduced species) originated in the 1970’s in the Krkono?e Mts. Analyses of chromosome numbers, reproductive biology, morphology, and ecology gave the following results: (1) Compared to the plants found in the 1970’s, the number of colour morphs have decreased and the types now prevailing in the field are morphologically closer toV. lutea subsp.sudetica, forming a continuum. (2) In the field, hybrids having approximately the same chromosome number as the primary hybrids are most common. Some plants of the hybrid swarm have certain characters unknown to their parents. (3) In the field, autogamous types similar toV. tricolor were found. (4) A limited number of plants from the field and culture have higher somatic chromosome numbers thanV. lutea subsp.sudetica; there was a tendency towards increasing chromosome numbers in their progeny (up to 2n=62). These plants have some new morphological characters (a small proportion of hexacolpate pollen) not found in the parents (nor in the other hybrids with prevailing tetracolpate and pentacolpate pollen grains) and higher pollen fertility in comparison to the other hybrids. These plants also have the highest germination rate. (5) There is a tendency for chromosome numbers to decline in the progeny of open pollinated hybrid plants in the lowland experimental graden. (6) The phenology of the plants in the mountain grasslands and the lowland garden is different; the parents behave in a totally contrasting manner. (7) In the field at least some hybrids extend outside the geographical and ecological ranges of the parental species, invading new communities. (8) Seeds ofV. tricolor do not show any dormancy and germinate in the year of production; most of theV. lutea subsp.sudetica seeds germinate during the spring of the following year. Hybrids with intermediate chromosome numbers had both types of germination strategy. The germination rate of intermediates with high chromosome numbers was even higher than that ofV. tricolor.     

Allelic interaction of F1 pollen sterility loci and abnormal chromosome behaviour caused pollen sterility in intersubspecific autotetraploid rice hybrids

The intersubspecific hybrids of autotetraploid rice has many features that increase rice yield, but lower seed set is a major hindrance in its utilization. Pollen sterility is one of the most important factors which cause intersubspecific hybrid sterility. The hybrids with greater variation in seed set were used to study how the F(1) pollen sterile loci (S-a, S-b, and S-c) interact with each other and how abnormal chromosome behaviour and allelic interaction of F(1) sterility loci affect pollen fertility and seed set of intersubspecific autotetraploid rice hybrids. The results showed that interaction between pollen sterility loci have significant effects on the pollen fertility of autotetraploid hybrids, and pollen fertility further decreased with an increase in the allelic interaction of F(1) pollen sterility loci. Abnormal ultra-structure and microtubule distribution patterns during pollen mother cell (PMC) meiosis were found in the hybrids with low pollen fertility in interphase and leptotene, suggesting that the effect-time of pollen sterility loci interaction was very early. There were highly significant differences in the number of quadrivalents and bivalents, and in chromosome configuration among all the hybrids, and quadrivalents decreased with an increase in the seed set of autotetraploid hybrids. Many different kinds of chromosomal abnormalities, such as chromosome straggling, chromosome lagging, asynchrony of chromosome disjunction, and tri-fission were found during the various developmental stages of PMC meiosis. All these abnormalities were significantly higher in sterile hybrids than in fertile hybrids, suggesting that pollen sterility gene interactions tend to increase the chromosomal abnormalities which cause the partial abortion of male gametes and leads to the decline in the seed set of the autotetraploid rice hybrids.     

Preferential elimination of chromosome 5R of rye in the progeny of 5R5D dimonosomics

Transmission of chromosome 5R of rye (Secale cereale L.) and chromosome 5D of common wheat (Triticum aestivum L.) through gametes of 5R5D dimonosomics (2n = 42, 20W″ + 5R′ + 5D′) was studied. Chromosome 5R was found to have lower competitiveness as compared to 5D. Gametes with the rye chromosome were two times less often involved in the formation of a progeny. The combined frequency of the karyotypes of wheat (5D5D) and wheat monosomics (5D) was 11.6-fold higher than the frequency of the karyotypes of substitution lines (5R5R) and monosomics for the rye chromosome (5R). The karyotypes of 10.38% of hybrid plants had aberrant 5R chromosomes with different translocations formed as a result of breakages in the centromere and in the proximal region of the long arm. Telocentrics for the short arm t5RS, i5RS isochromosomes, and chromosomes with a terminal deletion T5RS.5RL-del were identified. The absence of amplification of SSR markers mapped on 5RS and the detection of PCR products for a number of 5RL markers (including the genome-specific rye marker Xrms115) permitted nine plants carrying only the long arm of chromosome 5R to be revealed. Since t5RL telocentrics were not detected by the cytological analysis, the results obtained allow us to suggest the presence of small intercalary translocations of the long arm of chromosome 5R in chromosome 5D or in other wheat chromosomes.     

The effects of a morphactin,chlorflurenol, on organ redifferentiation from tobacco calluses culturedin vitro

A certain morphactin, chlorflurenol (methyl-2-chloro-9-hydroxyfluorene-9-carboxylic acid) was found to be effective in inducing organ redifferentiation (shoot formation) from calluses derived from the pith parenchyma ofNicotiana tabacum ‘Wisconsin No. 38.’ The regenerative ability of chlorflurenol was strongly intensified in combination with kinetin. A combination of 1 mg/l of chlorflurenol and 2 mg/l of kinetin resulted in shoot formation in 70% of inoculated calluses. One reason for this high frequency of regeneration seems to be the promotion of tracheary elements, probably due to some synergism of chlorflurenol and kinetin. Chromosome numbers of the root-tip meristematic cells in regenerated plants varied widely over a range of 6 to 98. One regenerated plant showed normal chromosome constitution in the first metaphase of meiosis, and showed normal pollen and seed fertility; but the others remained unstable and had low pollen and seed fertility.     

Z6／陕7859胚培养再生植株的细胞遗传学研究与易位系选育

二体附加系Ｚ６携带抗大麦黄矮病毒病基因,为了将其抗性导入小麦,将Ｚ６与普通小麦陕７８５９杂交,杂种Ｆ１经幼胚培养诱导形成再生植株,对再生植株及后代进行抗性鉴定,农艺性状考察及对ＳＣ２部分抗病植株花粉母细胞减数分裂期染色体行为进行了观察。结果表明,（１）ＳＣ２不同单株间存在染色体数目,结构的变异。（２）同一再生植株后代的不同单株,染色体数目可能相同,但染色体组成及减数分裂期行为可心不同,致使后代抗性