Monosomic Plants Research Articles

Microprotoplast-mediated transfer of single specific chromosomes between sexually incompatible plants

Microprotoplast-mediated chromosome transfer (MMCT) through fusion of small (subdiploid) microprotoplasts of a transgenic triploid potato (Solanum tuberosum) cell line with leaf protoplasts of tobacco (Nicotiana tabacum) and the wild tomato species Lycopersicon peruvianum is reported. The microprotoplasts contained one or a few chromosomes. Monosomic addition plants were produced from the fusion products. We employed mass-scale induction of micronuclei in donor suspension cells of potato using the microtubule inhibitor Cremart. Protoplasts were isolated from micronucleated cells after incubation in a cell wall digesting enzyme mixture. The microprotoplasts were isolated from the micronucleated protoplasts by high-speed centrifugation. By using sequential filtration, small microprotoplasts containing one or few chromosomes were separated from the bigger subdiploid microprotoplasts. These small microprotoplasts were fused with recipient protoplasts of tobacco or tomato using polyethylene glycol. The selectable marker kanamycin resistance (KanR) and the reporter gene β-glucuronidase (gus), carried by the donor potato chromosome, were used for the selection of fusion products and the isolation of hybrid calli. Several monosomic addition plants were obtained within the short period of 3-4 months after fusion. These contained one potato chromosome carrying a single copy of gus and one or two copies of the neomycin phosphotransferase (nptII) gene conferring KanR, and the complete set of chromosomes of tobacco or tomato, as revealed by genomic in situ hybridization and Southern blot hybridization. The alien genes, gus and nptII, were stably expressed in both the tobacco and tomato backgrounds. They were transmitted to the progeny after backcrossing to tomato. Monosomic and disomic additions, and some introgression plants showing integration of gus and nptII in the tomato genome, were recovered in the first backcross progeny. The potential value of MMCT for the transfer of economically important traits, genome analysis, and gene expression is discussed. Key words : chromosome transfer, microprotoplast fusion, monosomic-disomic additions, sexual transmission, DNA integration, alien gene expression.

Selection of monosomic addition plants in offspring families using repetitive DNA probes in Beta L.

The distribution of two repetitive DNA probes Sat-121 and PB6-4, specific for the section Procumbentes of the genus Beta, was tested in 16 B. patellaris monosomic addition families using a dot-blot hybridization procedure. All monosomic additions were accurately distinguished from diploid sib plants with both DNA probes. The probe PB6-4, with the strongest signal after hybridization, was selected for rapid screening of an extensive number of putative monosomic additions in B. patellaris or B. procumbens addition families using a squash-blot hybridization procedure. The probe PB6-4 detected 118 monosomic additions in 640 plants (18.4%) in eight different B. procumbens addition families. The addition family with chromosome 4 of B. procumbens was semi-lethal and could not be tested. The distribution of PB6-4 in B. patellaris addition families was confirmed in 63 addition families using the squash-blot procedure. In 4580 plants of these addition families, 628 individual monosomic additions (13.7%) were found. The relationship of the morphological characteristics of monosomic addition plants to the results of the squash-blot hybridization (plants with signal) using probe PB6-4 is quite rigorous but not complete. The correlation between plants with a signal and chromosome number (2n=19) is complete. These results indicate that sequences present on PB6-4 are probably present on all chromosomes of B. patellaris and B. procumbens. The possibility of utilizing the sequence information of Sat-121 for a PCR-based assay to screen for putative monosomic addition plants was also investigated as an alternative to chromosome counting. The DNA-amplification profiles using the primers REP and REP.INV clearly distinguished monosomic addition plants from their diploid sibs.

Selection of monosomic addition plants in offspring families using repetitive DNA probes in Beta L.

Selection of monosomic addition plants in offspring families using repetitive DNA probes in Beta L.

Intergeneric transfer of a partial genome and direct production of monosomic addition plants by microprotoplast fusion

Intergeneric transfer of a partial genome and direct production of monosomic addition plants by microprotoplast fusion

Intergeneric transfer of a partial genome and direct production of monosomic addition plants by microprotoplast fusion

Results are reported on the transfer of single, specific chromosomes carrying kanamycin resistance (Kan(R)) and β-glucuronidase (GUS) traits from a transformed donor line of potato (Solanum tuberosum) to a recipient line of the tomato species Lycopersicon peruvianum through microprotoplast fusion. Polyethylene glycol-induced mass fusion between donor potato microprotoplasts containing one or a few chromosomes and normal recipient diploid L. peruvianum protoplasts gave several Kan(R) calli. A high frequency of plants regenerated from Kan(R) calli expressed both Kan(R) and GUS, and contained one or two copies of npt-II and a single copy of gus. Genomic in situ hybridization showed that several microprotoplast hybrid plants had one single potato donor chromosome carrying npt-II and gus genes and the complete chromosome complement of the recipient L. peruvianum (monosomic additions). Several monosomic-addition hybrid plants could be regenerated within the short time of 3 months and they were phenotypically normal, resembling the recipient line. These results suggest that the transfer of single chromosomes is tolerated better than is the transfer of the whole donor genome. The unique advantages of microprotoplast fusion are discussed: these include the direct production of monosomic addition lines for the transfer and introgression of economically important traits in sexually-incongruent species, the construction of chromosome-specific DNA libaries, high-resolution physical mapping and the identification of alien chromosome domains related to gene expression.

Identification and breeding behaviour of a major deletion of chromosome 5D ofTriticum aestivum

A study was undertaken to evaluate the breeding behaviour and to identify a spontaneously produced putative chromosomal deletion in the winter wheat (Triticum aestivum L. em. Thell.) cv Norstar. Male and female transmission studies of plants heterozygous for the deletion chromosome indicated 9.5% and 48.8% transmission through the pollen and the egg, respectively. Meiotic analyses of progeny from deletion heterozygotes indicated that the deletion chromosome was eliminated from half of the plants, which agreed with the male and female transmission frequencies. Testcrosses of the deletion chromosome with telocentrics and nullisomic-tetrasomic combinations suggested that the deletion involved the long arm of chromosome 5D. This was confirmed by restriction fragment length polymorphism (RFLP) analysis. Also, monosomic plants obtained in progeny of deletion heterozygotes were shown to be monosomic for 5D. Studies of plants homozygous for the deletion showed relatively normal pairing between the deletion chromosomes, and with the short arm (5DS), but not the long arm (5DL). Deletion homozygotes were self-sterile, and morphologically similar to plants nullisomic for 5D, but plants that also contained 5DL, or a homoeologous chromosome were self-fertile and had normal morphology. Studies of chromosome morphology indicated that the deletion chromosome was metacentric, and the length of the long arm was reduced by approximately 60%. RFLP studies showed that, in terms of genetic distance, 90% of the arm was missing.

Meiotic and isozymic characterization of plants regenerated from euploid and selfed monosomic tall fescue embryos

Tissue culture of tall fescue (Festuca arundinacea Schreb., 2n=6x=42) would be enhanced by improving the callus induction and plant regeneration efficiency, and evaluating the meiotic and isozymic variation induced by culture. Mature embryos were cultured from four lines of Kenhy tall fescue and from the progeny of three selfed monosomics. Evaluation of six media-auxin combinations showed callus initiation was greatest on SH medium with 2.5 mg/l 2,4,5-T or 7.4 mg/l pCPA, while plant regeneration was greatest on SH medium with 0.5 mg/l 2,4-D. Cytological analyses of 27 plants derived from euploid parents showed a high frequency of aneuploidy (15/27). Chromosome numbers of aneuploids ranged from 36 to 41, with one plant having 80 chromosomes and two plants being asynaptic. Two of ten monosomic-derived plants were euploid, five were monosomic, one was monosomic with a fragment and two were double monosomic. Zymograms of the parents and regenerants were obtained for the enzymes ACPH, ADH, GOT, 6-PGD and PGI. Isozyme variation was observed for two groups of plants derived from the same Kenhy embryos. One group of four monosomic-derived plants differed for the enzymes GOT and ACPH, and all four plants had a PGI pattern. different from that of the parental monosomic plant. This indicated loss of a PGI allele, probably as a result of callus culture.

Male gametophyte development in monosomics of maize

The development of male gametophytes in diploid and monosomic-1, -2, -3, -4, -6, -7, -8, -9, and -10 maize plants was characterized. Developmental differences due to nullisomy in the gametophyte were evaluated by comparing the development of haploid and nullisomic microspores formed by monosomic plants, while differences due to gene dosage in the sporophyte were evaluated by comparing the development of haploid microspores in monosomic plants with those in diploids. These analyses show that (i) male gametophytes nullisomic for the chromosomes analyzed are developmentally delayed and eventually abort; (ii) male gametophytes nullisomic for chromosome 2 or 6 can reach the first mitosis, but those nullisomic for chromosomes 1, 3, 4, 7, 8, 9, or 10 do not reach the first division; and (iii) monosomy of chromosome 1, 2, 3, 4, 6, 7, 8, or 9 interferes with normal male gametophyte development, and monosomic-2 and -9 plants specifically cannot support pollen maturation.Key words: aneuploidy, monosome, nullisome, microspore, r-X1, deficiency.

Gametic selection during wheat anther culture

Gametic selection was investigated in a monosomic 1D line of wheat. Comparison of chromosome numbers in progenies from cross- or self-pollination and anther culture indicated the frequency of the different types of gametes acting in zygote formation. The progenies obtained from reciprocal crosses (monosomic × euploid) were different in terms of frequencies of monosomic plants. All the pollen grains do not have an equal probability of fertilization since a strong gametic selection for euhaploid male gametes was observed when the monosomic line was used as male parent; only 13% of the nullisomic survived to embryogenesis and seed germination. Regenerants from anther culture also demonstrated that the percentage of nullihaploids was reduced, indicating a strong selection for euhaploid gametes. A comparison between the frequency of male nullihaploid gametes in the cross disomic × monosomic and in the anther culture revealed that the two processes generate the same gamete transmission. The in vitro cultures do not induce more selection pressure than the embryogenic development even if they are together biased samples of the male gametic population.

Investigation into chromosomal stability of in-vitro growing cells from a transformed monosomic addition plant of beet

Crown galls were obtained from a monosomic addition plant of beet, carrying an extra chromosome of Beta procumbens, after transformation with a wild-type Agrobacterium tumefaciens strain. Analysis of peroxidase and esterase patterns obtained after cellulose acetate gel electrophoresis and iso-electric focusing respectively, gave evidence that no preferential loss of the alien chromosome on repeated subculture in-vitro from the crown galls occurred.

Cytogenetic studies of aneuploids in rapeseed. II. Morphology, fertility, and cytology of self-pollinated progenies of monosomic plants of Brassica napus L.

Cytogenic studies were conducted on self-pollinated progenies from two monosomic lines of Brassica napus to ascertain the mode of transmission of the monosomic chromosome. An approximately 1:1 ratio of disomic to monosomic plants was obtained. Only one nullisomic plant was found among a total of 71 plants analyzed. The 1:1 ratio and the anaphase I disjunction (19–18) suggest that the gametes (n) were twice as competitive or functional as their counterpart (n − 1). Since quadrivalents were observed in the disomic plants and only trivalents in the monosomics, the monosomic chromosomes were believed to be the ones that were involved in the formation of multivalents. The percentage of multivalent association in monosomic plants was almost double that in disomic plants. In the disomic plants, an average of 11.96% of pollen mother cells had one to two quadri-valents. The average frequency of trivalents in the monosomics was 23.63%. It was suggested that pairing of these homologs was genetically controlled with a dosage effect. The absence of one chromosome increased the frequency of multivalents. Key words: monosomic, Brassica, rapeseed, aneuploid.

Chromosome pairing at five ploidy levels in Elymus trachycaulus

An autoallooctaploid (2n = 56) form of Elymus trachycaulus (Link) Gould ex Shinners (previously Agropyron trachycaulum (Link) Malte ex H. F. Lewis) was induced by treating allotetraploid shoots with 0.2% colchicine. Successive backcrossing to tetraploid pollen parents was successful and yielded five hexaploid (2n = 42), one pentaploid (2n = 35), and three hyperploid (2n = 31, 32, 33) plants. Metaphase I of the tetraploids was normal and 14 II chromosomes were observed, almost all of which were ring bivalents. Chromosome pairing in one octaploid, four hexploids, and one pentaploid were 4.38 IV + 0.65 III + 17.84 II + 0.85 I, 13.16 III + 0.84 II + 0.84 I, and 5.82 III + 8.18 II + 1.18 I, respectively. Efficiency of chromsome pairing (chiasmata per chromsome) was highest in tetraploids (1.29), lowest in hexaploids (0.75), and intermediate in both octaploid (0.95) and pentaploid (0.93) plants. The octaploid produced longer and broader leaves than the tetraploid, although the total dry matter produced was 14.3% lower. Total dry matter yield of the hexaploid was on an average 30.04% higher than the tetraploid and the leaves were significantly larger. The hexaploid plants were taller than both the tetraploid and the octaploid plants. Metaphase I pairing in hyperploid 1 (2n = 33) was 4.34 III + 9.66 II + 0.66 I, hyperploid 2 (2n = 32) was 2.98 III 11.03 II + 1.00 I; hyperploid 3 (2n = 30 + 1 t) was 1.97 III + 12.05 II + 0.66 I + 0.33 t. The pattern of chromosome pairing in these hyperploids suggest that they are a quintupal trisomic, a quadrupal trisomic, and a triple trisomic respectively. Backcrossing these hyperploids to euploid pollen parents was successful. Backosses and their progeny should result in a series of primary trisomiclines and some monosomic plants, which would be useful for gene mapping. Key words: octaploid, hexaploid (double triploid), pentaploid, tetraploid, hyperploid, trisomic monosomic, Agropyron.

The cloning, genetic mapping, and expression of the constitutive sucrose synthase locus of maize

Two differentially expressed genes encode isoenzymes of sucrose synthase in Zea mays. A clone of the shrunken 1 (Sh1) locus, the structural gene for the major endosperm form of sucrose synthase, was used to isolate a genomic clone of constitutive sucrose synthase (Css), the structural gene for the isoenzyme expressed in embryo and other tissues. The Css clone was positively identified by RNA blot analysis of RNA from wild type and a sh1 deletion stock and by analysis of the in vitro translation product of hybrid-selected mRNA. Southern blot analysis of DNA from monosomic plants derived from an r-x1 stock, coupled with restriction fragment length polymorphism mapping, placed the Css gene 32 map units from Sh1 on chromosome 9. In seedling tissues, Css mRNA is present at higher levels than Sh1 mRNA. Expression of both Sh1 and Css in root tissue is enhanced by anaerobic conditions, although Css is induced to a lesser extent than is Sh1. Thus, Css appears to be expressed constitutively, whereas Sh1 is expressed at high levels only in response to specific developmental and environmental stimuli.

Cytogenetic stability of aneuploid maize tissue cultures

Monosomic maize tissue cultures might be used to select recessive mutations of cellular traits. This strategy would avoid some of the problems encountered with haploid cultures such as lack of vigor, sterility of regenerated plants, and uncontrolled diploidization. Monosomic and other aneuploid plants were selected among progeny of W22 R/r-x1 crossed with genetic stocks containing recessive markers. The r-x1 allele induces aneuploidy at a frequency of about 15%. Immature tassels of selected plants were used to initiate totipotent tissue cultures. Plants were regenerated from the cultures over a period of 3 to 17 months after culture initiation. Meiotic karyotypes of microsporocytes and pollen sterility were analyzed in regenerated plants. At least 40% of the 161 plants regenerated from aneuploid cultures had altered karyotypes. This frequency was not related to culture age. Most alterations involved chromosome breakage rather than changes in chromosome number. Types of alterations included heteromorphic pairs (18.1%), translocations (12.5%), addition (10.6%) or loss (1.4%) of chromosomes, and genomic doubling (2.8%). Four euploid cultures, including one with a translocation, were equally unstable (49% with alterations among 115 plants). Euploid cultures gave rise to plants with translocations (12.3%), heteromorphic pairs (8.8%), and genomic doubling (29.2%), but no single chromosome additions or losses. Plants that shared a common distinctive karyotype, such as a specific translocation, were probably derived from a common cell line. Tassels with sectors of two different karyotypes were frequent in plants regenerated from aneuploid (20%) or euploid (33%) cultures. Coenocytic microsporocytes, which lacked cell walls between nuclei, were found in plants from monosomic-2, deficient-2L, and monosomic-6 cultures. Another aberration (23% of 144 regenerants) was lack of cell wall formation after the first and (or) second meiotic division, which was often followed by nuclear fusion. Karyotypic changes observed in this study rarely involved the monosomic chromosome, which means that monosomic tissue cultures could be used to select recessive mutants. Further tests would be needed to demonstrate that the selected gene resides in the monosomic chromosome.Key words: Zea mays, monosomic, trisomic, chromosome, somaclonal variation, karyotype.

A cytogenetic study of monosomics in Brassica napus L.

Two types of monosomic plants of Brassica napus L. were discovered among the backcross progenies of crosses between Diplotaxis muralis L. and B. napus and between Brassica juncea L. and B. napus. These monosomics were designated mono-1 and mono-2, respectively. Morphologically the monosomic plants were virtually indistinguishable from their sib disomic plants. Seed production on both mono-1 and mono-2 plants was normal. Cytological examination revealed that most pollen mother cells (85%) of mono-1 plants formed 18 bivalents plus one univalent at diakinesis, while the remainder (15%) formed 17 bivalents plus a trivalent. The univaltn in mono-1 was submetacentric and its two arms were always stained more lightly than the centromeric region. Later meiotic stages in mono-1 plants appeared normal. The plants of mono-1 produced two types of pollen grains which were different in size. Both the large and small pollen grains of mono-1 were deeply stained with an I2–KI solution. Meiotic behavior of mono-2 plants was similar to that of mono-1 plants, but the frequency of trivalent formation was higher (62%). The univalent in mono-2 was longer than the two chromosomes it paired with to form a trivalent. Pollen produced on mono-2 plants was uniform in size and comparable to that of the normal disomics.Key words: Brassica napus, Brassica juncea, Diplotaxis muralis, aneuploidy, monosomics.

Location of the Benzoxazinless (bx) Locus in Maize by Monosomic and B‐A Translocational Analyses1

The r‐X1 deficiency in maize (Zea mays L.) produces large numbers of monosomics. These monosomics are a unique and useful tool for determining the chromosome location of specific genetic loci. In this study, maize monosomics generated using the r‐X1 deficiency were used to determine the chromosome location of the benzoxazinless (bx) locus. The bx locus is deficient in the production of benzoxazin‐3‐one glucosides, which have been positively correlated with resistance to first‐brood European corn borers, corn leaf aphids, and resistance to northern corn leaf blight. F1 progeny of R/r‐X1; Bx/Bx females crossed by r/r; bx/bx males were screened for monosomic plants expressing the bx phenotype. Karyotypic analysis indicated the monosomic chromosome in such plants was chromosome 4; thus, the bx locus is on chromosome 4. B‐A translocations were then used to confirm the above observation and to determine that the bx locus is in the short arm of chromosome 4. Conventional linkage analysis placed the locus as probably being near Rp4.

Genetic Analyses of Male‐Fertility Restoration in Wheat. II. Isolation, Penetrance, and Expressivity of Rf Genes1

A previous paper reported that the Rf1 and Rf4 genes (controlling male fertility) were located on chromosomes 1A and 6B, respectively, of an alloplasmic Triticum aestivum L. R‐line (R113) with T. timopheevii Zhuk. cytoplasm. This paper reports the development of wheat lines with individual Rf genes from RI13 and the relative penetrance and expressivity of the Rf1 and Rf4 genes. Male‐sterile ‘Chris’ (mst‐Chris) monosomic 1A and monosomic 6B (with T. timopheevii cytoplasm) were crossed with R113; and cytologically identified monosomic F1's were crossed as females with euplasmic Chris pollen. The number of malefertile and sterile disomic and monosomic segregants and the mean number of seed/spike of the fertile segregants were compared between and within the two sets of F1, F2, BCF1, BCS1, BCS2, and testcross progenies; i.e., mst‐Chris ✕ BCF1, mst‐Chris ✕ BCS1 and mst‐Chris ✕ BCS2 progenies. In the BCF1 monosomic IA and monosomic 6B progenies, most disomics were fertile because they retained the Rf1 gene on 1A or Rf4 gene on 6B, respectively. In contrast, the fertile monosomic plants in BCF1 monosomic IA progeny retained the Rf4 gene on 6B; but the Rf1 gene on 1A was deleted. Similarly, fertile monosomic plants in BCF1 monosomic 6B progeny retained the Rf1 gene on 1A; but the Rf4 gene on 6B was deleted. A substantially higher proportion (indicating greater penetrance) of BCF1 fertile monosomics with Rf4 were obtained than BCF1 fertile monosomics with Rf1. Also, fertile monosomic 1A with Rf4 had a greater number of seed/spike (indicating greater expressivity) than fertile monosomic 6B with Rf1. However, the expressivity of the Rf genes was reversed in the euploid testcross progenies. Thus, the expression of Rf4 was affected to a lesser extent by the deficiency of one homologue of 1A than the expression of Rf1 was affected by the deficiency of one homologue of 6B.

Cytogenetic observations on a monosomic inSesbania macrocarpa Muhl. (Leguminosae)

Karyotypic analysis of a spontaneous monosomic plant isolated from a population ofSesbania macrocarpa (2n=4x=24) revealed that one chromosome of the smallest set was missing. The absence of this chromosome caused a deleterious effect on the meiotic system of the plant, resulting in total male and female sterility. The origin of the species in this context is discussed.

Variation in DNA content of 21 individual chromosomes among six subspecies in common wheat.

In order to consider at chromosomal level the phylogeny of wheat, DNA content of 21 individual chromosomes was measured cytophotometrically at M1 or A1 of meiosis in monosomic plants and compared among seven strains belonging to six subspecies of common wheat, Triticum aestivum.1) Nuclear DNA content at pollen tetrad did not differ at all among seven strains.2) DNA content of individual chromosomes varied from chromosome to chromosome in any strain.3) Significant difference was found among three genomes, A, B and D, the ratio being 1.14:1.21:1.00. This ratio apparently differed from those of two earlier reports based on nuclear DNA content of putative diploid ancestors (T. monococcum: Aegilops speltoides: Ae. squarrosa). The authors suggested diploid species of section Sitopsis, with a little higher DNA content than Ae. speltoides as B genome donor to polyploid wheat.4) There was not significant difference among seven homoeologous groups, but homologous chromosomes.5) Total DNA content of seven strains calculated from DNA content of individual chromosomes was very similar to one another, being clearly compatible with the results of nuclear DNA content at pollen tetrad.

Monosomic analysis of stem rust resistance in the wheat cultivar Almus

Monosomic analysis of resistance to stem rust, race 11 (isolate G 425) was carried out in the cultivar Almus (GDR) possessing a 1B/1R translocation. F2 progenies of monosomic and disomic F1 plants of Almus crossed with 21 monosomic lines of Chinese Spring were tested. Two lines (1B and 6B) differed significantly from the disomic segregation ratio by a higher number of resistant plants and two other lines (1D and 6A) by a lower number of resistant plants. The results fitted a hypothesis comprising the interaction of two genes for resistance and two inhibitors.