Effect of selection and temperature stress on the production of 2n gametes in Lotus tenuis

Five Lotus tenuis genotypes showing 2n pollen production greater than 5% and producing 4x progenies in 4x(male sterile Lotus corniculatus) × 2x crosses were found in different natural populations. Meiotic analyses of these plants showed some second metaphases with disoriented spindles, i.e. parallel and tripolar spindles which resulted in diads and triads, respectively. Because useful genes for winter growth and resistance to powdery mildew from L. tenuis can be introgressed into L. corniculatus via 2n gametes, a programme of selection was started to increase the occurrence of 2n gamete production in L. tenuis. Controlled crosses were performed between genotypes producing 2n gametes and the progenies were checked for 2n pollen production and meiotic aberration, following the same methodologies as used for the parental plants. Although in some progeny plants the frequency of 2n gamete production increased remarkably, the overall progeny level of 2n gamete production remained unchanged. This suggested that different genes for meiotic aberration exist in natural populations and/or differential seed-setting ability exists between genotypes. Diallel crosses between selected genotypes confinned this hypothesis. Evaluation of 2n pollen production of clones grown under different temperature regimes (warm vs. temperate) showed that, in L. tenuis, the effects of genotype, temperature and genotype × temperature interaction are significant. In particular, high temperature increases the level of 2n gamete production in L. tenuis.     

Barriers to hybridization of Solanum bulbocastanum Dun. and S. Verrucosum Schlechtd. and structural hybridity in their F1 plants

Summary Results of reciprocal crosses between Solanum verrucosum (2n=2x=24) and S. bulbocastanum (2n=2x=24) are described in terms of pollen tube behaviour in styles, of berry and seed set, of fertility and of meiotic behaviour of the F₁ hybrids. Pollen tube growth of S. verrucosum is strongly inhibited in styles of S. bulbocastanum, whereas no inhibition is observed in the reciprocal cross. Therefore S. bulbocastanum x S. verrucosum fails to set berries or seeds, whereas the reciprocal cross produces both berries (54.4% berry set) and seeds (0.3 per berry). Only 14.6% of the seeds germinate. Both the diploid and corresponding tetraploid hybrid plants are vigorous, flower abundantly, have a rather regular meiosis (mainly rod bivalents), but show a high degree of cytoplasmic-genic male sterility. Crossability of the diploid hybrid plants is nil when used as pollen parents and near to zero when used as pistillate parents. The barriers to hybridization of the parent species (unilateral inhibition of pollen tube growth, somatoplastic sterility, cytoplasmic-genic male sterility and structural differences of the parental chromosomes) are discussed and methods are suggested to overcome these barriers.     

The inheritance of a basal branching type in guar

Summary Eight different genotypes of the F₁ hybrids between Oriental × Asiatic lily (Lilium) hybrids (2n = 2x = 24) were treated with nitrous oxide (N₂O) gas under pressure for 24 and 48 hours. At the time of treatment, all plants possessed early meiotic stages in the anthers of the oldest flower buds. The mature flowers from treated plants were monitored for fertility through pollen germination in vitro as well as by using them in crosses with diploid Asiatic hybrids (2n = 2x = 24) both as male and female parents. In five out of the eight genotypes of OA hybrids there was evidence for the production of 2n pollen which germinated in vitro from either one or both treatments. The 2n pollen from three genotypes was successfully used in crosses. In two cases, the treated plants were successfully used as female parents which indicated the formation of 2n (or 2x) egg cells. From an analysis of 41 sexual polyploid progenies obtained from N₂O treated plants it was shown that they were all euploids consisting of 34 triploids (2n = 3x = 36) and seven tetraploids (2n = 4x = 48). A detailed cytological analysis of 12 progeny plants through genomic in situ hybridization (GISH) proved that N₂O had induced first division restitution gametes in most cases and in two cases they produced gametes through indeterminate meiotic restitution. There was evidence for intergenomic recombination in three cases.     

Cytological mechanism of 2n pollen formation and unilateral sexual polyploidization in Lolium

Summary Plants of Lolium perenne, L. multiflorum and L. hybridum (331, 51 and 18, respectively) were screened for 2n pollen production. The screening was based on the size differences that are expected to be found between n and 2n pollen of a plant. It was found that 28 plants of perennial ryegrass-belonging to 13 cultivars-produced produced pollen grains of heterogenous size (big and small). The estimated frequency of big pollen grain production was higher than 10% in ten out of 28 plants and in three of them it reached a value of 100%. Eight plants obtained from two 4x × 2x crosses, in which the male parent had been previously identified as producing pollen grains of heterogenous size, was tetraploid. The cytological mechanism of big pollen grain formation observed in three plants consists in the lack of spindle formation and, consequently, of chromosome migration in anaphase II. From these breeding and cytological results, it was concluded that the big pollen grains observed were viable 2n pollen grains. The nuclei of the 2n pollen grains produced by this mechanism are genetically equivalent to those formed by the restitution of the second meiotic division. The value of these gametophytes in a breeding program of Lolium is discussed.     

The effect of colchicine treatment on Solanum acaule and S. bulbocastanum; A complete analysis of ploidy chimeras in S. bulbocastanum

Summary Seeds of tetraploid Solanum acaule (2n=48) and diploid S. bulbocastanum (2n=24) were germinated in petri-dishes on filter paper soaked in 0.3% colchicine. An additional treatment with 0.3% colchicine was applied one month after sowing at four successive days in the axils of the cotyledons of the seedlings. S. acaule appeared much more sensitive to colchicine (14 surviving seedlings from 500 seeds) than S. bulbocastanum (109 surviving seedlings from 450 seeds). Six S. acaule plants with 2n=96 chromosomes were obtained against 38 S. bulbocastanum plants with 2n=48 chromosomes.The ploidy level in each of the three germ layers L₁, L₂, L₃ was determined in 113 plants of S. bulbocastanum and the following results were obtained. Four of the eight possible ploidy types were detected, viz 2x-2x-2x (72 plants), 4x-2x-2x (3 plants), 2x-4x-4x (9 plants) and 4x-4x-4x (29 plants). Doubling the number of chromosomes resulted in a highly significant increase of the number of chloroplasts in the guard cells of stomata and a greatly significant decrease in the proportion of trimerous pollen, male fertility and leaf index. The variability for all characters studied, except for leaf index, was clearly lowest in the 2x-2x-2x group. All plants with a 2x-L₂ were highly male fertile and self-incompatible, also in the three bud stages tested. Male fertility of the plants with 4x-L₂ varied greatly: 12 plants had more than 90% stainability, 5 plants must be considered male sterile. All non-sterile plants with 4x-L₂ were found to be self-compatible, pointing to a gametophytic system of incompatibility in S. bulbocastanum.     

Studies of pollen grain germination,pollen tube growth,micropylar penetration and seed set in intraspecific and intergeneric crosses within three Cruciferae species

Summary Pollen grain germination, pollen tube growth and micropylar penetration were investigated in intraspecific and intergeneric crosses involving Brassica napus L. ssp. oleifera (Metzg.) Sinsk. (oil-seed rape or fodder rape), xBrassicoraphanus Sageret (Raparadish) and diploid (2x) and tetraploid (4x) accessions of Sinapis alba L.(white mustard). For the reciprocal intergeneric crosses between B. napus and xBrassicoraphanus no effective barriers to pollen tube growth on stigmata or in styles were observed. The resulting low frequency of hybrid plants was mainly associated with a low rate of ovules with micropylar penetration per siliqua or with embryo abortion. Hybrid plants could be obtained without use of embryo rescue. In reciprocal crosses between B. napus and S. alba 2x or 4x incongruity barriers were observed on the stigma, in the style, and in the ovary resulting in a low frequency of ovules with micropylar penetration per siliqua. Open flower-pollination compared to bud-pollination generally was the more favourable procedure for pollen grain germination and pollen tube growth in crosses involving S. alba, but for micropylar penetration and seed set no differences were observed. Crosses between S. alba 2x () and B.napus () were found to result in a higher frequency of ovules with micropylar penetration as compared to reciprocal crosses or crosses with S. alba 4x. All reciprocal crosses between B. napus and S. alba 2x or 4x were unsuccessful when no embryo rescue was applied. Embryo rescue shortly after pollination, i.e. 2 to 5 days, however, resulted in hybrid seeds and plants, but only when applied to crosses between S. alba 2x () and B. napus (). The possible effects of the genome constitution, taxonomic distance and the parthenogenetic and parthenogenesis inducing ability of the parental genotypes on the observed malfunctions at the pre-and/or post-zygotic stage of the pollen grain-pistil interactions are discussed.Abbreviations DAP Days After Pollination - IAA Indole-3-Acetic Acid - kin Kinetin     

Meiosis and fertility in interspecific hybrids of Nicotiana tabacum L. ×N. glauca Grah. and their derivatives

Nicotiana glauca is of potential interest to breeders as it carries resistance to black root rot of tobacco. Cytological investigations of sexual interspecific hybrids of N. tabacum T′T′TT (2n = 4x = 48) cv. ‘Wiślica’ × N. glauca GG (2n = 2x = 24) were carried out. The analyses of chromosome association at diakinesis and metaphase I in the PMCs of amphihaploid F₁ T′TG (2n = 3x = 36) revealed low variable pairing with 0–5 bivalents. The sterile amphihaploids F₁ were converted into partial female fertile amphidiploids T′T′TTGG by chromosome doubling. Among 36 mature plants obtained, 15 were found to have chromosome numbers (2n = 6x = 72) and were verified as amphidiploids, 9 had (2n = 6x = 70 or 71) chromosomes while the remaining 12 were haploid. True amphidiploids, in spite of quite high chromosome pairing during meiosis, were very different in pollen fertility, ranging from 0% to 85%. Male fertility disturbances did not correlate with the degree of female fertility upon pollination with N. tabacum. Sesquidiploids T′TG (2n = 5x = 60) obtained from backcrossing the amphidiploids to parental tobacco showed more than 22 bivalents, 10–12 univalents and occasional multivalents that indicated the possibility of interchange events between N. tabacum and N. glauca genomes.     

Induction of an alloplasmic male-sterile Brassica oleracea by substituting cytoplasm from ‘Early Scarlet Globe’ radish (Raphanus sativus)

Summary Alloplasmic male-sterile Brassica oleracea L. was synthesized in a backcrossing program through amphidiploid Raphanobrassica by using Early Scarlet Globe radish (Raphanus sativus L.) as the donor of cytoplasm and B. oleracea broccoli and cabbage as recurrent pollen parents. Persistence of radish chromosomes and high female sterility were encountered in the first four backcrosses. Following use of colchiploid 4x broccoli as pollen parent, a BC₅ plant was obtained that had 2n=3x+1=28 chromosomes, improved seed set, and no radish traits. The BC₆ with recurrent 2x broccoli contained male-sterile plants with 2n=18 or 19 chromosomes, increased seed set, and broccoli morphology. Subsequent generations segregated for male-sterile and restored male-fertile plants, some with variable development of stamens and pollen. Leaf color of the alloplasmic plants, especially seedlings, was lighter green than normal.     

Crosses between Beta intermedia bunge and B. vulgaris L.

Summary A tetraploid annual male sterile form of Beta vulgaris L. (2n=4x=36) was crossed with the wild beet species Beta intermedia Bunge (2n=36). The resulting F₁-plants were male sterile annuals being two or three times back-crossed to diploid and tetraploid sugar and fodder beets in the next years. Apart from tetraploid material (36 chromosomes) hexaploid (54 chromosomes) and a number of aneuploid plants developed.The results obtained justify the conclusion that, at a tetraploid level the material mostly propagates apomictically after the F₁ generation. The presence of penta-, hexa-, septa-and even octaploid plants might be explained by assuming that no meiosis has taken place in the crossing partners. Triploid plants are sometimes found in the progeny of hexaploid material and may presumably be considered haploids. Moreover some pentaploid plants were found in the progeny of the open pollinated F₁ which after two generations of bagging are still pentaploids although they produce no pollen. This is another clear indication of apomictic reproduction.The tetraploid generation from the cross between the hexaploid material and diploid sugar beets probably contains the best prospects for breeding.     

Transmission of bacterial wilt resistance by First Division Restitution (FDR) 2n pollen via 4x×2x crosses in potatoes

Summary Resistance to bacterial wilt (Pseudomonas solanacearum) found in tuber-bearing Solanum species was transferred into a diploid potato breeding population. Simultaneous selections were made for agronomic characters, production of first division restitution (FDR) 2n pollen, and resistance. Diploid resistant genotypes were identified via inoculation with a virulent isolate (CIP-204) of race 3 of Pseudomonas solanacearum. These resistant diploid genotypes were crossed to susceptible tetraploid potatoes. An investigation was made to assess whether resistant diploid genotypes transmit resistance to bacterial wilt, which is a quantitatively inherited trait, to tetraploid potatoes via FDR 2n pollen. Tetraploid seedlings from 4x×2x crosses were inoculated with the same isolate CIP-204, and the percentage of surviving seedlings was scored. Some 4x×2x families from resistant diploid genotypes demonstrated a high level of survival rate. The transmission of bacterial wilt resistance was achieved by the use of FDR 2n pollen. It was speculated that a female x male interaction effect on the survival rate of the evaluated progeny may exist. Selecting proper 4x and 2x parents would be important for obtaining a higher frequency of transmission of resistance to bacterial wilt in the progeny.     

Ploidy races in Actinidia chinensis

Summary Ploidy levels were examined in 26 accessions of Actinidia chinensis: 20 accessions were diploid (2n=2x=58) and 6 accessions were tetraploid (2n=4x=116). There was no evidence of variation in ploidy level within an accession. Our results are consistent with tetraploid A. chinensis coming from a restricted part of China.Interploid crosses within A. chinensis produced only low numbers of seedlings which were mainly triploid. Crossing hexaploid A. deliciosa with pollen of tetraploid A. chinensis produced a large family of plants and those checked were pentaploid.Counts on 83 genotypes of different ploidy levels (2x, 3x, 4x, and 5x) confirmed that the basic chromosome number in Actinidia is 29.     

Amphidiploids between Cyclamen persicum Mill. and C. purpurascens Mill. induced by treating ovules with colchicine in vitro and sesquidiploids between the amphidiploid and the parental species induced by conventional crosses

Summary We cultured colchicine-treated hybrid ovules in vitro to produce fertile amphidiploids of C. persicum (2n=2x=48. referred to as AA) × C. purpurascens (2n=2x=34, referred to as BB). Seedlings and mature plants were obtained from the ovules without colchicine and those exposed to 50 mg/l colchicine for 5, 10 and 15 days, whereas they were not obtained from the ovules exposed to 50 mg/l colchicine for 20 days and 500 mg/l for 5, 10, 15 and 20 days. Although 8 mature hybrids derived from the ovules without colchicine produced a few fertile pollen grains, they failed to produce viable seeds by self-fertilization. The hybrids had 41 somatic chromosomes. Four and 3 mature plants were derived from ovules exposed to 50 mg/l colchicine for 10 and 15 days, respectively. One each among 4 and 3 mature plants showed a high frequency of pollen grain fertility, produced several seeds by self-fertilization, and had 82 somatic chromosomes which is twice the number of hybrid chromosomes (2n=41, AB). These findings indicated that these plants are amphidiploids (2n=82, AABB) between C. persicum and C. purpurascens. Three and 2 viable seeds were derived by the conventional crosses of diploid C. persicum × the amphidiploid and the amphidiploid × C. purpurascens, respectively. Flowering plants that developed from the seeds of diploid C. persicum × the amphidiploid were barely fertile and had 65 somatic chromosomes (2n=65, AAB), whereas those that developed from the seeds of the amphidiploid × C. purpurascens were barely fertile and had 58 somatic chromosomes (2n=58, ABB). The somatic chromosomes indicated that these plants are probably sesquidiploids between the amphidiploid and either C. persicum or C. purpurascens. The interspecific cross-breeding of cyclamen using the amphidiploids and the sesquidiploids is discussed.     

Introgression of crown rust resistance from Festuca spp. into Lolium multiflorum

Crown rust, Puccinia coronata Corda, causes one of the most damaging foliar diseases in Italian ryegrass, Lolium multiflorum Lam. For introgression of crown rust resistance, highly resistant hybrids of the crosses Festuca arundinacea (2n = 6x = 42) ×L. multiflorum (2n = 4x = 28) and reciprocally, L. multiflorum (2n = 2x = 14) ×Festuca pratensis (2n = 4x = 28) and subsequently resistant recombinant individuals were used as female parents and susceptible cultivars of Italian ryegrass as male parents in three successive backcrosses. The BC₃ plants were selfed and crossed mutually. Uredospores of seven different crown rust isolates collected from plants of L. multiflorum, Lolium perenne, F. pratensis, F. arundinacea and L. multiflorum × F. pratensis hybrids were applied to identify the resistance or susceptibility of the parental species, backcrossed, selfed and intercrossed progenies. The various crown rust populations revealed a species-specific capability to infect plants of the Lolium-Festuca complex corresponding to the host species from which the spores originated. Selected BC⁹ plants, however, were found to be completely resistant to all crown rust populations tested. Successful introgression of the resistance was achieved from F. arundinacea as well as from F. pratensis. The resistance represents a dominant character, apparently based on a strong heterologous incompatibility between host and pathogen. In phenotype, bivalent formation during meiosis and in fertility, the novel germplasms are comparable with the L. multiflorum cultivars.     

Interspecific hybrids of Cyclamen persicum Mill. and C. purpurascens Mill. produced by ovule culture

Summary Interspecific crosses were made to introduce the scent of flowers of C. purpurascens into C. persicum cultivars and ovule culture was used to rescue the abortive hybrid embryos. Cultivars of C. persicum diploid (CPD, 2n=2×=48) and C. persicum tetraploid (CPT, 2n=4×=96) were the pistillate parents and wild species of C. purpurascens (CP, 2n=34) were staminate parents. After pollination, crossed ovaries were collected periodically and examined using paraffin sections. Histological observations suggested that both hybrid ovules of CPD x CP and CPT x CP should be transferred to culture medium 35 days after pollination. Based up on this observation, crossed ovaries were collected 28 days after pollination and ovules with placenta were transferred to MS (1962) medium containing 3% sucrose. These ovules were cultured in the dark at 25° C. The hybrids (2n=41) derived from CPD x CP had the scent of C. purpurascens, whereas the hybrids (2n=65) derived from CPT x CP had the scent of C. persicum. Although both hybrids had complete genomes from the parents and produced a few viable pollen grains, they failed to yield viable seeds by self- and cross-pollination with fertile pollen grains of C. persicum cultivars.Abbreviations CPD C. persicum diploid - CPT C. persicum tetraploid - CP C. purpurascens     

Genetics of self-compatibility in dihaploids of Solanum tuberosum L. I. Breeding behaviour of two self-compatible dihaploids

Summary Two highly fertile and self-compatible dihaploids (2ns=2x24) from Solanum tuberosum L. (2n 4x 48) were investigated to elucidate the genetic basis of their self-compatibility. To this end the two dihaploids were selfed and reciprocally intercrossed and the resulting I₁ and F₁ plants tested for self-compatibility. Reciprocal backcrosses of I₁-plants and F₁-plants were made. Complete diallels both within self-compatible and within self-incompatible F₁-plants were carried out as well as reciprocal matings between self-compatible and self-incompatible F₁-plants. From the wealth of data it could be concluded, that the dihaploids have two intact S-alleles, one being common to both. Six hypotheses were tested for explaining self-compatibility in these particular dihaploids. All but one had to be discarded. It is concluded that the self-compatibility most likely is brought about by the presence of an S-bearing translocation, which is not linked to the S-locus. The ratio sc :si in the F₁'s point either to certative disadvantage of translocation-bearing pollen or to lethality of translocation homozygotes. The importance of this self-compatibility mechanism for genetic and breeding research in potato is discussed.     

Intergeneric hybrids and an amphiploid between Elymus canadensis and Psathyrostachys juncea

Summary Fifty four hybrid plants between Elymus canadensis and Psathyrostachys juncea were obtained by handpollination and embryo culture. The average cross compatibility between both species was 31.2 percent. One amphiploid plant was induced by colchicine treatment. The hybrid and amphiploid plants resembled P. juncea in appearance but showed a higher plant height and dry matter yield than the parents. The hybrids showed extremely low pollen stainability and were completely sterile. With the exception of one plant (2n=3x+1=22), all hybrid plants were allotriploids (SHN, 2n=3x=21). The amphiploid plant (SSHHNN, 2n=6x=42) showed 58.9% pollen stainability and 11.6% seed fertility.Mean chromosome associations of the hybrids and amphiploid at metaphase I were 0.02IV+0.06III+2.03II+16.91I and 0.07III+18.00II+5.85I, respectively. Lagging chromosomes, chromosome bridges, abnormal cytokinesis, and micronuclei were occasionally observed at the anaphase, telophase, or tetrad stage.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. III. First backcross to parental species

Summary Atrazine resistant Brassica napus × B. oleracea F₁ hybrids were backcrossed to both parental species. The backcrosses to B. napus produced seeds in both directions but results were much better when the F₁ hybrid was the pollen parent. Backcrosses to B. oleracea failed completely but BC₁s were rescued by embryo culture both from a tetraploid hybrid (2n = 4x = 37; A₁C₁CC) and sesquidiploid hybrids (2n = 3x = 8; A₁C₁C). Progeny of crosses between the tetraploid hybrid and B. oleracea had between 25 and 28 chromosomes. That of crosses between the sesquidiploid hybrid and B. oleracea had between 21 and 27. A few plants that had chromosome counts outside the expected range may have originated from either diploid parthenogenesis, unreduced gametes or spontaneous chromosome doubling during in vitro culture. Pollen stainability of the BC₁s ranged from 0% to 91.5%. All the BC₁s to B. oleracea were resistant to atrazine.     

Identification and inheritance of male sterility in groundnut (Arachis hypogaea L.)

Summary Some plants without pods but with gynophores were observed in two F₄ progenies of two crosses of goundnut (Arachis hypogaea L.). The flowers on these plants had translucent white anthers with no or a few sterile pollen grains. Three such plants in the succeeding generation were hand pollinated with pollen from a short-duration Indian cv. JL 24. The resulting F₁ hybrid plants (male sterile x JL 24) were normal. Chi-square tests for segregation for male fertile and male sterile plants in F₂ and F₃ generations indicated that the male sterility in these crosses of groundnut is governed by two recessive genes. We designate these genes as ms₁ and ms₂ with ms₁ms₁ms₂ms₂ being a male sterile genotype.Submitted as ICRISAT J. A. No. 1812.     

Tracing of 2n egg occurrence in perennial ryegrass (Lolium perenne L.) using interploidy crosses

Several genotypes of Lolium perenne (2n = 2x = 14) were screenedfor the production of 2n eggs. In each cross a diploid genotype, used asfemale parent, was pollinated with a tetraploid genotype. All interploidycrosses were made in isolation cages. Flow cytometrical analysis of theprogeny of 154 diploid plants revealed 28 tetraploid descendants. Theseoffspring plants were harvested on 15 different diploid mother plants.Amplified Fragment Length Polymorphism (AFLP) markers were used tocheck the parenthood of these tetraploids. Two seedlings died, theremaining plants could, after AFLP fingerprinting, be identified as meiotictetraploids. The results obtained allowed to conclude that the tetraploidprogeny plants harvested on 15 diploid mother plants were the result ofthe fertilisation of an unreduced egg by a reduced pollen grain of thetetraploid plant.     

Aneuploids of perennial ryegrass (Lolium perenne)

Summary Aneuploid plants of perennial ryegrass (Lolium perenne L.) with 2n=15 to 30 chromosomes were obtained by crossing a near-triploid (2n=3x+1=22) with a diploid or on open-pollination with diploids and tetraploids. Aneuploids occurred with a frequency of 83% in near triploid × diploid progeny and 92% on open-pollination with diploid and tetraploid plants. Aneuploid plants with 15 to 18 chromosomes resembled diploids in morphology and those with 19 to 30 chromosomes were akin to tetraploids. Meiotic studies suggested that most aneuploid plants resulted from transmission of aneuploid egg cells (n=8 to 23). Aneuploid plants with 2n=27 to 30 chromosomes in the progeny of 22×14 cross originated from unreduced egg cells. Plants with 19 to 21 chromosomes were recovered only by immature seed culture. Aneuploid plants with 26 to 30 chromosomes and triploids (2n=21) had higher pollen fertility and bigger seeds than plants with 15 to 22 chromosomes.