Production and characterization of Raphanus sativus-Brassica rapa monosomic chromosome addition lines

Breeding of Raphanus sativus‐Brassica rapa monosomic chromosome addition lines (MALs, 2n = 19) was carried out by backcrossing the synthesized amphidiploid line, Raphanobrassica (R. sativus×B. rapa, 2n = 38, RRAA, line RA89) with R. sativus cv. ‘Shogoin’ (2n = 18, RR). In the first cross of Raphanobrassica× radish, four sesquidiploidal BC₁ plants (2n = 28, RRA, RA89‐36‐1, RA89‐31‐1, RA89‐31‐2, RA89‐31‐3) were successfully developed. In these plants, the chromosome configurations of 9II + 10I and 10II + 8I were observed frequently at first metaphase (MI) of meiosis in pollen mother cells (PMCs). The RA 89‐36‐1 plant produced many seeds in the reciprocal backcrosses with radish. About 50% of the BC₂ plants obtained from the cross of RA89‐36‐1 plant × radish were 2n = 19 plants, followed by 2n = 18 plants (24%) and 2n = 20 plants (19%). In the reciprocal cross, 2n = 19 plants were also developed at the rate of 40%. From analysis of specific morphological traits, 2n = 19 plants were classified into eight types (a‐h). When 25 selected primers were used in polyacrylamide gel electrophoresis, random amplified polymorphic DNA (RAPD) markers derived from B. rapa for each type of MAL were detected in numbers between three for e‐type and 16 for b‐type. RAPD markers specific for each type alone were from one (OPE 05‐344) for h‐type to nine for b‐type. In the g‐type, no marker specific to this type alone was observed. However, 19 bands were common between at least two types. These MAL plants exhibited predominantly the chromosome configuration of 9II + 1I at MI of PMCs, pollen and seed fertility being the same level as the radish cv. ‘Shogoin’. From the morphological traits and DNA markers, eight different MAL types among 10 expected were identified.     

Genetic stability and maintenance of Raphanus sativus lines with an added Brassica rapa chromosome

The genetic stability and maintenance of Raphanus sativus‐Brassica rapa monosomic chromosome addition lines (a‐h‐types MALs, 2n = 19, BC₂), developed by backcrossing the synthesized amphidiploid Raphanobrassica (Raphanus sativus × Brassica rapa, 2n = 38, RRAA) with R. sativus cv. ‘Shogoin’ (2n = 18, RR), was investigated. Transmission of the added alien chromosome through selected smaller seeds (SSS) and the inheritance of morphological traits and random amplified polymorphic DNA (RAPD)‐specific markers together with meiotic chromosome configuration and seed fertility were also investigated for three successive generations (BC₃ to BC₅). The distinctive traits and the RAPD‐specific markers of the eight types of MAL were substantially inherited and stably maintained throughout three generations, although a few variant plants (2n =18) resembling MALs (2n = 19) and hyperploidal plants (2n = 26 and 2n = 37) were generated in the earlier generations of BC₃ and BC₄ in comparison with BC₅. The average transmission rates for three generations ranged from 26% for both the b‐type and the d‐type to 44% for the e‐type through SSS. On the other hand, the transmission rates through randomly selected seeds (RSS) were lower, ranging from 6.5% for the f‐type to 23.5% for the b‐type. In meiosis, more than 90% of PMCs showed the 9II +1I pairing configuration at metaphase I throughout three generations. For seed fertility, when backcrossed with the radish cv. ‘Shogoin’, the values were approximately 180% to 500% with the mode around 300% with the seed harvested from a pod increasing with the advancing generations. Genetic recombination between the radish chromosomes and the added chromosome is probably rare, suggesting that the added chromosome is mostly maintained unaltered in the background of the radish genome.     

Homoeological relationships between the f chromosome of Brassica rapa and the e chromosome of Brassica oleracea

Eight plants of the putative double monosomic addition line (DMAL, 2n= 20) were developed by crossing a monosomic chromosome addition line of radish [f(A)‐type monosomic addition line (MAL) (2n= 19)] carrying the f chromosome of Brassica rapa (2n= 20, AA) with another [e(C)‐type MAL (2n= 19)] having the echromosome of Brassica oleracea (2n= 18, CC). The homoeological relationships between the two alien chromosomes were investigated by morphological, cytogenetic and random amplified polymorphic DNA (RAPD) analysis. Seventeen morphological traits that were not present in the radish cv. ‘Shogoin’ were observed in both MALs and these traits were substantially exhibited in DMAL plants. At the first metaphase of pollen mother cells (PMCs), the two parental MALs showed a chromosome configuration of 9II +1I, demonstrating impossibility of recombination between the R and the added chromosomes. The DMALs formed 10II in approximately 73% of PMCs, with one bivalent showing loose pairing between two chromosomes differing in size. In an attempt to identify the two MALs by RAPD‐specific markers using 26 selected random primers, 13 and 20 bands were specific for the f(A)‐type and the e(C)‐type MALs, respectively; 12 bands were common to both MALs (26.7%). In conclusion, the f chromosome of B. rapa is homoeologous to the e chromosome of B. oleracea. The genetic domain (genes) for 17 morphological traits are linked to each homoeologous chromosome bearing 27% of the corresponding RAPD markers.     

Production and characterization of intergeneric hybrids between Brassica oleracea and a wild relative Moricandia arvensis

Intergeneric crosses were made between Brassica oleracea and Moricandia arvensis utilizing embryo rescue. Six F₁ hybrid plants were generated in the cross‐combination of B. oleracea × M. arvensis from 64 pods by the placenta‐embryo culture technique, whereas three plants were produced in the reciprocal cross from 40 pods by the ovary culture technique. The hybrid plants were ascertained to be amphihaploid with 2n = 23 chromosomes in mitosis and a meiotic chromosome association of (0–3)II + (17–23)I at metaphase I (M I). In the backcross with B. oleracea, some of these hybrids developed sesquidiploid BC₁ plants with 2n = 32 chromosomes that predominantly exhibited a meiotic configuration of (9II + 14I) in pollen mother cells. The following backcross of BC₂ plants to B. oleracea generated 48 BC₃ progeny with somatic chromosomes from 2n = 19 to 2n = 41. The 2n = 19 plants showed a chromosomal association type of (9II + 1I) and a chromosomal distribution type of (9¹/₂ + 9¹/₂) or (9 + 10) at M I and M II, respectively. These facts might suggest that they were monosomic addition lines (MALs) of B. oleracea carrying a single chromosome of M. arvensis that could offer potential for future genetic and breeding research, together with other novel hybrid progeny developed in this intergeneric hybridization.     

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.     

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.     

Intergeneric Hybrids between Aegilops squarrosa and Secale cereale and their Meiotic Chromosome Behaviour

Three hybrid plants with different combinations of D and R genomes were produced from crosses between Aegilops squarrosa (2×: and 4×) and Secale cereale (2× and 4×) using embryo culture. Production frequencies of mature hybrids, having the genomes DR, DDR and DDRR, were 2.0%, 5.2% and 2.2 %, respectively, of florets pollinated. Amphidiploids were obtained directly from the cross between tetraploid parents. The majority of their morphological features were intermediate between those of the parents but, as with rye, the rachis was tough. Diploid and triploid hybrids were completely seed-sterile, whereas the amphidiploid had an average self-fertility of 4.5 % with a range of 0 to 45 %. The somatic chromosome numbers of diploids, triploids and amphidiploids were 2n = 14, 21 and 28, respectively. At meiotic metaphase I, the mean chromosome associations were 0.01 III + 0.26 II + 13.4 I in diploids, 6.96 II + 7.1 I in triploids, and 10.5 II + 7.9 I in amphidiploids. In diploids, pairing between D and R chromosomes was observed in the form of end-to-end types without chiasmata. Homologous pairing between D chromosomes was predominant in plants having two sets of D genomes. Amphidiploids showed a diploid-like chromosome behaviour. Homoeologous pairing was not observed in either triploids or amphidiploids.     

Cytogenetical and molecular investigations on somatic hybrids of Sinapis alba and Brassica juncea and their backcross progeny

Somatic hybrids of Sinapis alba+Brassica juncea (Sal Sal AABB) were synthesized by protoplast electrofusion. They were true genomic allopolyploids since they possessed 60 chromosomes, i.e. the sum of S. alba (2n= 24) and B. juncea (2n= 36) chromosomes. Chromosome pairing was predominantly bivalent with the occasional occurrence of multivalents in the pollen mother cells at diakinesis and metaphase I. Hybrids were completely pollen-sterile, but produced seeds on back-crossing with B. juncea and B. campestris. A total of 37 BC₁ plants were raised from two somatic hybrids (JS-1 and JS-2) and 24 of these were analysed cytologically. The 22 plants originating from the pollinations of somatic hybrids with B. juncea showed a chromosome configuration of 18II+12I and had 42–86% pollen fertility. Two plants from the backcrosses of the somatic hybrid with B. campestris formed 10II +20I, and had 0–4% fertile pollen. Total DNA analysis by probing with pTA71 carrying a full-length 18S–25S rDNA fragment of the wheat nuclear genome revealed that the two somatic hybrids possessed all the characteristic bands of both the species, confirming their hybridity. Probing with the mitochondrial coxI and atp9 genes indicated mitochondrial genome recombination in the hybrids. Hybridization with chloroplast-specific psbD indicated that both the somatic hybrids possessed the cp genome of S. alba origin.     

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.     

An Evaluation of Hordeum vulgare L. ×Psathyrostachys fragilis (Boiss.) Nevski Crosses for Doubled Haploid Barley Production

Investigations were carried out to assess the suitability of the intergeneric cross Hordeum vulgare×Psathyrostachys fragilis for haploid barley production. H. vulgare cvs. ‘Emir’ and ‘Vada’ were each pollinated with P. fragilis P.I. 343192 and plants regenerated from embryos cultured on a modified B5 medium. Seed sets on ‘Vada’ were significantly lower than on ‘Emir’, and all the planes from ‘Vada’×P. fragile remained hybrid. Several of these flowered but there was little pairing between the parental chromosomes. Most of the plants from ‘Emir’×P. fragilis died, as seedlings but 3 plants developed into haploid barley. Because of the practical limitations of pollen availability from P. fragilis and the inconsistencies in haploid plant formation, it is unlikely that the cross will prove as valuable as that between H. vulgare×H. bulbosum for a doubled haploid barley programme.     

Molecular and biological studies on male-sterile cytoplasm in the Cruciferae. IV. Ogura-type cytoplasm found in the wild radish, Raphanus raphanistrum

The orf138 gene, which is specific to Ogura male-sterile cytoplasm, was analysed in mitochondrial DNA (mtDNA) of the wild radish, Raphanus raphanistrum, by polymerase chain reaction (PCR), Southern hybridization and sequencing. The effect of R. raphanistrum cytoplasm on the expression of male sterility was also examined in progeny with R. sativus. A PCR-aided assay and Southern hybridization revealed that three out of six strains analysed included plants with orf138. The sequence of wild type orf138 was same as that of Ogura, except for one or two nucleotide substitutions. Southern hybridization showed a novel mtDNA configuration in R. raphanistrum, in addition to the normal and Ogura types identical to those in R. sativus. Among interspecific hybrids, all the F₁ had normal pollen fertility. In the F₂ progeny between female wild plants having orf138 and the maintainer of Ogura male sterility, male-sterile plants were segregated, fitting the ratio of 3 fertile: 1 sterile plant. R. raphanistrum has cytoplasm that induces male sterility in radishes, and contains a dominant fertility restorer gene.     

Meiotic chromosome behaviour in an allotriploid derived from an amphidiploid × diploid mating in Cucumis

A synthetic amphidiploid species Cucumis hyriviis Chen & Kirkbride (2n = 4x = 38; genome designated as HHCC) has recently been created from an interspecific mating between C. sativus L. (2n = 2x = 14: genome designated as CC) and C. hystrx. Chakr. (2n = 2x = 24; genome designated as HH). This amphidiploid is resistant to root knot nematode, tolerant to low irradiance, and has higher nutritional value than standard processing cucumber cultivars. An allotriploid (2n = 3x = 26; HCC) was derived from a cross between C. sativus L. and C. hytivus Chen & Kirkbride. Diploid meiotic behaviour in C. sativus and C. hystrix involves the development of seven and 12 metaphase bivalents respectively. In the derived allotriploid. univalents. bivalents, and trivalents (at relatively low frequency) were observed at metaphase I indicating that some homeologues from the C and H genomes can synapse. Based on a comparative karyotype analysis of cucumber (i.e. chromosome size and pairing behaviour) and aliotriploid plants, the seven bivalents observed at metaphase I were ascertained to be cucumber homologues, while the 12 univalents were of C. hystrix origin thus confirming the allotriploid karyotypic constitution to be HCC. On average, the frequency of trivalents was 0.24 at diakinesis and 0.22 in 100 meiocytes at metaphase I. indicating the possibility of genetic exchange due to the homoeology between the C and H genomes. After simultaneous cytokinesis, only polyads were observed in pollen mother cells (PMCs) at telophase II, which led to the production of sterile pollen grains. Multi‐polarization of chromosomes was dominant at anaphase II. However. in about 20% PMCs. chromosomes separated to form a 7C + 1‐2H complement, suggesting a possible method for the production of alien addition cucumber‐C hystrix lines through further backcrossing of the allotriploid to diploid cucumber.     

诸葛菜小孢子培养及其单倍体减数分裂染色体配对观察

诸葛菜是一种极有价值的观赏、蔬菜、饲料和油料作物种质资源。为建立诸葛菜小孢子胚状体诱导再生植株技术,并为诸葛菜染色体组的起源与进化研究提供相关数据资料,本研究通过对诸葛菜游离小孢子的培养,研究了热激培养时间和活性炭浓度对胚状体产量的影响,并采用常规压片法对诸葛菜单倍体减数分裂染色体配对行为进行了观察。结果表明,添加活性炭和热激培养对胚状体诱导是必需的。在直径6 cm培养皿中培养4 mL密度为1花蕾花粉mL?1的小孢子NLN悬液时,每皿添加1mg活性炭和32℃热激3d的培养条件下子叶形胚状体和总胚状体产量最高,分别为每花蕾0.92±0.18个和1.32±0.25个。子叶形胚状体在1/2 MS培养基上萌发率为27.73%。花粉植株中自然加倍率为25%,加倍植株染色体数为24,单倍体植株染色体数为12。诸葛菜单倍体减数分裂染色体的平均配对构型为n=12=6.352I+2.008II+0.384III+0.12IV,具有二价体及三价体和四价体的细胞比例高达96%,少量细胞的12条染色体联会形成3个四价体,说明诸葛菜很可能是起源于染色体基数x=3的同源八倍体。本试验结果对于诸葛菜新材料新品种选育...     

甘蓝型油菜三体、双三体和双四体的形态和细胞学特征

从甘蓝型油菜品种“Oro”×诸葛菜杂交后的一个混倍体杂种（2n=19~37）的小孢子再生植株中鉴定出一株2n=42的植株，此混倍体不含有诸葛菜染色体，故2n=42植株的细胞内没有诸葛菜染色体。用其花粉授予“Oro”后获得2n=39、40的植株。2n=39、40、42植株PMCs内染色体的主要配对方式分别为19Ⅱ+1Ⅰ、19Ⅱ+2Ⅰ和19Ⅱ＋2Ⅱ，后期Ⅰ的分离方式分别为20∶19、21∶19和21∶21。故2n=39、40、42为甘蓝型油菜的三体、双三体、双四体。最后讨论了这些种内非整倍体材料在油菜遗传研究中的意义。     

Morphology, Cytology and Fertility of Avena sativa L. ×A. magna Murph. et Terrell Amphiploids

The genotypes of Avena sativa were crossed to A. magna Murph. et Terrell, from the sterile pentapliod F₁ amphiploids were successfully developed after colchicine treatment. The C₁ plants were observed for their morphology, cytology and fertility. These plants were characterized by longer and broader glumes, swollen pedicel attachment, pubescent lemmas, bold seeds and persistent spikelets. Other characters, viz., final plant height, rachis length, peduncle length and spikelets per panicle were intermediate. All plants hail the chromosome number 2n = 70 or close to. 7C. Due to stickiness of chromosomes, meiosis of only two decaploid plants could be studied in detail. The average chromosome Association was 1.3 I + 28.56 II + 1.4 III + 1.77 IV + 0.07 VI and 0.13 I + 28.7 II + 0.14 III + 2.09 IV + 0.59 VI; but in some cells up to 35 bivalents were observed. Seed set as well as pollen fertility and size exhibited great variation.     

萝卜-芥蓝异源四倍体的合成及GISH分析

通过萝卜(Raphanus sativus L.,2n=18,RR)与白花芥蓝(Brassica alboglabra Bailey,2n=18, CC)杂交,F1经秋水仙碱加倍合成萝卜-芥蓝异源四倍体（Raphanobrassica, 2n=36, RRCC）。经F₄~F₁₀代连续育性选择,F₁₀单株种子产量达32.3 g,每角粒数达14.9。基因组原位杂交显示F₁₀减数分裂行为类似于二倍体物种,表明该异源四倍体的细胞学行为已经稳定。育性观察表明,可育花粉足够各代生产种子,但低世代杂种出现高频瘪粒种子,胚珠败孕可能是其主要原因。该萝卜-芥蓝异源四倍体可以用作向油菜（B. napus L.,2n=38,AACC）转移萝卜基因的遗传桥梁。     

Elymus canadensis×Secale cereale Amphiploids. II. Chromosome Constitutions and Pairing of Open-Pollinated Progeny

Eleven C₂ and two C₃ 0pen-pollinated plains from Elymus canadensis × Secale cereale amphiploid plants (2n = 6x = 42, SSHHRR) were examined for chromosome constitution and meiosis. Chromosome numbers of the progeny varied: 2n = 26, 27, 28, 36, 37, 39, 40, and 41. Elimination of portions of genome constituents were made at random and were irregular in all o the progeny. Monosomic (2n = 41) and nullisomic (2n = 40) plants lost one to two E. canadensis or S. cereale chromosomes and showed average of 17 to 18 bivalents and 4 to 5 univalents per cell at Ml. The C₂, aneuploid plants with 36 to 41 chromosomes seemed to result from selfing or intercrossing among; the C₁ amphiploid plants, while the plants of 2n = 26 to 2S (6–9 II + 10–141) might originate from outcrosses of the C_l amphiploid to S. cereale. Bivalent pairing might be preferentially intragenomic (S-S, H-H, or R-R). The occurrence of multivalents indicates a low potential of both intragenomic and intragenomic pairing; Pollen of the lour plants showed poor stainability (1 to 13 %) and no seed set in any of the progeny.     

Relative efficiency of maize and Imperata cylindrica for haploid induction in Triticum durum following chromosome elimination‐mediated approach of doubled haploid breeding

Intergeneric hybridization in seven diverse durum wheat genotypes was carried out using two composite varieties of Himalayan maize, viz., Bajaura Makka and Early Composite, and a wild grass, Imperata cylindrica, as pollen sources. Observations related to various haploid induction parameters put forth I. cylindrica as significantly better pollen source for haploid induction in durum wheat over maize in terms of pseudoseed formation (46.93%), embryo formation (38.06%), haploid regeneration (40.42%) and haploid formation efficiency (7.44%). The line x tester analysis revealed that both male and female genotypes had significant effects on all haploid induction parameters except haploid formation frequency in later. Among the pollen sources, I. cylindrica emerged as best combiner based on GCA values when compared with the two Himalayan maize composites. Durum wheat genotype, A‐9‐30‐1 was recognized as the best general combiner followed by PDW 314. The present investigation proposed durum wheat × I. cylindrica as a superior technique over maize‐mediated system, and its large‐scale use can open a new horizon in the sphere of durum wheat doubled haploidy breeding programme.     

Production of an interspecific hybrid between Brassica fruticulosa and B. rapa

An interspecific hybrid between a wild species, Brassica fruticulosa (2n = 16, FF) and a crop Brassica species, B. rapa (2n = 20, AA) was synthesized using sequential ovary‐ovule culture on MS medium supplemented with casein hydrolysate. Morphological, molecular and cytogenetic analysis confirmed the true hybrid nature of the offspring. The F1 plants (2n = 18) were intermediate in morphology, highly pollen‐sterile as well as self‐sterile. A maximum of three bivalents per PMC was recorded, but 14 I + 2 II was the most common meiotic configuration. Normal pollen fertility and regular bivalent (18 II) formation was observed in the amphiploid sectors of the hybrid plants. The F₁ hybrid harboured significantly lower aphid populations than the crop Brassica parent.     

Rapid introduction of disease resistance from rye into common wheat by anther culture of a 6x triticale × nulli-tetrasomic wheat

Powdery mildew (caused by Erysiphe graminis) and yellow rust (caused by Puccinia striiformis) are the two most serious wheat diseases found in China. Rye chromosomes, carrying genes for resistance to these diseases, were introduced into common wheat in two generations using chromosome engineering and anther culture. The F₁ hybrids from a cross involving a hexaploid triticale (×Triticosecale Wittmack) בChinese Spring’ nulli‐tetrasomic N6DT6A wheat aneuploid line were anther cultured and doubled‐haploid plants were regenerated. Using genomic in situ hybridization, C‐banding and biochemical marker analyses, one of the anther‐cultured lines (ZH‐1)studied in detail, proved to be a doubled‐haploid with one rye chromosome pair added (1R) and a homozygous 6R/6D substitution (2n= 44). The line was tested for expression of disease resistance and found to be highly resistant to powdery mildew and moderately resistant to yellow rust.