Interspecific hybridization between Brassica carinata and Brassica rapa

The crossability between Brassica carinata (BBCC, 2n=34) and Brassica rapa (AA, 2n=20), and the cytomorphology of their F₁ hybrids were studied. Hybrids between these two species were only obtained when B. carinata was used as the female parent. The hybrid plants exhibited intermediate leaf and flower morphology, and were found to be free from white rust and Alternaria blight diseases. One of the four F₁ plants was completely male sterile, while the remaining plants had 4.8, 8.6, and 10.9% stainable pollen, respectively. No seed was produced on hybrid plants under self pollination or in backcrosses; but seed was obtained from open pollination. The occurrence of the maximum of 11 bivalents as well as up to 44.8%) of cells with multivalent associations in the form of trivalents (0‐2) and a quadrivalent (0‐1) in the trigenomic triploid hybrid (ABC, 2n = 27) revealed intergenomic homoeology among the A, B and C genomes. Meiotic analysis of F₁ hybrids indicated that traits of economic importance, such as disease resistance, could be transferred from B. carinata to B. rapa through interspecific crosses.     

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.     

Cytogenetics of a new trispecies hybrid in cotton: [(Gossypium hirsutum L. × G. thurberi Tod.)2 × G. longicalyx Hutch. & Lee]

A three‐species hybrid named HTL including Gossypium hirsutum L. [2n = 4 x = 52, (AD)₁ genome] was created using the pseudophyletic introgression method with G. longicalyx Hutch. & Lee (2n = 2x = 26, F₁ genome) as donor parent and G. thurberi Tod. (2n = 2x = 26, D₁ genome) as bridge species. The new hybrid was totally self‐sterile and its interspecific status was confirmed using simple sequence repeat markers and cytogenetic analysis. Cytogenetic studies showed that its chromosome configuration was 2n = 52 = 14.13 I + 15.10 II + 1.03 III + 0.9 IV + 0.03 V + 0.13 VI (where I, II, III, IV, V and VI are univalents, bivalents, trivalents, tetravalents, pentavalents and hexavalents, respectively). Prospects for successfully exploiting the HTL hybrid in breeding programmes are discussed.     

Chromosome pairing in haploid plants of radish derived from alien monosomic addition lines

Six haploid plants of radish were obtained via alien monosomic addition lines (2n = 19). One plant was derived from anther culture of an Raphanus sativus‐Brassica oleracea addition line, one plant from the selected smaller seed of an R. sativus‐Sinapis arvensis addition line and four plants from an R. sativus‐B. rapa addition line. During metaphase I of pollen mother cells, two plants exhibited the chromosome pairing of (0‐3)II+ (3‐9)I, three showed (0‐1)III + (0‐3)II + (3‐9)I and the remaining plant (0‐2)II + (5‐9)I. Trivalents seemed to be formed by the pairing between two larger chromosomes and the smaller one with somewhat loose pairing. All haploid plants were inferior to the radish cv. ‘Shogoin’ (2n = 18) with respect to vegetative growth. Their flowers were smaller, with sterile pollen grains, but a few normal flowers with fertile pollen sporadically developed and then produced a few seeds. It is suggested that the radish genome (R, n = 9) might comprise three pairs of homoeologous chromosomes, with the remaining three chromosomes carrying the homologous region(s) that results in a trivalent formation.     

Genomic affinities between Brassica napus and Raphanus raphanistrum as revealed by meiotic GISH

An intergeneric hybrid between Brassica napus (AC, n = 19) and Raphanus raphanistrum (Rr, n = 9) was developed using the sequential ovary–ovule culture. Morphological, molecular, cytological studies and fluorescent genomic in situ hybridization were conducted to establish the hybridity and genomic relatedness. F₁ hybrid plants were male sterile and morphologically intermediate between the two parents. Cytological analysis of the hybrid plants revealed 10II + 8I as the predominant meiotic configuration with the mean bivalent frequency of 8.83. A maximum of 13II were observed in 8.24% of the pollen mother cells (PMCs). GISH studies facilitated the identification of two allopairs, confirming homoeologous pairing between A/C and Rr genomes. This may have significant implications in terms of transgene flow and possible introgression into R. raphanistrum, which is a common canola weed in Canada and Australia. The intergeneric hybrid B. napus × R. raphanistrum so produced also has the potential to be used as a bridging species for the transfer of desirable genes to amphiploid crop Brassica species carrying A/C genomes.     

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.     

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.     

Cytogenetic and morphological characteristics of new types of diploids (2n=22, 24, 40) derived from consecutive selfing of aneuploids in brassica crops

Summary New types of diploids in Brassica crops were synthesized after three consecutive selfing of aneuploids derived from backcrossing of sesquidiploids (2n=29, AAC) with B. campestris (2n=20, AA). The cytogenetic and morphological characteristics of plants with 2n=22, 24 and 40 in the S₃ generation were analyzed in order to establish the extent in which these addition and polyploid lines were stabilized. A high frequency of 11_II (79.7%), 12_II (84.6%) and 20_II (100%), were observed at metaphase _I of pollen mother cells in 2n=22, 24 and 40 plants, respectively. The chromosome configuration at methaphase _II also indicates that a certain level of stability has been attained cytogenetically. Although pollen stainability was relatively high, the seed set percentage was still low. Variation in morphological characteristics indicate the incorporation of one or more chromosome pairs from the C genome of B. oleracea. Other diagnostic characters such as the formation of determinate inflorescence, branching from the base of the stem, and the shift from self-incompatibility to self-compatibility must have resulted from the interaction between A and C genomes. Thus plants with 2n=22, 24 and 40 have been stabilized to some extent and can be developed into new breeding lines of Brassica. It is suggested that limited pollination could be effective in increasing the seed fertility of these plants.     

Expression of male sterility in alloplasmic Brassica juncea with Erucastrum canariense cytoplasm and the development of a fertility restoration system

An alloplasmic mustard, Brassica juncea, has been synthesized by placing its nucleus into the cytoplasm of the related wild species Erucastrum canariense to express cytoplasmic male sterility. To achieve this, the sexual hybrid E. canariense (2n=18, E^cE^c) ×Brassica campestris (2n= 20, AA) was repeatedly backcrossed to B. juncea (2n= 36, AABB). Cytoplasmic male‐sterile (CMS) plants were recovered in the BC₄ generation. These plants are a normal green and the flowers have slender, non‐dehiscing anthers that contain sterile pollen. Nectaries are well developed and female fertility is > 90%. The fertility restoration gene was introgressed to CMS B. juncea from the cytoplasmic donor E. canariense through pairing between chromosomes belonging to B. juncea with those of the E. canariense genome. The restorer plants have normal flowers, with well‐developed anthers containing fertile pollen. Meiosis proceeds normally. Pollen and seed fertility averaged 90% and 82%, respectively. F₁ hybrids between CMS and the restorer are fully pollen fertile and show normal seed set. Preliminary results indicate that restoration is achieved by a single dominant gene. The constitution of the organelle genomes of the CMS, restorer and fertility restored plants is identical, as revealed by Southern analysis using mitochondrial and chloroplast probes atp A and psb D, respectively.     

An ms2 male-sterile, female-fertile soybean sharing phenotypic expression with other ms mutants

Genetic and cytological studies of microsporogenesis and microgametogenesis were conducted with a natural male‐sterile, female‐fertile soybean mutant (BR97‐17971) found in an F₄ breeding line of the Brazilian soybean breeding programme. Allele tests with ms Type Collection from USDA/ARS showed that the gene was allelic to ms2. Conventional analysis of microsporogenesis revealed a differential meiotic behaviour from the normal one reported in ms2 male‐sterile plants. Sterile plants have different levels of meiotic abnormalities related to chromosome segregation. Despite segregational abnormalities in the first and second divisions leading to micronuclei formation, the main meiotic cause of pollen sterility was absent or the defective cytokinesis following telophase II. Absent or defective cytokinesis has never been reported in ms2 mutants, but it was reported in the ms1 and ms4 mutants. After telophase II, one to four nucleate microspores underwent degeneration. Cross‐sections of sterile plant anthers showed that, at telophase II, the tapetal cells with large vacuoles degenerated. Despite callose deposition around meiocytes, sterile plants did not form tetrads. They degenerated and collapsed after callose dissolution, forming an amorphous mass. Anthers from male‐sterile plants were shrivelled.     

Cytogenetics of Brassica juncea×Brassica rapa hybrids and patterns of variation in the hybrid derivatives

Interspecific hybridization is an important tool to elucidate intergenomic relationships, transfer characters across species and develop synthetic amphidiploids, and it has been widely applied for improving Brassicas. The objective of the present study was to create genetic variability in Brassica through interspecific hybridization. Crosses between Brassica juncea (AABB, 2n= 36), and Brassica rapa (AA, 2n = 20) vars toria, yellow sarson, and brown sarson were attempted, and the hybrid derivatives were advanced to the F4 generation. Hybrids were obtained from the crosses B. juncea× toria and B. juncea× yellow sarson. The F₁ plants were vigorous and intermediate to the parents in many morphological traits. The meiotic study of AAB hybrids showed 10 II + 8 I in the majority (71.8%) of cells analysed. A maximum of 12 and a minimum of seven bivalents were also observed in a few cells. The occurrence of multivalent associations (trivalents to pentavalents) at diakinesis/metaphase I and a bridge‐fragment configuration at anaphase I were attributed to homoeology between A and B genomes. A high percentage of plants resembling B. juncea was observed in the F₂ generation. Transgressive segregation in both directions was found for plant height, primary branches, main raceme length, siliquae on main raceme, siliqua intensity, seeds per siliqua and seed yield. There were significant differences for the 14 characters in the F₄ derivatives. Moderate to high estimates of phenotypic and genotypic coefficients of variation, broad‐sense heritability, and expected genetic advance were found for seed yield, 1000‐seed weight, siliquae per plant, seeds per siliqua and days to flowering. Intergenomic recombination, reflected as wide variation in the hybrid progenies, permitted the selection of some useful derivatives.     

N2O induces mitotic polyploidization in anther somatic cells and restores fertility in sterile interspecific hybrid lilies

Fertile plants undergoing male gametogenesis can be treated with nitrous oxide (N₂O) gas to obtain 2n male gametes. N₂O treatment is also expected to restore the fertility of interspecific hybrids through meiotic restitution or mitotic amphidiploidization. However, this technique has few applications to date, and it is un-known how N₂O treatment restores fertility in sterile hybrids. To establish optimal N₂O treatment conditions and determine its cytological mechanism of action, we treated various sized floral buds with N₂O gas at different anther developmental stages from fertile and sterile hybrid lilies. N₂O treatment using the optimal 1–4 mm floral buds induced mitotic polyploidization of male archesporial cells to produce 2n pollen in fertile hybrid lilies. In sterile hybrid lilies, N₂O treatment doubled the chromosome number in male archesporial cells followed by homologous chromosome pairing and normal meiosis in pollen mother cells (PMC), resulting in restoration of pollen fertility. Backcrossing the resultant fertile pollen to Lilium × formolongi produced many triploid BC₁ plants. Thus N₂O treatment at the archesporial cell proliferating stage effectively overcame pollen sterility in hybrid lilies, resulting in fertile, 2n pollen grains that could produce progeny. The procedure presented here will promote interspecific or interploidy hybridization of lilies.     

Synthesis and preliminary characterization of a new species (amphidiploid) in Cucumis

A successful interspecific hybridization between cucumber (Cucumis sativus L., 2n = 14) and a wild it Cucumis species, C. hystrix Chakr. (2n = 24) was made via embryo rescue. Hybrid plants (2n = 19; 7 from cucumber and 12 from C. hystrix) were sterile, but morphologically uniform. Self-pollination and backcrossing of F₁ hybrid plants to either parent confirmed presence of both male- and female-sterility that were likely caused by lack of homology and improper pairing during meiosis. While the multiple-branching habit, densely brown hairs (on corolla and pistil), orange-yellow corolla, and ovate fruit of F₁ hybrid plants were similar to that of the C. hystrix parent, the appearance of the first pistillate flower was more similar to that of C. sativus parent. The diameter and internode length of the stem, and the shape and size of leaves and flowers were intermediate when compared to the parents. The chromosome number in the hybrid was doubled through somaclonal variation during embryo culture and regeneration process to restore the fertility. Pollen grains were released and fruits with viable seeds matured on fertile, synthetic amphidiploid plants. The results from flow cytometry indicated that, on average, 7.3% of the morphologically unique regenerants had the 4C DNA content of 2.35 pg relative to the 2C DNA content of the original F₁ hybrid at 1.17 pg and, therefore, were likely chromosome-doubled F₁ hybrids (2n = 38). Nutrition alanalysis indicated that the synthetic species had higher protein (0.78%)and mineral (0.35%) content compared to the normal pickling cucumber(0.62% and 0.27%, respectively), and could be considered a new Cucumis crop having a special place in the future agriculture. Preliminarily evaluation indicates that C. hystrix possesses a high level of root-knot nematode resistance, and that this resistance is partially expressed in the interspecific F₁ and chromosome-doubled F₁. This and the fact that the fruit morphology of the fertile amphidiploid differs during the growing season (e.g., short and long fruit) suggest that it could be useful in broadening the germplasm base of cucumber. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genomic in situ hybridization analysis of Brassica napus × Orychophragmus violaceus hybrids and production of B. napus aneuploids

To further utilize the valuable germplasm Orychophragmus violaceus for Brassica genetics and breeding, a B. napus × O. violaceus cross was repeated with embryo rescue. All F₁ plants except one B. napus haploid were mixoploids (2n = 17–39 in ovaries) with 2n = 31, 37, 38 and 39 as the maximal chromosome numbers in individuals, but the higher numbers mostly appeared in pollen mother cells (PMCs) with a preponderance of 2n = 30, 37 and 38. Only one chromosome and one chromosome segment of O. violaceus were detected at a low frequency in some ovary cells and PMCs with 2n = 37, 38 and 39 as determined by genomic in situ hybridization analysis. The fatty acid profiles of seeds from the majority of the F₁ and F₂ plants were similar to those of female B. napus cv. ‘Oro’, but some were obviously different in the percentages of oleic, linoleic and erucic acids, and some F₂ plants (2n = 38) with good seed set had high percentages of oleic (>70.0%) or linoleic (to 38.3%) acids and low erucic acid (<1%). Subsequently, many kinds of B. napus aneuploids (2n = 28, 30, 34, 36, 37, 39 and 42), without O. violaceus chromosomes, were derived from F₂ progeny and microspores of partial F₁ plants. Finally, the cytological mechanisms behind the variations in chromosome numbers were discussed together with the implications of these aneuploids for Brassica genome research and of the plants with altered fatty acid profiles for improving the oil quality of B. napus.     

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.     

Recurrent Addition of the Pennisetum americanum Genome in a P. americanum×P. orientale Hybrid

An interspecific hybrid involving Pennisetum americanum (2 n = 14) and a diploid cytotype of P. orientale (2 n = 18) was backcrossed to P. americanum using the hybrid (2 n = 16, 7‘A’+ 9 ‘O’) as the female parent. Pollen mother cells of 13 BC₁ plants contained a complement of 14‘A’+ 9 ‘O’ chromosomes. Five BC₂ plants obtained through further backcrossing to P. americanum had 21‘A’+ 9 ‘O’ chromosomes revealing another addition of the P. americanum genome. The role of such recurrent additions of parental genomes in the evolution of polyploid species has been discussed.     

Interspecific hybrids between helianthus PetiolarisNutt. and H. Annuus L.: Effect of backcrossing on meiosis

Summary Interspecific substitutions of the nucleus of Helianthus annuus (2n=34) into the cytoplasm of H. petiolaris (2n=34) were obtained by successive backcrossing using cultivated sunflower, H. annuus, as the recurrent pollen parent.Meiosis in the F₁ was characterized by multivalents, suggesting that 10 of the 34 chromosomes were heterozygous for chromosomal interchanges. An additional pair of chromosomes also contained a paracentric inversion. Continued backcrossing resulted in rapid elimination of the meiotic aberrations evident in the F₁. In the BC₁, 1 of 11 plants had normal meiosis and by the BC₂, only 13 of 54 plants had meiotic aberrations similar to those of the F₁. However, trisomic progeny (2n=35) were found in 3 of the 11 BC₁ plants and 20 of the 54 BC₂ plants. No meiotic aberrations were observed in BC₃ or BC₄ plants. Plants with indehiscent anthers, and considered to be male sterile (M.S.), first occurred in the BC₁ and, by the BC₂, 51 of 54 plants were M.S. All 19 BC₃ and 16 BC₄ plants were M.S. Preliminary investigations suggest that the pollen from such plants is sterile and that the sterility is cytoplasmic rather than genetic.Disc-flower measurements were a useful technique for selecting samples at the correct stage of microsporogenesis, but could not be used to distinguish between successive backcrosses.     

Chromosome doubling and fertility study of Alstroemeria aurea × A. caryophyllaea

Self-pollinations of a diploid (2n = 2x = 16) interspecific hybrid Alstroemeria aurea × A. caryophyllaea, resulted in no seeds. Backcrosses of the hybrid with parent A. aurea did not produce any seeds. In an attempt to restore the hybrid fertility, an efficient in vitro procedure has been developed and applied effectively in the chromosome doubling of the diploid hybrid. Forty-one percent of the treated plants were proven to be truly tetraploid by chromosome counts and stomatal measurements after applying 0.2 to 0.6% colchicine for 6 to 24 hours. Over 87.5% of these colchicine-induced tetraploids were stable and retained their tetraploidy after one year of growth. The fertility of the hybrid was not restored although the pollen stainability was increased from 0 to 12% after chromosome doubling. Cytological studies on the pollen mother cells (PMCs) of the sterile diploid hybrids revealed abnormal meiotic behaviors. In addition, aneuploid chromosome numbers, ranging from 2n = 1 to 2n = 18, were observed in over 45% of the PMCs examined. PMCs of the colchicine-induced tetraploids showed that meiotic chromosome pairings were normal in most cases (1.59_I + 15.07_II + 0.05_III + 0.03IV). These results indicate that the sterility of this hybrid is not only caused by parental chromosome differences, but other complex fertility/sterility-regulating mechanisms are involved too. This revised version was published online in July 2006 with corrections to the Cover Date.     

An intersubgeneric hybrid between Glycine tomentella Hayata and the soybean,G. max (L.) Merr.

Summary The objective of the present paper is to provide information on the morphology and cytology of an intersub-generic hybrid (2n=59) between Glycine tomentella Hayata (2n=78) and G. max (L.) Merr. (2n=40) obtained through in vitro immature seed culture. The hybrid plant was slow in vegetative growth and twinning like the female parent but morphologically was intermediate between both parents for several traits. At metaphase I, the average chromosome associations and ranges for 25 cells were 44.0 I (37–51)+7.5 II (3–11). The plant was completely pollen and seed sterile. The present investigation suggests that wild perennial Glycine species can be exploited as either the male or female parent in wide hybridization programs with the soybean, G. max.     

Nucleus substitution of Brassica japonica Sieb. with Raphanus sativus L. and its resultant chlorophyll deficiency

Summary Nucleus substitution of Brassica japonica (2n=20) with Raphanus sativus (2n=18) was carried out by means of repeated backcrossing of Brassicoraphavus (2n=37) to R. sativus as a pollen donor. In the course of nucleus substitution, chlorophyll deficiency appeared. Plants with more than 28 chromosomes, like their parents, had green leaves and those with 24 to 26 chromosomes had yellowish green ones. Almost all plants with 18 to 23 chromosomes showed yellow or whitish yellow. The R. sativus with B. japonica cytoplasm (2n=18) was obtained after four successive backerosses. The completely substituted R. sativus showed the same fertility as the true R. sativus used as a recurrent parent. It is assumed that the chlorophyll deficiency is caused by disharmony between the B. japonica cytoplasm and the R. sativus nucleus. The chlorophyll deficiency is discussed in comparison with male sterility or other characters which sometimes occur in alloplasmic Raphanus and Brassica species.