Multiple chromosomal interchanges in pearl millet

Summary Intercrossing and irradiation were successfully used in pearl millet (Pennisetum typhoides) to develop multiple interchanges involving up to the total complement of all the chromosomes in one complex. In interchange heterozygotes showing 12 + 1 II and 14, 90.9 and 87.8 per cent of the cells, respectively, had chromosome configurations other than that of 12 and 14 chromosomes. In general, the frequency of such cells resulting from breakdown of the expected complex configuration increased with the increase in the number of translocated chromosomes in the complex. The higher the number of chromosomes involved in the interchange ring, the higher were the pollen and ovule sterility. The results indicated that meiotic instability, deficiency-duplication gametes, and unequal distribution of chromosomes account for increased sterility of multiple interchange heterozygotes. Even though interchanges in pearl millet predominantly show the alternate type of segregation, sterility seems to be the major barrier for the exploitation of the multiple interchange method for gamete selection and the establishment of homozygous lines in this plant species.     

SEMISTERILITY IN THE INTERSPECIFIC HYBRID MELILOTUS POLONICA × M. ALBA

Jaranowski , J. K. (Coll. of Agriculture, ul. Wojska Polskiego 71c, Poznan, Poland.) Semisterility in the interspecific hybrid Melilotus polonica × M. alba. Amer. Jour. Bot. 48(1): 28–35. Illus. 1961.—Interspecific hybrids between Melilotus polonica (n = 8) and M. alba (n = 8) are readily secured. The F₁ hybrids are intermediate between the parents and partially sterile with a mean percentage of 58.8 (ranging from 46.8 to 72.6) defective pollen grains. Six bivalents and a chain or ring of 4 chromosomes occur at diakinesis and metaphase-I of microsporogenesis. A crossshaped configuration characteristic of a reciprocal translocation is present at pachytene, indicating that one of the parents is homozygous for an interchange of relatively large section between two of the members of the chromosome complex. Chromosome bridges, lagging chromosomes, movement of the univalents to the same pole and precocious division of the univalents lead to aberrant chromosome distribution during the course of meiosis. Reduction in self-fertility indicates a corresponding aberrant distribution of chromosomes during megasporogenesis. Pollen sterility in the F₂ generation ranged from 24.8% to 72.5% with a mean value of 54.6%. Two plants in the F₂ generation which had relatively low pollen sterility proved to be aneuploids (2n + 1). Meiotic irregularities in the F₂ plants were comparable to those exhibited by the F₁ plants.     

Meiotic studies of the F1 hybrid between rice bean (Vigna umbellata) and its wild relative V. minima

The interspecific F₁ hybrid V. umbellata x V. minima, together with the parental species was investigated cytogenetically. Both parents showed normal meiosis, with 11 bivalents at diakinesis. The hybrid was completely pollen sterile. The cytological causes underlying sterility in the hybrid include an array of meiotic abnormalities ranging from non-pairing of chromosomes through the presence of univalents, chain and ring multivalents and anaphase bridges to the formation of micronuclei, during microsporogenesis. The presence of chain and ring multivalents at diakinesis not only demonstrates the structural hybridity of the F₁ hybrid, but also suggests that small interchanges and inversions played a significant role in the speciation within the genus Vigna.     

Intraspecific hybridization and its bearing on chromosome evolution inVicia narbonensis (Fabaceae)

Nine accessions ofVicia narbonensis, considered to be the wild progenitor of faba bean (Vicia faba), were investigated to ascertain the nature and extent of intraspecific karyotypic polymorphism. The chromosome complements resolved into four distinct types (A, B, C, D), and the meiotic data of F₁ hybrids (A × B, B × C, A × C) revealed that alteration in chromosome morphology is the result of segmental interchanges. The interchange complexes indicate that the parents differ from each other by 1 to 2 interchanges. It is also evident that karyotype B, and not A as previously reported, is the normal karyotype of the species, and A and C are single homozygotes for unequal interchange. The comparative karyomorphology of the parents and the hybrids, and of two interchange heterozygotes of four chromosomes each in F₁ hybrids of A × C shows that the chromosomes involved in the single interchange homozygotes (A, C) are not common and the breaks in both interchanges occurred in short and long arms of the involved chromosomes. Identification of the interchanged chromosomes in the complements and the frequency of ring and chain quadrivalents in the heterozygotes enabled location of the breakpoints. The present results provide probably the first example indicating that interchange homozygosity (A) is not only firmly established but also has enabled the species to spread further by adapting to a wide range of habitats. — The genetic relationships between A and D are very different. All seven chromosome pairs in D could be distinguished from A, and for that matter, B and C as well. From the meiotic pairing properties it is also amply clear that genome D is well differentiated from A and possibly B, and C, and deserves special status.     

An interchange heterozygote inThelepogon elegans roth ex roem et schult.

In a population of 231 plants of the diploidThelepogon elegans (n=5), seven were found to be interchange heterozygotes. A multiple of four, six or eight chromosomes was observed in 62.32% of the pollen mother cells at MI, the remainder having five bivalents. Chiasmata were mostly localised terminally. The orientation of the multiples was predominantly alternate. The subsequent course of meiosis was normal, and pollen fertility was high.     

Estimation of the efficiency of seed irradiation by thermal neutrons for inducing chromosomal aberrations in M<Subscript>1</Subscript> of cotton <Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.

Exposure of cotton seeds to thermal neutrons at doses of 15, 25, and 35 Gy was shown to induce many biomorphologically abnormal plants, including sterile and chimeric ones. Most of these phenotypic changes were shown to result from novel genomic, chromosomal, and desynaptic mutations. The presence of these mutations in the karyotype of M₁ plants often decreased meiotic index and pollen fertility. In translocation forms, the decrease in pollen fertility was caused by the prevalence of quadrivalents in form of rings and chains with adjacent segregation of chromosomes from the translocation complexes. Based on the shapes and sizes of multivalent associations, we performed preliminary localization of translocation breakpoints. A specific feature of the effect of thermal neuron irradiation in M₁ was induction of numerous unique chromosomal aberrations, consisting in the appearance in the same plant of several types of mutations (genomic and chromosomal), interchange complexes in the same nucleus, and multiple interchanges involving three nonhomologous chromosomes.     

The origin of tertiary monosomics in the tomato

Six aneuploid tomato plants with 2n–1=23 chromosomes were observed in populations grown from the seedlings treated with thermal neutrons and from seeds treated with X-rays. Four of the aneuploids were tertiary monosomics in which, as a result of centromeric interchanges between two different chromosomes, two whole arms were missing from the complement and two arms connected at the centromere. In one aneuploid, as a result of centromeric breakage, the two short arms of a homologous pair were missing from the complement and the two long arms connected to the long arm and the short arm respectively of another chromosome in which breakage had occurred also at the centromere. In one aneuploid, the interchange has occurred in the arms, and not in the centromere. Here the aneuploid condition is due to the loss of an arm with a centromere and a short piece of the other arm.In most of the tertiary monosomics the missing arms were either the short arms of sub-metacentric chromosomes or any of the arms of metacentric chromosomes. However, in one case the long arms of two submetacentric chromosomes were lost from the complement. That in spite of such large chromosomal deletions the sporophyte can survive, may be due to the fact that the aberrant plants are mostly chimeras.This study was part of a project resulting from a contract between the Association Euratom-I.T.A.L., and the Agricultural University of Wageningen.     

Außerkaryotische Vererbung von Pollensterilität bei Oenothera

Zusammenfassung Nach Kreuzungsversuchen erhielten wir Pflanzen, bei denen das B · l-Genom der Oenothera berteriana mit den außerkaryotischen Erbfaktoren der Oenothera blandina kombiniert war. Diese Pflanzen waren vollständig pollensteril. Die Fertilität des weiblichen Geschlechts war ebenfalls außerordentlich schwach.Auch nach erneuten Kreuzungsversuchen mit den Genomen B und l blieben die Fertilitätsverhältnisse unverändert.Die Ergebnisse zeigen, daß die Pollensterilität und die geschwächte Fertilität des weiblichen Geschlechts aus dem offenbar gestörten Zusammenwirken zwischen Oenothera berteriana-Genom und den außerkaryotischen Erbfaktoren der Oenothera blandina resultieren.

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Extrakaryotic inheritance of pollen sterility in Oenothera

Summary In crossbreeding tests we obtained plants having the B · l genome of Oenothera berteriana and the extrakaryotic genes of Oenothera blandina. The plants showed complete male sterility. The fertility of the female sex also was extremely low. The condition of fertility remained unchanged after further crossbreeding tests with the genomes B and l . According to these results the male sterility and the decreased fertility of the female sex is due to incompatible interaction between the karyotic genes of Oenothera berteriana and the extrakaryotic genes of Oenothera blandina.

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Angenommen durch R. Hagemann     

Ectopic expression of <Emphasis Type="Italic">MNX</Emphasis> gene from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> involved in auxin biosynthesis confers male sterility in transgenic cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) plants

A transgenic male sterile line of upland cotton was generated by the ectopic expression of the monooxygenase (MNX) gene from Arabidopsis thaliana via Agrobacterium-mediated transformation. The bacterium harbored a plasmid pBinplus carrying a 1.25-kb MNX coding sequence together with a GUS reporter gene; the former was driven by the MS2 promoter of a male sterility gene in Arabidopsis, and the latter was under the control of CaMV 35S promoter. Twenty-seven putative transgenic plants (T₀) were obtained, all of which showed GUS activity and positive signals of NPTII and MNX genes by PCR analysis, and also showed male sterility to some extent. It was further confirmed by Southern blotting that one copy of the NPTII and MNX gene was integrated in the genome of the plants which expressed male sterility to a higher degree. Northern blotting assay also demonstrated that the transgenes stably transcribed in the genome of the transgenic plants in F₄ generation. The male sterile plants usually display lower plant height, shortened internodes, shrunken anthers without pollen grains or with some abortive pollen grains, and unusual leaves with deeper multi-lobes. Microscope observations on the meiosis processes of pollen mother cells (PMCs) showed that the abortion of pollen grains mainly resulted from abnormalities of meiosis such as direct degeneration of PMCs, degenerations of dyad and tetrads, amitosis, lagging chromosomes, and the multi-polar segregations of chromosomes and so on. This study indicates a method of developing novel cotton male sterile materials for potential application in agriculture and for engineering of male sterility in other important crops.     

Cytogenetics of pearl millet

Summary The somatic karyotype of pearl millet Pennisetum americanum (L.) Leeke. (2n = 14) has been studied in several cultivars, but few cytological markers have been discovered which could help in the easy identification of the chromosomes. Analysis of pachytene bivalents permits such identification but is feasible only in a few cultivars. Recently, several lines having telocentric chromosomes have been produced and classified but their potentialities as cytogenetic tools have yet to be explored. Some African populations of pearl millet carry B-chromosomes in their karyotype. Cytogenetics of B-chromosomes has been reported in great detail. Bs undergo spontaneous changes to produce deficient- and iso-chromosomes. The main effect of B-chromosomes is on chiasma frequency which is exerted by the relative amounts of chiasma promoting euchromatin and the chiasma depressing heterochromatin in the Bs. Haploid plants occur occasionally and sometimes show a low degree of seed set, offering a possibility of establishing homozygous inbred lines. Cytogenetics of several spontaneous and induced autotetraploids have been reported. In general quadrivalent formation between the seven sets of four homologues was random. Seed set of the autotetraploids could be improved by selection; improved seed fertility was found to be associated with increased chiasma frequency, increased quadrivalent frequency and regular distribution of chromosomes at anaphase I. Genes controlling morphological characters of plant phenotype segregate independent of those controlling fertility and in pearl millet polyploidy per se is not limiting to plant vigour. Primary trisomics represent the best studied among the aneuploids of pearl millet. All the seven primary trisomics have been identified and described. Some were used in assigning genes to specific chromosomes but in general trisomies have poor vigour and fertility, and show low frequency of transmission. Apart from B-chromosomes, cytogenetics of interchanges has been the best studied aspect of pearl millet. The frequency of co-orientation of an interchange complex at metaphase I, which determines the fertility or sterility of the interchange heterozygote, is influenced by the genetic background and thus is theoretically amenable for selection leading to improved fertility of the heterozygote. Interchange tester-stocks have been assembled which can be used to identify the chromosomes involved in any newly obtained interchange. A complex interchange line involving all the chromosomes of the complement has also been produced, but the ring-of-fourteen produces total male and female sterility.Genotypic control of mitosis and meiosis has been reported, with reference to chromosome numerical mosaicism, multiploid sporocytes, desynapsis and chromosome fragmentation, and male sterility. Pearl millet being a largely outbreeding species, forced inbreeding was mainly found to result in loss of morphological vigour and reduction in mean chiasma frequency per PMC. Interspecific hybrids between pearl millet and several related species have been cytologically investigated and homology of the seven chromosomes of pearl millet with seven of the fourteen chromosomes of P. purpureum has been demonstrated. Cytogenetic evidence from haploids, autopolyploids and interspecific hybrids has indications to suggest that the haploid number of x = 7 is derived from x = 5, but the evidence is inconclusive and needs critical evaluation.     

Cyto-morphological studies op some species and hybrids in the Eu-Sorghums

Summary Important morphological features such as plant height, leaf size and number of leaves, shape of the panicle and sessile spikelets, staminate condition of the pedicellate florets, nature of lemma, colour of the stigmatic surface and seeds etc., were studied in 8 Sorghum species and 10 F₁ hybrids between them. Based on the data, interrelationship amongst the species are discussed.Pachytene pairing was complete and apparently normal, followed by regular meiosis at later stages resulting in high pollen stainability and good seed setting in all the parental species except the male sterile Kafir. Studies on the pairing properties of the differentially stained regions showed that synapsis starts from the proximal to the distal end and separation of the split chromosomes starts from the distal to the proximal.The interspecific hybrids studied are classified into four types based on pachytene pairing and pollen sterility. 1. normal pairing accompanied by high pollen fertility, 2. normal pairing accompanied by partial pollen sterility. 3. irregularities in the pachytene pairing followed by partial pollen sterility and 4. irregularities in the pachytene pairing accompanied by normal pollen fertility. Suitable explanations are advanced to explain the meiotic aberrations noted in some of the hybrids under study.Cytogenetical mechanisms underlying species differentiation in the Eu-Sorghum species are discussed.     

Meiotic behaviour,stability, transmission and recombination in four balanced tertiary trisomics of rye (Secale cereale L.)

The F₃ and F₄ progeny of four balanced tertiary trisomics (BTTs) of rye carrying the short translocation chromosomes of their corresponding interchanges were karyotyped and scored for the monogenic recessively inherited tigrina marker (ti) which is located terminally on the short arm of chromosome 5R. The segment carrying the dominant allele (Ti) was included in the extra translocation chromosome of each BTT, while the normal 5R pair carried the recessive allele. Recombination fractions between ti and the interchange breakpoint varied between 0.005±0.003 and 0.032±0.005 for the different BTTs. Interstitial chiasma formation could be demonstrated in only one BTT, in 1% of the first metaphase configurations without, however, resulting in recombination. Chiasma localization just proximal to the terminal C-band of 5RS was concluded to cause the low recombination observed. Less than 0.5% of the progeny consisted of plants with a deviant karyotype able to reach maturity, indicating a high level of meiotic stability of the trisomics. The results further suggest that selection towards low male and high female transmission rates of the extra chromosome is possible. The suitability of the BTTs investigated for use in hybrid breeding programs is evaluated.     

Introduction of a gene from fertility restored radish (Raphanus sativus) into Brassica napus by fusion of X-irradiated protoplasts from a radish restorer line and iodacetoamide-treated protoplasts from a cytoplasmic male-sterile cybrid of B. napus

To establish a cytoplasmic male-sterile/restored fertility (cms-Rf) system for F₁ seed production in Brassica napus, we transferred a gene from fertillity restored radish to B. napus by protoplast fusion. X-irradiated protoplasts, isolated from shoots of Raphanus sativus cv Kosena (Rf line), were fused with iodoacetamide-treated protoplasts of a B. napus cms cybrid. Among 300 regenerated plants, six were male-fertile. The fertile plants were characterized for petal color, chromosome number and the percentage of viable pollen grains. Three fertile plants had aneuploid chromosome numbers and white or cream petals, which is a dominant marker in radish. Of these three plants, one which had 2n = 47 chromosomes and white petals was used for further backcrosses. After two backcrosses, chromosome number and petal color became identical to that of B. napus. No female sterility was observed in the BC₃ generations.     

Effects of X-rays and Colchicine on Pollen of Trichosanthes anguina L. (Cucurbitaceae)

Pollen grains of M, and C, generation plants of Trichosanthes anguina L. were studied after treatment of seeds with X-rays and colchicine respectively. Pollen sterility increased with increase in X-ray dose and colchicine concentration. The polar axis of X-irradiated populations was shorter than that of controls. But colchicine treatment resulted in larger pollen grains than the controls, although the exine did not increase as much. Ex-perimentally-produced tetraploids had larger pollen grains than diploids and triploids. The triploids were characterised by changes in pore morphology. Of 293 treated plants examined, 11 aberrant plants were isolated in the M, and C, generations. These plants had large and small fertile pollen grains, and also showed treatment effects in external morphology and chromosomal aberration during meiosis. The changes in polar diameter were not associated with variation in chromosomal material.     

Morphologic,cytogenetic, and enzymatic variation in tissue culture regenerated plants of apomictic old-world bluestem grasses (Bothriochloa sp.)

Somaclonal variant plants may be of use in broadening the germplasm base of plant species and providing useful stocks for cytogenetic investigations. This study was conducted to compare morphologic, cytogenetic and enzymatic characteristics of 21 R₁ (initial regenerate) bluestem,Bothriochloa sp., plants, visibly identified in a field-grown population of 522 plants as probable variants, with their respective R₀ (explant donor) progenitor. An R₂ seedling population was grown to ascertain the transmission of the variant R₁ phenotypes. All R₁ plants differed from their respective R₀ progenitors in one or more morphological characters. Foliage colour was the most pronounced difference in most cases. Four of the plants, three of which were dwarfed, produced no inflorescences. The R₁ plants tended to be shorter than R₀ progenitors and had corresponding decreases in lengths on inflorescences and lowest racemes. All R₁ plants of accessions 8911C and 8793 had an increase in chromosome number from2n=4x=40 to2n=5x=50. Three dwarfed R₁ plants, derived from accession 8873B, were aneuploids, two having2n=48 chromosomes and the third being a probable mixoploid with 55–58 chromosomes. Other plants of accession 8873B had the R₀ chromosome number. Fertility, as estimated by pollen stainability and seed set, generally was reduced in R₁ plants relative to the R₀. This reduction was not drastic, however, with all flowering plants having 45% or higher seed set. Apomixis apparently maintained fertility in all R₁ plants, including those with a pentaploid chromosome number. All R₁ plants differed from their respective R₀ plants in peroxidase and esterase banding patterns. All R₁ plants of accessions 8911C, and 8793, respectively, had identical peroxidase and esterase bands. For both enzyme systems two banding patterns were present in R₁ plants of accession 8873B, with 12 of 13 plants exhibiting common patterns. Examination of R₂ progeny plants confirmed the genetic transmission of the variant phenotypes and, by virtue of uniformity, indicated apomictic reproduction in the R₁ plants. The results demonstrate the production of potentially useful genetic and cytogenetic variant plants via tissue culture in these apomictic species.     

The effects of X-rays on the chromosomes of locust embryos

The structure and frequency of chromatid interchange types in C-metaphase cells of embryos of Schistocerca gregaria are described at the first mitosis following irradiation at the G ₂ stage of interphase. The relationship between the different interchange types and the organisation of the interphase nucleus is discussed. It is concluded that most chromatid interchanges are induced between polarised chromosomes though the occurrence of loops and to a lesser extent of terminal overlapping may subsequently modify the appearance of these aberrations at metaphase.     

Cytogenetical studies onBougainvillea. I. a case of interchange heterozygosity

A case of interchange heterozygosity has been found inB. peruviana cv “Princess Margaret Rose” in which there is a regular formation of 15 bivalents and an interchange multiple of 4 chromosomes. The multiple is always associated with the nucleolus at diakinesis, indicating that one of the chromosome involved is nucleolar. The nucleolar pair of chromosomes shows a slight heteromorphicity which may be due to an unequal interchange. Although 80% of interchange multiples orientate non-disjunctionally, yet 65% pollen is stainable. The pollen is ineffective in self pollination, but highly effective in crosses with 2x and 3x cultivars ofB. spectabilis. The higher pollen stainability indicates that the deficiencies and duplications caused by non-disjunction do not have serious physiological offects on pollen grains and that its genome can withstand rearrangements.     

Engineered selective plant male sterility through pollen‐specific expression of the EcoRI restriction endonuclease

Unintended gene flow from transgenic plants via pollen, seed and vegetative propagation is a regulatory concern because of potential admixture in food and crop systems, as well as hybridization and introgression to wild and weedy relatives. Bioconfinement of transgenic pollen would help address some of these concerns and enable transgenic plant production for several crops where gene flow is an issue. Here, we demonstrate the expression of the restriction endonuclease EcoRI under the control of the tomato pollen‐specific LAT52 promoter is an effective method for generating selective male sterility in Nicotiana tabacum (tobacco). Of nine transgenic events recovered, four events had very high bioconfinement with tightly controlled EcoRI expression in pollen and negligible‐to‐no expression other plant tissues. Transgenic plants had normal morphology wherein vegetative growth and reproductivity were similar to nontransgenic controls. In glasshouse experiments, transgenic lines were hand‐crossed to both male‐sterile and emasculated nontransgenic tobacco varieties. Progeny analysis of 16 000–40 000 seeds per transgenic line demonstrated five lines approached (>99.7%) or attained 100% bioconfinement for one or more generations. Bioconfinement was again demonstrated at or near 100% under field conditions where four transgenic lines were grown in close proximity to male‐sterile tobacco, and 900–2100 seeds per male‐sterile line were analysed for transgenes. Based upon these results, we conclude EcoRI‐driven selective male sterility holds practical potential as a safe and reliable transgene bioconfinement strategy. Given the mechanism of male sterility, this method could be applicable to any plant species.     

A CYTOGENETIC ANALYSIS OF CERTAIN POLYPLOIDS IN GRINDELIA (COMPOSITAE)

Two diploid taxa, Grindelia procera and G. camporum, and 3 tetraploid ones, G. camporum, G. hirsutula, and G. stricta, have been studied to ascertain their interrelationships. Meiosis in diploid parental strains was regular, the common chromosome configuration being 5 rod bivalents and 1 ring bivalent. The average chiasmata frequency per chromosome was 0.60. Pollen fertility was about 90% in all strains examined. Diploid interspecific hybrids had normal meiosis with an average chiasmata frequency of 0.56 per chromosome. No heterozygosity for inversions or interchanges was detected, and pollen fertility was above 85%. Meiosis in parental tetraploid strains was characterized by the presence of quadrivalents in addition to a complementary number of bivalents. The average chiasmata frequency per chromosome was 0.59 and pollen fertility was generally about 80%. Tetraploid interspecific hybrids also had quadrivalents, normal meiosis, and high pollen fertility. Close genetic relationships between the diploids and between the tetraploids are indicated, and geographical, ecological, and seasonal barriers to gene exchange exist. Attempts to obtain hybrids between diploids and tetraploids were successful in a few cases. The hybrids were tetraploid and had normal meiosis and fertility similar to parental and F₁ tetraploids. Their origin was by the union of unreduced gametes of the diploid female parent and normal pollen from the tetraploid parent. On the basis of chromosome homology, normal meiosis, plus high fertility exhibited in the diploid, tetraploid, and diploid X tetraploid interspecific hybrids, these species of Grindelia are considered to be a part of an autopolyploid complex. Gene exchange between diploids and diploids, tetraploids and tetraploids, and diploids and tetraploids is possible. Tetraploid G. camporum may have originated by hybridization between G. procera and diploid G. camporum with subsequent doubling of chromosomes and selection for the combined characteristics of the diploids.     

Stable inheritance and expression of the CMS traits introduced by asymmetric protoplast fusion

The donor-recipient protoplast fusion method was used to produce cybrid plants and to transfer cytoplasmic male sterility (CMS) from two cytoplasmic male-sterile lines MTC-5A and MTC-9A into a fertile japonica cultivar, Sasanishiki. The CMS was expressed in the cybrid plants and was stably transmitted to their progenies. Only cytoplasmic traits of the male-sterile lines, especially the mitochondrial DNAs, were introduced into the cells of the fertile rice cultivar. More than 80% of the cybrid plants did not set any seeds upon selfing. Sterile cybrid plants set seeds only when they were fertilized with normal pollen by hand and yielded only sterile progenies. This maternally inherited sterility of the cybrid plants showed that they were characterized by CMS. The CMS of cybrid plants could be restored completely by crossing with MTC-10R which had the single dominant gene Rf-1 for restoring fertility. These results indicated that CMS was caused by the mitochondrial genome introduced through protoplast fusion. The introduced CMS was stably transmitted to their progenies during at least eight backcross generations. These results demonstrate that cybrids generated by the donor-recipient protoplast fusion technique can be used in hybrid rice breeding for the creation of new cytoplasmic male-sterile rice lines.