Sterile Anthers Research Articles

A new cytoplasmic male sterility system for hybrid seed production in Indian oilseed mustard Brassica juncea

We report a novel cytoplasmic male sterility (CMS) system in Brassica juncea (oilseed mustard) which could be used for production of hybrid seed in the crop. A male sterile plant identified in a microspore derived doubled haploid population of re-synthesized B. napus line ISN 706 was found to be a CMS as the trait was inherited from the female parent. This CMS, designated '126-1', was subsequently transferred to ten different B. juncea varieties and lines through inter-specific crosses followed by recurrent backcrossing. The F(1)s of inter-specific crosses were invariably partially fertile, but irrespective of the variety/line used, the recipient lines became progressively male sterile over five to seven generations and could be maintained by crossing the male sterile lines with their normal counterparts. The male sterile lines were found to be stable for the trait under both long and short day conditions. CMS lines when crossed with lines other than the respective maintainer line were restored for fertility, implying that any variety could act as a restorer for '126-1' cytoplasm in B. juncea. These unique features in maintenance and restoration of CMS lines coupled with near normal floral morphology of the CMS lines have allowed the use of '126-1' cytoplasm for hybrid seed production. The uniqueness of '126-1' has been further established by Southern hybridization with mitochondrial DNA probes and by a histological study of the development of male sterile anthers.

Homeotic-like modification of stamens to petals is associated with aberrant mitochondrial gene expression in cytoplasmic male sterile OguraBrassica juncea

We have previously reported correction of severe leaf chlorosis in the cytoplasmic male sterile Ogura (also called Ogu) Brassica juncea line carrying Ogura cytoplasm by plastid substitution via protoplast fusion. Two cybrids obtained from the fusion experiment, Og1 and Og2, were green and carried the plastid genome of B. juncea cv. RLM198. While Og1 displayed normal flower morphology comparable to that of its euplasmic B. juncea counterpart except for sterile anthers, Og2 retained homeotic-like floral modification of stamens to petal-like structures and several other floral deformities observed in the chlorotic (Ogu) B. juncea cv. RLM198 (or OgRLM). With respect to the mitochondrial genome, Og1 showed 81% genetic similarity to the fertile cultivar RLM while Og2 showed 93% similarity to OgRLM. In spite of recombination and rearrangements in the mitochondrial genomes in the cybrids, expression patterns of 10 out of 11 mitochondrial genes were similar in all the three CMS lines; the only exception was atp6, whose expression was altered. While Og1 showed normal atp6 transcript similar to that in RLM, in Og2 and OgRLM weak expression of a longer transcript was detected. These results suggest that the homeotic-like changes in floral patterning leading to petaloid stamens in Og2 and OgRLM may be associated with aberrant mitochondrial gene expression.

Cytochemical investigation of genic male-sterility in Chinese cabbage

A genic male sterile Chinese cabbage, Brassica campestris L. ssp. chinensis Makino, was examined using cytological and cytochemical methods to characterize the process of pollen abortion in this plant. Thick sections of both fertile and sterile anthers at different developmental stages were stained using Toluidine Blue O, Periodic Acid-Schiff’s (PAS) reaction and Sudan Black B to detect cytochemical changes that may occur in the distribution of insoluble polysaccharide and lipid storage bodies. Pollen abortion in sterile anthers occurs at an early stage of microspore development. During early microspore development, reductions in the number of starch grains in the connective tissue of fertile anthers coincide with the accumulation of starch grains in cells of the anther wall. In the late microspore stage, a large vacuole forms in the microspore, and tapetal cells synthesize and accumulate lipid droplets. The cellular organization of tapetal cells in sterile anthers appears similar to that in fertile anthers, except for the absence of lipid droplets in cells of sterile anthers and diffusely labeled tapetal polysaccharides, suggesting defects in nutrient storage.

Cell‐specific regulation of a Brassica napus CMS‐associated gene by a nuclear restorer with related effects on a floral homeotic gene promoter

Cytoplasmic male sterility (CMS) is a maternally inherited defect in pollen production specified by novel mitochondrial genes. It can be suppressed by nuclear restorer (Rf) genes which normally downregulate expression of a CMS-associated novel mitochondrial gene. Two forms of Brassica napus CMS, nap and pol, are associated with related chimeric genes, orf222 and orf224, respectively. We show that in pol and nap CMS, anther locule development is asynchronous and asymmetric, that one or more locules within each anther may fail to develop entirely and that CMS anthers display polarity in locule development. We show, by in situ hybridization, that orf222 transcripts accumulate in sterile anthers prior to development of morphological differences between CMS and restored stamens, and remain preferentially localized to microsporangia. In fertility-restored anthers, however, orf222 transcript levels remain low throughout development. Some sporogenous and meiotic cells differentiate within CMS anthers and form functional pollen despite retaining high orf222 transcript levels, suggesting that the effect of orf222 expression in blocking pollen development is limited to an early and specific stage. Transcripts of other mitochondrial genes, exemplified by atp6 and cob, and of the nuclear-encoded ATP synthase gamma subunit, accumulate preferentially in the microsporangia of both sterile and fertile anthers. Thus nuclear fertility restoration reduces orf222 transcript levels in a gene and tissue-specific manner. We observe differences between CMS and fertile plants in the timing and patterning of APETALA3 promoter activity that suggest a possible basis for the developmental abnormalities of CMS flowers.

Temporal and spatial distribution of pectin epitopes in differentiating anthers and microspores of fertile and sterile sugar beet.

We studied the possible involvement of several pectin epitopes in anther differentiation and microsporogenesis in fertile and cytoplasmically male sterile sugar beets. The spatial and temporal distribution of five structural motifs were traced with a panel of monoclonal antibodies in six stages: premeiosis, meiotic prophase, young and mature tetrads, young and expanding microspores. The composition of the walls of sporogenous cells and meiocytes differed than that in the tapetum, as evidenced by the presence of alpha-Fuc(1-->2)-beta-Gal and alpha-(1-->5)-L-Ara epitopes binding CCRC-M1 and LM6 antibodies. At meiotic prophase, the meiocyte walls were additionally marked by the appearance of poorly methyl-esterified domains of homogalacturonan and of (1-->4)-beta-Gal residues, detected by JIM5 and LM5. Some constituents of the meiocyte wall which reacted with JIM5 and JIM7 persisted on the surface of the special callose sheath during tetrad development. In newly formed primexine and exine layers of tetrads and microspores, epitopes that were bound by JIM5, JIM7 and LM5 were abundant. No differences in the deposition or relative abundance of pectins were found between fertile and sterile anthers until microspore release from the callose. Later, at the time of abortion, sterile microspores had much larger amounts of epitopes detected by JIM5 than their fertile counterparts.

Cytological screening of rice TGMS lines

AbstractAn investigation was undertaken to develop new stable thermo‐sensitive genic male sterility (TGMS) rice lines in intermated progenies (IMPs) of TGMS lines by using an anther clearing technique. The results indicate that both pre‐ and post‐meiotic genetic systems operate during anther development for the expression of sterility in TGMS lines. In all the TGMS lines, sterile anthers were small with empty pollen grains of irregular shape, except for TS 16, which showed pollen‐free anthers. This indicates that the sensitive stage of TS 16 is around stage IV (stamen and pistil primordia) of panicle development. Distinct differences were observed between sterile and fertile phases with respect to anther size, shape and colour of the pollen grains in TS 18 and TS 29. The pollen grains at the sterile phase were small and irregular in shape while in the fertile phase they were plump and larger with a yellow colour, establishing that the occurrence of sterility in TS 18 and TS 29 is post‐meiotic. Three distinct classes of pollen fertility percentage viz. <20%, 50‐70% and >90% were observed in IMPs. Anther clearing in IMPs showed distinct developmental patterns of pollen production with respect to distinct classes of pollen fertility. Less than 20% pollen fertility was observed in hybrids such as TS 15 × TS 16, TS 15 × Co 47, TS 18 × TS 16 and TS 18 × Co 47 which hold promise for developing new TGMS lines with a good plant type and acceptable quality.

Antisense inhibition of mitochondrial pyruvate dehydrogenase E1alpha subunit in anther tapetum causes male sterility.

We hypothesized that cytoplasmic male sterility (CMS) in sugar beet may be the consequence of mitochondrial dysfunctions affecting normal anther development. To test the hypothesis, we attempted to mimic the sugar beet CMS phenotype by inhibiting the expression of mitochondrial pyruvate dehydrogenase (PDH), which is essential for the operation of the tricarboxylic acid (TCA) cycle. Screening with a cDNA library of sugar beet flower buds allowed the identification of two PDH E1alpha subunit genes (bvPDH_E1alpha-1 and bvPDH_E1alpha-2). bvPDH_E1alpha-1 was found to be highly expressed in tap roots, whereas bvPDH_E1alpha-2 was expressed most abundantly in flower buds. Green fluorescent protein (GFP) fusion of bvPDH_E1alpha revealed mitochondrial targeting properties. A 300-bp bvPDH_E1alpha-1 cDNA sequence (from +620 to +926) was connected to a tapetum-specific promoter in the antisense orientation and then introduced into tobacco. Antisense expression of bvPDH_E1alpha-1 resulted in conspicuously decreased endogenous bvPDH_E1alpha-1 transcripts and male sterility. The tapetum in the male-sterile anthers showed swelling or abnormal vacuolation. It is also worth noting that in the sterile anthers, cell organelles, such as elaioplasts, tapetosomes and orbicules were poorly formed and microspores exhibited aberrant exine development. These features are shared by sugar beet CMS. The results thus clearly indicate that inhibition of PDH activity in anther tapetum is sufficient to cause male sterility, a phenocopy of the sugar beet CMS.

The implications of aberrant anatomical and histochemical features in the anthers of male sterile dsf-15 sunflower - Helianthus petiolaris Nutt.

Present investigation aims at determining the developmental stage at which the male sterile anthers of DSF-15 Helianthus petiolaris Nutt. deviate from their isogenic fertile counterparts and the underlying histochemical factors responsible for that deviation. The flower buds of fertile and male sterile H.petiolaris were fixed in the Carnoy’s fluid, dehydrated in ethyl alcohol series, embedded in paraffin and microtomed. The sections were stained for the localization of insoluble polysaccharides, RNA and total proteins using periodic acid Schiff’s toluidine blue and mercuric bromophenol blue reagents respectively. The results of the present investigation reveal that, during premeiotic stages, anthers of fertile and sterile lines are indistinguishable except in the occurrence of starch storage in the sterile anthers during the onset of meiosis. In the sterile anthers, precocious degeneration of callose failure of microscope wall information, persistent abnormal tapetum (hypertrophied or premature plasmodial formation), failure of formation of endothecial thickenings accompany the abortion process of microspores. The malfunctioning of the tapetum seems to be responsible for male sterility in DSF-15 H.petiolaris.

Dioecy in South American Deprea (Solanaceae)1

Abstract Dioecy in the Solanaceae is rare, occurring in <1 percent of the species worldwide and known in only two species from South America. We report the occurrence of cryptic dioecy in the Neotropical genus Deprea. Studying herbarium material of the Venezuelan endemic D. paneroi revealed morphological distinctions that tentatively served to divide collections into male and female morphs. This discovery prompted investigation of population structure, crossing studies, and morphometric analyses of 95 individuals from the largest known wild population and a small sample of greenhouse-grown plants. About 60 percent of the population was reproductive, and of these, nearly equal numbers exhibited female or male characteristics. Plants that bore fruit and small sterile anthers were characterized as female; those that lacked fruit but produced pollen in large anthers were characterized as male. The morphology, receptivity, and developmental phenology of pistils were identical in both forms. Crossing studies re...

Dioecy in South American Deprea (Solanaceae)1

Abstract Dioecy in the Solanaceae is rare, occurring in <1 percent of the species worldwide and known in only two species from South America. We report the occurrence of cryptic dioecy in the Neotropical genus Deprea. Studying herbarium material of the Venezuelan endemic D. paneroi revealed morphological distinctions that tentatively served to divide collections into male and female morphs. This discovery prompted investigation of population structure, crossing studies, and morphometric analyses of 95 individuals from the largest known wild population and a small sample of greenhouse-grown plants. About 60 percent of the population was reproductive, and of these, nearly equal numbers exhibited female or male characteristics. Plants that bore fruit and small sterile anthers were characterized as female; those that lacked fruit but produced pollen in large anthers were characterized as male. The morphology, receptivity, and developmental phenology of pistils were identical in both forms. Crossing studies re...

Morphological Analysis of Three Equivocal Sibling Species of Deprea (Solanaceae)

Taxonomic confusion among closely related and morphologically similar Deprea species has persisted in the literature and in the identification of species. Morphological variation among three closely related, monophyletic Deprea species was studied to determine if and how they can be distinguished. Their sympatric occurrence in Venezuela afforded an opportunity to couple field study with analysis of herbarium specimens representing their entire geographic range. An analysis of 94 morphological characters resulted in five vegetative and 13 reproductive taxonomically informative traits. Canonical variates analysis clearly separated the three species using six quantitative traits. We conclude that these taxa, although quite variable and similar morphologically, are taxonomically distinct. Results of character analysis indicated that D. orinocensis is morphologically more similar to D. bitteriana than either are to D. paneroi. In D. paneroi, small, sterile anthers on fruit-bearing plants and the absence of fruits on plants possessing large, plllen-bearing anthers, suggest cryptic dioecy. Based on these data, D. granulosa is considered to be a synonym of D. orinocensis: Athenaea bitteriana, a misapplied synonym, is the correct basionym and is applicable to many specimens identified as D. granulosa. We submit a new combination, D. bitteriana (Werderm.) Sawyer & Benitez, and designate a lectotype to accommodate these findings.

A New Species of Sorghastrum (Poaceae) from Isla Socorro, Colima, Mexico

A new species ofSorghastrum,S. pohlianum, from Mexico is described and illustrated. A numerical analysis comparing the new species to the closest species (S. nutans andS. nudipes) was undertaken. The first three principal components explain 84% of the variation of the taxa involved. In addition there is morphological evidence to distinguish the new species from its closest relatives. It differs fromS. nutans by showing smaller inflorescences, having sterile lemmas and anthers, and having somewhat smaller but wider blades and different flowering periods. In addition, it is distinguished fromS. nudipes due to the presence of auricles > 1 mm long and inflorescence branches bearing spikelets along their entire length.

MRNA differential display between sterile and fertile anther of rice and analysis of cDNA differential fragments

Rice anther and leaf mRNAs from two cytoplasmic male sterile lines (Maxie A and Congguong 41A) were compared with those from their maintainers and F(1) hybrids by using mRNA differential display to study gene expression pattern in sterile anther during pollen abortion. Anther cDNA differential bands between sterile and fertile plants were more than those of leaf. It was indicated that the expression of fertility gene(s) in the anther was more activable and sufficient than in the leaf. The gene transcript pattern in one of different types of anther was not only associated with its pollen sterility degree but also with its stage of pollen abortion. The anthers with full or partial sterility or with early abortion produced more cDNA differential bands than those fertile or late abortion anthers. In twelve recovered differential cDNA fragments, two were probably associated with male sterility, i. e. one is (AB(4)A(5)), which was specifically expressed in the sterile anther, and the other (AB(3)B(2)), which contained some sequence homologous to a mitochondrial gene (coxII) and whose expression was partially suppressed in the sterile anther.

Calcium distribution in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice

Potassium antimonate was used to locate Ca2+ in fertile and sterile anthers of a photoperiod-sensitive genic male-sterile rice (Oryza sativa L. japonica). During the development of fertile anthers, abundant calcium precipitates accumulated in the anther walls and on the surface of pollen grains and Ubish bodies at the late developmental stage of the microspore, but not in the cytoplasm of pollen grains. Following the accumulation of starch grains in pollen, calcium precipitates on pollen walls diminished and increased in parenchymatous cells of the connective tissue. In sterile anthers, calcium precipitates were abundant in the middle layer and endothecium, but not in the tapetum, as was found in fertile anthers. A special cell wall was observed between the tapetum and middle layer of sterile anthers that appeared to relate to distinctive calcium accumulation patterns and poor pollen wall formation in the loculi. The formation of different patterns of antimonate-induced calcium precipitates in the anthers of photoperiod-sensitive genic male-sterile rice indicates that anomalies in the distribution of calcium accumulation correlate with the failure of pollen development and pollen abortion.

The Mexican Chaptalia hintonii Is a Gerbera (Asteraceae, Mutisieae)

The Mexican Chaptalia hintonii Is a Gerbera (Asteraceae, Mutisieae)

Genetics and cytology of a new genic male-sterile soybean [ Glycine max (L.) Merr.

Genetic and cytological studies were conducted with a new male-sterile, female-fertile soybean [Glycine max (L.) Merr.] mutant. This mutant was completely male sterile and was inherited as a single-recessive gene. No differences in female or male gamete transmission of the recessive allele were observed between reciprocal cross-pollinations in the F1 or F2 generations. This mutant was not allelic to any previously identified soybean genic male-sterile mutants: ms1, ms2, ms3, ms4, ms5, or ms6. No linkage was detected between sterility and flower color (W1 locus), or between sterility and pubescence color (T1 locus). Light microscopic and cytological observations of microsporogenesis in fertile and sterile anthers were conducted. The structure of microspore mother cells (MMC) in male-sterile plants was identical to the MMCs in male-fertile plants. Enzyme extraction analyses showed that there was no callase activity in male-sterile anthers, and this suggests that sterility was caused by retention of the callose walls, which normally are degraded around tetrads at the late tetrad stage. The tapetum from male-sterile anthers also showed abnormalities at the tetrad stage and later stages, which were expressed by an unusual formation of vacuoles, and by accumulation of densely staining material. At maturity, anthers from sterile plants were devoid of pollen grains.

CHANGES IN THE MITOCHONDRIAL DOUBLE MEMBRANE IN TAPETAL AND SPOROGENIC CELLS IN CYTOPLASMIC MALE STERILE ANTHERS OF PETUNIA

Comparative histological and ultrastructural studies were made on the gametogenesis and microsporogenesis in isonuclear fertile and cytoplasmic male sterile petunia lines. Using electron microscopy, changes in the mitochondrial double membrane were observed in the tapetal and sporogenic cells of cytoplasmic male sterile anthers as the first cytological sign of breakdown of the process of microsporogenesis at early prophase I. These changes were manifested by a larger space between the outer and the inner membrane and fewer sites of close proximity (“contact sites”) in the sterile mitochondria, as compared to fertile mitochondria. The mitochondrial membrane of the parietal tissue in the cytoplasmic male sterile anthers was not affected in the same way. Even when total breakdown was already obvious in the tapetal and sporogenic cells, the mitochondria of the parietal layer remained intact.

Effects of Petunia Cytoplasmic Male Sterile (CMS) Cytoplasm on the Development of Sterile and Fertility-Restored P. parodii Anthers

The developmental defects causing cytoplasmic male sterility in Petunia parodii are described in isonuclear fertile, sterile, and fertility-restored plants using both light- and scanning electron microscopy. The aberrant development of the sporogenous tissue and tapetal layer caused by the cytoplasmic male sterile cytoplasm in both Petunia hybrida and P. parodii nuclear backgrounds is similar in onset and progression. The degeneration of the sporogenous tissue and tapetal layer of sterile anthers is first apparent late in meiosis and results in highly abnormal sterile sporogenous tissue by tetrad stage of fertile anthers. The stomium and endothecium do not show major developmental differences between fertile and sterile anthers, but the inner connective tissue of sterile anthers contained calcium crystals not found at high abundance in fertile anthers. Ovoid bodies containing magnesium and phosphorus were seen only in the vascular bundles of fertile anthers. Material prepared for the scanning electron microscope by freeze drying showed better retention of fragile morphological features, while critical-point drying permitted examination of nonvolatile structures, such as cell walls.

The TM5 MADS Box Gene Mediates Organ Differentiation in the Three Inner Whorls of Tomato Flowers.

The tomato MADS box gene no. 5 (TM5) is shown here to be expressed in meristematic domains fated to form the three inner whorls-petals, stamens, and gynoecia-of the tomato flower. TM5 is also expressed during organogenesis and in the respective mature organs of these three whorls. This is unlike the major organ identity genes of the MADS box family from Antirrhinum and Arabidopsis, which function in overlapping primordial territories consisting of only two floral whorls each. The developmental relevance of the unique expression pattern of this putative homeotic gene was examined in transgenic plants. In agreement with the expression patterns, antisense RNA of the TM5 gene conferred both early and late alterations of morphogenetic markers. Early defects consist of additional whorls or of a wrong number of organs per whorl. Late, organ-specific changes include evergreen, cauline, and unabscised petals; green, dialytic, and sterile anthers; and sterile carpels and defective styles on which glandular trichomes characteristic of sepals and petals are ectopically formed. However, a complete homeotic transformation of either organ was not observed. The early and late floral phenotypes of TM5 antisense plants suggest that TM5 mediates two unrelated secondary regulatory systems. One system is the early function of the floral meristem identity genes, and the other system is the function of the major floral organ identity genes.

Persistence of Callose and Tapetum in the Microsporogenesis of Genic Male Sterile Cajanus cajan (L.) Millsp. with Well Formed Endothecium.

The microsporogenesis and the male gametophyte development in the male fertile and male sterile anthers of Cajanus cajan (L.) Millsp. (ms Prabhat) were studied using the cytochemical staining technique for polysaccharides, ascorbic acid, RNA and proteins. The pre-meiotic development was identical in the anthers of both the lines. During post-meiotic stages, sterile anthers showed persistent callose and tapetum. Breakdown of microspores occurred at late tetrad stage. Sterile anthers had ascorbic acid storage in the connective, even at mature stage. Male sterility had no effect on the formation of endothecium. It is postulated that the malfunctioning of the tapetum is a cause for the induction of male sterility.