Sterile Buds Research Articles

Comprehensive Analysis of Genic Male Sterility-Related Genes in Brassica rapa Using a Newly Developed Br300K Oligomeric Chip

To identify genes associated with genic male sterility (GMS) that could be useful for hybrid breeding in Chinese cabbage ( Brassica rapa ssp. pekinensis ), floral bud transcriptome analysis was carried out using a B . rapa microarray with 300,000 probes (Br300K). Among 47,548 clones deposited on a Br300K microarray with seven probes of 60 nt length within the 3' 150 bp region, a total of 10,622 genes were differentially expressed between fertile and sterile floral buds; 4,774 and 5,848 genes were up-regulated over 2-fold in fertile and sterile buds, respectively. However, the expression of 1,413 and 199 genes showed fertile and sterile bud-specific features, respectively. Genes expressed specifically in fertile buds, possibly GMS-related genes, included homologs of several Arabidopsis male sterility-related genes, genes associated with the cell wall and synthesis of its surface proteins, pollen wall and coat components, signaling components, and nutrient supplies. However, most early genes for pollen development, genes for primexine and callose formation, and genes for pollen maturation and anther dehiscence showed no difference in expression between fertile and sterile buds. Some of the known genes associated with Arabidopsis pollen development showed similar expression patterns to those seen in this study, while others did not. BrbHLH89 and BrMYP99 are putative GMS genes. Additionally, 17 novel genes identified only in B . rapa were specifically and highly expressed only in fertile buds, implying the possible involvement in male fertility. All data suggest that Chinese cabbage GMS might be controlled by genes acting in post-meiotic tapetal development that are different from those known to be associated with Arabidopsis male sterility.

Sequence characterization and expression pattern of BcMF21, a novel gene related to pollen development in Brassica campestris ssp. chinensis

In this report a full length cDNA, Brassica campestris Male Fertile 21 (BcMF21) was successfully isolated from one of the cDNA-amplified fragment length polymorphism (cDNA-AFLP) transcript-derived fragments (TDFs), BBP10, that was found down-regulated in the flower buds of sterile plants in Brassica campestris L. ssp. chinensis Makino genic male sterile (GMS) AB line system (Bcajh 97-01A/B). BcMF21 protein structure analysis showed a signal peptide at the N-terminus; two protein kinase C phosphorylation sites, five N-myristoylation sites and one casein kinase II phosphorylation site. The promoter region of BcMF21, a 779 bp upstream of ATG was isolated by thermal asymmetric interlaced-PCR (TAIL-PCR). Bioinformatics analysis revealed that the promoter of BcMF21 contained several classical cis-acting elements and three pollen specific elements. Transient expression analysis showed that the promoter could drive green fluorescence protein (GFP) expression. Quantitative reverse transcript-PCR analysis revealed that BcMF21 was specifically expressed in flower buds. The transcript level of BcMF21 was much lower in the sterile flower buds than in the fertile flower buds in 'Bcajh 97-01A/B' system. In situ hybridization further showed that BcMF21 was only expressed in the tetrads and the microspores at the tetrad stage and the uninucleate stage. In addition, phylogenetic analysis demonstrated that the BcMF21 was relative conserved within family Crucifereae and might be originated from the ancestor diploid B. campestris within genus Brassica according to the Triangle of U theory.

The Acquisition and Expression Analysis of Polygalacturonase Gene MF6 Related to Fertility in Brassica napus L.

The purpose of this study is to analyze the differentially expressed genes in the buds of male sterile plants which are induced with a male sterilizing chemical Hua-Sha-Ling WP1 in Brassica napus L., R121. Firstly, we isolated the differentially expressed gene fragments from the male sterile buds through the suppression subtractive hybridization (SSH) and the reverse northern dot blotting. Then, homology comparisons and functional analysis were conducted through online database NCBI. Finally, the differentially expressed gene was further analyzed through reverse transcription quantitative PCR. In the results, we obtained a gene fragment which was 100% homologous to the polygalacturonase gene ( MF6 ) in Arabidopsis from the 6 positive clones. It is reported that the polygalacturonase gene ( MF6 ) is closely related to the development and maturation of pollen. The amount of MF6 gene expression in flower buds with different lengths and different fertilities was analyzed quantitatively. The results showed that the expression of MF6 gene in the male fertile flower buds was higher than that in the male sterile flower buds. And the differences are about 18.53 times and 43.15 times when the bud lengths are 1.00~1.50 mm and 3.5~4.0 mm, respectively. Therefore, Hua-Sha-Ling WP1 may inhibit the expression of the MF6 gene during the meiosis stages of pollen mother cell and cause the abortion of pollens. At the same time, the MF6 gene expression may also be associated with the elongation of filaments.

Construction and analysis of a suppression subtractive hybridization (SSH) library of genic multiple-allele inherited male-sterility in Chinese cabbage (Brassica campestris L. ssp. pekinensis)

Utilization of male sterility is a key method for producing crossbred Chinese cabbage (Brassica rapa L. ssp. pekinensis (Lour.) Olsson. In this study, suppression subtractive hybridization (SSH) was used to construct sterility and fertility cDNA libraries, which included differentially, expressed clones between fertile and sterile buds of the A/B line ‘AB01’. The positive clones were randomly selected by polymerase chain reaction amplification (PCR) and 25 high quality sequences (22 from the fertile-tester library and three from the sterile-tester libraries) were generated. The fragment lengths varied from 77 to 469 bp. Differential expression patterns between fertile and sterile buds were selected and verified using five expressed sequence tags (ESTs). Results indicated that, three ESTs were expressed only in fertile buds and two ESTs were down-regulated in sterile buds. According to the Basic Local Alignment Search Tool (BLAST) screening and functional annotation, the 25 ESTs were homologous to known sequences deposited in National Center for Biotechnology Information (NCBI). These genes had homology to known proteins such as flowers/buds development proteins, metabolic-related proteins, cell structure proteins, cell growth/division proteins and secondary metabolic-related proteins. The results suggested that, these proteins played a critical role in nuclear male sterility progression of genic multiple-allele inherited male-sterility in Chinese cabbage.

Isolation of fertility-related genes of multiple-allele-inherited male sterility in Brassica rapa ssp pekinensis by cDNA-AFLP

To better understand the molecular mechanisms of multiple-allele-inherited male sterility in Chinese cabbage (Brassica rapa ssp pekinensis), differentially expressed genes in fertile and sterile plants must be isolated. We used cDNA-AFLP analysis to isolate differentially expressed genes in fertile and sterile buds of the two-type line, AB01. Sixteen high-quality sequences were generated, 11 of which were up-regulated in fertile buds, and five of which were up-regulated in sterile buds. Based on BLAST screening and functional annotation, these genes have homology with genes encoding known flower- or bud-specific proteins, metabolism-related proteins and cell-structure proteins. In addition, the full-length cDNA sequences of the actin gene were cloned from the cabbage plants by RACE and used as an internal standard for semi-quantitative reverse transcription-PCR. Expression of three flower- or bud-specific differentially expressed transcript-derived fragments in fertile and sterile buds was examined using RT-PCR; the expression patterns of these genes were similar to the patterns observed in the cDNA-AFLP analysis.

Meiotic abnormality in dominant genic male sterile Brassica napus

Rs1046AB is a dominant genic male sterile (DGMS) Brassica napus line derived from Yi-3A. Until now the molecular mechanism of its male sterility is still unknown. In this paper, cytological observations demonstrated that all cells in sterile plants contained condensed nuclei at the beginning stage of meiosis; this implied that meiotic cells were degenerating. Although 31% (93/300) cells escaped from the state of nuclei condensation in buds about 3 mm in length (in such length, normal plants are at tetrade stage), no cells could pass the pachytene stage. Then pachytene or zygotene like chromatin/chromosomes sometimes congregated into two or more groups with different size, which resulted in the formation of micronuclei. Nucleoplasmic bridge could also be found in some meiotic cells. Even when the "microspore's analogue" appeared in sterile buds about 4 mm in length (in such length, mature pollens could be detected in normal buds), the nuclei condensation and escaped cells with pachytene like chromosome still could be found in the sterile anthers. So it could be concluded that male sterility was caused by meiotic abnormality. According to our previous research, four genes related to cell cycle/DNA processing were identified in fertile plants. RT-PCR further confirmed that three DNA repair genes were partially or completely repressed in the sterile plants, and were only expressed in the early stage fertile flower buds, i.e. the buds <3 mm in length. Therefore, DGMS of rapeseed was probably caused by the abnormality in DNA damage repair system during meiosis. According to these results, some possible mechanisms of fertility control were discussed.

A simple in vivo model of collagen degradation using collagen-gelled cotton buds: the effects of collagenase inhibitors and other agents.

A simple in vivo model of collagen degradation has been developed, and the effects of various agents have been tested. Type I collagen was prepared from rat skin and acetylated with either [3H]- or [14C] acetic anhydride. The radiolabelled collagen was added to sterile cotton buds and incubated at 37 degrees C to allow the collagen to form native fibrils that were firmly adsorbed to the cotton matrix. After subcutaneous implantation of the collagen-gelled cotton buds into rats, the radiolabelled collagen was progressively removed over a period of weeks by an infiltrating granuloma. Of the agents that were administered directly into the cotton buds using subcutaneously implanted osmotic mini-pumps, only the synthetic collagenase inhibitors CI-A (containing a hydroxamate moiety as a zinc ligand) and CI-C (containing a thiol moiety as a zinc ligand) were able to prevent the removal of collagen: their efficacy correlated with the level of collagenase inhibitory activity assayed in the exudate fluid sequestered within the cotton bud granuloma. Of the agents that were administered systemically, including anti-inflammatory drugs and other compounds used as therapies for arthritis, only hydrocortisone was able to inhibit the removal of radiolabelled collagen. These results suggest that, in this model, interstitial collagenase, a member of the matrix metalloproteinase family, comprised the major degradative pathway for collagen. The collagen-gelled cotton bud model is a useful test system for delineating those processes that result in collagen catabolism. In addition, the model can be used for testing agents, including those of limited or unknown systemic bioavailability, in order to discover novel therapeutic agents for preventing collagen degradation in connective tissue diseases such as arthritis.

Development of long-shoot terminal buds of western white pine (Pinus monticola)

Long-shoot terminal bud (LSTB) development in western white pine (Pinus monticola Dougl.) was studied throughout the annual growth cycle to determine the phenology of LSTB development and the time of cone-bud differentiation. Development of LSTB began in early April and cataphylls were initiated from mid-August until early November. Cataphyll initiation was slow during May and June when shoots were elongating and then rapid just after shoot elongation was completed. Proximal cataphylls were sterile, whereas more distal cataphylls began to initiate axillary buds by late June or early July. Axillary buds were initiated first in the proximal portions of the LSTB and then acropetally in rapid succession. The last cataphylls to be initiated in the fall remained as sterile bud scales enclosing the LSTB apex. Axillary buds initiated sterile cataphylls which functioned as bud scales. The number varied with the type of axillary bud. Proximal axillary buds initiated few cataphylls and began to differentiate into dwarf shoots or pollen cones in August. The more distal axillary buds differentiated into dwarf shoots during September and October, The most distal axillary buds initiated many cataphylls during September and October but did not differentiate into seed-cone buds or lateral branch buds until after winter dormancy. Consequently, attempts to induce or enhance seed-cone production in P. monticola would probably be most successful in the spring when seed-cone buds differentiate. LSTB bearing seed cones were larger, had broader apices, and produced more cataphylls during the growing season than did LSTB bearing pollen cones. The phenology of LSTB development in soft pines and hard pines is discussed in relation to reports available on the association of cone crops and climatic conditions in several species of Pinus.

Interrelationship of Abscisic Acid and Gibberellic Acid in the Promotion of Callus Formation in the Abscission Zone of Citrus Bud Cultures

AbstractThe mechanism of ABA‐induced callus formation was studied in sterile bud cultures of Citrus [Citrus sinensis (L.) Osbeck] on defined media. ABA was found to promote callus formation in the abscission zone between the petiole and the branch while inhibiting bud growth. The promoting effect of ABA was dependent on the physiological state of the shoot from which buds were excised, and on the size of the explant. Callus formation was highest in autumn and summer (i.e. younger) buds, and lowest in older buds excised from previous summer flush.GA was only slightly active in promoting callus formation when applied separately, but showed a highly synergistic effect when applied with ABA: maximal callus formation was attained at a combination of 10−5M ABA and 10−6 MGA in the medium. Subcultures of ABA‐induced callus revealed that ABA inhibited the growth of isolated subcultured callus, while IAA and kinetin, and especially GA, promoted its rapid proliferation. A general decrease in protein synthesis was found in the abscission zone during the first 5 days of induction, while total protein content changed only slightly.The results suggest that ABA‐induced callus formation in Citrus bud explants is a multiphasic phenomenon involving, at least, two stages: (1) activation of certain cells in the abscission zone by ABA, resulting in the formation of callus layers, and (2) subsequent proliferation of the callus tissue, which is dependent on the hormonal balance in the explant. This growth‐promoting effect of ABA seems to be a general phenomenon in explants exposing a cut‐surface.