A genetic study of the regeneration capacity of embryonic callus from the maize immature embryo culture.

We carried out a study of regeneration capacities of embryonic callus from maize immature embryo culture with 144 different inbred lines of natural groups from different countries, and found that the regeneration capacity was affected by three factors: environment, genotype and the interaction between the environment and genotype. We found that green embryonic callus rate (GCR), embryonic callus differentiating rate (CDR) and the plantlet number of embryonic callus regeneration (CPN) have significant positive correlations with each other, and they all have significant negative correlations with embryonic callus browning rate (CBR). Moreover, embryonic callus cloning index for the first subculture (CCI1) and embryonic callus cloning index for the second subculture (CCI2) have a significant positive correlation with each other, and CCI2 is positively correlated with green GCR, and is negatively correlated with CBR. Embryonic callus rooting rate (CRR) is positively correlated with GCR, CDR and CPN to some degree. Furthermore, we calculated Broad-Sense Heritability of each trait, and uncovered that the heritability index of CCI1, CCI2 and CRR was lower, and the heritability index of others was higher. In addition, by using the Ward method for two-way cluster analysis, we found eleven inbred lines with high regenerating abilities, and the rooting situation of regenerating plantlet was excellent by rooting culture, which could be used as the elite inbred lines of the maize transgenic receptor.

[Analysis on breeding potential of eight synthetic populations to improve a Chinese maize hybrid Zhengdan 958].

Maize (Zea mays L.) populations are potential sources of favorable alleles absent in parental inbred lines to improve elite hybrids. The maize hybrid Zhengdan 958 has been hampered by the lack of favorable new alleles for improving yield and commodity quality. In the present study, 16 testcrosses made by using eight synthetic populations as the donors and the two parental lines of Zhengdan 958 as the receptors were evaluated in 2009 and 2010 at Shunyi, Beijing and Xinxiang, Henan Province for grain yield and test weight. Four genetic parameters were used to determine the breeding potential of eight synthetic populations as the donors to improve the target hybrid. Several synthetic populations were identified as the potential sources of favorable alleles absent in the target hybrid for each trait evaluated. The two most promising germplasms, WBMC-4 and Shanxi Syn3, had the potential for simultaneously improving grain yield and test weight of the target hybrid, which could be used to improve the parental lines Zheng 58 and Chang 7-2, respectively, and further broaden the germplasm base of Chinese heterotic groups PA and Sipingtou.

Transcriptional responses of maize seedling root to phosphorus starvation.

Maize (Zea mays) is the most widely cultivated crop around the world, however, it is commonly affected by phosphate (Pi) deficiency and the underlying molecular basis of responses mechanism is still unknown. In this study, the transcriptional response of maize roots to Pi starvation at 3 days after the onset of Pi deprivation was assessed. The investigation revealed a total of 283 Pi-responsive genes, of which 199 and 84 genes were found to be either up- or down-regulated respectively, by 2-fold or more. Pi-responsive genes were found to be involved in sugar and nitrogen metabolic pathways, ion transport, signal transduction, transcriptional regulation, and other processes related to growth and development. In addition, the expression patterns of maize inorganic phosphorus transporters, acid phosphatase, phytase, 2-deoxymugineic acid synthase1, POD and MYB transcription factor were validated in 178 roots response to low phosphorus stress. of which, two genes encoding phytase and acid phosphatase were significantly induced by Pi deficiency and may play a pivotal role in the process of absorption and re-utilization of Pi in Maize. These results not only enhance our knowledge about molecular processes associated with Pi deficiency, but also facilitate the identification of key molecular determinants for improving Pi use in maize. Moreover, this work sets a framework to produce Pi-specific maize microarrays to study the changes in global gene expression between Pi-efficient and Pi-inefficient maize genotypes.

[The role of miR319 in plant development regulation].

MicroRNAs (miRNAs) are an extensive class of endogenous, non-coding, short (21~25 nt) RNA molecules, which regulate expression of target genes through miRNA-guided cleavage or translational repression of mRNAs. Plant miRNAs are involved in all aspects of regulation of plant growth and development. The miR319 was shown to regulate TCPs transcription factor controlling the fate of plant organ growth such as leaves and flowers and was involved in regulating part of hormone biosynthesis and signal transduction pathways. Thus, they play a key biochemical function in plant organs development. This review focused on the key roles of miR319 in regulation of the morphogenesis, development, and senescence of plant organs such as leaves and flowers.

[Genetic analysis of maize cytoplasmic male sterile mutants obtained by space flight].

Three maize male sterile mutants were obtained from the offsprings of two maize inbred lines 18-599 and 08-641, which were carried into space by the Shijian 8 Satellite. The stability of male sterile expression was observed in different locations, years, and seasons. In order to analyze the genetic characteristic of male sterility, testcross, backcross and reciprocal cross were made with these male sterile plants. The results showed that the male sterility character was stable in different locations, years, and seasons, and the sterility was inheritable. Because the maintainer lines and restorer lines for these sterile materials were found, and there was no male sterile plant separated among the reciprocal cross F2. Thus, we concluded that these mutants could be cytoplasmic male sterile. Combining the results of male fertility restoration test and PCR analysis, we could conclude that the three male sterile mutants were classified into the CMS-C type in maize. Owing to their difference in fertility restoration, these mutants may belong to different subgroups of CMS-C type. The discovery of the three male sterile mutants increased the genetic diversity of CMS-C type, improved the tolerance to Bipolaris maydis, and laid a foundation for extensive application of CMS-C in seeds production.

[Heavy metal-transport proteins in plants: a review].

The heavy metals in soil not only damage plant growth, but also threaten the health of human beings and animals through food chain. Heavy metal-transport proteins play crucial roles in the heavy metals uptake and tolerance of plants. Plant heavy metal-transport proteins can be classified as metal-uptake proteins and metal-efflux proteins. The metal-uptake proteins can transport essential heavy metals into cytoplasm, and also, transport toxic heavy metals into cytoplasm due to the absence of essential heavy metals or the competition among ions. The metal-efflux proteins are a group of detoxification proteins, which can efflux excess and toxic heavy metals from cytoplasm, or move these metals into vacuole. In recent years, the associations between elevated steady-state transcript levels of heavy metal-transporter genes and metal accumulation in plants have been revealed, and many heavy metal-transport proteins have been cloned and identified. In this paper, the metal affinity, tissue-specific gene expression, and cellular location of representative heavy metal-transport proteins were reviewed.

[Identification of known microRNAs in root and leaf of maize by deep sequencing].

microRNA (miRNAs) is a newly identified class of 20-24 nt non-protein-coding and endogenous small RNA, which plays an important role in plant growth, development and response to environmental stresses. Combined with bioinformatic method, the types, abundance, and targets of known miRNAs in root and leaf of maize (Zea mays L.) were analyzed by small RNA deep sequencing technology, which was based on Illumina/Solexa principium. The results indicated that 92 known miRNAs were detected in maize root, which were attributed to 18 miRNA families and their abundance ranged from 1 to 105,943 reads. Synchronously, 86 known miRNAs were detected in maize leaf, which were attributed to 17 miRNA families and their abundance ranged from 1 to 85,973 reads. The target gene prediction showed that 54 putative target genes as these known miRNAs were predicted. Some of them were involved in the following processes, such as transcription regulation, substance and energy metabolism, electron transport, stress response, and signal transduction through further function prediction. In conclusion, there were obvious differences in both types and abundance of known miRNAs between root and leaf in maize.

RNA editing of mitochondrial functional genes atp6 and cox2 in maize (Zea mays L.).

RNA editing of two mitochondrial or organs genes, atp6 and cox2, in different tissues were analyzed using homonucleic but alloplasmic, and homoplasmic but heteronucleic maize (zea mays L.) as experimental materials. A total of 18 and 26 editing sites for atp6 conservative region transcript were identified by direct and clone sequencing, respectively. By direct sequencing 23 and 22 editing sites for cox2 transcript were identified in 48-2 and Huangzaosi nuclear backgrounds, respectively. From the direct sequencing results, the occurrence rates of different transcripts generally increase in sterile lines. It is concluded that RNA editing of atp6 and cox2 might have a certain relationship with maize CMS.

[Advances in study of plant miRNAs under stressed environmental conditions].

Biotic and abiotic stresses influence plant growth and cause great loss to crop yield. In the long course of evolution, plants have developed intricate biological mechanism to resist stressed conditions. Under various stressed conditions, not only the protein-coding genes, but also the non-protein-coding genes were induced for response. More and more researches showed that the transcripts of these non-protein-coding genes played important role in regulation of gene expression. miRNA is one of the groups in these no-coding regulatory small RNAs. Recent findings showed that in order to resist the biotic and abiotic stresses, expression of microRNA (miRNA) genes will be induced and their transcripts (miRNAs) can regulate gene expression by guiding target mRNA cleavage or translation inhibition. This paper focused on the advances of plant miRNAs research in stressed conditions, especially induced expression of miRNA and target gene regulation and its role on adaptation under stressed conditions. Then, the methods of miRNA researches in stressed environments are discussed.

[Advances of microarray analysis on plant gene expression under environmental stresses].

Different stressed conditions impair plant growth and further, cause great loss of crop yield and even lead to lose production completely. Increasing resistance/tolerance of crops under stressed conditions is a major goal of numerous plant breeders, and many elegant works are focusing on this area to uncover these complicated mechanisms underlying it. However, the traditional strategies including physiological and biochemical methods, as well as studies on a few genes, can not well understand the overall biological mechanism. Microarray analysis opens a door to uncover these cryptic mechanisms, and has the ability of detecting gene transcription and regulation at genomic level in different plant tissues. And works in association with related methods of proteomics and metabolomics. Therefore, it is possible to locate genes in certain key metabolism pathways. Through these procedures, it is also possible to look for critical genes in the pathway and to well understand the molecular mechanism of resistance/tolerance. These results can be as a guidance for increasing the resistance/tolerance of stressed conditions using biotechnology methods in future. This paper mainly focused on and discussed the advances of microarray analysis of stressed conditions-related genes in plants.

[The editing sites in the transcripts of mitochondrial atp6 gene of maize sterile lines and maintainer line].

Using maize (Zea mays L.) cytoplasmic male-sterile lines T Huang Zao Si, C Huang Zao Si, S Huang Zao Si and maintainer line N Huang Zao Si as the plant materials, editing sites in the conservative area of mitochondrial atp6 gene transcripts of the 4 experimental materials' tassels, of which microspores had developed to uni-nucleate stage, were analyzed. The results showed that DNA sequences of the T, C and S male-sterile cytoplasms were completely unanimous, while being compared with the N-cytoplasm, all the sequences were similar except for the 27th and 28th nucleotides. However, the cDNA sequences of each cytoplasm were not always the same. By comparing DNA and cDNA sequences, we found that within the conservative area of atp6 gene transcripts there were 19, 22, 20 and 19 editing sites in the N, T, C and S cytoplasms, respectively. The 4 cytoplasms also shared 18 sites. The majority of the editings occurred at the 1st or the 2nd position of codons, which might alter the amino acid type. Most the shared editings were fully editing, and the 1st and the 19th sites were partially edited in nearly all cytoplasms, except for the 19th sites editing in the N-cytoplasm. The specific editings in each cytoplasm occurred in the form of partially editing. Thus the editing of atp6 gene in maize was not only sequence specific but also affected by cytoplasmic background. Furthermore, plant RNA editing was inclined to improve the predicted protein's hydrophobicity and enhance the conservation among species.

[Cytological observation on pollen abortion of genetic male sterile mutant induced by space flight in maize].

In order to understand the cytological mechanism of pollen abortion of genetic male sterile mutant induced by space flight in maize, the sister cross population were used for sterility analysis and cytological observation. Intact anther observation, isolated cells observation and paraffin section were adopted in this research. The results showed that pollen abortion occured mostly in dyad stage of meiosis in genetic male sterile mutant. The dyad were degenerated with abnormal shape. In late anther developing stage, the tapetal cells were giant vacuolated and delayed degeneration. The pollen mother cells (PMC) began to dissolve and degenerate in a few anther before meiosis.

[Initial identification of quantitative trait loci controlling resistance to banded leaf and sheath blight at elongating and heading date in maize].

The genetic linkage map was constructed with 146 SSR markers based on a maize population consisting of 229 F2 individuals from the cross R15 (resistant) x 478 (susceptible), covering 1666cM on a total of ten chromosomes, with an average interval length of 11.4 cM. The disease index from the population of 229 F24 lines were evaluated for BLSB resistance under artificial inoculation at elongating stage and heading date. With the method of composite interval mapping (CIM) described in QTL cartographer v2.0 procedure, 9 QTLs among of 17 QTLs were identified on 1, 2, 3, 4, 5, 6 and 10 chromosomes at Jointing Stage, accounting for 3.72% to 9.26% of the phenotypic variance. The other 10 QTLs were identified on 2, 3, 4, 5, 6, 8 and 9 chromosomes at elongating stage and heading date, accounting for 4.27% to 9.27% of the phenotypic variance. Among of them, two QTLs were detected at two stages, which were located between bnlgl600-umc1818 and umc1006-umc1723 on chromosome 2 and 6, respectively. The result indicated that the significant difference about QTL Controlling Resistance between the two stages had a close connection with Developing Stages, which was showed on the resistance of banded leaf and sheath blight in maize. So this result provided new information to the resistance breeding of maize.

QTL mapping of Fusarium moniliforme ear rot resistance in maize. 1. Map construction with microsatellite and AFLP markers.

To map the QTLs of Fusarium moniliforme ear rot resistance in Zea mays L., a total of 230 F2 individuals, derived from a single cross between inbred maize lines R15 (resistant) and Ye478 (susceptible), were genotyped for genetic map construction using simple sequence repeat (SSR) markers and amplified fragment length polymorphism (AFLP) markers. We used 778 pairs of SSR primers and 63 combinations of AFLP primers to detect the polymorphisms between parents, R15 and Ye478. From the polymorphic 30 AFLP primer combinations and 159 SSR primers, we scored 260 loci in the F2 population, among which 8 SSR and 13 AFLP loci could not be assigned to any of the linkage groups. An integrated molecular genetic linkage map was constructed by the remaining 151 SSR and 88 AFLP markers, which distributed throughout the 10 linkage groups of maize and spanned the genome of about 3463.5 cM with an average of 14.5 cM between two markers. On 4 chromosomes, we detected 5 putative segregation distortion regions (SDRs), including 2 new ones (SDR2 and SDR7). The other 3 SDRs were located near the regions where gametophyte genes were mapped, indicating that segregation distortion could be partially caused by gametophytic factors.

[Subcellular localization and tissues expression profile of hGPCRc: an orphan G protein-coupled receptor].

As a member of orphan G protein-coupled receptors (oGPCRs), hGPCRc was cloned from human colon tissue and analyzed by bioinformatic softwares. It was showed that the corresponding amino acids of hGPCRc formed seven-transmembrane domains as the key characteristic of GPCRs. Then, the recombinant GFP-hGPCRc was constructed by fussing hGPCRc into pEGFP-N1 carrying green fluorescent protein (GFP) gene, and CHO-K1 cells were subsequently transfected with the GFP-hGPCRc or pEGFP-N1. The green fluorescence protein expression in the two different transfected cells was observed under the laser scanning confocal microscopy (LSCM). It was showed that green fluorescence protein was distributed in the whole bodies of the cells transfected with pEGFP-N1, but mainly distributed on the plasma membrane and cytoplasm membrane transfected with GFP-hGPCRc. Thus, the localization on the membrane of hGPCRc was accorded with the predication by bioinformatic analysis. The expression analysis of hGPCRc by RT-PCR indicated that hGPCRc was abundantly expressed in heart, kidney, cerebel and colon etc., but absent in liver, cerebra, small intestine and muscle etc. The expressing profile of hGPCRc could provide some useful clues to understanding its effects on embryonic development and physiological functions.

[Locating maize male sterility gene induced by space flight using microsatellite markers].

Two F2 populations with different kinds of spike derived from maize male sterility materials RP(3)195 (A) x S37 (inbred line) ,which had been sib-bred for many generations,were used for sterility analysis and gene location. There were 138 and 247 plants in the two F2 populations respectively. Among the 326 pairs of microsatellite primers selected,56 were found polymorphic. Linkage analysis of F2 populations with the 56 pairs of primers showed that microsatellite markers bnlg197 and umc1012 were linked with the male sterility gene. The genetic distance between marker bnlgl97 and the male sterility gene in the two different F2 populations were 7 cM and 14.5 cM respectively. The genetic distance between marker umc1012 and the male sterility gene in the 138 plants was 28.5 cM. Thus the male sterility gene was located on chromosome 3L.

[The production and multi-color genomic in situ hybridization identification of maize-Z. perennis substituted material].

Genus Zea. L was composed of two sections: sect. Luxuriantes Doebley & Iltis including Z. dipperennis, Z. luxurians and Z. perennis, and sect. Zea. mays consisting only one species, annual Z. mays. To improve the biodiversity of germ-plasm in maize breeding, the study of transferring maize relatives gene into common maize were performed. Firstly, interspecific hybrids between maize (Zea. L) (2n = 20) and Z. perennis (2n = 40) were produced with the aim of transferring desirable horticultural traits from Z. perennis to maize. The F1 of maize x Z. perennis (2n = 30) plant had the most frequent configuration of 5 III + 5 II + 5 I, which were sterile and difficult to produce progeny because of genomic affinities. However,few F2 individuals of maize x Z. perennis could be obtained by some specially treatments, and one maize-like F2 plant were obtained, which were used as a female parent in backcrossing with maize parent. Twelve F2 x P1 ( BC1 F2 ) plants were obtained and then self-crossed to produce self-crossing generation of F2 x P1 (2n = 20) ( BC1 F3). The phenotypic characters of parents, F1 (2n = 30) hybrids, F2 and F2 x P1 were investigated, such as plant height, flowering, leaf shape and tillers. To further verify the genomic organization of maize-Z. perennis material, maize (inbred line 48-2) and BC1 F3 chromosomes, the root tip cells were analyzed by Multi-color GISH. We probed maize and BC1 F3 chromosomes with the probe mixture containing biotin-labeled Z. perennis genomic DNA and digoxigenin-labeled maize genomic DNA, the maize spread exhibited 10 chromosomes with yellow signals and the other 10 chromosomes carried green fluorescing bands. However, although the BC1 F3 was 2n = 20 in all cases. Multi-color GISH images revealed that 17 chromosomes had uniform signals similar to maize chromosomes, but dispersed red signals over the remaining three chromosomes were observed, which indicated that the 3 chromosomes originated from Z. perennis, and they were smaller than maize chromosomes. Data obtained from multi-color GISH images indicated that BC1 F3 was probably a substituted material from maize-Z. perennis.

[Genetic analysis about maize male sterile mutant obtained by space flight].

The seeds of maize single hybrid Chuandan No. 9 were carried into space in 1996 by satellite. After the seeds were planted in field in comparison with travel in space seeds which was not carried into space. Fortunately, male sterile plants were discovered in one of the ear rows. The stability of male sterile expression was observed in different years, different locations and different generations. In order to analysis the genetic characteristic of male sterility, test cross, sister cross, back cross, reciprocal cross and self-pollination were conducted with these male sterile plants. The results showed that the male sterility was stable in different years and different locations, it is inheritable from generation to generation. The sterility is controlled by a single nuclear recessive gene. The appearance of male sterile mutant is the conclusion of gene mutation which happened in nuclear by space flight. This mutant material always accompanies with lower plant height.

[A study of genetic stability of male sterile mutant of maize obtained from space flighted seeds].

OBJECTIVE: To select a male sterile mutant of maize through space flight for production application. METHOD: Air dried seeds of maize (Chuan Dan No. 9) were carried to space for 15 d. After returned to the earth, a male sterile mutant was selected and a sterile line was obtained through direction breeding. RESULT: It was found that the sterile material was thoroughly abortive. The sterile was trail stable and expressed a genetic feature of single recessive gene controlled nucleus sterility. CONCLUSION: The appearance of male sterile mutant was due to gene mutation caused by space conditions.