植物激素对体细胞胚胎发生的诱导与调节

以作者自己的工作为背景,结合国内外近几年的有关报道,综述了几种外源和内源激素对植物体细胞胚胎发生的诱导与调节作用。外源生长素和细胞分裂素是诱导离体培养细胞分化与增殖所必需的,2,4-D是诱导胚性愈伤组织的重要激素。在体细胞胚胎发生中内源激素含量和代谢的平衡起着关键的作用,而且外源和内源激素对诱导体细胞胚胎发生起相互调节作用。ABA在提高体细胞胚胎发生频率和质量上具有重要作用,同时,外源与内源ABA对体细胞胚胎发生起相互促进作用。本文还较为深入地讨论了这些激素诱导体细胞胚胎发生的可能作用机制。 Abstract:The paper summarizes the induced and regulatory effects of a few exogenous and endogenous hormones in plant somatic embryogenesis by our studies and related international reports.The exogenous auxin and cytokinin are necessary to induced differentiation and proliferation of cells of culture in vitro.2,4-D is an important hormone of induced embryogenic calluses.The contents and the metabolic balances of endogenous hormones have key effects for somatic embryogenesis.In addition,the exogenous and endogenous hormones have mutual regulatory effects for somatic embryogenesis.ABA has an important effect to improving the frequency and quality of somatic embryogenesis.Meanwhile,the exogenous and endogenous ABA have mutual promoted effects for somatic embryogenesis.The paper discusses possible mechanism of hormones-induced somatic embryogenesis in a deep-going way.     

Screening of <i>Bacillus subtilis</i> HAAS01 and Its Biocontrol Effect on <i>Fusarium</i> wilt in Sweet Potato

Fusarium wilt, a disease caused by Fusarium oxysporum f.sp batatas (Fob) is an important disease in sweet potato production. Using endophytic bacteria for biological control of sweet potato diseases is one of the important ways. A Bacillus subtilis with antagonistic effect on Fusarium wilt of sweet potato was isolated from soil by confrontation culture. According to the biological characteristics, 16S rDNA sequence analysis, and physiological and biochemical analysis, the Bacillus subtilis HAAS01 was named. A pot experiment was conducted for the biological control experiment of strain HAAS01, and the endogenous hormone content, antioxidant enzyme activity, soluble protein content, and related gene expressions of sweet potato plants were detected. The results showed that the HAAS01 strain could promote the production of endogenous hormones and resist the infection of plant diseases together with defensive enzymes and upregulation of related gene expressions. In summary, Bacillus subtilis HAAS01 was effective in controlling Fusarium wilt of sweet potato and has potential for application and development.     

The Regulatory Roles of microRNAs and Associated Target Genes during Early Somatic Embryogenesis in <i>Liriodendron Sino-Americanum</i>

Somatic cells respond to considerable stress, and go through a series of phytohormone pathways, then forming an embryo. The developmental process is recorded as somatic embryogenesis (SE). One of the key components regulating SE are the microRNAs (miRNAs). Despite previous studies, it is still not clear exactly how miRNAs exert their function of regulating targets during conditionally activated early SE. Here, we use Liriodendron sino-americanum as a model system and perform a combined analysis of microfluidic chips and degradome sequencing to study this process. We identified a total of 386 conserved miRNAs and 153 novel miRNAs during early SE. According to the ANOVA test, 239 miRNAs showed 12 distinct expression patterns. Through degradome sequencing, 419 targets and 198 targets were identified for 136 known miRNAs and 37 novel miRNAs, respectively. Gene Ontology (GO) and metabolism pathway enrichment analysis revealed that these targets were significantly involved in oxidation-reduction processes, calmodulin-mediated signal transduction pathways and carbohydrate metabolism. The genes that were related to stress responses, phytohormone pathways and plant metabolism were identified within the targets of miR319, miR395, miR408, miR472, miR482, miR390, miR2055, miR156, miR157, miR171, miR396, miR397, miR529, miR535 and miR159. According to promoter analysis, various cis-acting elements related to plant growth and development, phytohormones response and stress response were present in the promoter of the miRNAs. The differential expression patterns of 11 miRNA-target modules were confirmed by real-time quantitative PCR. The study demonstrated that the miRNA plays an important role in the early SE process by regulating its target and then participating in carbohydrate metabolism and stress response. It also provided a valuable resource for further research in determining the genetic mechanism of SE, and then facilitating breeding programs on plants.     

Effect of <Emphasis Type="Italic">NtDCN1</Emphasis> gene activated during embyogenesis on organogenesis in tobacco tissue culture

The role of NtDCN1 gene in organogenesis in the culture of tobacco (Nicotiana tabacum L.) somatic tissues was studied. This gene is specifically expressed in tobacco microspores induced for somatic embryogenesis. This gene knockout resulted in a disturbance of formation and development of embyoids from microspores. In leaf disks and calli derived from tobacco lines with active and inactivated NtDCN1 gene, we studied induction of shoots and roots. A comparative analysis of tobacco line morphogenetic responses in vitro showed that NtDCN1 gene inactivation enhanced shoot formation and suppressed rhizogenesis, whereas this gene reactivation returned organogenesis processes to control level. Difference between lines was manifested only at a definite ratio between exogenous hormones supplied. The involvement of NtDCN1 gene in line responses to exogenous auxin is discussed. The results obtained permit a supposition that the NtDCN1 gene is critical for regulation not only somatic embryogenesis but also organogenesis.     

AM Fungi and <i>Piriformospora indica</i> Improve Plant Growth of <i>Pinus elliottii</i> Seedlings

Pinus elliottii is an exotic afforestation pine extensively distributed in southern parts of China. In order to understand whether endophytic fungi can affect seedling growth of P. elliottii, Piriformospora indica (Pi), Funnelifcrmis mosseae (Fm), and Diversispora tortuosa (Dt) were inoculated respectively, and the non-inoculated group was set as control. The growth indexes, the contents of soluble sugar and soluble protein, and plant endogenous hormone levels in the leaves of P. elliottii, were analyzed. The results showed that Fm, Dt and Pi colonized the P. elliottii roots to form mycorrhizal structure and chlamydospores arranged in beads respectively. Three fungal inoculants exhibited the stimulated growth responses, whilst Dt illustrated the most positive effect on plant height, single fresh weight, trunk diameter and root system structure, compared with the control. On the other hand, the soluble sugar and soluble protein contents were increased distinctively in mycorrhizal plants. The endogenous IAA, GA3, ZR contents were increased, while the ABA contents were reduced in mycorrhizal plants versus non-mycorrhizal plants. The fungi-induced endogenous hormone changes triggered plant growth improvement of P. elliottii seedlings. This research unraveled the positive effect of AM fungi and P. indica on growth of pine seedlings, while, more application of endophytic fungi to fields needs to be explored.     

Genome-Wide Identification,Evolution and Expression Analyses of <i>GA2ox</i> Gene Family in <i>Brassica napus</i> L.

Gibberellin 2-oxidases (GA2ox) are important enzymes that maintain the balance of bioactive GAs in plants. GA2ox genes have been identified and characterized in many plants, but these genes were not investigated in Brassica napus. Here, we identified 31 GA2ox genes in B. napus and 15 of these BnaGA2ox genes were distributed in the A and C subgenomes. Subcellular localization predictions suggested that all BnaGA2ox proteins were localized in the cytoplasm, and gene structure analysis showed that the BnaGA2ox genes contained 2–4 exons. Phylogenetic analysis indicated that BnGA2ox family proteins in monocotyledons and dicotyledons can be divided into four groups, including two C₁₉-GA2ox and two C₂₀-GA2ox clades. Group 4 is a C₂₀-GA2ox Class discovered recently. Most BnaGA2ox genes had a syntenic relationship with AtGA2ox genes. BnaGA2ox genes in the C subgenome had experienced stronger selection pressure than genes in the A subgenome. BnaGA2ox genes were highly expressed in specific tissues such as those involved in growth and development, and most of them were mainly involved in abiotic responses, regulation of phytohormones and growth and development. Our study provided a valuable evolutionary analysis of GA2ox genes in monocotyledons and dicotyledons, as well as an insight into the biological functions of GA2ox family genes in B. napus.     

DDG1 and G Protein α Subunit RGA1 Interaction Regulates Plant Height and Senescence in Rice (<i>Oryza sativa</i>)

Many studies have already shown that dwarfism and moderate delayed leaf senescence positively impact rice yield, but the underlying molecular mechanism of dwarfism and leaf senescence remains largely unknown. Here, using map-based cloning, we identified an allele of DEP2, DDG1, which controls plant height and leaf senescence in rice. The ddg1 mutant displayed dwarfism, short panicles, and delayed leaf senescence. Compared with the wild-type, ddg1 was insensitive to exogenous gibberellins (GA) and brassinolide (BR). DDG1 is expressed in various organs, especially in stems and panicles. Yeast two-hybrid assay, bimolecular fluorescent complementation and luciferase complementation image assay showed that DDG1 interacts with the α-subunit of the heterotrimeric G protein. Disruption of RGA1 resulted in dwarfism, short panicles, and darker-green leaves. Furthermore, we found that ddg1 and the RGA1 mutant was more sensitive to salt treatment, suggesting that DDG1 and RGA1 are involved in regulating salt stress response in rice. Our results show that DDG1/DEP2 regulates plant height and leaf senescence through interacting with RGA1.     

Conservation Strategy for African Medicinal Species: <i>In Vitro</i> Biotechnological Approach

The use of medicinal plants for different therapeutic values is well documented in African continent. African diverse biodiversity hotspots provide a wide range of endemic species, which ensures a potential medicinal value. The feasible conservation approach and sustainable harvesting for the medicinal species remains a huge challenge. However, conservation approach through different biotechnological tools such as micropropagation, somatic embryogenesis, synthetic seed production, hairy root culture, molecular markers based study and cryopreservation of endemic African medicinal species is much crucial. In this review, an attempt has been made to provide different in vitro biotechnological approaches for the conservation of African medicinal species. The present review will be helpful in further technology development and deciding the priorities at decision-making levels for in vitro conservation and sustainable use of African medicinal species.     

Effect of polyamines on in vitro somatic embryogenesis in <Emphasis Type="Italic">Momordica</Emphasis><Emphasis Type="Italic">charantia</Emphasis> L.

The effects of exogenous polyamines (PAs) on enhancement of somatic embryogenic calli was investigated in Momordica charantia L. in vitro. Induction of somatic embryogenesis (SE) in leaf explants of M. charantia after 21 days of culture in Murashige and Skoog (MS) medium was determined using scanning electron microscopy. During induction of SE there were high titers of Putrescine (Put) as compared to Spermidine (Spd) and Spermine (Spm), a prerequisite for cell division. Addition of PAs to the embryogenic media resulted in an increase in fresh weights and number of somatic embryos of 21-day old embryogenic calli. Put at a concentration of 1 mM showed maximum increase in fresh weights of embryogenic calli (5 fold) and number of somatic embryos produced per 0.2 g of callus (2.5 fold). Moreover addition of PAs to the embryogenic media resulted in lowering of endogenous free PA level of 21-day old embryogenic calli. Thus, when the media was supplemented with exogenous PAs a positive correlation was found to exist between Somatic Embryogenesis enhancement and decrease in endogenous free PA levels.     

Analysis of expression profile of selected genes expressed during auxin-induced somatic embryogenesis in leaf base system of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) and their possible interactions

Somatic embryogenesis is a notable illustration of plant totipotency and involves reprogramming of development in somatic cells toward the embryogenic pathway. Auxins are key components as their exogenous application recuperates the embryogenic potential of the mitotically quiescent somatic cells. In order to unravel the molecular basis of somatic embryogenesis, cDNA library was made from the regeneration proficient wheat leaf base segments treated with auxin. In total, 1440 clones were sequenced and among these 1,196 good quality sequences were assembled into 270 contigs and 425 were singletons. By reverse northern analysis, a total of 57 clones were found to be upregulated during somatic embryogenesis, 64 during 2,4-D treatment, and 170 were common to 2,4-D treatment and somatic embryogenesis. A substantial number of genes involved in hormone response, signal transduction cascades, defense, anti-oxidation, programmed cell death/senescence and cell division were identified and characterized partially. Analysis of data of select genes suggests that the induction phase of somatic embryogenesis is accompanied by the expression of genes that may also be involved in zygotic embryogenesis. The developmental reprogramming process may in fact involve multiple cellular pathways and unfolding of as yet unknown molecular events. Thus, an interaction network draft using bioinformatics and system biology strategy was constructed. The outcome of a systematic and comprehensive analysis of somatic embryogenesis associated interactome in a monocot leaf base system is presented. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of Two Potential Allelochemicals on the Photosystem II of <i>Nitzschia closterium</i> and<i> Monostroma nitidum</i>

In aquaculture, high-density seaweed farming brings higher economic benefits but also increases outbreaks of diatom felt. The effective control of diatom felt in high-density seaweed farming has always been a research hotspot. This study selected two potential allelochemicals 2-hydroxycinnamic acid and quinic acid to explore their effects on a diatom Nitzschia closterium and an economic seaweed Monostroma nitidum. The results showed that 2-hydroxycinnamic acid had better inhibitory effects than quinic acid on the growth, pigment content and photosynthetic efficiency of N. closterium. Their half-maximal inhibitory concentrations at 120 h (IC_{50–120 h}) were 0.9000 and 1.278 mM, respectively. Additionally, these allelochemicals had limited inhibitory effects on the growth, pigment content and photosynthetic efficiency of M. nitidum before 24 h. To further explore the allelopathic effect of these chemicals, this study focused on the photosystem II energy fluxes of N. closterium. It was found that 3 mM 2-hydroxycinnamic acid could destroy the whole photosynthetic system by devastating the PSII reaction centre (RC) before 24 h; however, the same concentration of quinic acid could only down-regulate the electron transport efficiency by changing the effective antenna size of an active RC and downregulating the PSII reaction centre density. These experimental results are expected to provide a new strategy to control diatom felt blooms on the high-density seaweed farming areas.     

Polyamines affect the cellular growth and structure of pro‐embryogenic masses in Araucaria angustifolia embryogenic cultures through the modulation of proton pump activities and endogenous levels of polyamines

Polyamines (PAs) are abundant polycationic compounds involved in many physiological processes in plants, including somatic embryogenesis. This study investigates the role of PAs on cellular growth and structure of pro‐embryogenic masses (PEMs), endogenous PA and proton pump activities in embryogenic suspension cultures of Araucaria angustifolia. The embryogenic suspension cultures were incubated with putrescine (Put), spermidine (Spd), spermine (Spm) and the inhibitor methylglyoxal‐bis(guanylhydrazone) (MGBG), respectively (1 mM). After 24 h and 21 days, the cellular growth and structure of PEMs, endogenous PA contents and proton pump activities were analyzed. The addition of Spm reduced the cellular growth and promoted the development of PEMs in embryogenic cultures, which could be associated with a reduction in the activities of proton pumps, such as H⁺‐ATPase P‐ and V‐types and H⁺‐PPases, and alterations in the endogenous PA contents. Spm significantly affected the physiology of the A. angustifolia somatic embryogenesis suspension, as it potentially affects cellular growth and structure of PEMs through the modulation of proton pump activities. This work demonstrates the involvement of exogenous PAs in the modulation of cellular growth and structure of PEMs, endogenous PA levels and proton pump activities during somatic embryogenesis. To our knowledge, this study is the first to report a relationship between PAs and proton pump activities in these processes. The results obtained in this study offer new perspectives for studies addressing the role of PAs and proton pump on somatic embryogenesis in this species.     

Biological characterization of young and aged embryogenic cultures of <Emphasis Type="Italic">Pinus pinaster</Emphasis> (Ait.)

Pinus pinaster (Ait.) somatic embryogenesis (SE) has been developed during the last decade, and its application in tree improvement programs is underway. Nevertheless, a few more or less important problems still exist, which have an impact on the efficiency of specific SE stages. One phenomenon, which had been observed in embryogenic tissue (embryonal mass, EM) initiated from immature seed, has been the loss of the ability to produce mature somatic embryos after the tissue had been cultured for several months. In an attempt to get insight into the differences between young cultures of EM (3-mo-old since the first subculture) of P. pinaster that produced mature somatic embryos and the same lines of significantly increased age (18-mo-old, aged EM) that stopped producing mature somatic embryos, we analyzed in both types of materials the levels of endogenous hormones, polyamines, the global DNA methylation, and associated methylation patterns. In addition, we included in the analysis secondary EM induced from mature somatic embryos. The analysis showed that the two tested genotypes displayed inconsistent hormonal and polyamine profiles in EM cultures of a similar phenotype and that it might be difficult to attribute one specific profile to a specific culture phenotype among genotypes. Experiments were also undertaken to determine if the global DNA methylation and/or the resulting methylation pattern could be manipulated by treatment of the cultures with a hypomethylating drug 5-azacytidine (5-azaC). An aged EM was exposed to different concentrations and durations of 5-azaC, and its response in culture was established by fresh mass increases and somatic embryo maturation potential. All of the analyses are new in maritime pine, and thus, they provide the first data on the biochemistry of EM in this species related to embryogenic potential.     

Effect of Putrescine on Low-Temperature Acclimation in <i>Chlamydomonas reinhardtii</i>

Putrescine is reported to be necessary for cold acclimation under low-temperature stress. In this study, the effect of low-temperature on some physiological and biochemical parameters has been investigated using the green algae Chlamydomonas reinhardtii. The lipid peroxidation rate, amount of Rubisco protein, activities of antioxidant enzymes and gene expression of polyamine biosynthesis (odc2, and spd1), heat shock proteins (hsp70c, hsp90a, and hsp90c), and PSII repair mechanisms (psba, rep27, and tba1) were determined to understand the low-temperature response. Exogenous putrescine application significantly increased Rubisco protein concentration and catalase enzyme activities under low-temperature stress. Moreover, real-time RT-PCR results and gene expression analysis showed that polyamine metabolism induced gene expression at low-temperatures in the first 24 h. In the same way, the gene expression of heat shock proteins (hsp70c, hsp90a, and hsp90c) decreased under low-temperature treatment for 72 h; however, application of putrescine enhanced the gene expression in the first 24 h. The results obtained indicated that molecular response in the first 24 h could be important for cold acclimation. The psba and tba1 expressions were reduced under low-temperatures depending on the exposure time. In contrast, the exogenous putrescine enhanced the expression level of the psba response to low-temperature at 24 and 72 h. The results obtained in this study indicate that putrescine could play a role in the PS II repair mechanisms under low-temperature stress.