黄花菜不同外植体形成的愈伤组织再生苗观察

研究了黄花菜不同外植体愈伤组织的形成与植株再生。结果表明,出愈率是花柄>花茎>叶片。愈伤组织在MS+6-BA 2mg/L的培养基上出现致密愈伤组织颗粒,经增殖→分割→增殖的程序逐渐形成“球状体”似的愈伤组织。叶片、花茎形成的球状体经20代继代培养,再生能力没有减退,苗形态正常,根尖细胞染色体数目为2n=2x=22。该球状体经秋水仙素处理获得多种形态的四倍体苗和少数重复加倍现象。花柄球状体再生苗中出现了叶片花瓣状或类似花蕾状的形态异常苗,其频率与花柄所处的花期及花蕾大小有密切关系。再生异常苗的球状体继代培养,或者由异常苗叶片重新形成的球状体仍然再生异常苗。花柄球状体经秋水仙素处理,获得的变异株其染色体数目类型较多。因而不宜用花柄作黄花菜快速繁殖的外植体。     

Abnormal development of floral meristem triggers defective morphogenesis of generative system in transgenic tomatoes

Parthenocarpy and fruit malformations are common among independent transgenic tomato lines, expressing genes encoding different pathogenesis-related (PR) protein and antimicrobal peptides. Abnormal phenotype developed independently of the expression and type of target genes, but distinctive features during flower and fruit development were detected in each transgenic line. We analyzed the morphology, anatomy, and cytoembryology of abnormal flowers and fruits from these transgenic tomato lines and compared them with flowers and fruits of wild tomatoes, line YaLF used for transformation, and transgenic plants with normal phenotype. We confirmed that the main cause of abnormal flower and fruit development was the alterations of determinate growth of generative meristem. These alterations triggered different types of anomalous growth, affecting the number of growing ectopic shoots and formation of new flowers. Investigation of the ovule ontogenesis did not show anomalies in embryo sac development, but fertilization did not occur and embryo sac degenerated. Nevertheless, the ovule continued to differentiate due to proliferation of endothelium cells. The latter substituted embryo sac and formed pseudoembryonic tissue. This process imitated embryogenesis and stimulated ovary growth, leading to the development of parthenocarpic fruit. We demonstrated that failed fertilization occurred due to defective male gametophyte formation, which was manifested in blocked division of the nucleus in the microspore and arrest of vegetative and generative cell formation. Maturing pollen grains were overgrown microspores, not competent for fertilization but capable to induce proliferation of endothelium and development of parthenocarpic ovary. Thus, our study provided new data on the structural transformations of reproductive organs during development of parthenocarpic fruits in transgenic tomato.     

Characterization of the Arrest in Anther Development Associated with Gibberellin Deficiency of the gib-1 Mutant of Tomato

The role of gibberellins in flower bud development was investigated by studying the gib-1 mutant of tomato, Lycopersicon esculentum. This gibberellin-deficient mutant initiates flower buds, but floral development is not completed unless the mutant is treated with gibberellin. Treatment with other plant growth regulators does not induce normal flower development. Development of gib-1 flower buds, as measured by progress toward anthesis, ceases at a bud length of 2.5 millimeters; however, increase in size of the bud continues. Buds between 2.5 and 3.7 millimeters are developmentally arrested but still are capable of developing normally after treatment with gibberellic acid. Anthers of these developmentally arrested buds contain pollen mother cells that are in the G1 phase of premeiotic interphase. Following treatment of developmentally arrested buds with gibberellic acid, premeiotic DNA synthesis and callose accumulation in pollen mother cells are evident by 48 hours posttreatment, and within 66 hours, prophase I of meiosis- and meiosis-related changes in tapetum development are observable.     

PLANTLETS FROM PETAL SEGMENTS,PETAL EPIDERMIS,AND SHOOT TIPS OF THE PERICLINAL CHIMERA,CHRYSANTHEMUM MORIFOLIUM ‘INDIANAPOLIS'

Cultivated chrysanthemums, especially the greenhouse series of ‘Indianapolis’ cultivars, are probably periclinal chimeras for flower color. Therefore, in vitro propagation of chrysanthemum, which has recently been described, might produce plants not true to type. To test this, plantlets were generated from cultures of petal segments, petal epidermis, and shoot tips; these plantlets were grown to flowering to determine whether chrysanthemums with two genetically different chimeral layers in the petals are stable in tissue culture. Layer I displaced layer II in the formation of new meristematic areas in shoot tip and petal culture, showing that such chimeras are unstable in culture. Many more abnormal morphological types were exhibited by the plants which were regenerated from petal cultures rather than those from shoot tip cultures. Abnormalities included quilled and incised petal forms, as well as lack of anthocyanin pigmentation, characteristics which may not be attributable to the rearrangement of chimeral layers. Paramutation, true mutation, and environmenal effects are offered as possible explanations for this phenomenon.     

Importance of nuclear localization for the apoptosis-induced activity of a fungal galectin AAL (Agrocybe aegerita lectin)

Agrocybe aegerita lectin (AAL) was identified previously in our group as a novel galectin from medicinal fungi Agrocybe aegerita, and has been shown to effectively induce cancer cell cycle arrest and apoptosis in vitro and tumor regression in vivo. Here, AAL was observed to translocate into the HeLa cell nucleus and induce cell apoptosis when it was predominantly in the nucleus. The N-terminus and C-terminus of AAL were required for nuclear localization. Site mutated proteins were generated based on AAL structure. Dimer interface mutant I25G, carbohydrate recognition domain (CRD) mutant R63H, and loop region mutant L33A could not enter the nucleus and lost the ability to induce apoptosis. CRD mutant H59Q and loop region mutant I144G maintained nuclear localization activity, and H59Q retained residual bioability but I144G had no activity, indicating that nuclear localization is important but not sufficient for AAL to become apoptotically active. Our findings provide a novel antitumor mechanism of fungal galectin.     

Wild-type herpes simplex virus 1 blocks programmed cell death and release of cytochrome c but not the translocation of mitochondrial apoptosis-inducing factor to the nuclei of human embryonic lung fibroblasts

Programmed cell death activated by herpes simplex virus 1 mutants can be caspase dependent or independent depending on the nature of the infected cell. The recently discovered mitochondrial apoptosis-inducing factor (AIF) on activation is translocated to the nucleus and induces programmed cell death that is caspase independent. To assess the role of AIF and also to assay apoptosis-related events in primary human embryonic lung (HEL) fibroblasts, cells were mock infected or infected with wild-type virus previously shown not to induce apoptosis in continuous lines of primate cells or with the d120 mutant lacking infected cell protein no. 4 (ICP4) and were shown to induce apoptosis in all cell lines tested. Cells exposed to dexamethasone or osmotic shock induced by sorbitol were the positive controls. The results were as follows: (i) AIF was translocated to the nucleus in all infected cell cultures and in cells treated with dexamethasone or sorbitol, but cells infected with the wild type-virus showed no evidence of undergoing programmed death. (ii) Cytochrome c was released from mitochondria of cells infected with the d120 mutant or exposed to dexamethasone or sorbitol but not from mitochondria in cells treated with sorbitol and infected with the wild-type virus. (iii) Poly(ADP-ribose) polymerase was cleaved in mock-infected cells exposed to sorbitol or dexamethasone and in cells infected with the d120 mutant but not in either untreated cells infected with wild-type virus or cells exposed to sorbitol and then infected with wild-type virus. In contrast to HEp-2 cells, neither d120 infection nor treatment with dexamethasone or sorbitol caused fragmentation of DNA in HEL fibroblasts. Electron microscopic examination showed chromatin condensation and vacuolization in a fraction of cells infected with d120 but not in wild-type virus-infected cells or cells treated with dexamethasone or sorbitol. We conclude that AIF is translocated to the nucleus in infected cells but apoptosis does not ensue in wild-type-infected cells. HEL fibroblasts infected with the d120 virus exhibit symptoms of classical apoptosis, such as cytochrome c release and cleavage of poly(ADP-ribose) polymerase observed also in cells undergoing caspase 3-dependent programmed cell death in which AIF is either not involved or not a contributory factor.     

棉花叶皱小突变体的遗传分析及激素含量研究

在海岛棉(Gossypium barbadense L.)品种海7124的基因组总DNA导入陆地棉(G.hirsutum L.)品种石远321的后代中,发现了一个形态性状发生变异的突变体。与正常棉株相比,突变体叶片皱小、茎杆细弱,铃小但种子发育正常。叶表皮显微观察发现,突变体叶表皮细胞增大,突变体叶片变小是由于叶片细胞数目的减少。遗传分析表明,突变体为杂合基因型,突变性状受显性基因控制,并可能具有纯合致死效应。内源激素含量测定显示,突变体茎尖中IAA和ZR含量显著高于正常棉株,推测突变体的叶片皱缩变小可能与主茎顶芽中IAA和ZR含量的异常有关。     

Influence of stock plant pretreatment on gynogenic embryo induction from flower buds of onion

The gynogenic response of a range of onion genotypes to flower bud culture was compared using a two-step culture system. Embryogenic cultures and plantlets were produced from unpollinated ovules in whole flower bud explants 6 to 19 weeks after culture initiation. Preconditioning stock plants significantly influenced gynogenic embryogenesis. A ten-fold increase in embryogenesis was obtained when flower buds were cultured from stock plants maintained at 15 °C compared to 10 °C or the ambient temperature conditions of a glasshouse (maximum-minimum of 25–12.7 °C). A total of 49 embryos was obtained from 2660 cultured flower buds and 45% of plantlets were successfully acclimatised to glasshouse conditions. The majority of acclimatised plantlets were haploid (68%) but spontaneous double haploid plants (23%) were obtained from three genotypes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Induction of <Emphasis Type="Italic">in vitro</Emphasis> flowering in the orchid <Emphasis Type="Italic">Dendrobium</Emphasis> Sonia 17

In this study, Dendrobium Sonia 17 plantlets were used to induce in vitro flowering. Inflorescences were induced and rooting was inhibited in the half-strength Murashige and Skoog medium containing 20 μM N ⁶-benzyladenine (BA). The medium with high P and low N contents was effective to induce inflorescences while the medium with low P and high N contents was only effective to promote forming of shoots. In addition, the induced in vitro inflorescences were able to multiply and maintain without exhibiting a distinctive vegetative phase. Different morphologies of in vitro flowers such as incomplete flower structures, abnormal and unresupinated in vitro flowers were observed.     

Yellow and red pigment production by cell cultures of Carthamus tinctorius in a bioreactor

Segments taken from flower-stalk internodes of Oncidium Sweet Sugar formed somatic embryos and shoot buds directly from wound surfaces or via nodular masses proliferation within 1.5 months, when cultured on a Gelrite-gelled 1/2-MS basal medium supplemented with thidiazuron (0.1–3 mg l⁻¹) in darkness. In light, when subcultured, these nodular masses proliferated into green compact callus, and produced somatic embryos, shoot buds and/or yellowish abnormal structures spontaneously. Supplementing 0.1–1 mg l⁻¹ NAA enhanced embryo formation, but retarded proliferation of shoot buds and yellowish abnormal structures. Somatic embryos that directly formed from wound surfaces of flower stalk explants usually developed into abnormal structures, but the callus-derived embryos could germinate into PLBs and eventually developed to normal plantlets on a hormone-free basal medium for 3–4 weeks. Both the embryo-and shoot bud-derived regenerants developed into healthly plantlets when potted in sphagnum moss and acclimatized in the greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Control of leaf-tip necrosis of micropropagated ornamental statice by elimination of endophytic bacteria

Summary Leaf-tip necrosis of micropropagated statice plantlets is a serious problem in commercial laboratories in Taiwan. Endophytic bacteria were detected in plantlets obtained from commercial laboratories with a leaf-tip necrosis problem. Endophytic bacteria were detected in flower stalks collected from four different statice farms at frequencies ranging from 61 to 100%. All plantlets regenerated from flower-stalk explants that tested free of endophytic bacteria did not develop leaf-tip necrosis. The most frequently detected endophytic bacteria were Pasteurella multocida, Stenotrophomonas maltophilia, and Alcaligenes sp. Most endophytic bacteria in statice plantlets were eliminated by the subculture of plantlets on medium with augmentin, cefotaxime, or augmentin plus cefotaxime. Those plantlets freed from endophytic bacteria by subculture on antibiotic-amended medium did not develop leaf-tip necrosis. Our results show that leaf-tip necrosis of micropropagated statice plantlets is associated with endophytic bacteria, and that the disease can be controlled by using explants pre-tested to be free from endophytic bacteria or by the subculture of affected plantlets on antibiotic-amended medium.     

Arabidopsis lox3 lox4 double mutants are male sterile and defective in global proliferative arrest

Fertility and flower development are both controlled in part by jasmonates, fatty acid-derived mediators produced via the activity of 13-lipoxygenases (13-LOXs). The Arabidopsis thaliana Columbia-0 reference genome is predicted to encode four of these enzymes and it is already known that one of these, LOX2, is dispensable for fertility. In this study, the roles of the other three 13-LOXs (LOX3, LOX4 and LOX6) were investigated in single and double mutants. Four independent lox3 lox4 double mutants assembled with different mutated lox3 and lox4 alleles had fully penetrant floral phenotypes, displaying abnormal anther maturation and defective dehiscence. The plants were no longer self-fertile and pollen was not viable. Fertility in the double mutant was restored genetically by complementation with either the LOX3 or the LOX4 cDNAs and biochemically with exogenous jasmonic acid. Furthermore, deficiency in LOX3 and LOX4 causes developmental dysfunctions, compared to wild type; lox3 lox4 double mutants are taller and develop more inflorescence shoots and flowers. Further analysis revealed that developmental arrest in the lox3 lox4 inflorescence occurs with the production of an abnormal carpelloid flower. This distinguishes lox3 lox4 mutants from the wild type where developmentally typical flower buds are the terminal inflorescence structures observed in both the laboratory and in nature. Our studies of lox3 lox4 as well as other jasmonic acid biosynthesis and perception mutants show that this plant hormone is not only required for male fertility but also involved in global proliferative arrest.     

椰子幼花序离体培养的器官发生

椰子幼花序在改良的ＭＳ附加高浓度的ＮＡＡ与２,４－Ｄ的培养基产生愈伤组织,在逐渐降离ＮＡＡ和２,４－Ｄ浓度的过程中,愈伤组织产生结构紧密的结节组织,结节组织有输导组织,分生细胞团,根原基及不同发育阶段的胚状体,有些进而发育成正常的根和绿色芽状物,同时,某些幼花枝的基部直接长出绿芽,经继续培养形成具有正常根,叶结构的完整植株,但茎尖无顶端分生组织,无叶原基,有的发育成花枝,甚至在顶部长出几朵发育不正     

Genetic and cytological analyses of a partial-female-sterile mutant (PS-1) in soybean (Glycine max; Leguminosae)

Soybean partial-female-sterile mutant 1 (PS-1) was recovered from a gene-tagging study. The objectives were to study the inheritance, linkage, allelism, and certain aspects of the reproductive biology of the PS-1 mutant. For inheritance and linkage tests, PS-1 was crossed to flower color mutant Harosoy-w4 and to chlorophyll-deficient mutant CD-1, also recovered from the gene-tagging study. For allelism tests, reciprocal crosses were made with PS-1 and three other partial-sterile mutants (PS-2, PS-3, and PS-4) recovered from the same gene-tagging study. The PS-1 mutant is inherited in a 3:1 ratio and is a single recessive gene. Linkage results indicated that the gene for partial sterility in PS-1 is not linked either to the w4 locus or to the CD-1 locus. Allelism tests showed that the gene in PS-1 is nonallelic to the gene in PS-2, PS-3, and PS-4. Investigations of developing and mature pollen indicated no differences in morphology, stainability, or fluorescence between normal and partial-sterile genotypes. The PS-1 mutant is completely male fertile. Confocal scanning laser microscopy was used to determine that early embryo abortion in PS-1 is due indirectly to abnormal migration of the fused polar nucleus, which prevented it from being fertilized. Subsequent absence of endosperm development leads directly to abortion of the proembryo.     

Metabolism of Benzyladenine is Impaired in a Mutant of Arabidopsis thaliana Lacking Adenine Phosphoribosyltransferase Activity

Formation of the riboside-5′-monophosphate is a general feature of the metabolism of cytokinins in plants. As part of a study of the biological significance of the nucleotide form of cytokinins, we analyzed a mutant of Arabidopsis thaliana deficient in adenine phosphoribosyltransferase (APRT) activity for its ability to metabolize N⁶-benzyladenine (BA). Formation of N⁶-benzyladenosine-5′-monophosphate (BAMP) was assayed in vivo, by feeding tritiated BA to wild-type and mutant plantlets, and in crude plantlet extracts. Metabolites were separated by high performance liquid chromatography and quantitated by on-line liquid scintillation spectrometry. BA was rapidly absorbed by A. thaliana plantlets and primarily converted to BAMP and to BA 7- and 9-glucosides. BA was also rapidly absorbed by APRT-deficient plantlets, but its conversion to BAMP was strongly reduced. Formation of BAMP from N⁶-benzyladenosine was not affected in the mutant plantlets. In vitro conversion of BA to its nucleoside-5′-monophosphate was detected in crude extracts of wild-type plantlets, but not in extracts of APRT-deficient plantlets. Therefore, results of both assays indicate that APRT-deficient tissue does not convert BA to BAMP to a significant extent. Further, nondenaturing isoelectric focusing analysis of APRT activity in leaf extracts indicated that the enzyme activities which metabolize adenine and BA into their corresponding riboside-5′-monophosphate in extracts of wild-type plantlets have the same apparent isoelectric point. These activities were not detected in extracts prepared from APRT-deficient plantlets. Thus, these results demonstrate that APRT is the main enzyme which converts BA to its nucleotide form in young A. thaliana plants and that the ribophosphorylation of BA is not a prerequisite of its absorption by the plantlets.     

FLORAL DEVELOPMENT OF A FLOWER-STRUCTURE MUTANT IN SOYBEANS,GLYCINE MAX (L.) MERR. (LEGUMINOSAE)

A flower-structure mutant with cleistogamous flowers (but often with an exposed style and stigma) and very low seed set was found in soybeans (Glycine max (L.) Merr.). The mutant, assigned Genetic Type Collection Number T269, is controlled genetically by duplicate recessive genes, fs₁ and fs₂. A study of flower development in T269 plants was undertaken to determine the cause of the low seed set. Both normal and mutant flower buds were observed with a light microscope by using paraffin serial sections and with a scanning electron microscope. Measurements of various floral structures were taken to verify differences observed between mutant and normal flowers. Young mutant flower buds had longer carpels and larger receptacles than did normal flower buds. These two factors caused the sepals to be positioned abnormally, which, in turn, prevented normal development of the petals. The abnormal petal development prevented staminal tube elongation, and a spatial separation between the anthers and stigma existed at anthesis, preventing self-pollination. Observations of the gynoecium of mutant flowers revealed that megasporogenesis and megagametogenesis were normal but that other features of ovule ontogeny were abnormal. In all ovules examined, the outer integuments failed to form micropyles. In addition, many ovules were positioned abnormally. The degree of aberration varied even within a carpel, but we estimated that at least 75% of the ovules were too aberrant to be functional. Therefore, the low seed set on T269 plants was due both to a lack of self-pollination and to partial female sterility. It is the only naturally occurring structural sterile reported in soybeans to date.     

哈姆林甜橙与粗柠檬体细胞杂种的育性

对异源四倍体柑桔体细胞杂种“哈姆林甜橙+粗柠檬”及其亲本的花粉活力、花器官发育、花器官形态发生与花粉母细胞减数分裂四分体阶段进行了观测和统计.结果发现“哈姆林甜橙+粗柠檬”的花粉染色活力、萌发率、每花药中花粉粒数均居于其双亲之间,花器官发育及其形态发生具有双亲的特点.但小花粉及花粉母细胞减数分裂过程中形成的不正常四分体比率远远高于其双亲.以体细胞杂种“哈姆林甜橙+粗柠檬”为花粉亲本,与二倍体单胚类型宜昌橙与华农本地早的有性后代杂交,获得了110棵有性后代植株,其中三倍体82棵,二倍体和其它倍性的植株28棵.     

Cytokinin receptors in sporophytes are essential for male and female functions in Arabidopsis thaliana

Arabidopsis has three cytokinin receptors genes: CRE1, AHK2 and AHK3. Availability of plants that are homozygous mutant for these three genes indicates that cytokinin receptors in the haploid cells are dispensable for the development of male and female gametophytes. The triple mutants form a few flowers but never set seed, indicating that reproductive growth is impaired. We investigated which reproductive processes are affected in the triple mutants. Anthers of mutant plants contained fewer pollen grains and did not dehisce. Pollen in the anthers completed the formation of the one vegetative nucleus and the two sperm nuclei, as seen in wild type. The majority of the ovules were abnormal: 78% lacked the embryo sac, 10% carried a female gametophyte that terminated its development before completing three rounds of nuclear division, and about 12% completed three rounds of nuclear division but the gametophytes were smaller than those of the wild type. Reciprocal crosses between the wild type and the triple mutants indicated that pollen from mutant plants did not germinate on wild-type stigmas, and wild-type pollen did not germinate on mutant stigmas. These results suggest that cytokinin receptors in the sporophyte are indispensable for anther dehiscence, pollen maturation, induction of pollen germination by the stigma and female gametophyte formation and maturation.Key words: cytokinin, cytokinin receptor, female gametophyte, male gametophyte, stigma     

印度南瓜梭形果突变体的发现及遗传分析

从印度南瓜自交系HL中发现了一个梭形果突变体HL-fu。该突变体的花、果、茎等多器官的生物学特性同时发生了明显变异:雌花及雄花花器中异性花蕊的残存度较野生型高;果实为梭形,果形指数达野生型5倍;节间呈现长短交替分布的排列状态,叶序类似于对生。以突变体HL-fu与野生型HL杂交分别构建了正、反交F1,正交F2,BC1(P1)F1、BC1(P2)F1群体进行遗传分析,推测该变异类型为隐性多效单基因突变,突变性状受1对隐性核基因fu控制。探讨了梭形果突变体在南瓜育种及相关基础研究方面的潜在价值。     

Effects of PEG-induced water stress onin vitro hardening of ‘Valiant’ grape

Polyethylene glycol was added to the rooting medium ofmicropropagated grape shoots to induce water stress. At the end of the rooting stage, plantlets treated with 2% polyethylene glycol were compared with untreated control plantlets and greenhouse-grown plants. Leaves of treated plantlets had the highest deposition of epicuticular wax, followed by those of the greenhouse and control. Stomatal index did not vary among treatments. However, differences in leaf epidermal cell configuration were observed among treatments. The morphological changes of treated plantlets, including substantial deposition of epicuticular wax and modified leaf surface anatomy were associated with increasedex vitro survival after four weeks in the greenhouse.