Influence of the sex of flowers on androgenesis in Aesculus hippocastanum L. anther culture

Summary Flowers of Aesculus hippocastanum L. are bisexual and zygomorphic, and are positioned on a 20–30 cm long inflorescence. Those located in the basal part of the panicle are female and fertile (segment A), flowers in the middle are bisexual (segment B), and those on top of the panicle are male (segment C). Androgenesis was achieved in anther culture which originated from three types of flowers cultured on modified Murashige and Skoog medium containing 2,4-dichlorophenoxyacetic acid (4.5 μM) and kinetin (4.6 μM). Differences in viability of uninuclear microspores were found between female (90.0%) and other flowers (bisexual 61.1%; male 72.7%.). Both the percentage of embryogenic anthers and the number of androgenic embryos formed per inflorescence differed according to the segment of origin. The highest embryogenic response was obtained in segment A (47.3%) and the lowest in segment C (24.1%). A significant difference was found between the number of androgenic embryos formed per inflorescence in segments A (921.0) and C (286.7). The highest germination percentage (21.3%) and plantelet formation (41.0%) were obtained on woody plant WPM liquid medium supplemented with 1% activated charcoal. Acclimation and regeneration were best from plantelets originating from female flowers (62.5%). Plantlets originating from bisexual and male flowers have much poorer survival (29.3 and 22.2%, respectively).     

Secondary embryogenesis in androgenic embryo cultures of <Emphasis Type="Italic">Aesculus hippocastanum</Emphasis> L.

Secondary somatic embryos appeared on the cotyledons and radiculi of embryos derived from suspension and anther cultures of Aesculus hippocastanum L. The highest number of secondary somatic embryos formed on a hormone-free medium.This work was supported by the Ministry of Science and Environment Protection of Serbia, grant N₀. 1573.     

Variability and Bimodal Distribution of Size in Microspores of Aesculus hippocastanum

Size variability of uninucleate microspores was studied in horse chestnut (Aesculus hippocastanum L.). Microspores were isolated from buds of different size (3, 4, and 5 mm) taken from lower, middle and upper segments of inflorescences. All analyzed buds showed bimodal distribution of microspore size which confirmed the presence of pollen dimorphism. This revised version was published online in July 2006 with corrections to the Cover Date.     

The effect of low temperature on germination of androgenic embryos of <Emphasis Type="Italic">Aesculus hippocastanum</Emphasis> L.

Treating androgenic embryos of Aesculus hippocastanum L. with low temperatures (6 °C) improved their germination and regeneration into plantlets. The embryos derived from anther cultures showed better results than those derived from microspore cultures.This work was supported by the Ministry of Science, Technology and Development of Serbia, grant No. 1573.     

Comparison of Anther and Microspore Culture in the Embryogenesis and Regeneration of Rye (Secale cereale)

Anther culture in solid and liquid medium and isolated microspore culture were compared in rye genotypes with potential agronomic characteristics. Some important factors influencing androgenic capacity were optimised. Three weeks cold pre-treatment of spikes and two days mannitol pre-treatment of anthers maximized callus and green plant yield in both culture methods. Intensity order of the culture methods in callus and green plant production was: isolated microspore culture, anther culture in liquid medium and anther culture in solid medium. Genotype ability of embryogenesis followed the same pattern in both cultivation methods. Kinetin (BA) with genotype dependent concentrations created the most effective regeneration conditions.     

禾本科牧草和草坪草雄核发育与单倍体育种研究进展

禾本科牧草与草坪草在农业可持续发展、城市绿化和生态环境保护方面起着至关重要的作用。近年来, 随着生物技术的发展, 国内外在牧草及草坪草雄核发育与单倍体育种研究方面取得了较大进展。该文在归纳总结该领域研究成果的基础上, 对影响禾本科牧草及草坪草雄核发育与单倍体育种的几个主要因素进行了探讨。大量研究结果表明, 供试材料的基因型是影响培养效率的最主要因素。小孢子发育到单核中期至晚期时有利于提高培养效率。培养前花药经过低温和甘露醇等预处理不但可以提高愈伤组织的诱导效率, 还可提高愈伤组织的质量。适宜的激素种类和配比也是影响培养成败的关键因素。同时, 总结了雄核发育再生植株的倍性鉴定方法和加倍技术, 对单倍体育种技术在禾本科牧草及草坪草育种中的应用前景进行了展望。     

Toward Doubled Haploid Production in the Fabaceae: Progress,Constraints, and Opportunities

The Fabaceae species have a major role to play in sustainable farming systems, but they have lagged behind other families in respect to the development of doubled haploid protocols for plant improvement. Currently, no plant improvement program uses doubled haploids on a routine basis for any member of the Fabaceae. There has recently been renewed interest in haploid research as the usefulness of doubled haploid material in molecular mapping has become clear. This review provides a comprehensive account of the current information regarding the development of haploid protocols in the Fabaceae. In the Fabaceae crop species there have been isolated reports of haploid plant induction in the phaseoloid clade; soybean, cowpea and pigeonpea, as well as promising progress towards haploidy in peanut and winged bean. As yet there have been no reports of haploid plant production in the galegoid clade, but early stage haploid embryogenesis has been achieved in chickpea, field pea, and lupin. Success in the production of haploid plants has also been reported within the pasture genera Lotus, Medicago, and Trifolium and the arboreal genera Cassia, Peltophorum, and Albizzia. A review of the literature has enabled us to identify some general similarities between the protocols developed for haploid plant induction across the various legumes. These are the culture of intact anthers; use of a cold pretreatment to induce sporophytic development; targeting of microspores at the uninucleate stage of development; and use of MS (Murashige and Skoog, 1962 Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 473–497. [CSA][Crossref], [Web of Science ®] [Google Scholar]) based nutrient medium with plant growth regulators to encourage continued division following induction. These protocol commonalities will assist researchers to identify approaches suited to their target Fabaceae species. The paucity of research funding for haploid research in most Fabaceae species has highlighted the need for strong collaborative linkages between institutions and researchers.

     

黄瓜花器形态发生、小孢子发育与花药培养

孢子发育时期与花蕾形态特征、花药颜色具有相关性。本试验目的是确定黄瓜花药的最佳培养时期,并确定对应的选蕾标准。对基因型、预处理等花药培养主要影响因素研究结果表明：不同基因型小孢子单核中后期花蕾长度不同,形态上没有差异;同一基因型的小孢子发育不同时期的花器特征和诱导率均有差异,单核中后期花药诱导率最高;低温预处理有利于保持小孢子的活力;花药培养中以黑暗中4℃、48～72h低温预处理最有助于愈伤发生。实验结果表明,黄瓜花药培养中小孢子最佳培养时期为单核中后期,取蕾标准为：花蕾长度0．90～1．50cm,绿色,瓣尖未张开,花药白绿色或淡绿色。     

High efficiency adventive embryogenesis on somatic embryos of anther,filament and immature proembryo origin in horse-chestnut (Aesculus hippocastanum L.) tissue culture

Adventive embryogenesis was successfully induced in cultures of zygotic and somatic embryos on MS medium supplemented with BA and NAA. A procedure has been proved successful for the in vitro multiplication of somatic embryos regenerated at low frequencies from filament and callus cultures. The occurrence and rate of adventive embryogenesis did not depend on the origin of the primary embryos (zygotic and somatic), but did depend on the developmental stage. Primary embryos are capable of embryogenesis in each of the different phases of embryogenesis, though the rate is different. BA concentrations of 22–44 M increased the rate of adventive embryogenesis and accelerated the development of embryos. The highest proliferation rate (22–25x/5 weeks) was achieved at hormone concentrations of 44 M BA and 5.4 M NAA.Abbreviations BA benzyladenine - CH casein hydrolysate - CM coconut milk - 2,4-D dichlorophenoxyacetic acid - MS Murashige & Skoog medium - WPM woody plant medium - NAA 1-naphtaleneacetic acid     

Genetic Analysis of Null Alleles in Rice Doubled Haploid Plants from Anther Culture

In the research of constructing a rice(Oryza sativa) molecular map, 4 RFLP markers, i.e. RG 229, RG 419, RG 424 and RG 353, detected the null alleles in the indica and japonica parental rice. RG 229 indicated two null alleles in indica rice Gui 630, and each of the other markers revealed a null allele in japonica rice 02428. Genetic analysis in the doubled haploid (DH) population consisting of 81 plants showed that the linkage relationships between these null allele loci and the neighboring molecular markers shown in McCouch' s rice molecular map were changed. In addition, the marker RG 684 could detect its null alleles in some DH plants, though the RG 684 sequence did exist in the genomes of both parents. Appearence of null alleles might be induced by transpositional changes on chromosomes.     

In vitro induction of pollen embryos and plantlets in Aesculus carnea Hayne through anther culture

Uninuclear microspores in red horse chestnut anther cultures formed pollen embryos and plantlents in MS agar medium supplemented with varying 2,4-D concentrations (1.0, 1.5 or 2.0 mg l^-1) and 1.0 mg l^-1 Kin. The highest number of embryogenic anthers (38%) was obtained in MS medium containing 1.0 mg l^-1 of each 2,4-D and Kin. The ability of pollen embryos to germinate was closely correlated with normal embryo morphology and was influenced by hormone content in the medium (MS+;1.0 mg l^-1 IAA+1.0 mg l^-1 GA₃+0.1 mg l^-1 Kin+400 mg l^-1 glutamine). Pollen embryos and plantlets had the haploid chromosome number (x=n=40). Cytological examinations demonstrated pollen dimorphism of this Aesculus species.Abbreviations AC activated charcoal - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - Kin 6-furfurylaminopurine - GA₃ gibberellic acid - MS Murashige and Skoog     

Microspore embryogenesis in anther cultures of two Indian cultivars of Brassica juncea (L.) Czern.

Direct microspore-derived embryo formation in anther cultures of two cultivars of Brassica juncea was obtained. Preliminary culture of anthers at 35°C for 1–5 days prior to maintenance at 25°C stimulated embryogenesis. Embryogenesis was also stimulated by an initial culture at 5°C for 3 days. Analysis of squashed anthers revealed that approximately 10% of the microspores began dividing, but less than 1% developed into macroscopic embryos. All embryos transferred to embryo culture medium survived, but only 30% of these developed directly into normal plantlets. The androgenic plants were haploid (2n=18).     

Induction of Haploid Androgenesis in Pacific Oyster by UV Irradiation

Androgenesis, development from paternal but not maternal chromosomes, can be induced in some organisms including fish, but has not been induced previously in mollusk. In this study we investigated the induction of haploid androgenesis in the Pacific oyster by ultraviolet irradiation and observed nuclear behavior in the androgenetic eggs. Irradiation for 90 seconds at a UV intensity of 72 erg/mm² per second (6480 erg/mm²) was the optimal dose to achieve haploid androgenesis. The fertilization and development rates of D-shaped larvae decreased with increasing exposure time, and the development of the genetically inactivated eggs terminated before reaching the D-shaped stage. Cytologic observations showed that UV irradiation did not affect germinal vesicle breakdown or chromosomal condensation but caused various nuclear behavioral patterns during meiosis and first mitosis: 21.7% of eggs extruded all maternal chromosomes as 2 or 3 polar bodies, and 59.1% of eggs formed one female pronucleus. The maternally derived nucleus did not participate, or partially participated, in the first karyokinesis. The cytologic evidence demonstrates that the male genome is directing development in haploids produced by UV irradiation.     

Anther Culture of Naked Oat and the Establishment of Its Haploid Suspension Cell

This paper reported the production of haploid plants through anther culture in naked oat (Arena nuda). Calluses were induced from anthers of naked oat placed on various culture media. MS medium with 4% sucrose, 1% activated charcoal and no hormones gave the highest initiation frequencies (14.7%) of anther callus among media tested. Twelve green plants and one albino plant have been regenerated from anther calluses. Cytological examination of mitotic rooot tip ceils from three green anther plants showed that two of the plants were haploid (2n=3x=21) and one was diploid (2n=6x=42). The cell suspension cultures were established from pollen friable calluses in liquid medium. The suspension cells were cytologically stable during one year subcultures. Most of the ceils examined were haploid.     

菊科植物单倍体研究进展

单倍体培养是快速获得菊科纯合系的重要途径。目前已进行单倍体研究的菊科植物共有13个种,其中9个已成功获得单倍体植株。菊科中诱导单倍体的途径有花药培养、小孢子培养、离体雌核培养、远源杂交和辐射花粉诱导单倍体。本文详细论述了不同外植体发育时期、预处理、培养基、培养条件等因素对单倍体植株诱导再生的影响。对菊科植物单倍体诱导的几种途径进行对比总结,指出研究中存在的问题并提出思路和建议。     

A seasonal cycle of cell wall structure is accompanied by a cyclical rearrangement of cortical microtubules in fusiform cambial cells within taproots of Aesculus hippocastanum (Hippocastanaceae)

Aspects of the structure and ultrastructure of the fusiform cambial cells of the taproot of Aesculus hippocastanum L. (horse chestnut) are described in relation to the seasonal cycle of cambial activity and dormancy. Particular attention is directed at cell walls and the microtubule and microfilament components of the cytoskeleton, using a range of cytochemical and immunolocalization techniques at the optical and electron-microscopical levels. During the dormant phase, cambial cell walls are thick and multi-layered, the cells possess a helical array of cortical microtubules, and microfilament bundles are oriented axially. In the early stages of reactivation, vesicle-like profiles are associated with the cell walls, whereas arrangement of the cytoskeletal elements remains unchanged. In the succeeding active phase, the cell walls are thin, and cortical microtubules form a random array, although microfilament bundles maintain a near-axial orientation. The observations are discussed in relation to the seasonal cycle of wall structure and cortical microtubule rearrangement within the vascular cambium of hardwood trees. It is suggested that the cell-wall thickening at the onset of cambial dormancy, which is associated with the presence of a helical cortical microtubule array, should be considered to be secondary wall thickening, and that selective lysis of this secondary wall layer during cambial reactivation restores the thinner, primary wall found around active cambial cells.     

甘蓝花药培养胚状体诱导形成影响因子研究

用甘蓝F1、F2和自交系S33个世代6种基因型材料进行甘蓝花药培养诱导胚状体形成影响因子研究。结果显示:(1)高浓度蔗糖对甘蓝胚状体形成具有显著的诱导作用,6%蔗糖浓度是甘蓝花药培养的最适浓度,其胚状体的诱导率最高达12.2%;(2)材料基因型是影响花药培养的主要因素,F2和F1代材料胚状体诱导效果好,且胚状体诱导率F2代(F2P192和F2P194)18.9%比F1代(F1S17和F1S13)17.1%较高,但差异不显著,自交系S3代材料很难诱导出胚状体;(3)B5培养基比MS培养基更适合甘蓝花药胚状体的诱导培养。结果表明,甘蓝F2代是其花药诱导培养胚状体的最佳基因型材料,B5 2.0 mg/L 2,4-D 2.0 mg/L KT 6%Suc是甘蓝花药诱导培养胚状体的最适培养基。     

Microspore embryogenesis of high sn-2 erucic acid Brassica oleracea germplasm

Brassica oleracea accessions possess traits that would be useful in commercial Brassica crops. These traits can be studied more effectively through the production of doubled haploid plants. Nineteen B. oleracea accessions from several subspecies possessing significant sn-2 erucic acid were screened for suitability for microspore culture using techniques well established for Brassica. Fifteen of the 19 accessions produced embryos. Genotypic differences were observed with embryogenesis ranging from 0 to 3000 embryos/100 buds. Embryogenesis was improved for two of four accessions by initiating cultures in NLN medium with 17% sucrose, then reducing sucrose to 10% after 48 h. An increase in embryogenesis for the same two accessions was observed when microspores were cultured at a density of 100 000/ml rather than 50 000 microspores/ml. A culture temperature of 32 °C for 48 h was beneficial for three of the four accessions when compared to a longer incubation period (72 h) or a higher temperature (35 °C). One accession line, Bo-1, was found to produce microspore-derived embryos which contained triacylglycerols with significant proportions of erucic acid at the sn-2 position. This revised version was published online in June 2006 with corrections to the Cover Date.     

菘蓝花药培养及单倍体的诱导

探讨菘蓝花药处于单核晚期的形态指标,并以适宜发育时期的花药为外植体,进行花药培养及单倍体诱导。实验结果表明,4℃低温处理2d后,在含有6-BA0．5mg·L-1和NAA1．0mg·L-1。的Ms培养基上,花药愈伤组织的诱导率为23．35％;将其转接到Ms附加6．BA1．0mg·L-1,NAA0．5mg·L-1的分化培养基上,80．00％以上的愈伤组织可以诱导产生不定芽;再将分化出的试管苗转接到1／2MS＋NAA1．0mg·L-1的生根培养基上,3d左右即可获得完整植株。经叶边缘压片检查染色体数目,花药培养所得的弱小绿苗为单倍体植株。