番茄游离小孢子培养研究进展

雄核发育是小孢子或未成熟花粉细胞沿孢子体发育途径形成植株的过程，也是创制育种材料的有效途径之一。为建立重复性好、再生频率高的番茄小孢子诱导方法，文章通过回顾国内外研究进展，总结了番茄雄核番茄雄核发育各阶段（胚胎发生能力的获得、诱导小孢子分化形成胚状体以及胚状体再生形成单倍体植株）的主要影响因素，并简要归纳了现有的游离小孢子分离培养技术。分析表明，基因型是制约番茄小孢子诱导体系的关键因素，且需与发育时期、培养条件等多个影响因素同期发生，才能使雄核发育偏离配子体途径。最后，展望了番茄小孢子培养未来的研究方向与发展趋势，以期为番茄双单倍体技术的深入研究提供参考。     

不同预处理对萝卜花药组织结构的影响

研究了花枝低温预处理及常、低温下离体花药的甘露醇和秋水仙素预处理对萝卜花药组织结构的影响。结果表明:4℃低温条件下处理枝条,处理1,3,5 d的花药绒毡层细胞出现从排列整齐到逐步退化的现象,药室中的小孢子染色也逐渐加深,7 d后花药壁细胞退化严重,小孢子数目则减少,故枝条低温预处理合适的时间为3~5 d。4℃条件下离体花药经甘露醇和秋水仙素预处理3 d后,药室中小孢子数目多,染色深,处理超过3 d,小孢子数目急剧减少,活力下降。25℃条件下甘露醇和秋水仙素预处理不利于花药结构的维持与小孢子活力的保持。     

基因型和供体条件对油菜小孢子培养成胚率影响

利用正交设计对影响油菜小孢子培养的基因型、低温预处理时间、取材时期及小孢子发育时期（用花蕾花瓣花药比表示）等供体材料条件进行系统研究。结果显示,对小孢子培养成胚率影响最大的为基因型,其次依次为小孢子发育时期,取材时期和低温预处理时间。最佳处理以51039为材料,开花前5天取样并于4℃低温预处理2d,取花瓣花药比为0.5至1的花蕾培养成胚率最高,达10.33枚/蕾。     

在水稻极耐寒冷品系Norin-PL8的遗传背景下矮秆基因d18-κ对孕穗期冷害耐性的多效性

水稻对低温最敏感的生育期是孕穗期，特别是从四分体到早期小孢子阶段，主要受害特征是由花药发育不完全和花粉败育引起小穗授粉失败。孕穗期低温还能引起小穗退化。水稻对低温的第二个敏感时期是开花期；低温阻碍了开花，在此期间花药开裂和花粉萌发都受到了影响，导致了不育小穗的增加。在日本北部和其它国家，如加利福尼     

玉米基因雄性不育系（ms2/ms2）小孢子败育的细胞学研究

利用光镜和透射电镜对同一核背景的ｍｓ２不育花药和正常可育花药进行了小孢子发育的细胞学比较观察。结果表明，ｍｓ２不育花药小孢子在幼小孢子期至中小孢子期瓦解。小孢子从四分体释放后，原外壁上没有孢粉素沉积，花粉外壁不能发育。不育花药大多数绒毡层细胞在幼小孢子期显示出明显异常，表现为质体、线粒体等细胞器退化，内质网网络细而少，液泡膜内陷吞噬细胞质，细胞内侧没有乌氏体形成，细胞质内出现大量沉积有浓染颗粒的大     

不同基因型大麦离体培养的花药及小孢子对低温和NaCl预处理的反应

以探讨大麦单倍体细胞水平的耐冷、耐NaCl性与植株水平抗病、耐盐性的相关性为目的，用赤霉病抗性、耐盐性不同的大麦为供试材料，比较了它们的离体培养花药及小孢子对低温、NaCl预处理的培养反应。结果表明：感病材料低温（5℃）预处理17d的花药培养愈伤组织诱导率比未经低温预处理的低，抗病材料则有一定的升幅。用NaCl溶液直接处理游离小孢子，所有供试材料的小孢子存活率随处理浓度升高不断下降，耐盐材料的存活率下降幅度比盐敏材料的小，且抗病材料的降幅比感病材料的小。说明：供体植株水平的抗病、耐盐性在花药、小孢子水平上也能有所表现。     

茄子小孢子热激效应和发育潜能的细胞学分析

高温热激处理茄子小孢子是改变其离体发育方向的主要途径,但是热激后小孢子培养的成功率很低。本试验对热激处理0、2、4、6、8 d的小孢子进行离体培养和细胞学研究。结果表明,培养中膨大的小孢子活力最高,不膨大的小孢子逐渐死亡。热激6 d的小孢子群体的膨大率最高、活力最高。诱导小孢子离体形态发生有必要游离纯化膨大的小孢子,去除衰败小孢子的负面影响。     

不同基因型大麦离体培养的花药及小孢子对低温和NaCl预处理的反应

以探讨大麦单倍体细胞水平的耐冷、耐NaCl性与植株水平抗病、耐盐性的相关性为目的,用赤霉病抗性、耐盐性不同的大麦为供试材料,比较了它们的离体培养花药及小孢子对低温、NaCl预处理的培养反应。结果表明:感病材料低温(5℃)预处理17d的花药培养愈伤组织诱导率比未经低温预处理的低,抗病材料则有一定的升幅。用NaCl溶液直接处理游离小孢子,所有供试材料的小孢子存活率随处理浓度升高不断下降,耐盐材料的存活率下降幅度比盐敏材料的小,且抗病材料的降幅比感病材料的小。说明:供体植株水平的抗病、耐盐性在花药、小孢子水平上也能有所表现。     

雄性不育和可育大麦花药和花粉的细胞学比较研究

本文对大麦雄性不育和可育花药形态和花粉发育进行了细胞胚胎学比较研究,结论如下: 1.可育和不育大麦的花药在外部形态上有明显区别,主要在于不育花药基部成为戟形,且开花时花药变得瘦小。 2.可育大麦的花粉发育类同于一般的禾本科植物。而不育花粉不能发育到二核期,并很快解体形成“无花粉型”花粉;其小孢子和雄配子体发育过程中有几种异常现象:(1)早期发生异常;(2)绒毡层过早退化;(3)属绒毡层解体较晚者,小孢子能发育到二细胞阶段,但形态扭曲,内含物贫乏。 3.可育的药壁发育正常同于单子叶型,直到2细胞花粉粒时期绒毡层才全部退化。而雄性不育药壁发育异常,主要体现在绒毡层早期退化或药壁分化不完全。     

白芷小孢子发育时期与花器形态相关性分析

目的：研究3个白芷品系小孢子发育时期与花蕾形态和花药形态的相关性,以期直接从花蕾形态和花药形态判断小孢子的发育时期。方法：采用苯酚品红对白芷小孢子进行染色,对白芷小孢子发育的细胞学以及小孢子不同发育时期与花柄长、花蕾大小、花药直径、花蕾和花药颜色的关系进行研究。结果：白芷小孢子发育经过四分体时期、单核前期、单核中晚期、二核期和三核期共5个时期,各时期特征明显;3个白芷品系小孢子发育时期与花蕾的外部形态和花药颜色密切相关,其中以CX08-2的花柄长、花蕾直径和花药长度最大,其次为B-2,而B-6为最小;但单核中晚期与其相邻时期的花柄长、花蕾直径和花药长度未达到显著水平,花瓣颜色、花瓣是否开裂和花药颜色可以作为花药培养取材的直观依据。结论：通过花器官形态判断小孢子的发育时期可以为白芷花药培养接种材料的选择提供依据。     

番茄花药培养研究进展

对番茄花药培养中基因型、小孢子发育、培养基种类、碳源、激素种类和配比、添加物、预处理和培养条件等方面的国内外研究概况和进展做了综述。     

Assessment of different anther culture approaches to produce doubled haploids in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.)

Cucumber is one of the most important vegetable crops worldwide, which makes it a good candidate to produce doubled haploid (DH) lines to accelerate plant breeding. Traditionally, these approaches involved induction of gynogenesis or parthenogenesis with irradiated pollen, which carries some disadvantages compared to androgenesis. Despite this, studies on anther/microspore cultures in cucumber are surprisingly scarce. Furthermore, most of them failed to unambiguously demonstrate the haploid origin of the individuals obtained. In this work we focused on anther cultures using two cucumber genotypes, different previously published protocols for anther culture, different in vitro culture variants to make it more efficient, and most importantly, a combination of flow cytometry and microsatellite molecular markers to evaluate the real androgenic potential and the impact of anther wall tissue proliferation. We developed a method to produce DH plants involving a bud pretreatment at 4 °C, a 35 °C treatment to anthers, culture with BAP and 2,4-D, and induction of callus morphogenesis by an additional 35 °C treatment and sequential culture first in liquid medium in darkness and second in solid medium with light. We also found that factors such as genotype, proliferation of anther wall tissues, orientation of anthers in the culture medium and growth regulator composition of the initial anther culture medium have a remarkable impact. Our rate of chromosome doubling (81%) was high enough to exclude additional chromosome doubling steps. Together, our results present androgenesis as an improvable but yet more convenient alternative to traditional gynogenesis and parthenogenesis-based approaches.     

Die Nutzung der Antherenkulturmethode im Zuchtprozeß von Winterweizen

Anther culture in the breeding process of winter wheat. I. Ability of 1B—-1R wheat-rye translocation forms for androgenesis 45 winter wheat varieties or F₁ hybrids, F₂ populations and lines with 1B—1R wheat-rye translocation were tested for their anther culture ability. A total of 48058 anthers was cultured on Potato-2 medium. When averaged over all genotypes and the two experimental years frequencies of embryoid formation of 5.4—6.8 per 100 anthers were observed. Plant regeneration efficiency from embryoids ranged from 5.3—9.1 % or a mean of 4—5 green plants per 1000 anthers plated. The results confirmed the preferential regeneration frequency of gametes with the 1BL—1RS chromosome compared to the gametes with the 1BL—IBS chromosome. Multiple peroxidase were used as marker. The effect of cold pretreatment or of media on the androgenetic response and productivity was not important. On the contrary the variability between the anther response from single ears of the same genotype was noticeable. Examples are presented for the transferability of the androgenetic ability to breeding material. Most green plants obtained were haploid or spontaneous doubled haploid. By cloning it was guaranteed, that progenies were obtained from most of the haploids after colchicine treatment.     

Androgenetic green plants from winter rye, Secale cereale L., of diverse origin

Twenty varieties and breeders’ lines of winter rye, Secale cereale L., including Finnish, Polish, Russian, Swedish and Danish germplasm, were tested for their anther culture ability. To establish a reference point, the initial culture method was based on published results from rye anther culture. Following results from methodological studies, a modified protocol was tested on the same 19 winter rye lines. The modifications to the protocol included growth of mother plants under controlled greenhouse conditions, prolonged cold pretreatment of spikes for 3 weeks at 4°C, excision of anthers with microspores in the late uninucleate/binucleate developmental stage and induction on liquid medium supplemented with a buoyancy increasing component, Ficoll. With the modified protocol, the total production of green androgenetic plants increased nearly fivefold; it reached an average of 2.75% with the best line yielding 12.5%, while the proportion of green to albino plants increased by 50% to 25.1%. Eighteen rye lines showed green plant regeneration ability. Several genetically different winter rye lines with relatively good anther culture ability, yielding ≤ 1/% green plants per 100 anthers, were identified for the use in further methodical studies and for researching doubled haploidy in rye breeding.     

In vitro androgenesis of wheat: from fundamentals to practical application

Wheat anther cultures have a history of almost 30 years and are nowemployed efficiently in many countries of the world for the developmentof doubled haploid lines for breeding. The present paper discusses keyquestions of the elaboration and perfection of the method: cytologicalaspects of in vitro androgenesis, the conditions required for theembryogenic development of microspores and the applicability of anthercultures in the Martonvásár wheat breeding programme.     

Haploidy in tomato (Lycopersicon esculentum Mill.): a critical review

Results on the induction of haploidy in tomato via both gynogenesis and microspore embryogenesis in vitro are far from satisfactory. The number of reports available on the gynogenic induction via in vitro non-fertilized ovary culture, wide hybridization and the use of irradiated pollen are limited. The main reason for this may be the difficulty experienced in working with this species. Therefore, many failed attempts have not been reported. Non-fertilized ovary culture and wide hybridization using Solanum sisymbriifolium Lam. as the male parent seem to be promising (Bal and Abak, Pak J Biol Sci 6:745–749, 2003a, b). Further efforts in this line may improve results obtained earlier. Several reports (Gresshoff and Doy, Planta 107:161–170, 1972; Sharp et al., Planta 104:357–361, 1972; Zamir et al., Plant Sci Lett 17:353–361, 1980; Chlyah and Taarji, Proc. Int. Symp. Plant tissue and cell culture application to crop improvement. 24–29 Sept. 1984; Jaramillo and Summers, J Amer Soc Hort Sci 115:1047–1050, 1990, HortScience 26:915–916, 1991; Summers et al., HortScience 27:838–840, 1992) are available on anther culture of tomato but a working protocol is yet to be developed. For the induction of anther callus, anthers carrying microspores at the meiotic stages appear to be the most responsive. However, the callus and the regenerants obtained were mainly of somatic origin. Somatic tissues of tomato anthers carrying the meiotic stages are highly responsive to tissue culture manipulations in comparison to anther tissues of the later stages. Therefore, reports on the induction of callus from anthers carrying early microspore stages should be met with caution. If culturing young anthers is of any help then it may be that the anther tissues are nursing the microspores and bringing them to the responsive uninucleate stage. Following the first report by Sharp et al. (Planta 104:357–361, 1972) on the induction of microspore embryogenesis, using a modified version of the microspore culture, reports concentrated only on anther culture (reviewed by Chlyah et al., Haploids in crop improvement I. Biotechnology in agriculture and forestry 12. Springer-Verlag, Berlin, 1990). Based on findings reported by Yinnan et al. (J Agric Biotechnol, , 1999) and Bal and Abak (Biotechnol Biotechnol Equip 19:35–42, 2005) on the induction of symmetrical division of microspore nuclei from uninucleate microspores, the formation of multicellular structures and globular embryos, it is likely that the future of tomato haploidy lies in the technique of isolated microspore culture.     

Chromosomal regions controlling anther culturability in rice (Oryza sativa L.)

Diallel analysis has revealed that anther culturability in rice (Oryza sativa L.) is a quantitative trait controlled by the nuclear genome. Mapping of anther culturability is important to increase the efficiency for green plant regeneration from microspores. In the previous study, we detected distorted segregation of RFLP markers in rice populations derived from the anther culture of an F₁ hybrid between a japonica cultivar ‘Nipponbare’ and an indica cultivar ‘Milyang 23’. To clarify the association between chromosomal regions showing distorted segregation and anther culturability, the anther culturability of doubled haploid lines derived from the same cross combination was examined, and the association between alleles of the RFLP markers, which exhibiting the most distorted segregation on chromosomes 1, 3, 7, 10 and 11, and the anther culturability was evaluated. One region on chromosome 1 was found to control callus formation from microspores, and one region on chromosome 10 appeared to control the ratio of green to albino regenerated plants. In both regions, the Nipponbare allele had positive effects. Three regions on chromosomes 3, 7 and 11, however, showed no significant effect on anther culturability. This revised version was published online in August 2006 with corrections to the Cover Date.     

Intra- and interspecific transmission of androgenetic competence in diploid potato species

Summary In an attempt to determine the transmission of androgenetic competence, 10 families resulting from intra- and interspecific hybrids including three reciprocal hybrids were examined in anther culture. Hybrid families were generated between competent clones of Solanum phureja and incompetent clones of S. phureja, S. microdontum and S. berthaultii. S. phureja clones PP5 and A95 (derived by androgenesis of a 2n microspore of PP5) were found to be consistently competent for androgenesis. Androgenetic competence was observed to segregate in all hybrid families with some highly responsive and some unresponsive genotypes in all families. A total of 9,465 cultured anthers have yielded 936 embryoids and 91 plants, including 29 monoploids. The cytoplasm of species lacking competence appeared to have greater influence on the expression of androgenesis in intraspecific than in interspecific hybrids. Expression of androgenesis varied among half-sib hybrid families indicating that competence for androgenesis was influenced by the parents lacking competence. The anther culture data on a backcross between a highly responsive hybrid and its unresponsive parent indicated that competence may be under control of a single dominant gene.