用花药培养法由马铃薯雄性不育双半日倍体诱导—单倍体植株的研究

用花药培养法由雄性不育材料诱导单倍体的尚未见报道,在马铃薯中,所有双单倍体或一单倍体,也都是由有效花粉百分数较高的雄性可育材料诱导产生的,本文首次报道了用花药培养法由一个典型的雄性不育的双单倍体吕系诱导产生一单倍体的试验结果。从接种的1850个花药中,诱导出了28个胚状体和23块愈伤组织,并从它们分化出了24株绿苗,分化出的小植株生活力大部分较弱,在继代培养过程中逐渐死亡,但也有一些生活力较强的植株存活下来,经L1,L2和L3 3个胚层细胞的检查表明,它们具有典型的一倍体特征,体细胞染色体数目为2n=x=12,植株之间表现出明显的性状分离,说明它们均来自减数分裂后的小孢子,大部分植株的产量较低,但也有少数生活力强,单株块茎产量达0．5公斤左右的抗病,品质好的植株,所有株都具有不正常的减数分裂,无有效花粉形成,个别植株能结实,但无种子形成,用四倍体或双单倍体的可育花粉授粉也不能形成种子,进一步证明了它们具有一单倍体和持性,对放有导马铃薯一单倍体和利用雄性不育材料进行花药培养诱导倍体的意义进行了讨论。     

马铃薯未传粉子房离体培养诱导双单倍体植株

以MS为基本培养基,附加不同水平的生长素,培养未传粉马铃薯子房,经三年试验,从两个品种中获得了双单倍体的绿色小植株,(2n=2x=24);从分化绿苗力很强的球状愈伤组织中,又不断地分化出许多绿色小植株。绝大多数品种,都可以诱导出愈伤组织,一般诱导率为70％左右。品种的基因型和培养基中的生长素种类与水平在愈伤组织分化绿苗中起着重要作用。通过扦插和试管微型薯培养,可以大量繁殖试管苗,这为马铃薯单倍体育种提供了较有利的条件。     

激光诱导杨树花粉单倍体植株的研究

用Ｈｅ－Ｎｅ激光适宜剂量２０Ｊ．ｃｍ＾－２诱导杨树花药培养花粉单倍体植株及其无性系获得成功。     

花粉单倍体的遗传品质——花药培养是否降低了育种材料的农艺价值

自S.Guha和S.C.Maheshwari 1964年以曼陀罗(Datura)花药为材料,首次成功地诱导出离体幼胚以来,人们经过不断的发展和完善使之形成了一套完整的花药培养技术,即通过植物花药(或小孢子)的培养,大批创制单倍体并经染色体加倍进而获得双单倍体(Doubled haploid)植株。该技术被迅速而广泛地应用于作物育种程序之中。目前已有不少的栽培作物成功地获得了花粉植株,如:烟草、小麦、大麦、水稻、黑麦、小黑麦、玉米、甘     

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。     

水稻单倍体、二倍体和三倍体的同工酶研究

通过花药培养得到的单倍体植株生活力比较低,白苗率较高。在花药培养中有大量的愈伤组织甚至不分化出根和芽,不能形成植株,严重地妨碍了花药培养应用于育种实践。特别是秈稻品种通过花药培养得到的植株成功率很低,急待提高。     

大麦×小麦杂种愈伤组织及再生植株的细胞学观察

本文研究了Ant-13大麦×中国春小麦未成熟胚诱导的愈伤组织细胞,再生植株体细胞和花粉母细胞染色体的变化情况。发现在这三个不同分化和发育阶段中都存在着混倍体现象。但随着分化和发育过程的进行,混倍体程度越来越小,正常双单倍体细胞的比例越来越大。从再生植株花粉母细胞减数分裂前的有丝分裂开始到整个减数分裂过程中都可以看到染色体行为的异常现象,从而形成败育花粉,造成杂种不孕。花粉败育发生在单核小孢子时期。     

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。     

烟草花粉单倍体育种的几个遗传学问题研究

烟草花药培养成功之后,国内外育种工作者用这一方法在烟草育种上做出了贡献,并在培养技术、染色体加倍方法、单倍体和加倍单倍体特性、花粉单倍体植株性状遗传变异及生活力等方面都进行了不同程度的研究。然而对花粉单倍体植株的遗传学问题,目前报道的为数不多,尚有不少有待进一步探讨的问题。     

小麦花粉植株的遗传学研究

自1971年4月我们用花药培养方法诱导出小麦单倍体花粉植株以来,国内外学者在小麦花药培养研究中作了大量工作,诸如:离体条件下的雄核发育的研究,提高诱导花粉植株频率的各种因素的研究,其中包括培养基的改进、材料基因型差异在诱导中的作用和培养条件,以及供取花药植株的环境条件的研究等。本文拟在多年研究的基础上,对小麦花粉植株的遗传学表现,包括目前大家所关心的花粉植株的生活力,以及花粉植株的变异等问题做进一步的探讨。     

Chromosome doubling in monohaploid and dihaploid potatoes by regeneration from cultured leaf explants

Plants were regenerated from cultured excised leaf segments of monohaploid (2n=x=12) and diphaloid (2n=2x=24) potato (Solanum tuberosum L.) and a sample has been studied cytologically. In the case of monohaploids, a single leaf regeneration cycle resulted in almost total recovery of doubled monohaploid plants (2n=2x=24), whilst 50% of the plants regenerated from doubled monohaploid leaves had doubled again to the doubled double monohaploid (or homozygous tetraploid, 2n=4x=48) level. Regeneration from dihaploid leaf pieces also gave a good proportion (60%) of doubled genotypes. Very few mixoploids and very few aneuploids were found. These results, together with the general applicability of the method to a large number of potato cultivars, suggest that it can be used as a simple and reliable method of obtaining homozygous tetraploid potatoes.     

Production of potato monohaploids (2n=x=12) through prickle pollination

Summary Data are presented on the potential of gynogenesis for the production of monohaploids and on factors affecting their frequency and relative vigour. Diploid Solanum tuberosum L. and S. tuberosumxS. phureja Juz et Buk hybrids were used as maternal parents and selected S. phureja clones as prickle pollinators with embryo-spot and nodal band as dominant seed and plant marker. About 2 million seeds were screened for absence of embryo-spot. After raising plants from phenotypically spotless seeds, further screening for absence of nodal bands and for ploidy level was carried out. Finally more than 500 monohaploid plants from three genetically different groups of maternal parents were obtained. Frequency and vigour of the monohaploids were clearly dependent on their maternal genotypes. The data also indicated an effect of the pollinator genotype, the physiological stage of the maternal plant and the environment on monohaploid frequency. On the basis of these results the possibility of breeding for a higher monohaploid production rate and for more stable and vigorous monohaploids is discussed. Furthermore, gynogenesis and androgenesis are compared. It is suggested that both should be used in order to obtain monohaploids from sufficiently various diploid breeding material.     

Breeding for nematode and virus resistance in potato via anther culture

Summary In Solanum tuberosum the production by parthenogenesis of 2x plants with 24 chromosomes, and the regeneration of microspores of such dihaploids to yield monohaploid (1x) plants is reproducibly possible, at least for some specific genotypes. Experiments are described using tissue culture techniques in an applied breeding program with the main aim of increasing the level of resistance to the potato cyst nematode Globodera pallida (Stone) and to the potato viruses X, Y and leaf roll. These resistances follow quantitative as well as qualitative modes of inheritance. Using anther culture it is demonstrated that doubled monohaploid clones can be produced which possess the resistance in the homozygous condition. In both ways of inheritance the ratio of resistant clones is rather high. The genotype of the anther donor plant has, however, a strong influence on the total number of androgenetic plants which can be regenerated. Therefore, experiments were initiated with the aim of integrating this capacity for regeneration (tissue culture ability) into valuable genotypes. The results show that the potentiality for regeneration is under genetic control and can be utilized by combination breeding. Its inheritance and physiological basis, as well as the behaviour of complete homozygous clones, is discussed.Dedicated to Prof. Dr. Joseph Straub on the occasion of his 70. birthday     

Isolation and characterization of potato diploid clones generating a high frequency of monohaploid or homozygous diploid androgenetic plants

Summary Cultured microspores of diploid potato clones lead with high frequency to diploid regenerants. In this paper we report on the genetic variability for in-vitro monohaploid production from anthers of diploid plants. Three diploid genotypes have been isolated which combine the capacity to regenerate monohaploid plants with outstanding embryoid production. A trait of the anther-donor clones associated with the generation of monohaploid plants is the low production of 2n pollen grains. When present in anthers of diploid genotypes, diploid unreduced microspores are, in fact, derived mainly from a first division restitution mechanism leading to high heterozygosity of the derived embryoids, a state which apparently supports superior growth in-vitro. Also, reduced microspores have been found capable of generating diploid regenerants and the adoption of the RFLP technique allowed the isolation of such diploid plants, which can be considered to be pure lines. Donor clones with a low capacity to generate monohaploids are, as expected, poor producers of homozygous diploid plants. The selection of an anther donor producing a sufficient number of monohaploid or homozygous diploid regenerants fulfills the requirements of the first part of the analytical breeding scheme, i.e., the production of homozygous diploid clones.     

Induction conditions for somatic and microspore-derived structures and detection of haploid status by isozyme analysis in anther culture of caraway (Carum carvi L.)

Plant regeneration was obtained from cultured anthers and hypocotyl segments of caraway (Carum carvi L.). Microspore- and somatic tissue-derived embryos were compared by observation of the regeneration process under identical induction conditions. Fluorescent microscopy with DAPI staining showed initiation of cell divisions and formation of embryogenic callus and somatic embryos from anther sacs, with production of embryos of both microspore and somatic origin. Induction of somatic embryos from hypocotyl-derived callus was also demonstrated. Isozyme native polyacrylamide gel electrophoresis was used to identify haploids and doubled haploids, and to determine the frequency of spontaneous diploidization of regenerated plants of microspore origin. Donor plants (2n = 20) and their anther-derived derivative plants (n = 10, 2n = 20, 4n = 40) in callus stage or leafy rosette stage were compared. The esterase (EST) band patterns of regenerated plants differed from the heterozygous parental material, suggesting that the regenerated plants were microspore-derived haploid/doubled haploid plants. The similar profile of EST bands between the diploid anther-derived plants and a sample of the donor plants corresponded to a somatic regeneration pathway. Although the selected induction conditions revealed no preference for induction of microspore embryogenesis, the anther culture protocol established for caraway utilizing isozyme segregating EST loci markers is suitable for DH production.     

Polyploidization in leaf callus tissue and in regenerated plants of dihaploid potato

Cytological studies on leaf callus cells and regenerated potato plants suggest that it may be possible to utilize somatic chromosome doubling to obtain tetraploids from outstanding dihaploid breeding clones. The ploidy levels found in callus-derived plants were diploid, tetraploid, and octaploid, but the proportion of these was dependent on the donor genotype. L₁ and L₃ germ layers were studied in more than 300 plants; periclinal ploidy chimerism, an undesirable feature of colchicine doubling, was not found. Leaf callus was more efficiently induced using NAA than 2, 4-D as an auxin source in the Murashige and Skoog medium. A high proportion of dividing cells in young calli were polyploid. The frequency of doubled and octaploid plants regenerated was significantly dependent on donor genotype. The extent of polyploidization was marginally higher after callus growth on a medium containing 2, 4-D than in a medium containing NAA. In some genotypes the chromosome numbers of regenerated plants were variable, being less than tetraploid (mixohypotetraploid). After tuber propagation, the original ploidy level was maintained although mixohypotetraploidy persisted. In a few somatically doubled clones, male fertility was tested and found to be satisfactory with respect to seed-setting.     

烟草属花粉—叶肉原生质体的融合及杂种植株再生

用饥饿预处理分离烟草（Ｎｉｃｏｔｉａｎａ ｔａｂａｃｕｍ Ｌ．）品系Ｎ３６４ Ｋｍ ＋ 二核早中期的花粉原生质体,用ＰＥＧ－高钙高ｐＨ 法诱导其与黄花烟草（Ｎ．ｒｕｓｔｉｃａ Ｌ．）叶肉原生质体融合。通过抗性筛选再生的４ 株小植株,经过氧化物酶同工酶、根尖染色体计数鉴定均为配子－体细胞三倍体杂种。未经筛选处理再生的２１株小植株,经鉴定有６ 株为配子－体细胞杂种。     

Production of doubled-haploid plants from tritordeum anther culture

The effects of different media and cold pretreatment of spikes on the androgenic response and regeneration capacity from anther culture of tritordeum was studied. L5 medium gave the highest frequency of anther response. The frequency of cultures regenerating green or albino plantlets was not affected by the composition of the medium tested. Cold pretreatment of the spikes significantly increased the frequency of anther response and also the percentage of cultures giving albino plantlets. A mean of four green plants was obtained per 100 subcultured calli/embryos. The percentage of spontaneous chromosome doubling was only 1%. The addition of colchicine at 0.02% to the induction medium significantly increased the frequency of doubled haploids regenerated without any effect on regeneration capacity. This technique proved more efficient than a conventional chromosome-doubling method.     

Mixing of maize and wheat genomic DNA by somatic hybridization in regenerated sterile maize plants

Intergeneric somatic hybridization was performed between albino maize (Zea mays L.) protoplasts and mesophyll protoplasts of wheat (Triticum aestivum L.) by polyethylene glycol (PEG) treatments. None of the parental protoplasts were able to produce green plants without fusion. The maize cells regenerated only rudimentary albino plantlets of limited viability, and the wheat mesophyll protoplasts were unable to divide. PEG-mediated fusion treatments resulted in hybrid cells with mixed cytoplasm. Six months after fusion green embryogenic calli were selected as putative hybrids. The first-regenerates were discovered as aborted embryos. Regeneration of intact, green, maize-like plants needed 6 months of further subcultures on hormone-free medium. These plants were sterile, although had both male and female flowers. The cytological analysis of cells from callus tissues and root tips revealed 56 chromosomes, but intact wheat chromosomes were not observed. Using total DNA from hybrid plants, three RAPD primer combinations produced bands resembling the wheat profile. Genomic in situ hybridization (GISH) using total wheat DNA as a probe revealed the presence of wheat DNA islands in the maize chromosomal background. The increased viability and the restored green color were the most-significant new traits as compared to the original maize parent. Other intermediate morphological traits of plants with hybrid origin were not found.     

Tuber induction and initiation during production and early field growth of transplants from in vitro-derived potato plants

In transplants from in vitro‐derived plantlets from very early potato (Solanum tuberosum) cultivars, a lower degree of tuber induction at the time of field planting is thought to increase tuber production. Leaf‐bud cuttings were used to assess the progress to tuber induction in in vitro‐derived potato plantlets during the transplant production phase and after subsequent transplanting into the field. Induction and initiation of tubers on the same plants were assessed to study the effects of the duration of transplant production and conditions during transplant production for cv. Gloria (very early) and cv. Bintje (mid‐early). In vitro‐produced plantlets were not induced by the time of planting but rapidly progressed to the induced state thereafter. The progress in induction with time and the change in percentage of plants showing tubers fitted typical sigmoid curves. Plantlets achieved 50% induction ca 15 days after planting into in vivo conditions, and 50% tuber initiation usually occurred 10 days later. Shorter transplant production periods reduced the degree of induction of the transplants at field planting. Transplant production for more than 2 weeks was required to allow conditions during that period to affect induction or initiation. Long‐term non‐inducing conditions delayed the progress to tuber induction in cv. Gloria and delayed tuber initiation in both cultivars. Cv. Gloria showed no faster progress to induction than cv. Bintje but initiated tubers earlier. The results suggest that the relation between progress to induction and tuber initiation is cultivar dependent and that leaf‐bud cuttings can be used successfully in very young in vitro‐derived plants for assessing the progress to tuber induction.