通过矿盐浓度的调整筛选小麦花药培养基

近年来,利用作物花药离体培养法诱导花粉植株,为进行单倍体育种提供了一种新的技术方法,并在某些作物的育种方面取得了一定成绩。就小麦的花粉单倍体培养来说,提高花粉愈伤组织诱导频率和分化频率是关系到这一方法能否应用于育种实践的关键,而培养基筛选的研究,又是提高花粉愈伤组织诱导频率和花粉植株分化频率的重要因素之一。朱至清等通过氮源调整试     

黑麦(Secale cereale L.)花药培养及其雄核发育

离体培养黑麦花药,单核花粉可以通过不等分裂和均等分裂由营养性质的核发育成花粉胚状体或愈伤组织,并且最后形成了单倍体植株。接种前对穗子进行适当的低温处理,可以有效地提高胚状体和愈伤组织的生成率。根据多核和多细胞花粉的败育情况,认为新壁的形成和花粉外壁的适时破裂是已经启动分裂的小孢子能否发育成胚状体和愈伤组织的两个关键问题。     

冬小麦的不同生育条件对其花粉植株诱导频率的影响

对三种不同生育条件的冬小麦的花药培养进行了比较,观察到它们对培养的反应有明显的和稳定的差异。经春化处理的冬小麦顶凌春播,5月中旬取花药做离体培养,比适时秋播,4月下旬进行离体培养的花粉愈伤组织和花粉植株的诱导频率提高约一倍;比冬天温室栽培,于2月下旬至3月上旬取花药做离体培养的愈伤组织诱导频率提高约二倍。1978年接种顶凌播种的28个杂交组合的冬小麦花药100％的组合诱导出了花粉愈伤组织,92.3％的组合分化出花粉绿苗。分析讨论了冬小麦顶凌播种诱导频率高的原因和它在冬小麦花粉单倍体育种研究中的应用     

小麦(Triticum vulgare)花粉植株的诱导及其形态发生过程的研究

采用离体培养小麦(Triticum vulgare)花药的方法成功地诱导小麦花粉形成了单倍体植株。在附加2—20毫克/升的2,4-D 和各种有机附加物的 MS 培养基上,属于27个杂种或品种的21094个花药中有103个花药产生了愈伤组织,这些愈伤组织均着生在裂开的药室内,显微观察证明它们是由花粉经过多次细胞分裂形成的。花粉愈伤组织转移到含有0.2—2毫克/升的萘乙酸和0.2—2毫克/升的激动素或含有0.5毫克/升的吲(口朶)乙酸和15%椰乳的 MS培养基上培养5天以后,即陆续分化出幼苗。此外还发现接种在含有20%椰乳或吲(口朶)乙酸及激动素各2毫克/升的 MS 培养基上的花药直接从药室内产生出幼苗。已检查的6株幼苗的根尖细胞的染色体数均为21,表明它们是单倍体。单倍体植株能够抽穗而不结实。     

从“湖北光敏感核不育水稻”的未受精子房和花药培养出单倍体植株

采用10种诱导培养基,培养湖北光敏感核不育水稻农垦58品种的未受精子房和花药。共培养未受精子房2790个,获得胚囊愈伤组织17块,最高诱导频率达3.33%,其中2块分化出绿苗。培养花药16740个,获得花药愈伤组织15块,最高诱导频率为0.92%,其中3块分化出苗,2丛白苗,1株绿苗。胚囊植株和花粉植株经根尖染色体检查为单倍体,2n=x=12。实验证明,液体培养、2,4-D0.2-0.5 mg/1、低温预处理对诱导胚囊愈伤组织及花粉愈伤组织的形成具良好效果。     

平贝母花粉植株的诱导及无性系的建立

采用附加植物激素的MS、N_6、百合、米勒和改良怀特培养基,培养平贝母单核中期的花药,诱导出一些愈伤组织(平均诱导频率为0.20％),其中MS培养基的诱导效果最好。绝大多数再生植株出现在不加任何激素的1/2MS(其大量元素的含量是MS的一半)培养基上。再生植株的根尖细胞的染色体计数表明,约有1/4的细胞2n=12,证明再生植株是来源于花粉细胞的单倍体植株。在愈伤组织的分化培养中,建立起了能继代繁殖、不断保持绿苗分化能力的愈伤组织无性系。     

从一粒花粉到一棵植株

花粉单倍体育种中,花药离体培养能否成功的关键之一是花粉的年龄。自然条件下花粉最初只有一个核,称为单核花粉,以后经过一次有丝分裂产生一个营养核和一个生殖核,称为两核花粉。营养核不再分裂,生殖核再经一次有丝分裂成两个精子,称为三核花粉。诱导单倍体最好用单核中晚期(单核靠近质膜时)的花粉。取下合适的花蕾或幼穗先经消毒灭菌,再在无菌条件下取出花药,接种到培养基上,培养条件对花粉的发育有十分明显的影响,一般进行花药培养的温度约在23—28℃之间,需给于适当的光照。由花粉长成单倍体植株一般有两条途径:一是由花粉分裂形成愈伤组织,再将愈伤组     

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。     

从大麦雌配子体诱导单倍体植株

近年来,应用离体培养花药的方法诱导花粉单倍体植物的研究得到迅速发展,但从雌配子体系统诱导单倍体植物的研究却作得很少,进展也不大。在裸子植物上,Tulecke曾离体培养未受精的银杏雌配子体,得到单倍体愈伤组织,但没有分化成植株。在被子植物上,用未受精的胚珠或子房进行培养,Uchimica 得到茄子胚珠的单倍体愈伤组织,Jensen 则在棉花的胚珠中看到极核的融合及二倍体胚乳的分裂,但都没有进一步的结     

改进水稻花药培养方法的途径

用液面漂浮方式培养水稻花药,研究了培养基的蔗糖浓度、激素成分、附加秋水仙碱对花粉愈伤组织的诱导、分化及花粉植株倍性的影响。提出改进水稻花药培养方法的途径,其要点是:用液体培养基培养花药;在培养初期,调整培养条件以增加对等核分裂花粉数和提高染色体加倍频率;在多细胞团花粉突破花粉壁生长之前,将其从开裂的花药中分离出来进行单花粉固着培养形成愈伤组织,并使愈伤组织在生长的早期转向分化。     

Production of haploids of neem (<Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss.) by anther culture

Androgenic haploids of the neem tree (Azadirachta indica A. Juss.) were produced by anther culture at the early- to late-uninucleate stage of pollen. Haploid formation occurred via callusing. The best medium for inducing callusing in the anther cultures was Murashige and Skoog's basal medium (MS) (9% sucrose) supplemented with 1 microM 2,4-D, 1 microM NAA and 5 microM BAP, while anther callus multiplied best on MS medium supplemented with 1 microM 2,4-D and 10 microM Kn. These calli differentiated shoots when transferred to a medium containing BAP; 5 microM BAP was optimum for young calli (75% cultures differentiated shoots), but older calli showed the best regeneration with 7.5 microM BAP. Shoots elongated at a lower concentration of BAP-0.5 microM. These shoots were multiplied by forced axillary branching and rooted in vitro. The plants were subsequently established in soil. Of the plants that regenerated from anther callus 60% were haploid, 20% were diploid and 20% were aneuploid.     

Improved callus formation and plant regeneration for shed microspore culture in asparagus (Asparagus officinalis L.)

 To establish an efficient asparagus microspore culture system, experiments were conducted to investigate the effects of medium components, period of cold pretreatment for flower buds, and period of anther co-culture on culture response. All factors affected the frequency of asparagus microspore division and the yields of microspore-derived calli. The best results were obtained by pretreating genotype G459 flower buds at 4  °C for 7–9 days, co-culturing anthers with shed microspores for 14 days, and including 6% sucrose, 2 mg l^–1α-naphthaleneacetic acid and 1 mg l^–1 N⁶-benzylaminopurine in the culture medium. After 4 days of culture, most shed microspores contained starch-like bodies and died. The 2% of shed microspores lacking these structures divided to produce microcalli. For the best treatments in the different experiments, about 140 calli per 100 anthers were recovered. Cultured on four different regeneration media, 19.6–21% and 3.9–8.0% of microspore-derived calli produced shoots and embryos, respectively, and ultimately plantlets, among which 49% were haploid, 34% diploid, 4% triploid and 11% tetraploid. Received: 3 September 1998 / Revision received: 25 November 1998 / Accepted: 5 December 1998     

High frequency production of haploid embryos in asparagus anther culture

Summary A method for obtaining a high frequency of haploid asparagus embryos through anther culture was developed. Flowers collected from plants in the field in July, August and September 1990, for the genotype G203, were stored at 5°C for 24 h. Anthers were placed on Murashige and Skoog medium (MS) containing 500 mg l ^–1 casein hydrolysate, 800 mg l^–1 glutamine, 2 mg l ^–1 NAA, 1 mg l ^–1 BA and 5 % sucrose at 32 °C in the dark for three to four weeks to induce calli. Calli were then grown at 25 °C with a 16 h photoperiod for three to four weeks. Developing embryos and calli were transferred to embryo maturation medium, MS containing 6% sucrose, 0.1 mg l ^–1 NAA, 0.1 mg l ^–1 kinetin and 0.65 mg l ^–1 ancymidol, for four weeks. More than 50% of the recovered mature embryos germinated on MS containing l mg l ^–1 GA₃. Anthers with microspores at the late-uninucleate stage had the highest frequency of total and embryogenic calli formation, 40% and 15%, respectively. Each embryogenic callus usually produced 10–15 embryos. Aproximately 75 plants per 100 anthers cultured were recovered: 76% haploid, 22% diploid and 2% triploid. High temperature was critical for the induction of embryogenic callus.Abbreviations NAA naphthaleneacetic acid - BA 6-benzylaminopurine - MS Murashige and Skoog (1962)     

In vitro induction of androgenic haploids in Safflower (Carthamus tinctorius L.)

The influence of culture medium on induction of androgenic calli was examined with five different basal media. MS medium was the most responsive in inducing callus. Differences in induction of calli among ten genotypes revealed that the most responsive genotype was a local cultivar, Mangira, with 48.6% anthers initiating callus formation. The influence of temperature pre-treatment (5°±1°C) for varying periods (0 to 15 days) on immature capitula prior to inoculation of anthers on the medium revealed that the percentage of anthers inducing callus increased till 3–5 days of pre-treatment. The effect of physiological conditions of anther donor plants grown in the field and in green houses on induction and re-differentiation have shown that the field grown anther donor plants exhibited optimum response. Shoot regeneration was observed on MS supplemented with BAP (2.0 mg/l) and NAA (0.5 mg/l) and rhizogenesis on MS (half-strength) medium, supplemented with NAA (0.1 mg/l) and 1% sucrose. Cytological studies of anther derived plants showed two ploidy levels, where the haploids were predominant (64%).     

Studies on the Induction of Pollen Sporophyte of Coix lacryma Jobic L.

The developmental pathways of pollen sporophyte in anther culture of Coix were observed. The types of androgenesis are different, and are relative to the degree of the differentiation in pollen cells. Pollen-inductors develop via multicellular mass into embryoids or calli. Both of them can develop into plantlets, but the frequency of the regeneration of the plantlets in calli is higher than that of in embryoids, because there are a lot of aberrant embryoids in the latter, which cannot develop further. It was found that the induction frequency of the polleninductors can be increased apparently by the pretreatment in a short time with a hypertonic sucrose solution. The chromosomes of the somatic cells in l0 plantlets were examined. It was found that all the plantlets derived from the embryoids are haploids, while there are haploid, diploid and mixoploid in the plantlets from the calli. The effects of anther wall cells and the stability of haploid cells were discussed.     

Efficient production of doubled haploid plants through colchicine treatment of anther-derived maize callus

Summary A chromosome doubling technique, involving colchicine treatment of an embryogenic, haploid callus line of maize (Zea mays L., derived through anther culture), was evaluated. Two colchicine levels (0.025% and 0.05%) and three treatment durations (24, 48, and 72 h) were used and compared to untreated controls. Chromosome counts and seed recovery from regenerated plants were determined. No doubled haploid plants were regenerated from calli without colchicine treatment. After treatment with colchicine for 24 h, the callus tissue regenerated about 50% doubled haploid plants. All of the plants regenerated from the calli treated with colchicine for 72 h were doubled haploids, except for a few tetraploid plants. No significant difference in chromosome doubling was observed between the two colchicine levels. Most of the doubled haploid plants produced viable pollen and a total of 107 of 136 doubled haploid plants produced from 1 to 256 seeds. Less extensive studies with two other genotypes gave similar results. These results demonstrate that colchicine treatment of haploid callus tissue can be a very effective and relatively easy method of obtaining a high frequency of doubled haploid plants through anther culture.     

Haploid and diploid plant regeneration from protoplasts of anther callus in rice

Summary The regeneration of haploid and diploid plants was demonstrated from protoplasts that were isolated from cell suspensions of anther callus in rice. The cell suspension in the AA medium that contained 4 amino acids as the sole nitrogen source was friable, finely dispersed, and readily released a large number of protoplasts. These protoplasts, subsequently cultured in NO₃ medium that contained nitrate as the sole nitrogen source, formed compact calli. The compact calli produced green plants with a frequency of 24%. Out of 15 flowering plants, 4 were haploids, the others were diploids which showed a uniform morphology but varied in seed fertility from 95 to 0%.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid     

骆驼蓬的组织培养及植株再生

以骆驼蓬(Peganum harmala L)无菌苗下胚轴切段为材料,在不同的培养基上进行愈伤组织的诱导,发现在MS基本培养基附加2．0mg/L 2,4—D、0．5mg／L 6—BA和3％蔗糖时,可100％的诱导出愈伤组织。愈伤组织在附加2．0mg／L 6—BA、0．5mg／L NAA、500mg／L CH和3％蔗糖的MS培养基上诱导出丛生芽,进而发育成苗,苗的分化频率在30％左右。分化苗或其茎切断在附加0．2mg／L IBA、0．2mg/L NAA和3％蔗糖的l／2MS培养基上出现根的分化,分化频率在90％以上。再生植株经炼苗后移栽成活,成活率在80％以上。     

Assay for doubled haploid sunflower (Helianthus annuus) plant production by androgenesis: fact or artifact? Part 1. In vitro anther culture

Sunflower anthers placed on solid medium developed calli and embryos after 12 days. Embryogenesis was improved by the addition of 0.1% polyvinylpyrrolidone (PVP) that alleviated anther and medium browning. As in other species, genotypic variability was an important parameter in the anther response and a medium genotype interaction was suggested with a different PVP effect depending on the genotype. Embryo germination was largely increased by the successive use of germination media with decreasing sucrose concentrations (10%6%3%). Histological examination of the anthers during the first ten days of culture showed that, under our conditions, the embryos were of somatic origin, arising directly from the anther wall on the outside or inside of the anther loculus, or indirectly from proliferating anther wall- or connective tissue-derived callus. Finally, the ploidy status of 78 embryo-derived plants was determined by Feulgen stain or flow cytometry: all plants were diploid (2n=34).Abbreviations PVP polyvinylpyrrolidone     

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.