粳稻花药培养基优化试验研究

培养基的筛选研究,是提高花药培养率的关键问题之一.国内外不少研究人员进行了大量工作,取得了一定的进展.我们自1986年开始以 N_6培养基为基础,对培养基原的各个组分进行全面的变量分析和主次因子的研究。我们已报道1987、1988年二个试验周期的结果.本文报道1989、1990年第三、四周期的试验结果。     

籼稻花药培养中简化培养基的研究

花药培养作为一种新的育种技术,已在我国广泛地进行了研究,但常用的合成培养基成份复杂,其中有些药物价格昂贵,且不易得到,加之配制手续繁琐,从而大大地限制了这一技术的应用与推广。为了大搞群众运动,使这一技术更好地运用于育种实践,为农业大干快上服务,我们在1974年早造开始进行籼稻花药培养的简化培养基研究,现将试验结果简要报道如下。     

不同培养基对187菌的生长发育及其对蚊幼虫毒力的影响

为了筛选适合187菌生长发育和发酵液高毒力效价的培养基,我们开展了不同培养基对187菌生长发育及其毒力的影响试验。其结果报道如下。     

无菌试验用培养基灵敏度比较试验

生物制品的无菌试验,是保证制品质量的重要措施之一。所用培养基灵敏与否对检查生物制品是否染菌十分重要。为比较不同蛋白胨不同基础液对培养基灵敏度的影响,一九七三年曾用5种细菌及12种培养基进行过灵敏度比较试验。此后,按照一九七三年全国生物制品无菌试验及试验用培养基经验交流会议建议,逐年分别在各所进行无菌试验用培养基质量汇检,为了配合这一工作,三年来,我们陆续进行了需气菌培养基及霉菌培养基的比较试验,结果归纳如下:     

低温预处理对籼稻花粉植株诱导的效应

为了提高籼稻花粉绿苗诱导率,我们于1974年开始在籼稻花药培养中进行低温预处理试验。先后对177个籼稻杂交组合花药低温预处理1—2天,除个别材料外,绝大多数材料在各种培养基上绿苗诱导率均有所提高。1978—1980年对接种材料低温预处理2—33天进行系统观察。其研究结果报道如下。     

小麦叶肉原生质体在马铃薯简化培养基上的分裂

近年来,在小麦、水稻花药培养的研究中, 使用了马铃薯简化培养基,已取得很大成 效^[1-3]。我们在大麦、小麦叶肉原生质体培养的 研究中,为了寻找能促使其再生细胞进行连续 的细胞分裂,除了采用各种合成培养基外,也使 用了马铃薯培养基。本文简要报道小麦叶肉原 生质体在马铃薯简化培养基中能有规则地进行 有限的几次细胞分裂的结果     

介绍一种玉米花药培养基

1978年我们在研究国内外优良培养基的各种成分配比的基础上,对培养基中5种大量元素进行了筛选。采用了5种元素4种水平的正交设计法试验[L_(16)(4~5)]。经过两年来的6次重复,41个处理的多次试验,筛选出一种适于花药培养的优良培养基,定名为“正14”。 1978年在正交试验的16种培养基上,接种玉米品种海淀八趟白,其结果以“正14”培养基的诱导频率最高,在试验的所有培养基中占首位。 1979年用“正14”培养基对玉米单三91,九单1×京黄13等11份材料进行试验,其平均诱导率为15.4％,绿苗诱导率为2.1％,而对照培养基N_6的愈伤组织诱导率和绿苗诱导率分别为5.4％和1.3％,     

低温预处理对釉稻花粉植株诱导的效应

为了提高釉稻花粉绿苗诱导率,我们于 1974年开始在釉稻花药培养中进行低温预处 理试验。先后对177个釉稻杂交组合花药低温 预处理1-2天,除个别材料外,绝大多数材料 在各种培养基上绿苗诱导率均有所提高。1978 一1980年对接种材料低温预处理2-33天进行 系统观察。其研究结果报道如下。     

幽门螺杆菌在不同培养基的生长情况

1983年Warren[1]和Marshall［2］报道了从慢性胃炎病患者冒粘膜中成功地培养出被命名幽门螺杆菌（HelicobacterPyloryHP）,引起了许多国家微生物学、病理学工作者的关注,并进行了大量的研究,同时建立了许多证明HP的方法。我们对HP培养基选择及方法进行了初步探讨,现将结果报道如下。1材料与方法1．1材料1．1．1培养基A:自配幽门螺杆菌培养基牛肉蛋白际及进口胰陈。抗菌素含量每100ml培养基中加TMP0．sg,多粘菌素B0．259,万古霉素1．og,两性霉素0．Zmp,加抗凝血或脱纤维羊血7～glnl。B：厌氧菌琼脂培养基（上海生物所出品）。…     

小麦根系联合固氮微生物的研究——1.菌种的分离和鉴定

我们从1979年研究小麦根系联合固氮的微生物,结果发现小麦根系固氮微生物有较高的固氮酶活力。本文报道从小麦根系固氮微生物中分离的菌株及其鉴定。材料和方法1.培养基:修改的Dbereiner无氮培养基     

Elevated carbon supply altered hypericin and hyperforin contents of St. John's wort (Hypericum perforatum L. cv `New Stem') grown in bioreactors

For the high frequency selection of salt-tolerant doubled haploids (DHs) using rice anther culture, the efficiency of anther culture was investigated with different genotype, media condition and NaCl concentrations. The six F₁ hybrids obtained by backcross or three-way cross between indica and japonica differed in salt tolerance. The efficiencies of callus induction and plant regeneration was decreased by NaCl concentration and salt tolerance of donor variety, and those in japonicas were higher than those in indicas. The percentages of callus induction in Gyehwa 5 (japonica, tolerant) and IR61633-B-2-2-1 (japonica, sensitive) were 21.1 and 13.5% on agar medium containing 0.3% NaCl, respectively. The plant regeneration of callus derived from anther floating culture in liquid media was less than that on solid medium. In four F₁ hybrids, the frequencies of high salt-tolerant DHs were 21.4 and 8.9% in 0.3% NaCl medium and the control, respectively. The high frequency of salt-tolerant DHs could be selected in the callus induction medium (0.3% NaCl) and in the combinations crossed with salt-tolerant japonica as the third parent.     

Induction of in vitro androgenesis in anther and isolated microspore culture of different spelt wheat (<Emphasis Type="Italic">Triticum spelta</Emphasis> L.) genotypes

This is the first report on isolated microspore culture—derived spelt wheat. The efficiency of anther- and isolated microspore was compared using four genotypes (‘Franckenkorn’, ‘GK Fehér’, ‘Mv Martongold’, ‘Oberkulmer Rotkorn’). In anther culture, genotype dependency was observed, and cold pre-treatment enhanced the efficiency of the method. In isolated microspore culture, the ovary co-culture supported the development of embryo-like structures. The presence of growth regulators (0.5 mg/l 2,4-D and 0.5 mg/l kinetin) were not essential for the induction of androgenesis, but these increased the production of embryo-like structures, green and albino plantlets. The low plant regeneration rate and high number of albinos hinder the practical application of isolated microspore culture while anther culture was efficient for in vitro green plantlets production in spelt wheat. The mean of green plantlets production was 41.45/100 anthers (from 20.93 to 83.07 depending on genotype). The phenomenon of albinism was mitigated in anther culture (3.48 albinos/100 anthers). Altogether, 1720 anther culture—derived green plantlets were produced from the four genotypes.     

Enhanced post-germinative growth of encapsulated somatic embryos of Siberian ginseng by carbohydrate addition to the encapsulation matrix

Based on a protocol for microspore culture in apple (Malus domestica Borkh.), the embryo induction phase has been improved with regard to pretreatment of microspores for initiation of microspore embryogenesis, the concentration of carbon source in the induction medium and the microspore density in the suspension. Furthermore, the effect of the genotype was studied. To determine the efficiency of in vitro androgenesis, both methods, via anther and microspore culture, were investigated using the same bud material. A comparison of the efficiency of embryo induction in anther and microspore cultures showed that microspore culture resulted in an increase up to 10 times, depending on the genotype. The regeneration route in microspore culture is similar to that of androgenic embryos via anther culture and showed adventitious shoot formation in most cases after a long period of secondary embryogenesis.Communicated by H. Lörz     

白桦单细胞培养体系的建立

以白桦花药（单核靠边期）、种子（萌发）及再生苗的茎段诱导愈伤组织,在NT1、NT2和B5三种不同的培养基中进行悬浮培养,筛选出优良的白桦悬浮体系,而后进行单细胞分离的结果表明,白桦花药愈伤组织在B5培养基上生长迅速,分散性好,适合于细胞分离。分离得到的单细胞用不同方式培养,建立了白桦单细胞两步培养法。     

Effects of n-butanol on barley microspore embryogenesis

Doubled haploid (DH) production is an efficient tool in barley breeding, but efficiency of DH methods is not consistent. Hence, the aim of this study was to study the effect of n-butanol application on DH barley plant production efficiency. Five elite cultivars of barley and thirteen breeding crosses with different microspore embryogenesis capacities were selected for n-butanol application in anther and isolated microspore cultures. Application of 0.1 % n-butanol after a mannitol stress treatment in anther culture significantly increased the number of embryos (up to almost twice) and green plants (from 1.7 to 3 times) in three low-responding cultivars: Albacete, Astoria and Majestic. No significant differences on microspore embryogenesis efficiency were observed in medium and high responding cultivars. The application of n-butanol treatment to isolated microspores from cold treated spikes in thirteen spring breeding crosses with a low or very low androgenetic response did not have a significant effect on the overall number of green plants. Nevertheless, an increase in the number of green plants was observed when 0.2 % n-butanol was applied in four out of seven low-responding crosses. Therefore, application of n-butanol could be routinely applied to anther cultures using mannitol treatment, in low-responding material. However, further studies are needed to determine optimal conditions in protocols using cold treatment and isolated microspore cultures.     

Increased doubled haploid plant regeneration from rice (Oryza sativa L.) anthers cultured on colchicine-supplemented media

Plating rice anthers on a semisolid induction medium containing 250 or 500 mg/l colchicine for 24 or 48 h-incubations followed by transfer to colchicine-free medium and standard anther culture procedures resulted in overall 1.5- to 2.5- fold increases in doubled haploid green plant productions compared to control anther cultures. The addition of colchicine had no detrimental effects on the different anther culture efficiency parameters, but in some treatments led to significant enhancement of anther callusing frequency or callus green plant regenerating ability. The most efficient treatment raised doubled haploid plant recovery from 31% to 65.5%. These results suggest that post-plating colchicine treatment of anthers, since it was found to improve both anther culture efficiency and doubled haploid plant recovery frequency, could be integrated into rice doubled haploid plant production programmes.Abbreviations DH doubled haploid - NAA naphthalenacetic acid - PAS periodic acid Schiff     

The influence of several factors on the efficiency of androgenesis in carrot

The influence of cultivar, donor plant and culture procedure on the efficiency of androgenesis was studied in carrot anther culture. Experiments were carried out on five carrot cultivars: CxC 9900 F1, Lucky B F1, HCM, Beta III and Perfekcja, which were chosen because of their high carotene contents. Two procedures of anther culture were compared: (1) incubation in darkness for two weeks, followed by exposure to continuous light and transfer onto a fresh medium of the same composition; and (2) incubation in darkness until embryos appeared, without transfer onto a fresh medium. Temperature was +27 degrees C all the time. Genotype played an important role in the process of androgenesis in carrot anther culture.The efficiency was the highest in cv. HCM - 5.6 embryos per 100 anthers. Considerable differences in the capacity for androgenesis were observed between individual donor plants. The ratio of embryos obtained per 100 anthers for cv. HCM varied from 0.0 to 48.9. The second procedure of anther culture proved to be more efficient, cheaper and less complicated.     

Antimitotic and hormone effects on green double haploid plant production through anther culture of Mediterranean japonica rice

Rice double haploid (DH) plants are produced mainly through anther culture. In order to improve the anther culture protocol, microspores of two japonica rice genotypes (NRVC980385 and H28) were subjected to three growth regulator combinations and four colchicine treatments on induction medium. In addition, a post anther culture procedure using colchicine or oryzalin was tested to induce double haploid plantlets from haploid plantlets. A cold pre-treatment of microspores for 9 days at 10 °C increased callus induction 50-fold in the NRCV980385 genotype. For both genotypes, 2 mg L^?1 2,4-D and 1 mg L^?1 kinetin on colchicine-free induction medium gave the best culture responses. The culturability of both genotypes changed on colchicine-supplemented induction media. A high genotype dependency was recorded for callus induction, callus regenerating green plantlets and regeneration of green double haploid plantlets. Colchicine at 300 mg L^?1 for 48 h enhanced callus induction 100-fold in H28. Colchicine-supplemented media clearly improved green double haploid plantlet regeneration. We showed that the post-anther culture treatment of haploid plantlets at 500 mg L^?1 of colchicine permitted fertile double haploid plantlets to be generated. Finally, an enhanced medium-throughput flow cytometry protocol for rice was tested to analyse all the plantlets from anther and post anther culture.     

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。     

Role of Cysteine in Enhancing Androgenesis and Regeneration of Indica Rice (Oryza sativa L.)

The effects of amino acid cysteine to culture systems of microspore-derived callus induction as well as plantlet regeneration were studied. Isolated pollen along with anther walls of basmati cultivars, Pusa basmati 1, Basmati 370 and Basmati 386 were cultured in a medium based on N₆ salts supplemented with or without cysteine following pollen embedment in agarose. The induction and regeneration medium with cysteine gave twice as effective androgenesis and plantlet regeneration in recalcitrant basmati rice cultivars as compared with medium lacking cysteine. Unlike the highly responsive model systems, most of the indica cultivars responded rather poorly in anther culture. So the study may accelerate the introgression of desirable genes into basmati rice using anther culture as a breeding tool. Response of microspores in androgenesis, plant regeneration and albinism was genotype specific. Regeneration of Indica rice varieties remains a limiting factor for researchers undertaking transformation experiments.