小麦遗传转化受体系统建立的研究

选用‘小偃22’和‘宁春16’小麦品种的成熟胚和幼胚进行培养,研究不同种类的胚和培养因子对愈伤组织诱导和分化的影响。结果表明,幼胚和成熟胚的愈伤组织诱导率无明显差异,但较高浓度的2,4-D有利于成熟胚的诱导,而幼胚培养时2,4-D浓度的影响效果因品种而异;两种外植体分化率的高低与KT/IAA的配比均有密切关系,但高浓度的激素水平不利于成熟胚的分化;诱导培养基中低浓度的2,4-D有利于所诱导的愈伤组织的分化。同时,在诱导培养基中添加低浓度的KT能显著提高两品种成熟胚愈伤组织的分化率;各种培养基处理与品种间都存在显著的互作效应,‘小偃22’成熟胚培养的最佳培养基组合为MSD 3.0 mg/L 2,4-D和MSD 0.5 mg/LIAA 1.0 mg/L KT,幼胚培养为MSD 4.0 mg/L 2,4-D和MSD 0.5 mg/L IAA 1.0 mg/L KT;‘宁春16’成熟胚培养为MSD 4.0 mg/L 2,4-D和MSD 1.0 mg/L IAA 1.0 mg/L KT,幼胚培养时为MSD 1.0 mg/L 2,4-D和MSD 2.0 mg/L IAA 2.0 mg/L KT。     

植物体细胞胚状体与器官发生的激素调节

研究了外源激素对胡萝卜、矮牵牛、石刁柏以及烟草、曼陀罗、芹菜等植物体细胞胚状体与器官(主要是不定芽)发生的调节作用。1.胚状体的诱导在基本培养基中所需生长素都比不定芽分化所需生长素浓度高,或再需补加少量细胞分裂素。不定芽的分化在基本培养基中所需细胞分裂素都比胚状体的诱导所需细胞分裂素浓度高,或再补加适量生长素。少量细胞分裂素对石刁柏E_3无性系胚状体发生有强烈的促进作用。2.在一定的激素条件下,石刁柏E_3、B_2茎尖无性系可以在同一块愈伤组织上发生胚状体、不定芽,有时还有根。改变培养基中激素的量,可使愈伤组织只发生胚状体或芽。3.IAA、NAA和KT、BA、Z(玉米素)对诱导石刁柏E_3系体细胞胚状体与不定芽都有效,2,4-D和KT对诱导石刁柏B_2系体细胞胚状体与不定芽都有效。4.石刁柏个体之间胚状体与不定芽发生能力差异很大,有三种以上不同的类型。胡萝卜下胚轴只能发生胚状体,球形胚发生的愈伤组织既能产生胚状体又能发生不定芽。     

甘露糖对大麦品种不同外植体生长的影响

以大麦栽培品种的茎尖、成熟胚为外植体,研究了不同甘露糖浓度对这些外植体愈伤组织诱导和生长的影响。结果表明:甘露糖浓度为20 g/L时,茎尖的叶片伸长和生根受到明显抑制;甘露糖浓度为10 g/L或15 g/L时,成熟胚的愈伤组织诱导率降低50%,甘露糖浓度为20 g/L或25 g/L时,成熟胚愈伤诱导和生长完全受到抑制,因此在以大麦茎尖和成熟胚为外植体的磷酸甘露糖异构酶(PM I)/甘露糖筛选中,可分别以20 g/L、25 g/L甘露糖作筛选压。另外,在培养早期阶段筛选比较适宜。     

单倍体水稻体细胞组织的植株再生及其激素调节

本文报道激素对调节单倍体幼穗组织去分化与分化的方向以及器官形成的影响。发现在试验浓度内(2毫克/升),2,4-D诱导组织去分化,NAA诱导根的大量形成,KT抑制愈伤组织形成和器官分化。KT2毫克/升 NAA2毫克/升使外植体不经过愈伤组织阶段就直接分化大量的苗。当KT/NAA=2:2时,直接分化苗的频率较高,达76％,不同浓度的2,4-D试验表明,2,4-D2毫克/升或4毫克/升时,愈伤组织诱导率高达94％以上。固体和液体继代培养中,低浓度的2,4-D(0.5—0.1毫克/升)加0.1毫克/升的KT,对愈伤组织保持旺盛的生长和后来的分化有良好的作用。发现单倍体体细胞组织再生的植株,白苗很少。讨论了单倍体体细胞愈伤组织无性系用于诱发突变和体细胞遗传研究的可能性。对于愈伤组织的再分化,不仅需要细胞分裂素,而且诱导培养基中生长素浓度的影响也是显著的。     

水稻组织培养中影响植株再生因素的研究

水稻组织培养开始于幼胚培养,一般认为禾谷类的组织和细胞培养要比双子叶植物困难,但有关水稻组织培养成功的例子却不少,不少人用去壳的种子、胚、根、和花粉诱导发生愈伤组织,获得了再生植株。日本古桥等人报导从根、胚乳、茎节诱导发生愈伤组织,并分化出植株,印度Bajaj等从水稻未成熟和成熟胚乳培养物获得三倍体植株。在我国,对水稻幼茎、幼穗、叶鞘、枝梗和茎尖的组织培养开展了研究,并获得成功。本试验对水稻的幼穗、幼茎、幼根和幼叶进行了离体培养,并研究了影响愈伤组织发生和分化的若干因素。     

蔗糖与激素对小麦幼穗体细胞无性系形成及生长特性的影响研究

通过在诱导培养基中添加不同浓度的蔗糖和激素,研究了蔗糖与激素对小麦幼穗体细胞无性系形成及生长特性的影响。结果表明;较高浓度的2,4-D有利于小麦幼穗愈伤组织的形成与生长,但低浓度的2,4-D更有利于胚性愈伤组织的形成,低浓度的KT能显著促进小麦幼穗愈伤组织的生长和胚性愈伤组织的形成,而高浓度的KT不利于小麦幼穗体细胞无性系的形成和生长;蔗糖有利于小麦幼穗胚性愈伤组织的形成,在2.5%-5.5%浓度范围内,随蔗糖浓度的提高,胚性愈伤组织的形成率也随之提高。     

一品红体细胞胚胎发生与植株再生

一品红不同部位愈伤组织诱导能力存在差异,嫩茎>幼花序>嫩叶。愈伤组织的长势主要受生长素的影响,细胞分裂素对愈伤组织生长有促进作用;但在含6-BA和NAA的培养基中诱导出的愈伤组织,其胚性明显强于单独用NAA诱导出的愈伤组织。液体悬浮培养是一品红体细胞胚胎高频发生的中间步骤。不同浓度BA对一品红体细胞胚的萌发率影响不大,萌发培养基中KNO₃含量加倍可提高萌发率。     

影响水稻幼穗培养体细胞胚胎发生因素的研究

用水稻幼穗为外植体进行组织培养,研究了影响其体细胞胚胎发生的有关因素,建立了高频率体细胞胚胎发生的培养程序。结果表明不同基因型之间体细胞胚胎发生率的差异达到61．2％;生长素2,4－D对诱导水稻体细胞胚起重要的调控作用,细胞分裂素BA有一定的协同促进作用。干燥处理可提高愈伤组织体细胞胚胎发生率,愈伤组织含水量在60％～80％范围的培养效果较好;外源DNA溶液浸泡发育早期的心状体细胞胚．其进一步发育时异常胚的频率显著增加．成苗率降低。     

提高春小麦花粉植株诱导率的研究

本文研究了影响春小麦花粉植株诱导率的几种主要因素。供试材料为1990年温室种植的杂种F_1代,试验结果表明,诱导培养基中附加细胞分裂素BA或BA与KT相结合能显著提高愈伤组织分化率;0.17M的葡萄糖加0.17M的蔗糖比用0.26M的蔗糖愈伤组织诱导率高60％以上;愈伤组织在18～25℃比在23～25℃下培养绿苗率显著提高;接种前2～3℃预处理幼穗72小时对提高出愈率和绿苗分化率有明显效果。     

小麦成熟胚愈伤组织诱导及分化研究

以2个小麦品种成熟胚为外植体进行离体培养,研究了不同预处理、不同2,4-D浓度及与KT组合、不同蔗糖浓度等因素对愈伤组织诱导及分化的影响。结果表明:4℃低温预处理可提高愈伤组织的出愈率及再生苗率,2个材料的出愈率及再生苗率均达到90%和30%以上;在不同预处理条件下,2,4-D浓度对出愈率及再生苗率的影响与基因型有关,2,4-D浓度为1~2 mg/L更有利于愈伤组织诱导及分化;附加KT能缓解高浓度2,4-D对再生苗率的抑制作用,而对于在1、2 mg/L 2,4-D的培养基中附加KT则不表现这种作用;蔗糖浓度则在30 g/L条件下更有利于愈伤组织诱导。因此通过4℃低温预处理,在MS基本培养基中附加1~2mg/L 2,4-D及30 g/L蔗糖亦可促进小麦成熟胚愈伤组织的诱导和分化。     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench) using an improved in vitro regeneration system

An improved regeneration protocol suitable for transformation of sorghum was developed. The improvements focused on limiting the production of phenolic compounds and the use of suitable culture vessels for each developmental stage in plant regeneration from immature embryo derived calli. The addition of activated charcoal in the callus induction medium reduced the production of black pigments, however it also inhibited the callus formation on immature embryo explants. Cold pre-treatment of the immature seeds from which embryo explants were excised had a positive effect on both explant survival and callus formation. A one-day 4°C treatment of immature seeds significantly improved the callus formation from immature embryos and reduced the need for frequent subculture. Petri dishes with ventilation were suitable for the callus induction phase, but not for plant regeneration. Regeneration of plants could be improved by using disposal plastic boxes (250 ml volume) instead of Petri dishes. Agrobacterium-mediated transformation using the improved regeneration protocol and the hygromycin phosphotransferase gene as selectable marker resulted in the recovery of 15 transgenic plants from 300 initial immature embryos (5% efficiency). The transgenic nature of the obtained plants was demonstrated by Southern hybridisation and progeny analysis. The transgenes were inherited in a Mendelian fashion and were integrated at a single locus in the majority of the analysed lines.     

Plant regeneration by somatic embryogenesis from callus cultures of sweet sorghum

Tissue culture methods were developed for the induction, maintenance, and regeneration of embryogenic callus in sweet sorghum (Sorghum bicolor) cultivars Keller, Rio, and Wray. No significant differences were observed in production of embryogenic callus in cultures established from developmentally immature or mature embryo explants cultured on LS medium with 2 mg/1 2,4-D plus 0.5 mg/1 kinetin. Prolific callus production did not occur until the third four-week culture period. Long-term maintenance of embryogenic callus was dependent upon the selective transfer of embryogenic callus, with other callus types discarded. High-frequency plant regeneration was achieved and quantified on a fresh weight basis of embryogenic callus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - LS Linsmaier and Skoog basal medium (Linsmaier and Skoog, 1965)     

Adventitious shoot regeneration from immature embryos of sorghum

Eleven genotypes of sorghum were examined for their response in tissue culture, and the tissue culture system was optimized. The cultures were initiated from immature embryos taken approximately two weeks after flowering. The response of immature embryos varied with the genotype. `C. Kafir' and `PE932 025' showed the highest frequency of callus induction and regenerable callus formation under appropriate culture conditions. Regeneration occurred at high frequencies when cytokinins (kinetin or 6-benzyladenine) had been added in the callus induction medium, followed by regeneration medium devoid of growth regulators. The addition of proline and polyvinylpyrrolidone also enhanced shoot formation, but the addition of cytokinins to regeneration media did not improve shoot formation. On the revised culture medium, plants were regenerated from up to 100% of sorghum immature embryos.     

Callus Induction and Plant Regeneration from Embryo of Elaeis guineenis Jacq

This paper deals with the preliminary results on callus induction and plant regeration from embryo in vitro of Elaeis guineenis. When mature embryos were cultured on dedifferen- tiation medium they proliferated calli during 30-90 days of culture. Among auxins applied, 2,4-D was more important for callus induction however 2,4-D and NAA combination gave bet- ter result. On the contrary, kinetin inhibited callus formation and growth. These experimental results explain that a higher callus induction frequency depends not only on the constituents of the medium used but also on the genotype of donor plants. After transfering the calli onto kinetin-containing media for a peirad, embryoids, which showed typical configuration of zygotic embryo, could be obtained. The embryoids can further develop into whole plants on a shoot induction medium. Some embryoids have subjected to srveral generations of subculture and still retained the ability to embryoid multification and plant regeneration.     

应用正交设计建立青花菜植株的再生体系

通过L16(4 5)正交试验 ,研究最适合青花菜 2周龄下胚轴愈伤组织诱导和不定芽发生的植物生长调节物质的种类和浓度组合。结果发现在NAA、6 BA、TDZ和KT四种激素中 ,NAA对下胚轴愈伤组织发生指数、不定芽发生频率的影响作用最大 ,确定以MS +NAA 0 .2 5mg/L +6 BA 0 .2 5mg/L +TDZ 0 .0 1mg/L(琼脂 0 .8% ,蔗糖 3 % ,pH5 .8)作为单因子试验的培养基。NAA、6 BA和TDZ浓度的单因子试验结果表明最适合下胚轴的培养基配方为 :MS +NAA 0 .1mg/L +6 BA 1 .0mg/L +TDZ 0 .0 6mg/L(琼脂 0 .8% ,蔗糖 3 % ,pH5 .8)。     

Embryo rescue and plant regeneration in banana (<Emphasis Type="Italic">Musa</Emphasis> spp.)

An efficient regeneration protocol for zygotic embryos at varying maturity stages was developed for wild banana (Pisang Jajee (AA)). Embryo ontogeny was studied to determine the best maturity stage for embryo rescue, suitable media and culture conditions (light and dark) for germination and regeneration. The conversion of endosperm from transparent fluid into a semi-solid state was followed by visible embryo development, which commenced only after 70% embryo maturity. Zygotic embryos of Pisang Jajee at different maturity levels were excised and cultured on medium fortified with different concentrations of 6-benzyl adenine (BA) and indole acetic acid (IAA). Zygotic embryos produced callus or plantlets 25 days after initiation. The frequency of callus induction was greater in immature embryos irrespective of the media composition and decreased with increasing maturity. Fully matured embryos regenerated directly into plantlets without producing callus. Immature embryos required medium supplemented with plant growth regulators (PGRs) for successful regeneration. Although the culture conditions had no influence, dark conditions favoured callus induction and plant regeneration.     

High frequency plant regeneration from immature embryos of an elite barley cultivar (Hordeum vulgare L. cv. Morex)

An efficient plant regeneration system was developed for Hordeum vulgare L. 'Morex' barley, an important United States malting cultivar. The protocol was based on a series of experiments involving the sizes of immature embryos and the culture media. We found that the embryo size is critical for the establishment of embryogenic callus. Smaller embryos (0.5-1.5 mm) showed a much higher ability to produce embryogenic callus capable of regenerating green plants with fewer albinos than did the larger embryos (1.6-3.0 mm). Either 3 mg/l 2,4-dichlorophenoxyacetic acid or dicamba in modified Murashige and Skoog's (MS) medium was optimum for the induction of embryogenic callus. The embryogenic callus maintained high regeneration during six subcultures in the callus induction medium. Efficient shoot regeneration was obtained on modified MS medium containing 0.5-1.0 mg/l 6-benzylaminopurine (BA). Regenerated shoots were rooted on half-strength MS medium containing 0.2 mg/l IBA. Plants were successfully transferred to soil and grown to maturity in the greenhouse. This efficient plant regeneration system provides a foundation for generating transgenic plants of this important barley cultivar.     

Studies on Cell Suspension Culture and Plant Regeneration in Rice

The present paper reports the establishment of rice cell suspension culture system, including callus induction and proliferation, isolation of single cells and small aggregates, cell suspension culture and callus re-formation, as well as regeneration of plantlets. The results have been obtained as follows: 1. The compositions of the different media used for callus induction, callus proliferation, cell suspension and plant regeneration are summarized in Table 1.2. Two kinds of disifectants, mercuric chloride and sodium hypochlorite, were used for surface sterilization of brown rice. The percentage of callus formation and callus yields were much higher when sodium hypochlorite was used (Fig. 3). We suggest that the disinfactant is one of the important factors that affect callus formed at the initial stage has an influence upon subsequent isolation of cells and suspension culture and even plant regeneration. 3. Table 3 shows that addition of yeast extract to the medium improves callus yield greatly and the efficiency of callus formation to a lesser extent. 4. Both medium Ⅱ (modified B5 medium) and N6 medium were suitable for cell suspension culture, but medium II was more effective for cell growth and callus re-formation (Fig. 4 and Table 4). 5. Effect of 2, 4-D on cell growth was tested at the concentration range among 0, 10-6, 10-5, 10-4 to 10-3 M. The results indicated that 10-5 M of 2,4-D was most effective for induction of rice callus. It has also been found that absence of 2,4-D increased callus re-formation in suspension culture, but no plant regeneration was observed. 6. By using 7% sucrose in differentiation medium, for all the three varieties, the plant regeneration frequency was raised up to 3 or 4 times than those of the 3% ones (Table 6). Occurrence of albino plants is often reported as one of the problems in rice anther culture. It is, however, no problem in seed-derived rice cell culture.     

不同浓度激素对野生细叶结缕草愈伤组织的诱导与分化的影响

以野生的细叶结缕草的匍匐茎为外植体,MS为基本培养基,研究2,4-D和BA及2,4-D和IAA不同浓度组合对其愈伤组织诱导与分化的影响。研究表明:(1)5.0mg/L的2,4-D对愈伤组织诱导有很大的促进作用;(2)匍匐茎诱导愈伤组织宜采用MS 2.0mg/L 2,4-D 4.0mg/L IAA,产生的愈伤组织分化情况也较好。     

Efficient callus induction and plant regeneration from mature embryo culture of winter wheat (Triticum aestivum L.) genotypes

Immature and mature embryos of 12 common winter wheat (Triticum aestivum) genotypes were cultured in vitro to develop an efficient method of callus formation and plant regeneration from mature embryo culture, and to compare the responses of both embryo cultures. Fifteen days after anthesis, immature embryos were aseptically dissected from seeds and placed with the scutellum upwards on a solid agar medium containing the inorganic components of Murashige and Skoog (MS) and 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were moved slightly in the imbibed seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg/l 2,4-D for callus induction. The developed calli and regenerated plants were maintained on 2,4-D-free MS medium. Plants regenerated from both embryo cultures were vernalized and grown to maturity in soil. Regenerated plantlets all maintained the hexaploid chromosome number. A strong genotypic effect on the culture responses was found for both explant cultures. Callus induction rate, regeneration capacity of callus and number of plants regenerated were independent of each other. Mature embryos had a high frequency of callus induction and regeneration capacity, and therefore, being available throughout the year, can be used as an effective explant source in wheat tissue culture. Received: 4 February 1997 / Revision received: 1 April 1997 / Accepted: 5 May 1997