诱导突变高粱愈伤组织初探

本实验以突变体高粱的不同外植体 ,成熟种子、成熟胚、茎尖、和幼胚等为材料 ,诱导愈伤组织。经出愈率和生长状况观察 ,幼胚最好 ,成熟胚较好 ,成熟种子和茎尖略差。对于茎尖 ,2 ,4 D与KT搭配较好 ;成熟胚而言 ,不加细胞分裂素 ,加适量 2 ,4 D浓度效果较好 ,幼胚愈伤组织诱导和培养不需要细胞分裂素 ,加浓度为 2 .0mg/L2 ,4 D效果较好 ;若加KT ,同时要提高 2 ,4 D浓度 ;成熟种子培养 ,需要细胞分裂素     

几种匍匐翦股颖成熟胚愈伤组织诱导及植株再生

利用成熟种子作为外植体,分析了2,4-D对匍匐翦股颖胚性愈伤组织诱导与植株再生体系的影响,并对体细胞胚的发生过程进行了观察.实验结果表明,在2.0 mg/L 2,4-D 0.1 mg/L 6-BA时胚性愈伤组织的诱导频率最高.随着2,4-D浓度的增加,愈伤组织的诱导和分化能力明显下降.在再生过程中采用1.0 mg/L的6-BA达到了比较好的效果,愈伤组织的再生频率大部分在90%以上.同时发现适当提高肌醇浓度可以使苗长得较为粗壮.在实验中发现匍匐翦股颖的体细胞胚发生过程为球形胚、心形胚、鱼雷胚和子叶胚.     

影响小麦成熟胚再生频率因素的研究(简报)

以小麦成熟胚为外植体,研究了基本培养基、预处理类型、接种方式、植物激素的浓度和不同组合以及分化培养基中是否添加抗生素对愈伤组织诱导和分化的影响．在此基础上建立了一套高效的小麦成熟胚植株再生系统。经过试验,我们选择在MS培养基上接种经无菌水预处理的纵切成熟胚作为起始的试验条件。在含2mg／L2,4-D的MS培养基上．初级愈伤组织的诱导频率可达80％以上．在继代培养基中添加0．5mg／L6-BA和0．2mg／LNAA可以显著提高胚性愈伤组织的产生。而在再生培养基中加入适当浓度的头孢霉素可以有效提高胚性愈伤组织再生出小植株的比例。利用该再生系统,我们从5个小麦优良主栽品种的成熟胚再生出了可育的植株,再生频率达15．3％-34．5％。     

杉木未成熟胚胚性愈伤组织诱导影响因素探析

该研究从基因型、6-BA浓度、外植体接种方式和合子胚发育阶段等方面,分析杉木未成熟胚胚性愈伤组织诱导的影响因素。结果表明:基因型、6-BA浓度、外植体接种方式和合子胚发育阶段均对胚性愈伤组织诱导频率有不同程度影响。6种基因型中,有3种基因型诱导出胚性愈伤组织,其中基因型S18胚性愈伤组织诱导频率最高,为11.7%。6-BA浓度在1.0~1.5 mg·L~(-1)范围内时,基因型S18的胚性组织诱导频率较高。以在去皮种子的一端切开一个小口的接种方式为最优,将合子胚剥出的方式易造成合子胚褐化死亡,将未剥皮的种子切开一个小口后直接接入培养基的方式不利于愈伤组织生成。适合胚性愈伤组织诱导的合子胚发育阶段为受精至胚器官分化阶段,合子胚进入成熟阶段后不利于胚性愈伤组织诱导,合子胚易生长成完整植株。     

‘SK4—316’胡萝卜体胚的诱导和培养

以'SK4-316'胡萝卜无菌苗的下胚轴为外植体,研究不同培养基配方和培养条件对愈伤组织诱导、体细胞胚间接发生及其同步化培养的影响,以及不同脱分化时间、脱分化培养基及外植体续存时间对体细胞胚直接发生的诱导及其培养的影响.结果表明:含3%蔗糖、0.8%琼脂的1/2MS + 2,4-D 2.5 mg/L + 6-BA(或KT)0.5 mg/L + CH 300 mg/L是诱导愈伤组织的良好培养基;1/2MS + 2,4-D 1.25 mg/L + KT 0.25 mg/L + 6-BA 0.25 mg/L(含3%蔗糖)适于愈伤组织分化并诱导体胚发生,0.02% ABA对体胚的诱导有促进作用,0.06% ABA或15% PEG能促进体胚成熟;外植体在MS + 2,4-D 1.0 mg/L固体培养基上脱分化培养48 h,再转入MS + CH 300 g/L液体培养基中可诱导体胚直接发生,但随着外植体续存于诱导培养基中时间的延长,体胚发生变异的几率也渐增.     

大麦成熟胚愈伤组织的诱导和植株再生的研究

以10个大麦优良品种为实验材料,成熟胚为外植体,研究基因型、种子的不同切割方式、培养基、激素等对大麦成熟胚愈伤组织的诱导及植株再生的影响.结果表明,种子纵切后接种出愈率显著高于横切;改良MS培养基能提高出愈率;在愈伤组织诱导过程中,不同品种对激素2,4-D与Dicamba的反应表现不同;初代愈伤组织经过3次继代培养后会转变为两种类型的胚性愈伤组织;不同品种的植株再生在不同浓度有机添加物的分化培养基上表现不同;长时间的继代培养,一些品种在植株再生过程中出现一定数量的白化苗.供试材料均能进行愈伤组织诱导,但是只有部分品种能再生植株.本实验筛选出愈伤组织诱导频率和绿苗分化率均较高,适合于遗传转化的受体材料,如87-3175、87-0053、97-4010、97-6004及208813-509.     

羊草成熟胚诱导愈伤组织及植株再生系统的优化

羊草(Leymuschinensis)为异源四倍体禾本科牧草,利用成熟胚诱导愈伤组织获得再生植株的效率极低,难以运用遗传转化方法进行品种改良。我们以羊草成熟胚为外植体,使用适宜羊草愈伤组织生长的新型培养基配方,筛选诱导愈伤组织、不定芽分化及生根阶段的最适植物激素浓度、光照和温度条件,从而优化羊草成熟胚的组织培养方案。研究结果表明,羊草成熟胚诱导阶段2,4-D的最适浓度为2.0mg·L~(-1),变温暗培养,诱导率可达74.1%;分化阶段6-BA和NAA的最适浓度均为1.0 mg·L~(-1),分化率可达57.1%;生根阶段NAA的最适浓度为0.25 mg·L~(-1),移栽后成活率为100%。     

胡桃楸胚性愈伤组织诱导与体细胞胚胎发生

胡桃楸是东北东部山地阔叶红松林的重要组成树种。因其被大量采伐,资源日趋枯竭。体细胞胚胎发生是快速繁殖和人工种子研制的基础,对遗传改良有重要意义。为探讨不同外植体、植物生长调节物质种类及配比对胡桃楸培养物的影响,建立了胡桃楸体胚发生及再生植株体系。结果表明:合子胚为外植体时最易形成胚性愈伤组织,外植体最佳取材时期为5～6月。胡桃楸胚性愈伤组织最适诱导为MS+1.0mg·mL-12,4-D+0.5mg·mL-16-BA;体细胞胚的诱导、发育和分化的适宜的培养基为附加蔗糖60g.L-1、水解酪蛋白700mg·mL-1时不添加任何生长调节物质的MS培养基。     

低温与植物生长调节剂预处理对小麦成熟胚培养特性的影响

为提高小麦成熟胚愈伤组织的分化率,以3个黄淮麦区冬小麦品种(系)‘小偃22’、‘西农1013’和‘千斤早’为材料,研究了4℃低温和3种植物生长调节剂(TDZ、2,4-D、多效唑)预处理小麦种子对小麦成熟胚愈伤组织分化特性的影响。结果表明:4℃低温和植物生长调节剂预处理对小麦成熟胚出愈率无显著影响,但能够显著促进愈伤组织的分化,并表现出处理及基因型间差异。‘小偃22’、‘西农1013’、‘千斤早’感应低温预处理的最好时间节点分别为6h、12h、12h,其成熟胚愈伤组织的分化率比对照分别显著提高16.2%、14.2%、12.8%;3个材料分别在10mg/L、5 mg/L、20 mg/L TDZ预处理下成熟胚愈伤组织分化率最高,比对照分别显著提高39.1%、29.7%、16.7%;3个材料的最佳2,4-D预处理浓度均为10mg/L,其愈伤组织分化率分别比对照显著提高17.3%、11.2%、25.2%;3个材料的成熟胚愈伤组织分化率分别在10mg/L、20mg/L、20mg/L多效唑预处理时最高,分别比对照显著提高6.2%、11.6%、7.2%。研究表明,低温和植物生长调节剂预处理均可有效提高小麦成熟胚愈伤组织的分化率,但促进植物生长的调节剂(TDZ和2,4-D)对小麦成熟胚愈伤组织再分化的效应远大于抑制植物生长的调节剂(多效唑)。     

羊草成熟胚诱导愈伤组织及植株再生系统的优化

羊草(Leymus chinensis)为异源四倍体禾本科牧草, 利用成熟胚诱导愈伤组织获得再生植株的效率极低, 难以运用遗传转化方法进行品种改良。我们以羊草成熟胚为外植体, 使用适宜羊草愈伤组织生长的新型培养基配方, 筛选诱导愈伤组织、不定芽分化及生根阶段的最适植物激素浓度、光照和温度条件, 从而优化羊草成熟胚的组织培养方案。研究结果表明, 羊草成熟胚诱导阶段2,4-D的最适浓度为2.0 mg·L ^-1, 变温暗培养, 诱导率可达74.1%; 分化阶段6-BA和NAA的最适浓度均为1.0 mg·L ^-1, 分化率可达57.1%; 生根阶段NAA的最适浓度为0.25 mg·L ^-1, 移栽后成活率为100%。     

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     

A comparison of callus induction and plant regeneration from different embryo explants of triticale (x Triticosecale wittmack)

Immature, mature and endosperm-supported mature embryos of six triticale cultivars (BDMT-98-8S, Melez-2001, Mikham-2002, Presto, Tacettin Bey and Tatlicak-97) were cultured in vitro to compare the levels of callus induction and plant regeneration. Immature embryos, 15-18 days after anthesis, were aseptically excised and placed with the scutellum upwards on a callus culture medium consisting of Murashige and Skoog (MS) mineral salts supplemented with 2 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were aseptically excised from the imbibed seeds and placed scutellum up on MS medium supplement with 2 mg l(-1) 2,4-D. Endosperm-supported mature embryos were moved slightly in the imbibed mature seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg l(-1) 2,4-D for callus induction. The developed calli and regenerated plants were maintained on hormone-free MS medium. Variability among the genotypes was observed for all the types of embryo culture. Immature embryos from "Presto" and endosperm-supported mature embryos from "Mikham 2002" had excellent regeneration capacities (92.0% and 97.3%, respectively) and the highest number of plants regenerated growing in soil (9 and 13, respectively). A comparison of the responses of the three explants used indicated that the endosperm-supported mature embryo was the most useful explant for plant regeneration in triticale.     

温度和水分对不同基因型小麦未成熟胚体细胞胚胎发生以及分化能力的影响

对小麦未成熟胚盾片组织离体再生途径中,未成熟胚发育时期以及不同小麦品种的体细胞胚发生能力和体细胞胚的分化能力进行了研究,在所 试的14个小麦品种中,筛选出具有很强的体细胞胚发生能力和体细胞胚分化能力的4个品种,西农1376、盐2号、85＋1－3和宝丰7228。为进一步给小麦离体遗传操作打下基础,研究还对温度的影响进行了分析。通过低温手段解决了胚性愈伤组织随继代天数的延长体细胞胚分化能力快速降低的问题,同时研究还首次分析了干燥处理对小麦体细胞胚转换能力的影响,建立起一套高效的小麦离体培养再生体系,而且该体系从接种未成熟胚到再生植株移至土壤只需10－12周时间,避免了长期培养过程中存在的体细胞变异问题。     

Plant regeneration via somatic embryogenesis of Elymus sibiricus cv. ‘chuancao No. 2’

The mature seeds, mesocotyls, and young leaf tips of Elymus sibiricus L. cv. ‘chuancao No. 2’ were cultured on Murashige and Skoog (MS) medium supplemented with 5.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-d) and 0.05 mg/L kinetin in the dark at 26°C, the calluses were produced. The rate of callus regeneration depended on the explants source and plant growth regulators. Plants regenerated from whitish-yellow-coloured compact nodular callus formed after subculturing for 8 weeks. Higher frequency (54%) of shoot differentiation was obtained from the embryo tissues of mature seed than from either mesocotyls (24%) or young leaf tip tissues (6%) when these calluses from different types of explants were cultured on plant regeneration medium containing half strength MS salts supplemented with 0.1 mg/L kinetin, 1.5 mg/L 2,4-D and 20 g/L sucrose. The green plants were rooted within 6 weeks in the root regeneration medium, and over 97% of these soil-established plants were obtained in the greenhouse when potted in a sand and peat mixture medium.     

Development of an efficient and reproducible regeneration system in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The availability of reproducible regeneration system through tissue culture is a major bottleneck in wheat improvement program. The present study has considered to develop an efficient callus induction and regeneration system using mature and immature embryos as explants in recently released agronomically superior spring wheat varieties. An efficient sterilization process was standardized using 0.1% HgCl₂ and 70% ethanol for both seeds and embryos. The maximum possible combinations of plant growth regulators (PGRs) were evaluated for their effect on different wheat regeneration processes through tissue culture starting from callus to root induction. Picloram is found as an effective auxin with 87.63–98.67% callus induction efficiency in both explants. Supplementation of CuSO₄ along with 2,4-D, zeatin in regeneration medium significantly enhanced the multiple shoot induction. The shoot development was achieved using full strength Murashige and Skoog’s (MS) medium and root induction using half MS medium without PGRs. The optimized medium and method has resulted up to 100% regeneration irrespective of the genotype used with high reproducibility. Thus, the standardized regeneration system can be used in the regeneration of healthy plants from embryos rescued from interspecies crosses, transgenic production, induced mutation breeding and recently developed genome editing techniques for the procreation of wheat plants having novel traits.     

小麦幼胚培养再生单性雌花的研究

通过45个基因型的小麦(Triticum aestivum L.)幼胚培养,发现有11.1%的基因型从靠近再生芽基部的愈伤组织上分化形成花器官.再生花芽呈裸露的、多子房丛生的、具有茂盛羽毛状柱头而缺乏雄蕊、外稃、内稃和颖片的单性雌花. 组织切片观察发现,其雌蕊起源于再生芽附近的分生组织细胞,并通过次生雌蕊再生的方式形成丛生状,其羽毛状结构的发育先于子房中胚珠的分化. 除正常的单胚珠外,还发现双生胚珠分化.χ2独立性检验结果显示,花芽再生率存在强烈的基因型效应.小麦品种YA-1 表现突出(44.4%),其花芽再生潜力能在不同年份间较好地再现,说明YA-1的花芽再生能力具有相对稳定性.与脱分化培养基的效应相比,YA-1的花芽再生效率主要受继代培养基成分的影响. 其中,6-BA、NAA和加倍无机铁盐的配比较2,4-D和正常浓度无机铁盐的配比更有利于YA-1的幼胚培养再生花芽.同时,外植体实验表明,YA-1的幼穗和成熟胚培养无任何成花反应,而其幼胚外植体具有特异的花芽再生能力.据此认为,YA-1的幼胚培养有助于为小麦花发育机理研究建立理想的实验系统.     

An efficient and rapid regeneration via multiple shoot induction from mature seed derived embryogenic and organogenic callus of Indian maize (Zea mays L.)

An efficient method for in vitro micro propagation and genetic transformation of plants are crucial for both basic and applied research. Maize is one of the most important cereal crops around the world. Regeneration from immature embryo is hampered due to its unavailability round the year. On the contrary mature embryo especially tropical maize is recalcitrant toward tissue culture. Here we report a highly efficient regeneration (90%) system for maize by using 2 different approaches i.e., embryogenic and organogenic callus cultures. Seeds were germinated on MS medium supplemented with 5 mg/l 2,4-D and 3 mg/l BAP. Nodal regions of 2 wks old seedlings were longitudinally split upon isolation and subsequently placed on callus initiation medium. The maximum frequency of embryogenic callus formation (90%) was obtained on MS medium supplemented with 2 mg/l 2,4-D and 1 mg/l BAP in the dark conditions. The compact granular organogenic callus formation (85% frequency) was obtained on MS medium supplemented with 2.5 mg/l 2,4-D and 1.5 mg/l BAP at light conditions. MS medium supplemented with 2 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA promoted the highest frequency of shoot induction. The highest frequency of root formation was observed when shoots were grown on MS medium. The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. The important advantage of this improved method is shortening of regeneration time by providing an efficient and rapid regeneration tool for obtaining more stable transformants from mature seeds of Indian tropical maize cultivar (HQPM-1).     

Callus Formation and Plant Regeneration from Cultured Embryos of Diploid and Tetraploid Winter Ryes (Secale cereale L)

The ability of immature, mature and endosperm-supported mature embryos of diploid and tetraploid winter ryes (Secale cereale L) was tested to compare the callus induction and plant regeneration. Immature embryos were obtained from field grown rye. Immature embryos were aseptically excised and placed, with the scutellum upwards, on the callus culture medium consisted of Murashige and Skoog (MS) mineral salts supplemented with 2 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D). Mature embryos were aseptically excised the imbibed seeds and placed, scutellum up, on MS medium supplement with 2 mg l^?1 2,4-D. Endosperm-supported mature embryos were moved slightly (not set free) in the imbibed mature seeds. The seeds with moved embryos were placed furrow downwards in dishes containing 8 mg l^?1 2,4-D for callus induction. The developed calli and regenerated plant were maintained on hormone free MS medium. Comparison of the responses of the three explants used indicated that endosperm-supported mature embryo was the most useful explant for plant regeneration in both diploid and tetraplold ryes. This is the first report of winter ryes plants having been regenerated from endosperm-supported mature embryos.     

Effects of support medium on embryo and plant production from cultured anthers of soft-red winter wheat (Triticum aestivum L.)

The present study was designed to examine the effects of media support on the frequency of embryo and plant production from cultured anthers of soft-red winter wheat. Approximately twice as many embryos were produced when anthers were cultured in a liquid as compared to an agar-solidified medium. Upon transfer to regeneration medium, a significantly lower percentage of the embryos produced in liquid regenerated plants. The addition of activated charcoal to an agar-solidified medium resulted in a considerable increase in embryo production, however, plant regeneration from embryos produced on charcoal-containing medium was significantly lower than those produced on agar only. Embryo production frequencies ranged from 2.4–13.2 and 2.5–32.2 embryos per 100 anthers on media with and without charcoal, respectively. Plant regeneration frequencies from embryos produced in the presence of activated charcoal ranged from 0–5.5% as compared to 0–39.1% from embryos produced in the absence of charcoal. More than twice as many embryos produced on Ficoll-containing liquid medium regenerated plants when compared to embryos produced in liquid only. The results from this study suggest that cultural modifications designed to maximize embryo production must take into account the quality of the resulting embryos as they relate to plant regeneration.     

Efficient in vitro direct shoot organogenesis and regeneration of fertile plants from embryo explants of Bambara groundnut (Vigna subterranea L. Verdc.)

An efficient protocol has been developed for direct shoot organogenesis from embryo axes derived from mature seeds of two different landraces of Bambara groundnut. Multiple shoots were initiated on several media containing different concentrations and combinations of benzylaminopurine (BAP) or thidiazuron (TDZ). Efficient regeneration occurred when the embryo axes were first plated for 6 days on a medium containing high concentrations of BAP (1 mg/l) and alpha-naphthaleneacetic acid (NAA, 1 mg/l) and then cut transversely and transferred onto a medium containing 1.5 mg/l BAP. Shoot regeneration frequency was 100% and from five to eight shoots per explant were obtained. The importance of using embryo explants and cytokinins in the culture media, with respect to controlling the development of a highly organogenic system, was demonstrated. Histological studies revealed that proliferating buds originated directly from the superficial layers of the explants without an intermediate callus phase. The regenerated shoots were rooted on a medium containing 1 mg/l NAA and then transferred to the greenhouse. Flow cytometric analyses and chloroplast counts of guard cells suggested that the regenerants were diploid. All were morphologically normal and fertile. The short duration, high efficiency and low frequency of somaclonal variation of this system make it well suited for wider biotechnological applications of Bambara groundnut-a neglected and under-utilized crop.