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。     

几种影响籼稻成熟胚愈伤组织诱导及再生的因素

建立了适合5个籼稻品种成熟胚籼稻遗传转化的高效植株再生体系.N6基本培养基有利于籼稻愈伤组织的诱导和继代培养;N6大量元素和MS微量元素有利于愈伤组织的分化.降低分化培养基中蔗糖含量,加入适量山梨醇、Cu2 、Ag 和玉米素(2T)均可明显提高水稻愈伤组织的再生植株频率,5个品种分化频率均达到75%以上.     

新疆天山雪莲体胚诱导与分化研究

以新疆天山雪莲的叶片为外植体,分别用不同配方培养基诱导愈伤组织,后进行体胚诱导和分化培养形成再生雪莲植株.结果表明,诱导愈伤组织的最适培养基为MS 2,4-D 0.5 mg/L BA 1.5 mg/L,诱导率可达到100%;愈伤组织转移至MS 2,4-D 0.5 mg/L BA 1.5 mg/L培养基进行继代培养,增殖后的愈伤组织转移到MS 2,4-D 0.2 mg/L的液体培养基后成功诱导出雪莲体胚,出胚率达40%;将体胚接至MS ABA 0.5 mg/L培养基后,结果分化生长出大量的再生雪莲幼苗.     

安祖花胚性愈伤组织诱导及植株再生研究

为构建安祖花(Anthurium andreanum)胚性愈伤组织再生体系,以3个盆栽品种幼嫩叶片和叶柄为外植体,分析了基本培养基、植物生长调节剂组合和培养条件等因素的影响。结果表明,安祖花胚性愈伤组织诱导的最佳培养基为改良MS3+1.5 mg L–1 2,4-D+0.5 mg L–1 KT+4%蔗糖+2%葡萄糖+0.25%Phytagel,且胚性愈伤组织诱导能力差异显著,表现为?粉冠军??罗宾奴??冠军?和叶片叶柄,其中?粉冠军?叶片的胚性愈伤组织诱导率可达57.9%。胚状体分化的最佳培养基为1/2改良MSa+2%蔗糖+0.25%Phytagel,其中?粉冠军?叶片诱导的胚状体分化率可达31.6%,且在光、暗下分化率的差异不显著。分化苗移栽后的成活率可达100%。     

籼型杂交稻亲本成熟胚愈伤组织再生体系的建立

本文给出了6个籼型杂交稻亲本成熟胚愈伤组织再生体系建立优化措施。采用6个有重要育种价值的杂交籼稻亲本成熟胚盾片诱导愈伤组织作为分化再生的外植体,通过调节2,4-D浓度、培养基成分、接种方式、激素组合和愈伤组织分化途径,建立适合籼稻遗传转化的再生体系。结果表明,MB培养基是较为合适的愈伤组织诱导培养基类型,6个品种在MB培养基上的愈伤组织诱导率均在60％-80％之间。半粒米的接种方式能够明显提高愈伤组织诱导率,提高幅度达到28．2％。通过调节2,4-D浓度和激素组合,愈伤组织诱导率最高可达到97．9％,两步分化法和适当干燥处理能够提高愈伤组织的分化效率,6品种愈伤组织分化率均在50％-90％之间。初步建立了6个杂交籼稻亲本品种成熟胚愈伤组织的再生体系,为以后遗传转化奠定基础。     

不同培养基对水稻胚性愈伤组织诱导及分化的影响

以水稻成熟胚为材料诱导愈伤组织,统计在不同基本培养基上的愈伤诱导率以及绿苗分化率,分析不同基本培养基及外源激素的含量和比例对愈伤组织生长及分化的影响。结果表明,试验材料对基本培养基具有选择性,MS培养基对籼稻种胚愈伤的诱导培养效果较好,NB培养基则更适合粳稻种胚愈伤的诱导培养;诱导继代培养基中加入多种氨基酸组合可有效提高出愈率和分化率,特别是粳稻的愈伤组织的诱导和分化需要多种氨基酸的共同作用;不同基因型水稻材料对激素和氨基酸组合的需求不同。     

白刺花胚性愈伤组织诱导及体细胞胚发生和萌发

探讨不同因素对白刺花下胚轴、子叶2种外植体胚性愈伤组织诱导及体细胞胚发生和萌发的影响。以B5和MS为基本培养基,研究2,4-D、6-BA和TDZ对白刺花下胚轴和子叶胚性愈伤组织的诱导;在MS培养基上添加不同浓度2,4-D,研究胚性愈伤组织增殖情况;采用ABA,探究对体细胞胚发生的影响。结果表明:下胚轴比子叶更易诱导胚性愈伤组织,筛选出2种外植最佳的胚性愈伤组织诱导培养基均为MS+2.0 mg/L 2,4-D+0.5 mg/L TDZ+0.5 mg/L 6-BA,胚性愈伤组织诱导率分别为77.3%和41.0%。15.0 mg/L ABA、0.2 mg/L 2,4-D和2.0 mg/L 6-BA有利于体细胞胚发生,1/3MS+0.2 mg/L NAA+0.1 mg/L 6-BA+2.0 g/L活性炭+25 g/L蔗糖+7 g/L琼脂的培养基可使体细胞胚萌发率达80%以上,再生植株移栽成活率高达90%。白刺花外植体种类及培养基类型均会影响胚性愈伤组织的诱导,其中下胚轴诱导效果优于子叶;MS培养基较适合启动细胞脱分化形成愈伤组织,2,4-D对胚性愈伤组织的增殖保持有调控作用,ABA有利于体细胞胚的发生。     

诱导茶树成熟胚培育成幼苗的研究

以茶树品种——农抗早的成熟胚为外植体离体培养,初次研究了2,4-D、6-BA等不同植物激素对茶树成熟胚诱导成幼苗的影响。结果表明,在合适的激素组合条件下,愈伤组织诱导为97%,在附加2.5mg/L 6-BA 0.5mg/L NAA的MS培养基上,愈伤组织的芽分化率为14.2%,附加1.5mg/L NAA的1/2 MS培养基,幼苗的根分化率为5%。     

扁桃愈伤组织诱导及分化的研究

以扁桃优良品种'Naporeil'的茎段、叶片和花药作为外殖体,分别对其进行愈伤组织诱导和分化研究,以筛选愈伤组织的最佳诱导增殖培养基、分化培养基和生根培养基.结果表明,该品种以茎段、花药作为外殖体最易诱导获得愈伤组织,叶片不适宜作为外殖体诱导愈伤组织;愈伤组织的最佳诱导增殖培养基均为B5+0.5 mg/L 2,4-D+0.2 mg/L 6-BA+1.0 mg/L NAA,愈伤组织诱导率为100%,增殖倍数最高可达7倍;茎段愈伤组织的分化培养基为MS+0.2 mg/L NAA+0.8 mg/L 6-BA+0.5 mg/L ZT,分化率为71%;花药愈伤组织未见分化.由茎段愈伤组织再分化获得的不定芽在1/2MS+0.5 mg/L IBA培养基上诱导生根,并给以黑暗预处理可使生根率达80%以上.     

盐肤木体细胞胚胎发生及植株再生

以盐肤木(Rhus chinensis Mill.)幼胚为外植体,研究不同植物生长调节剂组合对其愈伤组织诱导及体细胞胚胎发生的影响,以建立盐肤木体细胞胚胎发生及植株再生体系。结果表明,最适愈伤组织诱导培养基为MS+6-BA 0.2 mg/L+2,4-D 1.0 mg/L,诱导率为84.57%,诱导出的初代愈伤组织白色或淡黄色,质地疏松,表面光滑,为非胚性愈伤。初代愈伤组织转移到1/2 MS+6-BA 2 mg/L+NAA 0.5 mg/L培养基上培养1个月后,长出淡黄色质地紧密的胚性愈伤组织,诱导率高达100%,在此培养基上胚性愈伤组织增殖倍数为854.73%。所获得的胚性愈伤组织转接到1/2 MS+6-BA 2 mg/L+NAA 0.5 mg/L+蔗糖4%的培养基上培养1个月后可诱导体细胞胚胎发生,诱导率可达32.67%。诱导得到的体细胞胚胎经历球形胚、心形胚、鱼雷胚、子叶胚进一步分化发育成苗。无菌苗炼苗后栽种到泥炭土∶蛭石∶珍珠岩为2∶1∶1的生长基质上,能100%稳定成活。经过细胞学观察分析,体细胞胚的发育与合子胚相似。     

Effects of different combinations of benzyl adenine and indole acetic acid concentrations on in vitro plant regeneration in hexaploid wheat

Development of a reliable in vitro plant regeneration procedure for hexaploid bread wheat (Triticum aestivum L.) is a prerequisite for its improvement by genetic transformation. Here, we report the effects of two growth regulators, benzyl adenine (BA) and indole acetic acid (IAA) on callus induction and plant regeneration from scutellum cultures of two commercial bread wheat cultivars: Giza 164 and Sakha 69. Callus induction was obtained from isolated embryos cultured on modified Murashige and Skoog (MS) basal medium. After four weeks of callus induction, all calli were plated on MS basal medium for regeneration. Wheat genotype and callus induction medium played a dominant role in plantlet regeneration. 2.0 mg/L BA and 0.2 mg/L IAA were the best combinations for inducing callus and let to highest regeneration frequency (81.67%) across the cultivars. Overall, based on our medium conditions, Giza 164 displayed higher regeneration frequency (81.11%) than Sakha 69. These results will facilitate genetic transformation for the economic varieties Giza 164 and Sakha 69.     

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.     

多年生黑麦草成熟胚再生体系的建立及基因枪转化

目的:建立以多年生黑麦草成熟胚为起始材料的再生体系,用于基因枪转化。方法:多年生黑麦草成熟种子在附加 5mg L 2,4 D的MS培养基上诱导愈伤组织,转至新继代培养基上产生胚性愈伤组织。分化培养基为无激素MS培养基。再生植株在培养基成分减半的无激素MS培养基生根,之后移栽至土壤。基于这一再生体系,用含有水稻几丁质酶基因RC2 4的质粒pARN6和含有草丁膦乙酰转移酶基因Bar的质粒pDB1,通过基因枪轰击胚性愈伤组织。用附加PPT的继代培养基进行转化植株的抗性筛选。结果:共获得 2 4 3株再生植株。通过PCR进行检测,获得1 8株整合有RC2 4基因的植株,1 5株整合有Bar基因的植株,同时转入 2个基因的植株 2株。     

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.     

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     

Somatic embryogenesis and plant regeneration in inflorescence and seed derived callus cultures of Poa pratensis L. (Kentucky bluegrass)

Callus induction and plant regeneration were studied in 15 cultivars of the facultative apomictic species Poa pratensis L. (Kentucky bluegrass).The tissue culture responses of mature seeds and immature inflorescences were compared. Murashige and Skoog's (MS) medium, supplemented with 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) was used for callus induction and maintenance. Plants could be regenerated from compact and friable callus on MS medium devoid of 2,4-D. Plants were recovered from 14 cultivars at a high frequency (up to 79% of the callus cultures) when young inflorescences were used as the explant material and from only 3 cultivars, at a low frequency (up to 3%), with seeds. Somatic embryos were observed in callus cultures of many cultivars. Fully developed germinating somatic embryos were occasionally observed. Plant regeneration appeared to take place both via somatic embryogenesis and organogenesis. Plants were generally green but albino shoots developed at a low frequency from friable callus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium - IAA indole-3-acetic acid - N₆ medium of Chu et al. (1975)     

Factors influencing induction,propagation and regeneration of mature zygotic embryo-derived callus from Allium cepa

A systematic study on the effects of subspecies, cultivar, basal medium, sucrose concentration and 2,4-dichlorophenoxyacetic acid concentration on callus induction, propagation and subsequent plant regeneration in Allium cepa has been carried out. Mature zygotic embryos from two onion (cvs. Sturon and Hyton) and two shallot (cvs. Tropix and Atlas) varieties were used as explants. After callus initiation and growth on both Murashige and Skoog (MS) and Gamborg's B5 modified by Dunstan and Short (BDS) basal media with different 2,4-dichlorophenoxyacetic acid and sucrose concentrations for eight weeks, lines were identified on which compact or friable callus was induced. Callus induction and propagation were largely determined by the concentration of 2,4-dichlorophenoxyacetic acid whereas subspecies, cultivar, sucrose concentration and basal media were of less importance. After callus propagation for twelve weeks, 315 lines from a total of 3348 embryos initially subcultured were selected to test their regeneration capacity on growth regulator-free medium. It was found that shallot formed more shoots and roots than onion. The MS basal medium proved to be more beneficial for shoot regeneration and root formation than the BDS basal medium. There were no differences in plant regeneration among selected calli which had been previously subcultured on different concentrations of 2,4-dichlorophenoxyacetic acid and sucrose. The results show that plant regeneration strongly depended on the line: 45.4% from 315 tested lines could produce shoots while 93.0% formed roots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Establishment of oil palm cell suspensions and plant regeneration

Primary globular callus from immature zygotic embryos and friable embryogenic tissue derived from mature zygotic embryos were used to establish suspension cultures. Callus cultures were established either on modified Y3 or MS medium containing 475–500 M 2,4-D or 250 M picloram and 0.3% (w/v) activated charcoal. Suspension cultures of both cell lines were established in modified Y3 medium containing 10 M 2,4-D. The establishment of cell suspensions from friable embryogenic tissue took only 2 months, in contrast with suspensions from primary globular callus which took 3–5 months to establish. Embryo differentiation was observed only in cell suspensions derived from the friable embryogenic tissue after plating aliquots on regeneration medium. Germinated embryos were recovered and plantlets were successfully established under greenhouse conditions.Abbreviations CET compact embryogenic tissue - FET friable embryogenic tissue - CIM callus induction medium - PGC primary globular callus - 2,3-D 2,4-dichlorphenoxyacetic acid Y3-Eeuwens' medium - MS Murashige & Skoog medium - PVP-40 polyvinylpyrrolidone - KM Kao & Michayluk vitamins - ABA abscisic acid     

中国木薯栽培种通过器官发生再生植株研究

本文研究了中国木薯栽培种四种外植体通过器官发生再生植株的条件。结果表明:在MS附加0.05mg/L TIBA,1mg/L BA的培养基上“NZ 188”初步的萌发胚状体“切头”后切口处可直接产生丛芽,出芽率为43%。“SC201”胚状体子叶块在MS附加0.5 mg/L NAA,0.5mg/L BA的培养基上可直接出芽,出芽率为42%,在MS附加0.5mg/L IBA,1.5mg/L BA培养基上·出芽率为31%,AgNO_3和ABA单独使用或配合使用均不利于芽的再生。“NZ188”胚状体下胚轴在MS附加0.5mg/LNAA,0.5mg/L BA的培养基上形成的愈伤组织转入MS附加1mg/L NAA,2mg/L BA的培养基上,3周后大多数愈伤组织有绿点出现、仅4.4%外植体分化出芽。“HZ188”无菌苗茎段接种在MS附加0.05mg/L TIBA,2mg/LBA的固体培养基上,2周后形成大量愈伤组织,4周后仅见一块愈伤组织分化出芽。     

An efficient and reproducible method for regeneration of whole plants from mature seeds of a high yielding Indica rice (Oryza sativa L.) variety PAU 201

Tissue culture is one of the tools necessary for genetic engineering and many other breeding programs. Moreover, selection of high regenerating rice varieties is a pre-requisite for success in rice biotechnology. In this report we established a reproducible plant regeneration system through somatic embryogenesis. The explants used for regeneration were embryogenic calli derived from mature seeds cultured on callus induction media. For callus induction mature seeds were cultured on MS medium containing 30 g/l sucrose combined with 560 mg/l proline and 1.5-3.5 mg/l 2,4-D and 0.5-1.5 mg/l Kin. For plant regeneration, embryogenic calli were transferred to MS medium containing 30 g/l sucrose, supplemented with 1.0-3.0 mg/l BAP, 0.5-1.5 mg/l Kin and 0.5-1.5 mg/l NAA. The highest frequency of callus induction (44.4%) was observed on the MS medium supplemented with 2.5 mg/l 2,4-D, 0.5 mg/l Kin, 560 mg/l proline and 30 g/l sucrose. The highest frequency of shoot regeneration (42.5%) was observed on the MS medium supplemented with 2.0 mg/l BAP, 0.5 mg/l NAA and 0.5 mg/l Kin. The plantlets were hardened and transferred to soil in earthen pots. The developed method was highly reproducible. The in vitro developed plants showed normal growth and flowering under glasshouse conditions.