Screening wheat genotypes for high callus induction and regeneration capability from anther and immature embryo cultures

Plant regeneration via tissue culture varies with the genotype and is an important factor in establishing cell selection and genetic transformation systems. To select genotypes – especially Japanese ones – with a high regeneration capability, we screened 107 wheat genotypes (78 domestic, 29 foreign) for callus induction and regeneration capability from anther and immature embryo cultures. For anther culture, 83 of 107 genotypes tested induced calli and 45 regenerated plants. Only 9 genotypes, however, produced green plants, 25 produced only albino plants, and 11 produced both green and albino plants. Glennson 81 was the highest in callus induction, followed by Orofen, Danchi–komugi and Chris. The genotypes with a relatively high regeneration capability were Framala 80 at 24% and Glennson 81 at 19%, these two genotypes produced only green plants. For immature embryo culture, 97 genotypes showed a 90% callus induction rate and 74 genotypes regenerated plants. Very few genotypes produced albino plants. The genotypes with a high regeneration capability were Genaro 81 at 90%, Chinese Spring at 80%, and Norin 75 at 75%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of silver nitrate on the tissue culture of immature wheat embryos

The immature embryos of four common wheat (Triticum aestivum L.) genotypes with desirable agronomic traits were evaluated for their tissue culture response to ethylene antagonist, silver nitrate, added to callus-inductive and subculture media at six concentrations. The addition of AgNO₃ significantly improved embryogenic callus frequency and callus growth, but reduced the necroses and almost did not affect callus induction frequencies. Strong genotypic effects on callus induction, embryogenic callus formation, and necrosis frequency were observed. It was also found that there were significant interactions between genotype and AgNO₃ concentrations affecting embryogenic callus frequency and callus growth index. In general, 10 mg/l concentration may be considered most favorable for embryogenesis and prevention of necroses; at the same time, it did not reduce callus induction and promoted callus growth. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 4, pp. 592–596. The text was submitted by the authors in English.     

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     

Heritable somaclonal variation in gliadin proteins of wheat plants derived from immature embryo callus culture

Summary Fertile r₀ plants of the winter wheat line ND7532 (Triticum aestivum L.) were regenerated from callus tissue after 60–190 days in culture. Seeds produced from these self-pollinated plants were planted in the field. Of the 5586 R₁ plants, 32 differed for one or more agronomic traits from plants not passed through tissue culture process. Gliadin electrophoregrams were prepared from bulk samples of R₂ seed from these 32 plants. Four of the 32 produced gliadin patterns different from controls, so 12 seeds of each of these four lines were examined individually. Three of the four mutant lines were fixed for the presence of a mutant protein of 50 relative mobility units (RMU) and the corresponding loss of a parental protein of 26 RMU. The remaining line segregated for the presence/absence of band 50 and the corresponding loss/retention of band 26. The mutant protein of 50 RMU was never seen in control plants. This indicated that either band 50 was coded for by a mutant gene allelic to the gene that coded for band 26 or that bands 26 and 50 were coded for by two different structural alleles under the control of a common regulatory locus. Each of the 12 seeds from the four mutant lines contained a prominent protein band at 30 (RMU), which was only observed as a faint band in one control seed. The types of variation in gliadin patterns observed in somaclones of ND7532 were similar to those reported for the line Yaqui 50E, except that, gliadin changes occurred less frequently in ND7532.This article is submitted in partial fulfillment of the requirements for the Ph.D. in Agronomy for the senior author, D. B. CooperContribution 85-239-J from the Kansas Agricultural Experiment Station. Research was supported by the Science and Education Administration of the U.S. Department of Agriculture under Grant No. 59-22201-1-1-639-0 from Competitive Research Grants Office to R.G.S.     

Effects of low temperature on induction and differentiation of wheat calluses

Young inflorescences and long-term culture calluses of wheat (Triticum aestivum L.) were used to investigate the effect of low temperature treatment on the potential of plant regeneration. The results indicated that the frequency of callus induction from immature inflorescences was decreased when treated for a long time at 5 °C. However, it was found that a 5 °C treatment significantly improved the regeneration frequency of calluses. The clear difference of peroxidases of wheat calluses was apparent after the cultures were treated at the low temperature. The isozyme band 8 became clearly faint, however, bands 6 and 9 became intense. This revised version was published online in June 2006 with corrections to the Cover Date.     

基因枪转化小麦幼胚的再生培养与转基因植株的获得

以小麦幼胚为受体,用基因枪法对Trx-S反义基因 目的基因 和Bar基因 标记基因 进行了共转化,以轰击后的小麦幼胚为实验材料,对幼胚培养的基本培养基、分化和生根培养基进行了筛选优化.结果表明:4种基本培养基中,L3培养基的成愈率最高,且增殖速度快;MS培养基次之.以L3为基本培养基,分化培养基中添加NAA1mg·L-1和ZT2mg·L-1配比对愈伤组织诱导分化的效果最好,分化率达到50%以上.1/2MS培养基中添加IAA0.8mg·L-1的生根效果好,且移栽成活率高.以优化的培养方案对来自7个小麦品种的幼胚进行转化与再生培养,多数品种的出愈率都达到90%以上,分化率在40%以上,并在5个品种上获得再生植株,经检测证实在4个品种上获得转基因再生植株.     

Plant regeneration from immature and mature embryo derived calli of Hordeum marinum

Callus cultures were obtained from immature and mature embryos of Hordeum marnium on MS media containing 0.5 mg l^-1 parachlorophenoxyacetic acid or 2 mg l^-1 2,4-dichlorophenoxyacetic acid. Regeneration occurred after transferring calli to MS either devoid of hormones or supplemented with 1 mg l^-1 indole-3-acetic acid and 1 mg l^-1 zeatin. The regeneration capacity of the immature embryo derived calli (94%) was about 5 times higher than that of mature embryo derived calli (17%). A total of 30 and 964 plantlets were obtained from 21 mature and 59 immature embryo derived calli, respectively. Low frequency (less than 1%) of albino plantlets was obtained from both explants after 3–9 months in culture. Plants expressing transient chlorophyll deficiency were produced from immature embryo derived cultures at a frequency of 10%. However, when transferred to soil, these plantlets became green.     

不同杂种优势类群玉米幼胚愈伤组织的诱导及植株再生特性的研究

以Reid、唐四平头和其它种质等3个杂种优势类群共19份玉米自交系为试验材料,以玉米幼胚作为外植体,研究了基因型、培养基和激素对玉米幼胚愈伤组织的诱导及植株再生的影响,结果表明供试材料均能进行愈伤组织的诱导,但是仅有12个自交系能再生植株。N6和改良N6培养基有助于提高愈伤组织的质量及其生长速度,2,4-D在愈伤组织的诱导中起着关键性作用。在诱导培养基中添加0.2mg／L的6-BA或KT会使胚性愈伤组织的诱导频率下降以及降低愈伤组织的质量。在胚状体诱导培养基中添加1mg／L的KT能促进绿苗的分化,但是浓度过高会使丛生苗分化过多。此外,通过对不同杂种优势类群自交系玉米幼胚培养特性的分析,发现在唐四平头类群的4个自交系中,黄早四的绿苗分化率仅为0.5％,其它3个自交系不能再生植株。但是,从Reid和其它种质类群的供试自交系中筛选出了胚性愈伤组织的诱导频率和绿苗分化率均较高的、适合于遗传转化的受体材料,如3189／4380、4380／陕综5、8103、先早17、18-599红、18-599白、501、178和冀53。     

The production of callus capable of plant regeneration from immature embryos of numerous Zea mays genotypes

In the summer of 1983, immature embryos from 101 selfed inbred lines and germplasm stocks of Zea mays L. were examined for their ability to produce callus cultures capable of plant regeneration (regenerable cultures) using a medium with which some limited success had previously been obtained. Forty-nine of the genotypes (49%) produced callus which visually appeared similar to callus previously cultured and shown to be capable of plant regeneration. After five months, 38 of these genotypes were alive in culture and plants were subsequently regenerated from 35 (92%) of them. No correlation was observed between plant regeneration and callus growth rate, the vivipary mutation (genes vp1, 2, 5, 7, 8 and 9), or published vigor ratings based on K⁺ uptake by roots. When F₁ hybrid embryos were cultured, 97% of the hybrids having at least one regenerable parent also produced callus capable of plant regeneration. No regenerable cultures were obtained from any hybrid lacking a parent capable of producing a regenerable callus culture.In the summer of 1984, immature embryos from 218 additional inbred lines and germplasm stocks were plated and examined for their ability to produce regenerable callus cultures on media containing altered micronutrient concentrations, 3,6-dichloro-o-anisic acid (dicamba), glucose, and elevated levels of vitamin-free casamino acids and thiamine. Of these genotypes 199 (91%) produced callus that was regenerable in appearance. In the 1984 study, plant regeneration was noted in many commercially important inbreds, including B73, Mo17, B84, A632, A634, Ms71, W117, H99³H95 and Cm105. Thus tissue-culture techniques are now available to obtain callus cultures capable of plant regeneration from immature embryos of most maize genotypes.Abbreviations trade names 2,4-D 2,4-dichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid     

小麦幼胚培养高效成株系统的建立

研究探讨了不同的基因型、幼胚取材时期、4℃处理时间、盾片接种方式、分化及生根条件等对小麦幼胚培养再生成株特性的影响,并在此基础上建立了一套高效、可靠、重复性好的小麦组培再生系统。优化条件下,该系统从幼胚诱导致密愈伤组织的频率为89％,致密愈伤组织的分化频率诱导2周时为95％,培养近3个月时仍可达50％以上。此外还发现部分叶状结构当转至新鲜的分化培养基上时能够进一步发育成为芽苗。分化的芽苗在生根培养基上大多生成丛生苗。从基部切开后,每棵芽苗／分蘖均可独立成株。组培苗均可正常地开花结实。     

银杏胚的培养及不定芽的产生

通过对银杏未成熟胚和近成熟胚的培养,研究其不定芽的发生情况.结果表明:(1)银杏的种胚存在生理后熟现象,10月上旬,胚未成熟,在360个种核中,小子叶胚数(1.0～3.0 mm)为26.1％,大子叶胚(3.0～5.0 mm)的种子数占总种核数的42.2％,种核的出胚率为71.1％;(2)大子叶胚和小子叶胚接种在不同的培...     

Influence of 2,4-D, IAA, and duration of callus induction in anther cultures of spring wheat

Culture media and environmental factors may significantly influence the yield of haploid plants from anther cultures. Our objectives were to identify a combination of 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA) concentrations which produce the maximum number of haploid plants, and to evaluate the effects of duration in induction medium on calli induction, plant regeneration, and green plant production from anther cultures in spring wheat. Significant (P ≤ 0.01) plant growth regulator concentration effects (2,4-D and IAA) were observed on the number of calli, green plants and albino plants produced, and on direct plant regeneration. Addition of 2,4-D to the induction medium resulted in significantly (P ≤ 0.01) higher means for all anther culture components compared to IAA> While addition of 2,4-D significantly (P ≤ 0.01) reduced plant regeneration, it substantially increased green plant percentage at a 0.3-mg l⁻¹ concentration of IAA. Use of response functions to estimate the maximum effective 2,4-D × IAA combination implied that higher 2,4-D levels in the induction medium should be investigated, and that the optimum hormone combination differs for plant regeneration and green plant percentage. Significant (P ≤ 0.01) effects of duration on callus induction medium were observed for plant regeneration and green plant percentage.     

Effects of light regimes on anther culture response in bread wheat

This experiment was initiated to further test the effects of light regimes during callus induction and plant regeneration on anther culture response of spring wheat (Triticum aestivum L.). Spring wheat cultivars 'Edwall' and 'WA 7176' with high callus induction from anther culture but low green plant production were used. Different gro-lux light and dark regimes during callus induction, and gro-lux light and fluorescent light regimes during plant regeneration were used. Callus induction decreased significantly at relatively high light intensity (315 μmol m⁻² s⁻¹) applied at any period of culture when compared to continuous dark. Light regimes used continuously and from the 15^th to the last day of callus induction also had a significant negative effect on plant regeneration compared to continuous dark and light application in the first half of callus induction. During plant regeneration, '15 day dark + 7 day gro-lux light' significantly increased plant regeneration compared to both 'gro-lux' and 'fluorescent light' regimes. Light regimes during both callus induction and plant regeneration and their interaction effects were found to be highly significant on green plant proportion and green plant yield. 'Continuous light' application during callus induction increased green plant proportion more than other applications in contrast to its negative effect on plant regeneration. During plant regeneration, '15 day dark + 7 day gro-lux light' had the higher green plant proportion compared to only 'fluorescent light' and only 'gro-lux light'. The highest green plant yields were obtained from '15 day dark + 7 day gro-lux light' during plant regeneration in combination with either 'continuous dark' or 'continuous light' regimes during callus induction. This revised version was published online in June 2006 with corrections to the Cover Date.     

Tissue culture and plant regeneration from immature embryo explants of Calotropis gigantea (Linn.) R. Br.

Callus cultures were established from immature embryos of Calotropis gigantea (Linn.) R. Br. on a modified basal medium of Murashige & Skoog supplemented with 1 mgl^-1 2,4-D. In addition to 0.1 mgl^-1 of NAA the optimal BAP concentration for promoting shoot bud formation and growth was 2 mgl^-1. Rooting was induced when shoots were transferred to auxin-supplemented Bonner's solution or half-strength MS basal salt solutions.Abbreviations NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-butyric acid - BAP 6-benzylaminopurine - Kin kinetin     

玉米幼胚高效再生系统的建立

建立了玉米幼胚高效再生系统.经研究发现,苏玉1号、农大3138、农大108的幼胚培养在含有2,4-D(2 5 mg/L)的IM培养基上后,大多数幼胚能愈伤化并增大,形成基部相连、上部分开的微芽结构;微芽结构在转移到BM培养基上后,形成小植株;进一步转移到RM培养基上,它们长根并形成完整植株.玉米幼胚高效再生植株与下列因素有关玉米基因型、幼胚大小、幼胚长芽至分化时间、6-BA、IBA、Gelrite.不同品种玉米再生能力有显著差异,幼胚大小在1～2mm之间再生能力强,幼胚长芽至分化时间4～6 d最好.激素6-BA浓度在0.5～0.6 mg/L之间有利于微芽形成小植株,IBA浓度在0.6～1 0 mg/L促进生根.Gelrite可代替琼脂粉用于玉米生根.     

Control of direct and indirect somatic embryogenesis by exogenous growth regulators in immature zygotic embryo cultures of rose

Immature zygotic embryos of rose (Rosa hybrida L.; cv. Sumpath) did not form somatic embryos or embryogenic calluses when cultured on half-strength Murashige and Skoog's medium supplemented with various con-centrations of 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole growth regulator. However, the zygotic embryos produced somatic embryos without an intervening callus phase at a frequency of 27.3% on medium with 4.44 M 6-benzyladenine (BA) alone. Immature zygotic embryos formed embryogenic calluses at a frequency of 25% on medium with a combination of 1.36 M 2,4-D and 4.44 M BA. Upon transfer to medium without growth regulators, embryogenic calluses produced numerous somatic embryos that subsequently developed into plantlets. Somatic embryos were induced directly from immature zygotic embryos, or indirectly via an intervening callus phase, by manipulating the exogenous growth regulators. Plantlets were successfully transplanted to potting soil and grown to maturity in a greenhouse.     

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%。     

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.     

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.     

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.