Effect of 2,4-dichlorophenoxyacetic acid on callus induction and plant regeneration in anther culture of wheat (Triticum aestivum L.)

 Anthers from a doubled-haploid line of spring wheat (Triticum aestivum L.) cv. Pavon 76 were plated in liquid P-4 medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at four concentrations (0.5, 1.0, 2.0, 4.0 mg/l) for 5, 10, 15, and 25 days before being transferred to another medium with the same or reduced 2,4-D concentrations for the remainder of the induction phase for a total of 45 days. Incubation with 0.5 mg/l 2,4-D for 45 days produced lower callus yield and plant regeneration, indicative of insufficient auxin for callus induction. Callus yield and regeneration frequencies were higher with 1.0 mg/l 2,4-D. With 2.0 or 4.0 mg/l 2,4-D, an induction period of 10 or 15 days was sufficient for initiation of callus development. The extended presence of 2–4 mg/l 2,4-D in the medium beyond the initiation phase was detrimental to plant regeneration. Thus optimal callus induction and plant regeneration could be obtained through manipulating the 2,4-D concentration and the duration of its presence in the induction medium. Received: 1 December 1997 / Revision received: 15 February 1999 / Accepted: 26 February 1999     

Auxin and sugar effects on callus induction and plant regeneration frequencies from mature embryos of wheat (Triticum aestivum L.)

Summary Genetic engineering of cereals currently depends on the use of tissue culture and plant regeneration systems. In wheat (Triticum aestivum L.), immature embryos are the most widely used explant to initiate cultures, but they are inconvenient due to their temporal availability and production requirements. Mature embryos are easily stored and are readily available as mature seeds. However, plant regeneration frequencies from cultures derived from mature embryos are generally low. This research was undertaken to improve callus induction and plant regeneration from wheat mature embryos of cultivar ‘Bobwhite’. The effects of four auxins [2,4-dichlorophenoxyacetic acid (2,4-D): 3,6-dichloro-o-anisic acid (dicamba); 4-amino-3,5,6-trichloropicolinic acid (picloram): and 2-(2-methyl-4-chlorophenoxy) propionic acid (2-MCPP)], and the effect of maltose vs. sucrose under filter sterilized and autoclaved conditions were evaluated. All auxin treatments resulted in callus induction except 2 MCPP. A highly significant effect of auxin type on both callus and plantlet production was detected, though interactions were observed. The effect of sugar type was dependent on the type of auxin used. Substitution of sucrose by maltose enhanced the regenration ability of callus from embryos cultured on media containing 2,4-D and picloram, but caused an opposite effect on media containing dicamba. Picloram significantly enhanced callus growth, however, embryogenic response and plant regenerability were low. Relative to 2.4-D, dicamba (18μM) resulted in a twofold increase in the number of plants regenerated per embryo and reduced the amount of time required for plant regeneration by 3–4 wk. Mention of a trademark or proprietary product does not constitute a guarantce or warranty by the University of Wisconsin and does not imply its approval to the exclusion of other products that may be suitable.     

In vitro development of wheat (Triticum aestivum L.) from zygote to plant via ovule culture

Ovules of the wheat breeding line Veery #5 were excised and transferred to culture within 24 h after pollination. When ovules were cultured on Phytagel-solidified medium, and the pericarp removed exclusively at the micropylar tip and the abaxial side, zygotes from up to 79.2% of the ovules underwent embryogenesis with the same developmental pattern as found in planta. Embryos from more than 50% of the cultured ovules germinated when transferred to regeneration medium. More than 100 plantlets were randomly chosen for transfer to soil, all of which developed to phenotypically normal and fertile plants. With this system, the entire process of zygotic embryogenesis can be studied using living material. Furthermore, the method could be used as an embryo rescue technique for plant breeding purposes. Received: 17 June 1996 / Revision received: 22 October 1996 / Accepted: 15 December 1996     

Monosomic analysis of tissue culture response in wheat (Triticum aestivum L.)

Summary The ability of immature embryos of wheat (Triticum aestivum L.) to respond in cell culture was examined in crosses between the Wichita monosomic series and a highly regenerable line, ND7532. Segregation in disomic controls and 13 monosomic families showed a good fit to a monogenic ratio indicating a qualitative mode of inheritance. Segregation in the cross involving monosomic 2D showed a high frequency of regeneration (93.6%) and high callus growth rate (1.87 g/90 days) indicating that 2D is a critical chromosome. Modifying genes may be located on other chromosomes. Substitution of chromosomes from a low regenerable cultivar Vona further indicated that the group 2 chromosomes, in particular chromosome 2D, possess genetic factors promoting callus growth and regeneration.     

Control of tissue culture response in wheat (Triticum aestivum L.)

Summary The ability of immature embryos of wheat (Triticum aestivum L.) to respond to tissue culture has been shown to involve the group 2 chromosomes. The available group 2 ditelosomic and nullisomic-tetrasomic lines of Chinese Spring wheat were used to determine the chromosome arm location and chromosome dosage effect associated with the expression of tissue culture response (TCR). Significant differences were found between the aneuploid lines and the euploid control for the expression of both regenerable callus formation and callus growth rate. A model is proposed suggesting that a major TCR gene is located on 2DL and that 2AL and 2BS possess minor TCR genes. Furthermore, a major regulatory gene controlling the expression of TCR genes may be located on chromosome 2BL.     

Culture of and fertile plant regeneration from regenerable embryogenic suspension cell-derived protoplasts of wheat (Triticum aestivum L.)

Summary Regenerable embryogenic cell suspensions initiated from immature embryo-derived friable, fast growing, embryogenic calli of GK Ságvári winter wheat (Triticum aestivum L.) served as sources of protoplasts, which were cultured in different liquid or agarose-solidified media. Protocallus formation was best on KM_8p (Kao and Michayluk 1975) and GM (Li and Murai 1990) media, and protocallus growth on MS (Murashige and Skoog 1962) callus growing medium. Green shoot/plant regeneration occurred on MS regenerating medium, and rooting on MS or N6M (Mórocz et al. 1990) hormone-free media. Protocalli maintained their morphogenic capacity over 4 months, and with multiple subcultures on half-strength MS regenerating medium, the total number of regenerants could be increased. Approximately 1000 shoots/plants were regenerated and over 500 plants were transplanted in the greenhouse. The majority of them had an abnormal chromosome number and low viability, however, one plant grew to maturity and set seed.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - ECS embryogenic cell suspension - GA₃ gibberellic acid - GM General medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog medium - NAA 1-naphthaleneacetic acid - RECS regenerable embryogenic cell suspension     

不同小麦品种愈伤组织诱导和再生体系建立

为了筛选适合组织培养的小麦基因型,建立一套有效的小麦诱导再生体系,以24个小麦品种的幼胚为研究材料,选用4种诱导培养基和3种分化培养基,研究了影响小麦组织培养的各种因素。结果表明：①培养基之间存在显著差异,MM2培养基的诱导效果最好,平均诱导率为98．5％,M5B培养基的分化效果最佳,平均分化率为39．8％。②不同品种在诱导愈伤和分化再生上都有显著的基因型差异。③愈伤组织诱导率和分化率之间无显著相关性。     

Substitution analysis of callus induction and plant regeneration from anther culture in wheat (Triticum aestivum L.)

The genetic determination of callus induction, total plant regeneration and green plant regeneration from anther culture were studied using a Chinese Spring/Cheyenne substitution series. All the three characteristics were found to be polygenically determined, but their inheritance was independent from one another. The 7A and 18 chromosomes had a considerable effect on callus induction. In the case of total plant regeneration the most influential chromosome as the 3A while the 2D chromosome showed a definite influence on green plant regeneration. The interaction between the genetic background of the recipient plant and the substituted chromosome plays an important role in the manifestation of the studied features.     

Cytoplasmic effects on the tissue culture response of wheat (Triticum aestivum) callus

Summary Calli were initiated from immature embryos of eight lines of hexaploid wheat (Triticum aestivum L. em. Thell) with different cytoplasms, the euplasmic nuclear donor Chinese Spring and seven alloplasmic lines derived from wild relative species of the genera Triticum and Aegilops. The calli were found to differ in their initial growth rates, their sensitivity to 2,4-D and their ability to organise shoot primordia, demonstrating that the cytoplasm can significantly affect the behaviour of tissues in culture. The potential for improving the responses of tissues in culture by cytoplasmic changes is noted.     

Combining abilities and heritability of callus formation and plantlet regeneration in wheat (Triticum aestivum L.) anther cultures

Summary Frequency of callus formation in wheat (Triticum aestivum L. em. Thell) anthers cultured in vitro and the frequency of subsequent plantlet formation from such calli were examined in a diallel population produced from five inbred spring wheat cultivars. Two of the five cultivars were believed to possess relatively high frequencies of response and the other three relatively low response frequencies, based on previous studies. General and specific combining abilities were estimated and found to be highly significant for both traits. Reciprocal effects were also estimated and were highly significant for both traits. Of the 25 entries, the largest mean callus formation frequency was observed on anthers of Kitt x Olaf, while the largest mean plantlet formation frequency was observed using anthers of the cultivar, Fielder. No significant correlation was observed between the two traits. Heritability estimates in the range of 0.6–0.7 suggested, however, that both traits were highly heritable, so that rapid gain from selection for these traits should be possible. Current limitations due to genetic variation in responses therefore may not constitute a major obstacle to application of in vitro techniques by wheat breeders.Scientific Article No. 3710 Contribution No. 6686 of the Maryland Agric. Exp. Stn., Dept. of Agronomy, College Park, MD 20742, and USDA-ARS, Beltsville, MD 20705, USA     

Fertile wheat (Triticum aestivum L.) regenerants from protoplasts of embryogenic suspension culture

We report regeneration of fertile, green plants from wheat (Triticum aestivum L. cv. Aura) protoplasts isolated from an embryogenic suspension initiated from somatic early-embryogenic callus. The present approach combines the optimization of protoplast culture conditions with screening for responsive genotypes. In addition to the dominant effect of the culture media, the increase in fresh mass and the embryogenic potential of somatic callus cultures varied considerably between the various genotypes tested. Establishment of suspension cultures with the required characters for protoplast isolation was improved by reduction of the ratio between cells and medium and by less frequent (monthly) transfer into fresh medium. A new washing solution was introduced to avoid the aggregation of protoplasts. However, the influence of the culture medium on cell division was variable in the different genotypes. We could identify cultures from cultivar Aura that showed approximately a 9% cell division frequency and morphogenic response. The protoplast-derived microcolonies formed both early and late-embryogenic callus on regeneration medium and green fertile plants were obtained through somatic embryogenesis. The reproducibility of plant regeneration from protoplast culture based on the cultivar Aura was demonstrated by several independent experiments. The maintenance of regeneration potential in Aura suspension cultures required establishment of new cultures within a 9-month period.     

Pollen callus culture in Triticum aestivum

Summary Pollen shed between 4–8 d from anthers of Triticum aestivum cultured in liquid medium gave rise to calluses. Tillers were harvested at the mid-to late-unicellular pollen stages and chilled for 8 d at 4–5 °C before the anthers were dissected out. Pollen cultures gave about 6 times as many calluses on a per anther basis as anthers cultured on solid medium. With the most productive of 5 cultivars tested, pollen culture results in roughly one callus for each anther used, though the calluses formed by pollen culture were less productive for the regeneration of shoots than calluses derived from anthers cultured on solid medium. The ratio of green to albino shoots is roughly 1 1 for anther cultures but considerably less for pollen cultures.     

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.     

Somatic embryogenesis and plant regeneration from protoplasts isolated from embryogenic cell suspensions of wheat (Triticum aestivum L.)

We describe the early formation of somatic embryos followed by plant regeneration from protoplasts isolated from an embryogenic wheat cell suspension, which was initiated from small granular (0.2 to 1 mm in size) embryogenic calli. These granular calli formed embryogenic cell suspensions within 20 days in liquid culture, and were selected gradually from young inflorescence-derived nodular embryogenic calli of the winter wheat cv. Kehong 1041. The division frequency of protoplasts was 11 to 16%, and the frequency of differentiation into plants was about 0.001% (number of plants formed divided by the total number of protoplasts plated). About 20% of somatic embryos present in the culture formed directly from protoplast-derived cells within 15 days of cultures.     

Plant regeneration through callus cultures derived from immature-cotyledon explants of oleaster (Elaeagnus angustifolia L.)

Efficient plant regeneration was achieved from callus derived from immature-cotyledon explants of oleaster (Elaeagnus angustifolia L.). Calli were obtained on MS media containing 3% sucrose and different concentrations of TDZ. The highest rate of green, compact and nodular callus was formed on MS medium supplemented with 1 mg/l of TDZ. Shoot organogenesis was achieved when the callus was transferred onto MS media containing 3% sucrose and BA alone (05–4 mg/l) or BA (0.5 and 1 mg/l) combined with NAA or IAA (0.5 and 1 mg/l). Maximum organogenesis was obtained with 1 mg/l BA in combination with 0.5 mg/l NAA. Rooting of the shoots was achieved on MS medium supplemented with 0.2 mg/l IBA. Regenerated plantlets were acclimatized and successfully transplanted to soil.     

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.     

渗透胁迫条件下小麦成熟胚愈伤组织的诱导及植株再生的研究

用不同浓度的PEG6000及NaCl对5个小麦品种的成熟胚组织培养物进行处理,研究在渗透胁迫条件下基因型和激素对成熟胚愈伤组织的诱导及分化的影响。结果表明,小麦整株水平与细胞水平的抗性存在一定相关,不同基因型对干旱与盐胁迫的敏感程度不同,成熟胚愈伤组织的诱导率和植株再生率表现出明显的差异。初步得到了晋麦47、长武134、红芒麦的耐旱愈伤组织以及晋麦47、长武134的耐盐愈伤组织,并获得了晋麦47和长武134具有一定抗性的再生芽。     

In vitro plant regeneration from callus of shoot apices in apple shoot culture

A new reliable protocol for the induction of adventitious shoot formation and plant regeneration from apple callus has been developed. High regeneration frequency was obtained with this method in four different genotypes (Jork9, M26, Gala and McIntosh) and callus maintained regeneration ability for several months. The procedure consists of inducing vegetative shoot apices, excised from in vitro shoots, for 20 days in darkness on an MS medium without glycine, supplied with 17.8 μM BA, 2.7 μM NAA and 250 mg l⁻¹ cefotaxime. The explants are then transferred to a fresh auxin-free medium and given light. Histological studies revealed that all the regenerated shoots originated from callus. Regenerated shoots were multiplied, rooted and successfully established in soil. Received: 2 April 1999 / Revision received: 10 November 1999 / Accepted: 15 November 1999     

Reciprocal substitutions analysis of embryo induction and plant regeneration from anther culture in wheat (Triticum aestivum L.).

Reciprocal substitutions for all chromosomes between the hard red winter wheat cultivars Wichita and Cheyenne were used to investigate the effects of individual chromosomes, as well as their interactions with the genetic background, on androgenesis. Duplicate lines for each chromosome were included to check background homogeneity. Six experiments, two for each genome, were performed. In each experiment, 14 substitution lines, their 14 duplicate lines, and the two parental genotypes ('Cheyenne' and 'Wichita') were studied. The experimental design was a randomized block with three replications. 'Wichita' and 'Cheyenne' differed significantly in embryo yield and green plant regeneration (except green plant regeneration for the B-genome tests) and were equal for albino and total plant regeneration. Embryogenesis was influenced by some chromosomes of the A, B, and D genomes; green plant production was influenced by all chromosomes of the A and D genomes except 5D; albino and total plant regeneration were affected by some chromosomes of the B and D genomes. Reciprocal effects were obtained with chromosomes 1A, 7A, 1B, 5B, 1D, and 2D for embryogenesis, chromosomes 2D and 7D for green plant regeneration, and chromosome 2D for total plant regeneration. Reciprocal substitution lines revealed reciprocal effects of homologous chromosomes, as well as interactions between substituted chromosomes and their specific genetic background.     

Genetic variability in callus formation and regeneration of garlic (Allium sativum L.)

Twenty garlic Allium sativum (L.) genotypes were analysed for genetic variation in their ability to form callus (in one medium) and regenerate shoots (in four different media). Genotypes showed significant differences in the analysis of variance of all the traits tested. The accession Printanor showed the best general behaviour, with 83% callus-producing explants, 44.7% organogenic explants, and 15.35 shoots/g of callus. The best regeneration medium was MBO, without growth regulators. Shoot production capacity was examined with the additive main effects and multiplicative interaction model that proved to be a powerful tool for analysis and easy comprehension of the strong genotype×medium interactions frequently observed in in vitro culture systems. Received: 5 February 1998 / Revision received: 11 May 1998 / Accepted: 1 June 1998