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.     

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.     

Regeneration from long-term embryogenic callus of the <Emphasis Type="Italic">Rosa hybrida</Emphasis> cultivar kardinal

Summary Media components used for three stages of development: (1) callus maintenance, (2) maturation of embryos, and (3) conversion of embryos to plants were shown to affect regeneration of plants for the commercially important red rose cultivar Kardinal. Embryogenic callus was maintained for 5yr on either Schenk and Hildebrandt’s basal salts medium (SH) supplemented with 13.6 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or Murashige and Skoog’s basal salts medium (MS) supplemented with 18.1 μM dicamba and 0.46 μM kinetin. Maturation of embryos was three times higher using callus maintained on the SH medium supplemented with 2,4-D while conversion of cotyledonary-stage embryos to plants was significantly higher (10 times) using callus that had been maintained on MS medium with dicamba and kinetin. Maximum maturation (13.5%), and conversion (15.2%), occurred when callus was cultured on MS maturation medium without hormones. Cotyledonary-stage embryos cultured on MS conversion medium supplemented with abscisic acid (5–20 μM) produced plants that survived at a significantly higher rate (two times) in the greenhouse than when embryos were cultured without abscisic acid. The highest rate of plant regeneration occurred when embryogenic callus of ‘Kardinal’ was maintained on MS medium supplemented with dicamba and kinetin, maturation of embryos occurred on MS maturation medium without hormones, and conversion of cotyledonary-stage embryos occurred on MS conversion medium supplemented with abscisic acid.     

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.     

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     

Efficient and rapid plant regeneration of oil palm zygotic embryos cv. ‘Tenera’ through somatic embryogenesis

An efficient and rapid plant regeneration system through somatic embryogenesis was developed using 13-week-old zygotic embryos of oil palm (Elaeis guineensis Jacq.) cv. ‘Tenera’. Zygotic embryos were cultured on MS and N6 media supplemented with 2.0 mg L⁻¹ picloram, 2,4-D and dicamba. The highest embryogenic callus formation (32%) was observed on N6 medium with 2,4-D after 3 month culture on callus induction medium. Somatic embryos were continuously formed from nodular calli on embryo maturation medium [N6 + 0.1 mg L⁻¹ 2,4-D, 0.16 g L⁻¹ putrescine, 0.5 g L⁻¹ casein amino acids and 2.0 g L⁻¹ activated charcoal(AC)] for 3–5 months. Histological analysis confirmed that embryo development occurred via somatic embryogenesis. For plant regeneration, modified N6 medium (MN6) with AC (0.5 g L⁻¹) without growth regulators, induced both shoot and root formation simultaneously with the highest regeneration rate of 56%. This combined shoot and root induction protocol shortened the culture time to 9–12 months. Furthermore, after acclimatization, more than 85% of transferred plants from our protocol developed successfully in the soil.     

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 from immature and mature embryos of a minor millet Paspalum scrobiculatum L.

Immature and mature zygotic embryos of Paspalum scrobiculatum L. cv. PSC 1 cultured on MS or N₆ nutrient medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), formed embryogenic callus. Induction of embryogenic callus and subsequent somatic embryogenesis was possible at a lower concentration of 2,4-D on N₆ than MS medium. Immature embryos were highly totipotent, forming somatic embryos at a higher frequency than mature embryos. Addition of amino acids (L-proline or L-tryptophan) to 2,4-D medium resulted in significant enhancement of embryogenesis on culture of mature embryos. Silver nitrate also supported an increased frequency of embryogenesis. Thus it is possible to have high frequency of somatic embryogenesis on culture of mature embryos, which are available in abundance and with ease than immature embryos. The somatic embryos readily germinated and formed plantlets on hormone-free regeneration medium. The regenerated plantlets were successful on transfer to soil and set seed.     

Effect of 2,4-Dichlorophenoxyacetic Acid and NaCl on the Establishment of Callus and Plant Regeneration in Durum and Bread Wheat

Optimal callus induction and plant regeneration were obtained in bread and durum wheat by manipulating the NaCl concentration in the induction medium. Immature embryos from a high regeneration line of spring wheat (Triticum aestivum L.), 'MPB-Bobwhite 26', and an elite durum wheat (Triticum turgidum var. durum L.), 'Mexicali', were cultured in E3 induction medium consisting of Murashige and Skoog (MS) medium, 2.5 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 2% sucrose and 0.9% Bacto agar. The treated embryos were transferred to E3 liquid medium supplemented with various levels of 2,4-D and NaCl. Incubation on medium containing 2.5 mg l^–1 2,4-D for 45 days produced callus and plant regeneration in 'MPB-Bobwhite 26', but lower callus yield and plant regeneration in 'Mexicali', indicating that 2,4-D alone was not sufficient for callus induction and plant regeneration in this durum variety. Callus yield and regeneration frequencies were higher in 'Mexicali' embryos that were incubated in media containing 2 mg l^–1 2,4-D and 2 mg l^–1 NaCl. The presence of NaCl in the medium beyond the initiation phase was detrimental to plant regeneration. The use of NaCl in the callus formation could form the basis for improved transformation of durum wheat varieties.     

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.     

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.     

Plant regeneration from immature embryos of 48 elite CIMMYT bread wheats

Forty-eight bread wheat (Triticum aestivum L.) released cultivars and elite advanced lines were evaluated for their ability to produce embryogenic callus using three different media. Basal N6 medium supplemented with dicamba (E1), MS medium containing 2,4-D (E3) or MS medium containing 2,4-D plus different amino acids (E5) were used for callus initiation and maintenance. Plant regeneration was achieved on basal MS medium with indole-3-acetic acid (IAA) and 6-benzylamino purine (BAP) and rooting on MS with 1-naphthaleneacetic acid (NAA). Percentage regeneration varied widely with both genotype and initiation medium, with values ranging from 2% to 94%. The number of plantlets produced per embryo ranged from 6 to 42. Thirteen genotypes showed at least 50% regeneration after culture on E5 medium; 3 genotypes after culture on E3 initiation medium and 1 after initiation on E1. After four subcultures, over a 16-week period, 41 genotypes (85%) lost their ability to regenerate plants while the remaining 7 lines (15%) retained plant regeneration potential but at reduced levels. E3 medium was found to be the best for maintaining regeneration potential after four subcultures.     

节节麦×普通小麦杂种的胚援救和胚愈伤组织再生植株

通过活体-离体胚培养和胚愈伤组织培养有效地克服了节节麦(Aegilops tauschii Cosson.)×小麦(Triticum aestivum L.)杂种幼胚的败育,产生了大量的杂种植株。采用活体-离体胚培技术,节节麦×小麦三个组合杂种幼胚的成苗率为55%,是前人所用传统胚培方法成功率的5—20倍。杂种幼胚在添加有2 mg/L 2,4-D的MS培养基上诱导为愈伤组织,经继代产生全能性愈伤组织,继而分化出再生植株。愈伤组织经继代保存150天仍不丧失分化能力。本文还对两种产生杂种的组织培养方法进行了比较研究。     

High frequency androgenesis from isolated microspores of maize

Anthers from a highly androgenic genotype of maize (139/39-02), when cultured in a modified, liquid YP medium, dehisced within 2–7 days resulting in a stationary suspension of microspores. After 12–15 days, the microspore suspension was found to contain multicellular masses which went on to produce macroscopic embryo-like structures within 20–25 days of culture initiation. Embryogenic callus could be obtained by transferring microspore-derived embryos onto a modified N6 medium supplemented with 2.5 mg/l dicamba and 0.1 mg/l 2,4-D. Subculture onto hormone-free medium resulted in plant regeneration. Over 400 embryo-like structures per 100 anthers cultured have been obtained from liquid induction medium as compared to 55 embryos per 100 anthers cultured on an agar-solidified medium. Approximately 5–25% of these embryo-like structures went on to produce callus from which plants could be recovered. Mechanical isolation of microspores from anthers precultured for 0, 3, and 7 days also resulted in embryo production and plant regeneration. This represents the first report of plant recovery from isolated maize microspores. The use of a liquid induction medium applied to a highly androgenic genotype allows for the production of large numbers of microspore-derived plants and provides a single, haploid cell regeneration system for maize.     

骨干玉米自交系丹598遗传再生体系的建立

目的:以玉米骨干自交系丹598的幼胚为外植体,诱导愈伤组织建立遗传再生体系。方法:探讨胚龄、培养基种类、2,4-D浓度对愈伤组织诱导的影响。结果:在授粉后16～18 d,2,4-D浓度为2.0 mg/L时诱导最佳;设置N6、NB、改良NB、MS、MB等5种培养基,筛选出改良NB培养基为最佳诱导培养基;分化培养基中添加1 mg/L激动素、0.5 mg/L 6-卞基嘌呤和0.5 mg/L萘乙酸能促进绿苗分化和根系生长。结论:建立了玉米自交系丹598的优良再生体系,为以后的基因转化工作打下了良好基础。     

The effect of auxin on plant regeneration of wheat,barley and triticale

One of the basic components of a medium influencing somatic embryogenesis of cereals from immature embryos is the type of auxin. According to some researchers, phytohormones can also play an important role during Agrobacterium-mediated transformation. In this first part of research, the influence of three types of auxins used alone or in combination of two on somatic embryogenesis and plant regeneration in three cereal species has been tested. Eight cultivars of barley, five cultivars of wheat and three cultivars of triticale have been used. Efficiency of plant development on two regeneration media, with and without growth regulators has been compared. Efficiency of regeneration characterized by frequency of explants that form embryogenic callus ranged from 25% for wheat cultivar Torka to 100% for two barley cultivars. Mean number of plantlets regenerating per explant differed significantly (from 2 to 58) depending on the type of auxin in inducing media, the type of regenerating media as well as cultivar. The biggest differences in regeneration efficiency were observed between barley cultivars, however regeneration of plants occurred in all combinations tested. The best regeneration coefficients for most barley cultivars were obtained after culture on dicamba or dicamba with 2,4-D. However, in the case of highly regenerating cv Scarlett, the most effective culture media contained picloram or 2,4-D alone. The highest values of regeneration coefficients for two triticale cultivars (Wanad and Kargo) were obtained on picloram (26.1 and 21.4, respectively) and for `Gabo' on picloram with dicamba (12.6). The range of mean number of regenerated plantlets was from 12 to 30. Dicamba alone or lower concentrations of picloram with 2,4-D were the best media influencing embryogenic callus formation in five wheat cultivars. However, the highest values of regeneration coefficients ranging from 10.6 to 26.8 were obtained at lower concentrations of picloram with 2,4-D or picloram with dicamba. R2 regeneration medium containing growth regulators was significantly better for plantlet development in several combinations (cultivar and induction medium) than the one without growth regulators. Generally, regeneration coefficients for all tested cultivars of three cereal species on the best media were high, ranging from 5.5 for barley cultivar Rodion to 51.6 for another barley cultivar Scarlett. Plantlets developed normally, flowering and setting seed.     

Plant regeneration in vitro from embryogenic cultures of spring- and winter-type barley (Hordeum vulgare L.) varieties

Summary Immature embryos of 41 lines of barley were screened in vitro for callus induction and somatic embryogenesis on different media to establish totipotent cultures. The use of modified MS and CC media, both supplemented with 1 g/l casein hydrolysate, and the substitution of agarose for agar resulted in the highest frequencies of somatic embryo induction. Embryogenic callus was induced and plants regenerated from 23 of the lines tested. The auxins 2,4-D, dicamba, picloram and 2,4,5-T were suitable for embryogenic callus induction. High frequencies of somatic embryo germination occurred on CC medium supplemented with 1 mg/l IAA and 0.05 mg/l zeatin. A strong genotypic effect on the capacity and frequency of embryogenic callus formation was found. Cultivar Golden Promise always gave the best results. Experiments with field grown material in 3 consecutive years showed that environmental factors also strongly influenced the induction of somatic embryogenesis and plant regeneration.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid - picloram 4-amino-3,6,6-trichloropicolinic acid - NAA naphtaleneacetic acid - IAA indole-3-acetic acid - ABA abscisic acid - BAP 6-benzyl amino purine - 2iP 6-(3-methyl-2 butenyl 1-amino)purine - GA₃ gibberellic acid     

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.     

Embryogenesis and plant regeneration from tissue culture of Canada wildrye,Elymus canadensis L.

Somatic embryogenesis and plant regeneration of Canada wildrye (Elymus canadensis L.) from tissue culture was investigated by culturing immature embryos and inflorescences on MS medium containing 2 mg/l 2,4-D. The optimum size of explants for maximum embryogenic callus formation was 1.0 to 1.5 mm for embryos and 4 to 6 cm for inflorescences. Plant regeneration from the subcultured embryogenic callus was attempted monthly using hormone-free MS medium or MS medium with 0.5 mg/1 2,4-D and 0.3 mg/l GA3. Three hundred and fifty seven plantlets were regenerated from the callus cultures of both explant sources during a six month period. Ten chlorophyll deficient plants accounting for 2.8% of the total regenerants were observed. One plant with white striped leaves survived and was found to be an octoploid.Abbreviations GA3 gibberellic acid - MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid     

Enhanced production and germination of somatic embryos by temporary starvation in tissue cultures of Daucus carota

Embryogenic callus was induced from cotyledonary explants of Daucus carota L. cultured on solidified MS medium supplemented with 1 mg l^-1 2,4-D. Following callus initiation somatic embryos were developed from the callus on MS medium without 2,4-dichlorophenoxyacetic acid. To stimulate the production and germination of somatic embryos we cultured the callus under physically and chemically modified conditions during subculture. When the embryogenic callus was cultured on half-strength MS medium or MS medium without sucrose or cultured under conditions of reduced humidity (69.3%), the production of embryos increased 3.4- to 4.5-fold compared to culture on MS medium containing 3% sucrose (control). Embryogenic callus cultured on MS medium after 5 days of starvation (by being placed in empty 12-well tissue culture plates) showed a 20-fold increase in somatic embryo production and enhanced maturation and germination of embryos. An important point is that the germination of somatic embryos with cup-shaped cotyledons, after a period in culture without medium, was remarkably improved (92%) compared to that of the controls (23%).Thus, we were able to show that stress by starvation without medium led to the enhanced production and increased germination of somatic embryos.