Efficient plant regeneration system for immature embryos of triticale (x Triticosecale Wittmack)

A range of tissue culture conditions were tested to improve embryo culture frequency, and to develop an efficient plant regeneration system for triticale. Immature embryos (14–21 days post-anthesis) from two triticale genotypes (Hx87-139 and Tahara) were cultured on a commonly used Murashige and Skoog (MS) and on Lazzeri's (L1) basal medium with varied carbon sources, and two different plant growth regulators; 2,4-Dichlorophenoxyacetic acid (2,4-D) and 3,6-Dichloro-2-methoxybenzoic acid (dicamba). Although embryos could be cultured on both media types, L1 based medium was better than MS basal salts for callus induction and somatic embryogenesis, with plant regeneration frequencies up to 11 fold greater on L1 media types. In the presence of dicamba, callus induction was more rapid, that resulted in subsequent regeneration of up to 2 fold more plantlets than from callus induced on medium containing 2,4-D. Maltose appeared to be a superior carbon source during differentiation of callus. Genotype Tahara showed a better regenerative response than Hx87-138, with up to 23 normal, fertile plants being produced from a single embryo when cultured on L1MDic medium, containing maltose (5%) and dicamba (20 mg l^–1). Applications of this tissue culture procedure in triticale improvement through genetic engineering are also discussed.     

Comparison of callus induction and plant regeneration from different explants in triploid and tetraploid turf-type bermudagrasses

The turf-type bermudagrasses are genetically variable and do not respond uniformly to tissue culture and plant regeneration protocols. We evaluated the callus induction response of two explant types, young inflorescences and nodes, from multiple genotypes including triploid TifSport, TifEagle, and Tift97-4 and tetraploid Tift93-132, Tift93-135, Tift93-156 and Tift93-157 on MS medium supplemented with 1–1.5 mg l⁻¹ 2,4-D + 0.01 mg l⁻¹ BA + 1.16 g l⁻¹ proline. Four types of callus were observed. Type I was fluffy, soft, and white non-embryogenic callus, common to all cultures. Type II was globular, transparent, and hard, but sticky callus, which was pre-embryogenic and could be selected for subculture. Type III callus was transparent, compact, and embryogenic. Type IV callus was opaque white and compact. Both Type III and Type IV calluses were embryogenic and regenerative. A combination of gelling agents in the medium (2 g l⁻¹ Gelrite and 5 g l⁻¹ agar) improved callus quality and increased the rate of compact callus formation during subculture. Embryogenesis from compact callus was observed in TifEagle, TifSport and Tift93-132, and shoots were regenerated on MS medium with 0.1 mg l⁻¹ 2,4-D + 0.5–4.0 mg l⁻¹ BA. Low intensity light treatment (30 μmol m⁻2 s⁻¹ of cool white fluorescence) to callus before regeneration greatly enhanced regeneration frequency from 6.7% to 40% in recalcitrant TifSport.     

海边香豌豆胚性愈伤组织的诱导和体细胞胚发生

将生长１４ｄ的海边香豌豆（Ｌａｔｈｙｒｕｓ ｍａｒｉｔｉｍｕｓ（Ｌ．）Ｂｉｇｅｌ）无菌苗下胚轴切成０．５ｃｍ左右的片段,置于含有１ｍｇ／Ｌ２,４－Ｄ,０．５ｍｇ／Ｌ ＢＡ和０．５％ＮａＣｌ的ＭＳ培养基中,２８ｄ后诱导出胚性愈伤组织。将其转入含有适当浓度２,４－Ｄ的ＭＳ培养基上,又２８ｄ后可得到大量球形胚和心形胚以及极少量鱼雷胚和子叶胚。诱导体细胞胚适合的２,４－Ｄ浓度为０．５ｍｇ／Ｌ。较高浓度的２     

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     

In vitro plant regeneration from callus derived from root explants of Lathyrus sativus

Protocols have been developed for the in vitro production of plants from callus derived from root explants of Lathyrus sativus cv. P-24. Callus and shoot regeneration were achieved only in MS medium supplemented with 10.7 M naphthaleneacetic acid and an increased concentration of kinetin (0.9 M for 14 days to 1.4 M for 18 days) during callusing. The shoots obtained rooted in 1/2 MS supplemented with 0.5 M indolebutyric acid. During the year plants have been regenerated several times. The requirement for growth regulators is very specific and narrow.Abbreviations IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid - BA benzyladenine     

High efficiency plant regeneration by somatic embryogenesis from callus of mature embryo explants of bread wheat (Triticum aestivum) and grain sorghum (Sorghum bicolor)

Summary Tissue culture methods were developed for reproducible induction and maintenance of embryogenic (E) callus established from developmentally mature embryo explants of bread wheat (Triticum aestivum) and grain sorghum (Sorghum bicolor). Embryogenic callus was obtained by culturing seeds and mature embryos of wheat on Linsmaier and Skoog’s (LS) medium containing 5 or 2 mg/liter 2,4-dichlorophenoxyacetic acid (2,4-D), respectively, and for sorghum mature embryos on LS medium containing 2 mg/1 2,4-D plus 0.5 mg/liter kinetin. Plant regeneration from E callus was achieved for several months and quantified on a fresh-weight basis of E callus. Phenotypically normal plants were regenerated from E callus cultured on LS medium supplemented with 0.1 mg/liter IAA plus 0.5 mg/liter benzyladenine (BA) for wheat and 1.0 mg/liter IAA plus 0.5 mg/1BA for sorghum. Wheat research was funded by the United States Agency for International Development, Washington, DC, cooperative agreement DNA-4137-A-00-4-53-00. Sorghum research was supported by the Gas Research Institute, Chicago, IL, contract 5084-260-0973. Expert technical asistance was provided by Nitschka S. ter Kuile, Barbara J. Ashton, Laurie Osborne, Erin Scott, and Kathleen M. Petersen.     

Callus induction and plant regeneration from immature embryos of rye and triticale

Callus cultures were established from the scutellum, scutellar node and radicle region of immature embryos of rye and octoploid triticale on modified Murashige-Skoog basal medium supplemented with various growth regulators. 2, 4-D, 2, 4, 5-T and 2, 4, 5-Cl, POP were found suitable for initiation and maintenance of callus cultures. Cytokinins had no or inhibitory effect on callus induction and growth. On basal medium containing 5 mg/l of 2,4,5-Cl₃ POP, 16% of triticale and 17% of rye primary cultures exhibited shoot bud regeneration after 3–4 weeks. Transfer of such cultures to basal medium supplemented with zeatin or zeatin in combination with IAA further promoted shoot elongation and plantlet formation. Plantlets were rooted on basal medium containing 1 mg/l NAA and were eventually transferred to soil. Chlorophyll variants were observed in about 6% of triticale cultures.     

In vitro callus induction and plant regeneration from leaf explants of Ruta graveolens L.

A protocol for multiple shoot bud induction and plant regeneration from leaf segment-derived callus of Ruta graveolens has been developed. Maximum organogenic callus induction frequency (70.6 ± 2.33%) was observed on Murashige and Skoog (MS) medium supplemented with 10 µM 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Multiple shoot induction was achieved from the surface of the callus when transferred to shoot induction media (MS nutrients supplemented with 6-benzyladenine (BA), kinetin (Kn), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA) in various concentrations and combinations). The highest shoot multiplication (92.3%) was observed on MS medium with 7.5 µM BA and 1.0 µM NAA. Regenerated shoots were rooted in vitro on MS containing 0.5 µM IBA. Plantlets with well developed root and shoot systems were successfully acclimated (90%) and established in earthen pots containing garden soil; they exhibited normal morphology and growth characteristics.     

Significance of different carbon sources and sterilization methods on callus induction and plant regeneration of Miscanthus x ogiformis Honda `Giganteus'

Different carbon sources, sterilized by autoclaving or filter-sterilization, were tested during induction, maintenance, and plant regeneration of embryogenic Miscanthus x ogiformis Honda `Giganteus' callus, derived from various explant types. Explants from small immature inflorescences, between 2.5 and 8 mm, produced more embryogenic callus than explants from shorter or longer inflorescences, shoot apices or leaf explants. On medium containing mannitol or sorbitol, only small amounts of callus were induced and no embryogenic callus was formed. Callus induction and embryogenic callus formation on shoot apices and immature inflorescences did not differ significantly between media containing sucrose, glucose, fructose, maltose or a mixture of glucose and fructose. However, callus induction and embryogenic callus formation from leaf explants were best on glucose. A higher percentage of leaf explants formed callus on autoclaved sucrose, as opposed to the other carbon sources where filter-sterilization in general resulted in a higher callus percentage. The growth rate of embryogenic callus was influenced both by carbon source and sterilization method when less than 1 g of callus was inoculated. None of the tested carbon sources could considerably improve plant regeneration from M. `Giganteus' callus, but a higher number of plants tended to be regenerated per callus piece from filter-sterilized carbon sources. This revised version was published online in June 2006 with corrections to the Cover Date.     

陆地棉中棉所24胚性愈伤组织的诱导及植株再生

以陆地棉“中棉所24”为材料进行了全固体体细胞培养,获得了愈伤组织和再生植株。愈伤组织诱导阶段采用0．01IAA 0．01KT 0．012,4-D的培养基效果好,继代时间多为30～50d;激素由高到低的继代可明显提高胚性愈伤分化率,IAA和KT含量均较低,IAA／KT比例为1：1～1：6,胚性愈伤最高分化率为50．22％。     

Genetic mapping of a 7R Al tolerance QTL in triticale (x Triticosecale Wittmack)

Triticale (x Triticosecale Wittmack) is a relatively new cereal crop. In Poland, triticale is grown on 12 % of arable land (http://www.stat.gov.pl). There is an increasing interest in its cultivation due to lowered production costs and increased adaptation to adverse environmental conditions. However, it has an insufficient tolerance to the presence of aluminum ions (Al³⁺) in the soil. The number of genes controlling aluminum tolerance in triticale and their chromosomal location is not known. Two F2 mapping biparental populations (MP1 and MP15) segregating for aluminum (Al) tolerance were tested with AFLP, SSR, DArT, and specific PCR markers. Genetic mapping enabled the construction of linkage groups representing chromosomes 7R, 5R and 2B. Obtained linkage groups were common for both mapping populations and mostly included the same markers. Composite interval mapping (CIM) allowed identification of a single QTL that mapped to the 7R chromosome and explained 25 % (MP1) and 36 % (MP15) of phenotypic variation. The B1, B26 and Xscm150 markers were 0.04 cM and 0.02 cM from the maximum of the LOD function in the MP1 and MP15, respectively and were highly associated with aluminum tolerance as indicated by Kruskal–Wallis nonparametric test. Moreover, the molecular markers B1, B26, Xrems1162 and Xscm92, previously associated with the Alt4 locus that encoded an aluminum-activated malate transporter (ScALMT1) that was involved in Al tolerance in rye (Secale cereale) also mapped within QTL. Biochemical analysis of plants represented MP1 and MP15 mapping populations confirmed that the QTL located on 7R chromosome in both mapping populations is responsible for Al tolerance.     

Plant regeneration from apple seedling explants and callus cultures

Leaf, cotyledon, and hypocotyl explants were obtained from 3-week-old seedlings of open-pollinated ‘Golden Delicious’ (Malus domestica bork H.) grown in vitro. They were placed on modified Murashige and Skoog (MS) medium containing B5 vitamins, sucrose and agar, supplemented with 6-benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA), and maintained at 25°C±2 in the light or in the dark to assess morphogenetic responses. Leaf and cotyledon explants cultured in the dark for an initial 3 weeks, then transferred to light for 4 weeks, produced 5- to 20-fold more adventitious shoots than those cultured for 7 weeks in the light. Conversely, light did not significantly influence the number of adventitious shoots formed on hypocotyl explants. Five-minute daily exposures of leaf explants to red light (651 nm) suppressed adventitious shoot formation by 80%; five-minute exposure to far-red light (729 nm) immediately following the red light counteracted the red suppression. Seedling explants, immature fruit halves and immature embryos were also cultured on Schenk and Hildebrandt (SH) medium containing 2, 4-dichlorophenoxyacetic acid (2, 4-D), p-chlorophenoxyacetic acid (CPA) and kinetin. Light inhibited callus formation on leaf and cotyledon explants, but not on hypocotyl explants. The derived callus was placed on MS + BAP or MS + BAP + NAA for shoot regeneration. Both shoots and roots regenerated from callus placed in the dark but not in the light; the frequency of shoot regeneration was 5% or less. Regenerated shoots were rooted on MS macronutrient salts (1/3 concentration), micronutrients, i-inositol, thiamine HCl, sucrose and agar with or without indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or NAA under a light intensity of 5.0 W.m^-2 (16 h per day). Auxin concentration strongly influenced root morphology.     

Development of a highly efficient callus induction and plant regeneration system for Dendrocalamus sinicus using hypocotyls as explants

Plant Cell, Tissue and Organ Culture (PCTOC) - Dendrocalamus sinicus is the largest bamboo species in the world. To aid its rapid propagation and explore its desired traits, we identified the...     

Plant regeneration from callus and explants of Sesbania spp.

Root, hypocotyl and cotyledon explants of Sesbania bispinosa, Sesbania cannabina, Sesbania formosa, and Sesbania sesban were cultured on Murashige and Skoog medium with benzyladenine (BA; 2.22, 4.44, 8.88 M) in combination with 2,4-dichlorophenoxyacetic acid (2,4-d; 2.26, 4.52, 9.05 M), indolebutyric acid (IBA; 0.25, 0.49, 4.92 M) or naphthaleneacetic acid (NAA; 2.69, 5.37, 10.74 M). Although all explant types developed some callus, callus occurred earliest and continued to grow fastest with hypocotyls. Media including 2.4-d or NAA gave the fastest growing callus. Callus was subcultured up to 10 times at 20-day intervals and retained a rapid growth rate. Shoots regenerated readily from both hypocotyls or cotyledons but not from roots. Shoot organogenesis was most frequent with IBA (0.25–4.92 M) in combination with BA (4.44–8.88 M) and did not occur with 2,4-d. With each species at least one medium induced shoot differentiation from more than 50 percent of the callus pieces. With one exception, media containing IBA that induced shoot organogenesis on explants also did so in callus, but media containing NAA, even when effective with explants, did not cause differentiation of callus. Shoots that differentiated were excised and cultured on MS medium without growth regulators or with IBA (2.46, 4.92, 9.84 M). Roots developed after 3–8 days on an appropriate rooting medium, often without IBA. Rooted plantlets were transplanted to pots in a greenhouse and developed into normal plants. Suitable media and protocols for initiating and subculturing callus and regenerating whole plants in vitro from callus and explants have thus been established for four species of Sesbania.     

不同生长调节剂对马蹄金愈伤组织诱导的影响

马蹄金是一种优良的地被兼观赏草坪植物。采用正交设计试验法 ,研究了四种不同生长调节剂对马蹄金子叶、叶片、叶柄和下胚轴愈伤组织诱导的影响。结果表明 :生长调节剂是诱导愈伤组织的关键 ,2 ,4 D对愈伤组织诱导具有显著的影响 ,适宜于马蹄金愈伤组织诱导的培养基及生长调节剂为MS +1 .0mg/L 2 ,4 D +0 .2mg/L 6 BA +0 .2mg/LKT +1 .0mg/Lα NAA。     

High-frequency callus induction and plant regeneration in Tripsacum dactyloides (L.)

Summary A protocol for high-frequency callus, somatic embryogenesis, and plant regeneration for Tripsacum is described. Plants were regenerated from complete shoot meristems (3–4 mm) via organogenesis and embryogenesis. In organogenesis, the shoot meristems were cultured directly on a high cytokinin medium comprising 5–10 mgl⁻¹ (22.2–44.4 μM) 6-benzyladenine (BA). The number of multiple shoots varied from six to eight from each meristem. The time required for production of plants from organogenesis was rapid (4–6 wk). In contrast, callus was induced on an auxin medium and continuously cultured on an auxin medium for production of somatic embryos. Prolific callus with numerous somatic embryos developed within 3–4 wk when cultured on an auxin medium containing 5 mgl⁻¹ (22.6μM), 2,4-dichlorophenoxyacetic acid (2,4-D). The number of shoots induced varied from two to five per callus. Regardless of the cultivars used, the frequency of callus induction and plant regeneration was between 48% and 94%. The seed germination procedures also were modified and resulted in a maximum of 60–80% seed germination. Finally, the rate of T-DNA transfer to complete shoot meristems of Tripsacum was high on the auxin medium and was independent of whether super-virulent strains of Agrobacterium were used or not.     

Tissue culture and plant regeneration from immature embryo explants of Barley,Hordeum vulgare

Summary Immature embryo explants taken 8 days after anthesis were used to establish callus cultures of spring barley. Two types of calli were observed. A soft, watery callus produced a limited number of shoots and a harder, more compact, yellowish callus gave rise to numerous green primordia and shoots. Gamborg's B5 basal medium supplemented with either 2,4-D (2,4-dichlorophenoxyacetic acid) or Cl₃ POP (2,4,5-trichlorophenoxypropionic acid) was found to give good callus growth and shoot initiation. Media containing 2,4-D at 1.0 mg L^–1 or Cl₃ POP at 5.0 mg L^–1 produced numerous cultures resulting in regeneration of plants. Plantlets developed roots on basal medium with Cl₃ POP at 1.0 mg L^–1 or on auxin-free medium. Twenty genetically diverse genotypes were screened to determine if these techniques were suitable for a wide range of spring barley cultivars. Regeneration of plantlets was obtained for 19 of the 20 genotypes approximately 4 months after culture initiation. Lines differed in the ability to develop vigorously growing calli and in the ability of calli to develop large numbers of shoots and regenerated plantlets.Contribution from Department of Crop Science, Oregon State University, Corvallis, OR 97331. Oregon Agric. Exp. Stn. No. 7582     

Effects of explants and growth regulators in garlic callus formation and plant regeneration

We intended to evaluate the effects of different explants and growth regulators on callus induction and plant regeneration in garlic (Allium sativum L.). Furthermore, we intended to differentiate among different morphological types of callus by light microscopy and to relate them with their abilities to regenerate plants in the red-garlic cultivar 069. A factorial design with BDS—basal Dunstan and Short (1977)—medium, as a control and supplemented with 0.042, 0.42 and 4.24 μM picloram or with 0.045, 0.45 and 4.5 μM 2,4-D, in both cases with and without 4.43 μM N⁶-benzylaminopurine (BAP), was used. The cultures were grown in darkness at 25 ± 2°C and they were subcultured over a 6-month period. Basal plates and meristems were highly responsive explants, while immature umbels and root-tips were less responsive ones, as indicated by percentage of induced callus, growing callus and regenerating callus. The best response was 41% regenerating callus with 0.045 μM 2,4-D and BAP from basal plates while 57, 56 and 20% regenerating callus were obtained with 0.45 μM 2,4-D from meristems, root-tips and immature umbels, respectively. Also, these treatments showed a higher percentage of nodular and embryogenic callus (type I). Thus, it can be concluded that the use of meristems and 2,4-D will enhance callus production and quality, increase plant regeneration and allows to develop a protocol suitable for further transformation experiments in garlic.     

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。     

Callus induction and plant regeneration from hypocotyl explants of the forage legume Astragalus adsurgens

An efficient procedure was developed for inducing callus and plant regeneration using hypocotyl segments of Astragalus adsurgens. The combinations and concentrations of different growth regulators were shown to be critical factors for both the frequency and the type of callus formation as well as for the potential of callus differentiation. Of the four morphologically distinct types of calli that were induced, a friable, yellow callus, i.e. type I, induced on MS medium supplemented with 9.0 μM 2,4-dichlorophenoxyacetic acid and 2.2 μM N⁶-benzylaminopurine (BA), and then transferred to MS medium containing 0.5 μM α-naphthaleneacetic acid and 8.9 μM BA, exhibited the maximum frequency of shoot regeneration (75%). After regenerated shoots were transferred onto half-strength MS medium without growth regulators, they rooted and complete plants were obtained. Plantlet regeneration from callus cultures required 7–8 weeks. Received: 26 February 1997 / Revision received: 28 August 1997 / Accepted: 13 September 1997