Micropropagation of wild beet (Beta maritima) from inflorescence pieces

In order to investigate the regeneration of wild beet (Beta maritima) from inflorescence pieces, the effects of growth regulator, genotype, explant source and stage of plant development on adventitious shoot formation and rooting in vitro and subsequent transplanting in the glasshouse were tested. Inflorescence tips produced more adventitious shoots than sub-apical segments and the best micropropagation was achieved on a Murashige and Skoog (MS) medium supplemented with 1.0 mg l^–1 BAP. Addition of auxin was not beneficial. The induction rate of adventitious shoots was genotype-dependent and influenced by the stage of plant development. Adventitious shoots were produced from the base of the flower buds, i.e. from the receptacle, not from axils or stalks and only a few buds on inflorescence tip explants produced adventitious shoots. Rooting was increased by using a MS medium with 3% sucrose supplemented with 1.0 mg l^–1 NAA. There was no variation in leaf morphology of the transplants. This work shows that inflorescence tips can be used successfully as explants for in vitro multiplication of sugar beet and wild beet.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

Embryogenic callus induction and plant regeneration media for bentgrasses and annual bluegrass

Summary Embryogenic callus induction and plant regeneration systems have long been established for creeping bentgrass (Agrostis palustris Huds.), but little research has been reported on optimal medium for embryogenic callus induction and plant regeneration in velvet bentgrass (Agrostis canina L.), colonial bentgrass (Agrostis capillaries L.), and annual bluegrass (Poa annua L.). The present study compared 14 callus induction media and eight regeneration media for their efficacies on embryogenic callus induction and plant regeneration in these four species. The embryogenic callus initiation media contained the Murashige and Skoog inorganic salts and vitamins supplemented with 2,4-dichlorophenoxyacetic acid or 3,6-dichloro-anisic acid and 6-benzyladenine. l-Proline or casein hydrolyzate was included in some media to stimulate embryogenic callus formation and plant regeneration. The frequencies of embryogenic callus formation ranged from 0% to 38% and exhibited medium differences within each of the four species. Callus induction media, plant regeneration media, and genotypes affected plant regeneration rates, which varied between 0% and 100%. The embryogenic callus induced on Murashige and Skoog medium supplemented with 500 mgl⁻¹ casein hydrolyzate, 6.63 mg l⁻¹ (30 μM) 3,6-dichloro-anisic acid and 0.5–2.0 mg l⁻¹ (2–9 μM) 6-benzyladenine had much higher regeneration rates than those formed on other callus induction media. Embryogenic callus of annual bluegrass had higher regeneration rates than those of bentgrass species. MSA2D, a media containing 2 mgl⁻¹ (8 μM) 2,4-dichlorophenoxyacetic acid, 100 mgl⁻¹ myo-inositol, and 150 mgl⁻¹ asparagine, was effective in promoting embryogenic callus formation in creeping bentgrass but not in colonial and velvet bentgrasses and annual bluegrass.     

Tissue culture of Hypericum brasiliense Choisy: Shoot multiplication and callus induction

Hypericum brasiliense, a non-domesticated plant has been shown to have useful medicinal properties. This plant has not been cultivated so a protocol for mass propagation based on selection of superior clones was developed and a protocol established for the culture of callus cells that could be used for in vitro metabolite production. A micropropagation method based on amplification of nodal buds was developed, by selection, from ten seedling clones that were examined for growth rate, multiplication rate and rooting. The effect of various basal media, growth regulator types and concentrations were examined for optimal callus induction. Optimal callus induction occured on either Murashige and Skoog's or Gamborg's media supplemented with 1 to 2 mg l^–1 of 2,4-dichlorophenoxyacetic acid.Abbreviations B5 Gamborg's medium - 2,4-Dscd 2,4-dichlorophenoxyacetic acid - IAA indolacetic acid - MS Murashige & Skoog's medium - NAA naphtaleneacetic acid     

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.     

Measurement and effects of gel matric potential and expressibility on production of morphogenic callus by cultured sugarbeet leaf discs

During studies to optimize production of morphogenic callus from cultured leaf discs of sugarbeet (Beta vulgaris L.) large differences were observed associated with the gelling agent employed. Water availability, as determined mainly by gel matric potential, was found to be the dominant factor. A simple method was devised to measure the relative matric potential of different gels. A precisely moistened filter-paper disc was placed on the gel surface, allowed to equilibrate, removed and weighed. The relative gain or loss of water from the paper disc was a measure of the matric potential of the gel and varied with both gel type and concentration. Leaf disc expansion and production of callus-derived embryos and shoots were shown to be directly proportional to gel matric potential. Water availability may also be affected by the ease with which liquid is expressed from gels in response to localized pressure caused by explant expansion and contortion. This property, called gel expressibility, was easily measured with a weight and capillary pipette and shown also to vary with gel type and concentration. Validity of the technique for measuring relative matric potential was verified physiologically by culturing leaf discs on filter-paper overlays to eliminate expressibility differences among gels. Additionally, comparison of leaf disc growth on uncovered gel surfaces versus filter-paper overlays demonstrated the contribution of liquid expression to overall water availability. Expression of liquid by explants on uncovered gel surfaces greatly enhanced the production of morphogenic callus.     

Effects of genotype and plant growth regulator on callus induction and plant regeneration in four important turegrass genera: A comparative study

Summary Turfgrass, like other major crop species, is recalcitrant to manipulation in vitro. To perform efficient genetic transformation of turfgrass, it is necessary to optimize tissue culture conditions. In most reports, plant tissue culture techniques have been applied to propagate a single cultivar or several cultivars in one species of turfgrass. In this experiment, four turfgrass genera were used, namely common bermudagrass, Cynodon dactylon [L.] Pers. (California origin); red fescue, Festuca rubra L. var. rubra ‘Shadow’; perennial ryegrass, Lolium perenne L. ‘Barbal’; and Kentucky bluegrass, Poa pratensis L. ‘Merion.’ Mature seeds were surface-sterilized and cultured on basal Murashige and Skoog (MS) media supplemented with 30–250 μM 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction. Regeneration media consisted of MS supplemented with 5–10 μM 6-benzyladenine (BA). Among the genera, Poa had the higest callus induction percentage (CIP) regardless of 2,4-D concentration, followed by Cynodon, Lolium, and Festuca, respectively. Cynodon and Lolium had the highest callus regeneration percentage (CRP) and overall regeneration rate (ORR). Festuca had a poor CIP, CRP, and ORR compared to other studied genera. Cynodon produced the highest shoot number per explant. Based on the results of the present study, MS medium supplemented with 60 μM 2,4-D (for callus induction) and 7.5 μM BA (for regeneration) can be used in multi-generic transformation studies with the genera used.     

In vitro culture of petioles and intact leaves of sugar beet (Beta vulgaris)

Methods are described for obtaining explants which produce adventitious shoots, for subsequent stimulation of rooting and then transplanting using six commercial sugar-beet cultivars. The rate of adventitious shoot regeneration from petioles or intact leaf explants was affected by the source of donor plants, cytokinin type (BAP or Kin) and concentration and cultivar. Increasing the sucrose concentration of the medium from 3% to 5% or 8% had no apparent effect. Adventitious shoots could be produced directly from callus formed on the base of the petioles. In general adventitious shoots were produced on either the concave surface of the petiole or from the callus, occasionally simultaneously on both, and on the convex surface of the petiole in intact leaf explants. The highest rooting rate with 3% sucrose and 1.0 mg l^–1 NAA was obtained using half-strength MS medium. There was considerable variation in the propagules from petioles or callus indicating that this system may provide valuable somaclonal variation.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

Disturbed sugar metabolism in a fully habituated nonorganogenic callus of Beta vulgaris (L.)

Habituated (H) nonorganogenic sugarbeet callus was found to exhibit a disturbed sugar metabolism. In contrast to cells from normal (N) callus, H cells accumulate glucose and fructose and show an abnormal high fructose/glucose ratio. Moreover, H cells which have decreased wall components, display lower glycolytic enzyme activities (hexose phosphate isomerase and phosphofructokinase) which is compensated by higher activities of the enzymes of the hexose monophosphate pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase). The disturbed sugar metabolism of the H callus is discussed in relation to a deficiency in H₂O₂ detoxifying systems.Abbreviations 6PG-DH 6-phosphogluconate dehydrogenase - G6P-DH glucose-6-phosphate dehydrogenase - H fully habituated callus - HK hexokinase - HMP hexoses monophosphate - HPI hexose phosphate isomerase - N normal callus - PFK phosphofructokinase     

Somatic embryogenesis and plant regeneration of turf-type bermudagrass: Effect of 6-benzyladenine in callus induction medium

In order to optimize tissue culture conditions for bermudagrass, an important warm-season turfgrass species, tissue culture responses of young inflorescences of a hybrid bermudagrass cultivar `Tifgreen' (Cynodon dactylon×Cynodon transvaalensis) and a common bermudagrass cultivar `Savannah' (Cynodon dactylon) were investigated. When cultured on Murashige and Skoog medium with 4.52 to 13.57 μM (1–3 mg l^-1) 2,4-D, young inflorescence segments yielded non-embryogenic calli which were unorganized and had loosely associated, long tubular cells on the surface. However, inclusion of 6-benzyladenine (BA) in callus induction medium at a level of 0.044 μM (0.01 mg l^-1) induced formation of a compact, nodular embryogenic structure on approximately 20% of the calli. Calli with such a compact embryogenic structure were highly regenerable. When young inflorescences smaller than 0.75 cm were cultured, the embryogenic structure yielded green plantlets with regeneration rates of 79.5% and 83.3%, respectively for the two cultivars. All 96 plants regenerated from calli induced in the BA-containing medium were green and morphologically normal. The embryogenic nature of the compact structure was confirmed by scanning electron microscopy. This revised version was published online in June 2006 with corrections to the Cover Date.     

A full set of monosomic addition lines in Beta vulgaris from Beta webbiana: morphology and isozyme markers

Summary Nine different monosomic additions in Beta vulgaris from Beta webbiana were characterized through morphological characters and isozyme markers. The effect of the alien chromosome on the morphology of the recipient species is chromosome specific, and nine morphotypes could be distinguished. The added chromosome caused a growth reduction in the recipient plants. Eleven isozyme systems were used as marker systems. A 6PGDH band was found as a marker for chromosome 7, which contains a resistance gene for the beet cyst nematode in monosomic additions from Beta procumbens and Beta webbiana. A difference in the 6PGDH zymogram pattern between the two species with respect to this chromosome has been noted.     

Phenotypic and karyotypic status of Beta vulgaris plants regenerated from direct organogenesis in petiole culture

Summary A method for high frequency in vitro regeneration from petiole explants was tested on nine breeding lines of Beta vulgaris L. from the haploid, diploid and tetraploid levels. Regenerants could be obtained without a callus step, from excised petioles derived either from axillary buds sprouted in vitro or from field grown plants, by plating the explants on MS medium supplemented with TIBA (2,3,5-triiodobenzoïc acid) and BAP (6-Benzylaminopurine). The multiple shoots obtained were then rooted in vitro and transferred to soil. In some cases, these adventitious shoots were also used as a petiole explant source for further petiole culture cycles, and the phenotypic characteristics and ploidy status of the regenerants were investigated after one or three petiole culture cycles. Conventional shoot apex culture was used as an in vitro control. Phenotypic variations such as differences in morphology and changes in in vitro growth behaviour, were noticed. Chloroplast and chromosome counts indicated that the alterations in morphogenetic pathway could not be explained by the occurrence of gross cytogenetic abnormalities such as aneuploidy or myxoploidy. Our results suggest that the altered morphology is caused by the presence of the exogenous antiauxin (TIBA) during the in vitro phase. Following transfer to the greenhouse, none of these variations persisted and cytogenetic analyses revealed karyotypic stability in all the plants studied, even after three petiole culture cycles. An assessment of the in vitro petiole culture method as a true-to-type multiplication method for Beta vulgaris is made.     

A proline-rich chitinase from Beta vulgaris

A gene (Chl) encoding a novel type of chitinase was isolated from Beta vulgaris. The Ch1 protein consists of an N-terminal hydrophobic prepeptide of 25 amino acids followed by a hevein-like domain of 22 amino acid residues, an unusually long proline-rich domain of 131 amino acid residues with 90 prolines, and finally a catalytic domain of 261 amino acid residues. Proteins with similar proline-rich domains are present in some other plants. The Chl gene shows a transient expression in response to fungal infection.     

Relationship between type and age of the inoculum cultures and betalains biosynthesis by Beta vulgaris hairy root culture

From a study of the relationship between the type and age of the inocula, and the growth and biosynthesis of betalains in a Beta vulgaris hairy root culture, the best results were achieved with a 14 d inoculum grown in submerged culture giving 42 mg betalains (16 mg betacyanins and 26 betaxanthins) and 1.5 g dry biomass in 40 ml medium.     

The initiation of callus and regeneration from callus culture ofTulipa gesneriana

Intergeneric Fragaria vesca x Potentilla fruticosa hybrids were produced using in vitro culture. Hybrid plants were not obtained by direct embryo rescue, but were regenerated from cotyledon-derived callus. Experiments with F. vesca indicated that using cotyledon halves was not more productive than using entire cotyledons. A polarity was observed in cotyledons and in cotyledon halves, with callus and regenerated shoots produced more frequently from proximal ends. Cotyledons from 17% of hybrid embryos produced callus and regenerated mature plants. The technique enabled rapid multiplication of some embryos, with the production of more than one hybrid plant. In some cases more than 100 shoots were obtained from one embryo, demonstrating the potential usefulness of this technique for the production of intergeneric hybrids.Abbreviations BA 6-benzylaminopurine - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid     

Abnormal growth of habituated sugarbeet callus and cell suspensions

Summary Habituated sugarbeet callus and cell suspension cultures derived therefrom, compared to hormone-dependent normal cultures, exhibit a shorter linear growth phase, although they divide actively. Microscopic observations indicate deficiencies in cell expansion and absence of cell differentiation. Cell expansion apparently is interrupted by a cell “budding” process. Some of the cells seem to be empty due to ballooning out of the protoplasm and the bursting of the cell membrane by defective cell wall development. A low amount of cellulose was confirmed by microspectrophotometric estimation. Such cultures exhibit all the characteristics of vitrified tissue.     

In vitro gynogenesis in Beta vulgaris L.: Effects of plant growth regulators,temperature, genotypes and season

In order to increase the yield of haploid sugarbeet plants obtained from in vitro culture of unfertilized ovules, we have studied several factors: (1) the nature and concentration of various growth hormones, (2) the temperature for the cultured ovules, (3) genotypes and (4) seasonal effects. We found N6 medium [7] supplemented with 2.85 M of indoleacetic acid (IAA) and 0.88 M of 6-benzylaminopurine (BAP) or N6 medium containing 2.3 M of kinetin (KIN) to give the best qualitative and quantitative results for the culture of ovules. A two-step process for the induction and the development of gynogenic embryos was used. For the initiation of the embryos from the cultured ovules, the temperature of 27°C was favoured to that of 24°C. A pronounced and very significant seasonal effect was observed; the month of June was favoured to that of the other summer months tested. The effect of genotype was significant only for certain media. Using the above concentrations and combinations, a yield of viable gynogenic plants (plants/100 cultured ovules) between 6 and 10% (depending on the genotype) was obtained. In each case, 81% of these were haploids.     

Shoot-bud regeneration in subcultured callus of engelmann spruce

Summary Callus cultures ofPicea engelmannii (Parry, Engelmann spruce) were initiated and established from mature embryos cultured on von Arnold and Eriksson’s medium (AE) supplemented with N⁶-benzyladenine (10μM) and naphthalene acetic acid (10 μM). Cultures were maintained by subculture at 3-to-4-wk intervals. After three subcultures, callus was transferred to AE medium with only N⁶-benzyladenine (25 μM). Adventitious buds appeared on the surface of the callus after 2-to 4-wk and grew to adventitious shoots on AE medium without growth hormones or on AE medium with kinetin (0.1 μM). Shoot-forming capacity was maintained through 7 further subcultures. This study was supported by the Natural Sciences and Engineering Research Council of Canada grant G1438 to T. A. Thorpe and D. I. Dunstan.     

Monosomic additions in beet (Beta vulgaris) carrying extra chromosomes of Beta procumbens

Summary Eleven isozyme systems were used to identify the extra chromosomes, originating from Beta procumbens, in progenies of 33 monosomic additions in beet (B. vulgaris). Nine groups of monosomic additions could be distinguished, representing the nine different chromosome types of B. procumbens.     

Somatic embryogenesis and plant regeneration from callus culture of Acacia catechu-a multipurpose leguminous tree

Plant regeneration via somatic embryogenesis was achieved from callus derived from immature cotyledons of Acacia catechu Willd. on Woody Plant Medium (WPM) supplemented with 13.9 M kinetin and 2.7 M 1-naphthaleneacetic acid. The addition of 0.9–3.5 mM L-proline to the medium influenced development of somatic embryos and also promoted secondary somatic embryogenesis. The light-green somatic embryos germinated on half-strength MS medium supplemented with 2% (w/v) sucrose. Somatic embryos germinated into plantlets that were acclimatized in the greenhouse and subsequently transferred to the field.     

High frequency <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation and plant regeneration via direct shoot formation from leaf explants in <Emphasis Type="Italic">Beta vulgaris</Emphasis> and <Emphasis Type="Italic">Beta maritima</Emphasis>

We have developed a new procedure for Agrobacterium-mediated transformation of plants in the genus Beta using shoot-base as the material for Agrobacterium infection. The frequency of regeneration from shoot bases was analyzed in seven accessions of sugarbeet (Beta vulgaris) and two accessions of B. maritima to select materials suitable for obtaining transformed plants. The frequency of transformation of the chosen accessions using Agrobacterium strain LBA4404 and selection on 150-mg/l kanamycin was found to be higher than that in previously published methods. Genomic DNA analysis and -glucuronidase reporter assays showed that the transgene was inherited and expressed in subsequent generations. In our method, shoot bases are prepared by a simple procedure, and transformation does not involve the callus phase, thus minimizing the occurrence of somaclonal variations.