Subculture Of Callus Research Articles

Rapid production of transgenic turfgrass ( Festuca rubra L.) plants

Red or golf course fescue is a major turfgrass in the temperate regions. Regenerable mature embryo-derived callus cultures could be initiated from six out of seven European turf-type red fescue cultivars with significant genotypic differences in the callus induction- and -regeneration frequency and the number of regenerated plantlets per explant. The highly responsive cultivar Borfesta was chosen to develop a rapid transformation system. Biolistic gene transfer of a constitutive npt II expression cassette in freshly established callus was followed by paromomycin selection during callus subculture and regeneration. Transgenic plants were transferred to soil less than 5 months from the excision of explants. Between 3 and 5 % of the bombarded call i produced independent transgenic plants, which converts into 1 to 1.7 independent transgenic plants per bombardment. Transgene integration was confirmed by Southern blot analysis. No nontransgenic plants that escaped the selection process were regenerated, indicating a tight selection scheme. Transgene copy number and transgene expression were variable from line to line, as indicated by Southern- and Western blot analysis respectively. A transgenic plant with a high transgene copy number showed the highest transgene expression level. This is the first report on transgenic turf-type red fescue plants.

Structure and development of somatic embryos formed inArabidopsis thaliana pt mutant callus cultures derived from seedlings

Seeds of theArabidopsis thaliana mutant primordia timing (pt) were germinated in 2,4-dichlorophenoxyacetic acidcontaining liquid medium. The seedlings formed somatic embryos and nonembryogenic and embryogenic callus in vitro in a time period of approximately two to three weeks. Embryogenesis and callus formation were monitored with respect to origin, structure, and development. Ten days after germination globular structures appeared in close vicinity of and on the shoot apical meristem (SAM). Somatic embryos formed either directly on the SAM region of the seedling or indirectly on embryogenic callus that developed at the SAM zone. Globular structures developed along the vascular tissue of the cotyledons as well, but only incidentally they formed embryos. Upon deterioration, the cotyledons formed callus. Regular subculture of the embryogenic callus gave rise to high numbers of somatic embryos. Such primary somatic embryos, grown on callus, originated from meristematic cell clusters located under the surface of the callus. Embryos at the globular and heart-shape stage were mostly hidden within the callus. Embryos at torpedo stage appeared at the surface of the callus because their axis elongated. Secondary somatic embryos frequently formed directly on primary ones. They preferentially emerged from the SAM region of the primary somatic embryos, from the edge of the cotyledons, and from the hypocotyl. We conclude that the strong regeneration capacity of thept mutant is based on both recurrent and indirect embryogenesis.

330 Plant Regeneration via Organogenesis and Embryogenesis from Different Tissues of Sweetpotato

The objective was to determine optimum conditions for embryogenic callus, embryo, organogenesis, and embryogenesis developed from leaf, petiole, stem, and tip tissues of the sweetpotato `Jewel' cultivar and from subcultured callus. Embryogenic callus was developed from stem and tip tissues on MS medium containing combinations of BA and NAA only under light conditions. Plant regeneration via organogenesis was developed from stem and tip tissues on medium including 1, 3 and 4 mg/L BA under dark and light conditions, while no plant regeneration via organogenesis was developed from leaf and petiole tissues. Frequencies for plant regeneration via organogenesis from the tissues were very low. No plant regeneration via embryogenesis was developed from the four tissues on medium having any combinations of BA+NAA and of kinetin+NAA. Embryogenic callus was observed in the subculture of callus developed from petiole and tip tissues on medium containing 0.2 and 2 mg/L 2,4-D only under dark conditions. Embryo was found in the subculture of callus from the tissues on medium containing 0.2 mg/L 2,4-D only under both conditions. Plant regeneration via embryogenesis was obtained in the subculture of callus from the tissues. Plant hormones and other factors affecting plant regeneration from the four tissues of the `Jewel' cultivar and other elite cultivars are currently being investigated at our lab for its application in transformation.

Thidiazuron-induced highly morphogenic callus and high frequency regeneration of fertile peanut (Arachis hypogaea L.) plants

An efficient regeneration system was developed by culturing immature cotyledons and embryo axes of Arachis hypogaea L. cv. Georgia Green on Murashige and Skoog basal medium (MS) supplemented with various concentrations of thidiazuron (TDZ; 1, 5, 10, and 15 µM). Highly morphogenic callus was produced from 100% of the explants comprising the cotyledon with attached embryo axis when cultured in the dark on 10 µM TDZ. Upon excision and continued culture in the dark on 10 µM TDZ, morphogenic callus grew repetitively during monthly subcultures and retained its regeneration potential. For organogenesis, a gradual reduction in TDZ concentration and exposure to light were necessary before transfer to MS basal medium. Inclusion of indole-3-butyric acid in liquid MS medium favored rooting of recovered shoots. A distinct feature of this investigation is the induction of highly morphogenic callus by TDZ and regeneration of morphologically normal, fertile peanut plants after 8 months of callus subculture.

Plantlet regeneration via somatic embryogenesis in anther callus of Vitis latifolia L.

Anthers of Vitis latifolia L. (wild grape) cultured on Nitsch and Nitsch medium supplemented with 20 μM 2,4-D and 9 μM BAP produced callus after 4–6 weeks. Subculture of callus onto Nitsch and Nitsch medium containing 10 μM NAA produced somatic embryos within 6 weeks. On growth regulator-free Nitsch and Nitsch basal medium somatic embryos converted to plantlets in 6–8 weeks. One gram of callus produced more than 400 somatic embryos with 13.7% being converted to complete plantlets, which were subsequently established in soil. Regenerated plants were found to have mixoploid populations of cells, 2n = 38 and n = 19.

Callus initiation and subculture ofTaxus chinensis

Conditions have been established for the callus initiation and subculture ofT. chinensis. The calli were induced by the explants cultured first on the medium MS supplemented with 1.0 mg/L 2,4-D, 2g/L CH, and 25g/L sucrose, then on The medium: MS+1.0 mg/L NAA+0.5 mg/L BA+2g/L CH+25g/L sucrose. When the callus was subcultured and tamed several times, it could grow fast and stable on the medium: MS+0.2mg/L 2,4-D+0.5 mg/L NAA+0.5 mg/L BA+2g/LCH+25 g/L sucrose. The contamination of explants was a result of endophytic microbes ofT. chinensis. This could be avoided by adopting the tender shoots 3–5 cm long collected in early spring as the source of explants. The browning of the cultures could be prevented and controlled by means of the selection of a suitable explants, hormonal regime in the medium, culture methods and the use of antioxidants.

Plant regeneration of mangosteen via nodular callus formation

Nodular callus was induced at a high frequency on young purple red, 5–15 mm long laminae taken from in vitro grown plants of mangosteen. The optimal medium was composed of Murashige and Skoog (MS) nutrients supplemented with 2.22 μM benzyladenine (BA), 2.25 μM thidiazuron (TDZ), 500 mg l-1 polyvinylpyrrolidone (PVP 360 000) and 3% sucrose. A multiplication rate of two–three was obtained by subculture of the nodular callus at 3–4-week intervals. Plantlet regeneration from the nodules was achieved by transfer to woody plant medium (WPM) with 500 mg l-1 PVP, 0.4 μM BA and 3% sucrose and overlaying with half strength liquid MS containing 0.32 μM naphthaleneacetic acid (NAA), 0.13 μM BA and 3% sucrose. Elongated shoots were rooted to 100% when wounded at the base of shoot, dipped in 4.4 mM indolebutyric acid (IBA) solution in the dark for 15 min and cultured on WPM supplemented with 1.11 μM BA, 0.25% activated charcoal, 34.5 μM phloroglucinol (PG) and 3% sucrose.

Clonal propagation byin vitro culture of Corchorus (jute)

Callus was produced on cotyledon, shoot tip, hypocotyl and root explants of twoCorchorus species on several media. Cytokinin was necessary for callus production on cotyledon explants. BothC.olitorius genotypes produced most callus on media with zeatin and either NAA or IAA, and theC.capsularis genotype produced most callus on media with IAA and either zeatin or BA. High frequencies of regenerated shoots were obtained from shoot tip explants of both species, from the apical meristem and from callus. Media with 2.0 mg 1−1 BA were superior for both species, and media with zeatin were equally good forC.capsularis only. More regeneration was obtained for all genotypes after subculture of callus on media with 2.0 mg 1−1 zeatin. Cotyledon callus produced less regeneration, also with differences between genotypes; explants of both genotypes ofC.olitorius produced regeneration on a medium with NAA and zeatin, and theC.capsularis genotype produced regeneration on a medium with IAA and BA. Limited regeneration from root explant callus was obtained forC.capsularis only on medium with BA and IAA. Regeneration was not obtained from hypocotyl callus. Further regeneration of shoots of both species was obtained from secondary callus after subculture, and from nodal segments of regenerated shoots and of seedling shoots cultured on basic MS medium without growth hormones. Roots were produced on about 80% of all shoots after transference to medium with 0.2 mg 1−1 IBA, and rooted plantlets survived and flowered normally after transference to compost.

Plant regeneration from callus cultures of Lithospermum erythrorhizon

We previously studied the production of shikonin derivatives by cell lines of Lithospermum erythrorhizon. As a result, we have obtained a cell line LE 87, which exhibited high cell growth and high shikonin production. In the present study, the effects of auxins (2,4-D, IAA, picloram, and NAA) and cytokinins (BAP and kinetin) on organogenesis and somatic embryogenesis in this shikonin-producing cell line were investigated. The highest organogenic and embryogenic efficiency was obtained on MS medium supplemented with 10 μM NAA and 0.3 μM kinetin. Subcultured calli showed different morphogenic frequencies depending on the NAA and kinetin concentration. Morphologically normal plants have been regenerated via mostly organogenesis. Shoots subsequently produced roots on plant growth regulator-free MS medium and developed into plantlets. In most cases, a few thin roots were formed at the bases of the shoots after four weeks on the rooting medium. More than fifty green plantlets were transplanted to soil in pots and developed into phenotypically normal plants 8 weeks after being transferred to soil. The regenerated plants grew to maturity, flowered, and set seeds by only artificial pollination.

Plant regeneration from callus cultures ofLithospermum erythrorhizon

We previously studied the production of shikonin derivatives by cell lines ofLithospermum erythrorhizon. As a result, we have obtained a cell line LE 87, which exhibited high cell growth and high shikonin production. In the present study, the effects of auxins (2,4-D, IAA, picloram, and NAA) and cytokinins (BAP and kinetin) on organogenesis and somatic embryogenesis in this shikonin-producing cell line were investigated. The highest organogenic and embryogenic efficiency was obtained on MS medium supplemented with 10 µM NAA and 0.3 µM kinetin. Subcultured calli showed different morphogenic frequencies depending on the NAA and kinetin concentration. Morphologically normal plants have been regenerated via mostly organogenesis. Shoots subsequently produced roots on plant growth regulator-free MS medium and developed into plantlets. In most cases, a few thin roots were formed at the bases of the shoots after four weeks on the rooting medium. More than fifty green plantlets were transplanted to soil in pots and developed into phenotypically normal plants 8 weeks after being transferred to soil. The regenerated plants grew to maturity, flowered, and set seeds by only artificial pollination.

Organ Formation and Plant Regeneration from Internodal Segments of Actinidia kolomikta Maxim. in in-vitro Culture

To culture internodal segments of Actinidia kolomikta Maxim. and establish an efficient method for vegetative propagation and possibly for genetic improvement, BW medium (Sugawara et al., 1994, a combined medium of half strengths of both broad-leafed tree medium and woody plant medium) and Miller's medium supplemented with 16 combinations of CPPU (0, 0.01, 0.1 and 1 μM) and IBA (0, 0.01, 0.1 and 1 μM) were used.1. Callus formation was promoted by increasing CPPU and IBA concentrations. In BW medium, the rate of shoot formation was 100% with CPPU above 0.1 μM, and IBA of 0.01 μM. The rate of shoot formation and the number of shoots per segment were higher in the BW medium than were those in Miller's medium.2. The callus actively proliferated in Miller's medium containing 1 μM NAA and 10 μM BA. The rate of shoot formation was 100% in the culture of internodal segments and the 1st subculture of callus. The number of differentiated shoots per segment increased significantly, because multiple shoots were formed in the 1st subculture of callus.3. When the whole shoots were transferred to the medium supplemented with combinations of NAA (0, 0.1, 1 and 10 μM) and BA (0 and 1 μM), 100% rooting was obtained in a medium containing 1 μM NAA, and the plantlets obtained were healthy and grew vigorously. When the plantlets were transplanted into a soil: vermiculite mixture (4 : 1, v/v) and acclimated, nearly all plantlets developed into nursery stocks 40 days later.

Somatic embryogenesis and plant regeneration in Floribunda rose ( Rosa hybrida L.) cvs. Trumpeter and Glad Tidings

Somatic embryogenic callus was initiated from in vitro-derived petiole and root explants, but not leaves, of the Floribunda rose cultivars Trumpeter and Glad Tidings following repeated subculture of callus on Schenk and Hildebrandt (SH) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). The use of a high auxin pretreatment increased the frequency of somatic embryogenesis, whilst l-proline, as a media supplement, had a critical role in enhancing somatic embryogenesis in the early stages of culture but increased the frequency of abnormal embryo formation when used in the later stages of culture. Maturation of somatic embryos was achieved by transfer to SH medium with reduced concentrations of 2,4-D and incorporation of abscisic acid (ABA) and 6-benzylaminopurine (BAP). The use of SH medium containing BAP and indole-3-butyric acid (IBA) facilitated embryo germination which was also enhanced by cold treatment. Phenotypically normal plants were recovered from germinated somatic embryos and successfully transferred to the glasshouse for flowering.

Competence for callus induction on mature pepper leaves depends upon specific developmental stages of the donor plant

Mature leaf expiants of two pepper ( Capsicum annuum L.) cultivais, ‘Edino’ and ‘Brujo’, were cultured on Murashige and Skoog medium supplemented with 12.9 μM 6-benzyladenine and either 4.9 μM indolebutyric acid or 13.6 μM 2,4-dichlorophenoxyacetic acid. Callus induction depended very significantly upon the position of the leaves on the donor plants. Callus tissue induced on responding expiants showed two distinct morphological types which were further subcultured on induction media or on Murashige and Skoog medium supplemented with either 2.6 μM 6-benzyladenine and 2.5 μM indolebutyric acid or 25.8 μM 6-benzyladenine and 9.8 μM indolebutyric acid. Proliferation of subcultured callus tissue depended significantly on callus morphology, genotype, developmental stage and the type and concentration of auxin used. The possible relationship of the observed results to callus habituation in culture is discussed.

Selection of large quantities of embryogenic calli from indica rice seeds for production of fertile transgenic plants using the biolistic method

The microprojectile bombardment of immature embryos has proven to be effective in transforming many indica rice varieties. One of the drawbacks of using immature embryos is the requirement of a large number of high quality immature embryos, which itself is a tedious and laborious process. To circumvent these problems, we have developed a procedure, using indica variety TN1 as a model that generates highly homogenous populations of embryogenic subcultured calli by selectively propagating a small number of regeneration-proficient calli derived from seeds. Thousands of embryogenic calli were produced from 50 seeds within 10 weeks. Ten to 20 independent R0 transgenic lines were regenerated per 500 embryogenic calli bombarded. The convenience and reliability offered by this transformation system has made transformation of indica rice a routine procedure.

Regeneration of plantlets from petiole callus of wild viola (Viola patrinii DC.)

Plantlets were regenerated from 5-year subcultured compact callus derived from petiole tissues of wild viola (Viola patrinii DC.) but not from 5-year subcultured friable callus. Regeneration occurred most efficiently on medium that contained two-fold diluted basal salts of Murashige and Skoog's (MS) medium, 5 × 10(-6) M 1-naphthaleneacetic acid and 10(-6) M kinetin. The effect of dilution of MS basal salts could also be achieved solely by two-fold dilution of the potassium dihydrogen phosphate in the mixture.The present study revealed that dilution of MS basal salts, in particular of potassium dihydrogen phosphate, was important for the regeneration of wild viola. Moreover, although the callus had been subcultured for 5 years, regeneration of plantlets from callus was still possible. In addition, scanning electron microscopy revealed that details of the process of plant regeneration from subcultured callus varied with the age and source of callus and differed from that reported in rice.

Somatic embryogenesis and plant regeneration from immature seed-derived calli of rugosa rose ( Rosa rugosa Thunb.)

A procedure for plant regeneration from immature seed-derived calli of rugosa rose ( Rosa rugosa Thunb.) via somatic embryogenesis is described. Embryogenic calli were initiated from immature seeds 2–3 weeks after anthesis on Murashige and Skoog (MS) medium without growth regulators. Induced calli had a white, friable and nodular appearance with several proembryos. These calli were subcultured at 20-day intervals on MS medium containing 0.1–0.2 M galactose on which they grew rapidly; but somatic embryogenesis was inhibited. Somatic embryos were again induced from the subcultured calli after transferring to MS medium containing 0.1 M M fructose or sucrose but lacking growth regulators. After transferring these embryos (1–2 mm) to MS medium containing 0.1 M sorbitol, 3% of them germinated and grew into plantlets which showed sustained growth on the MS medium containing only 0.1 M sorbitol as the sole carbon source.

Metabolism of Arginine and Ornithine in Suspension-Cultured Cells and Aseptic Roots of Intact Plants ofAtropa belladonna

Suspension-cultured cells and aseptically cultured roots of intact plants of Atropa belladonna L. removed tropane alkaloid precursors arginine (Arg) and ornithine (Orn) at nearly an equal rate from the feeding medium. A great part of Arg- and Orn derived 14C-label was found in ethanol-insoluble compounds, mostly in proteins already after 2 h feeding. Ethanol-soluble label in the roots was found mainly in amino acids (e.g. glutamine, Gin) after 2 h feeding, and after 20 h also in some intermediates of the urea cycle (e.g. argininosuccinate). In suspension cultures, subculturing of the initiation callus decreased both the uptake of the basic amino acids tested and their binding on to the apoplastic space. After 20 h feeding with Arg more label was found in organic acids in stationary phase suspension cultures with repressed alkaloid synthesis than in roots producing alkaloids. The growth phase and passage number also alfected into which amino acids the label was incorporated. When the initiation callus was young (the 3rd passage), the intermediates of the urea cycle were actively labelled, but when the initiation callus was older (the 8th passage) and the suspension formed roots, especially Gin was labelled. Only traces of u-A-methylornithine were detected in feeding experiments with Orn and Arg. Considerable arginase activity with a high pH optimum was observed in cell suspensions and roots of A. belladonna.

In vitro culture of coconut endosperm: callus induction and its fatty acids

Successful induction of callus from coconut endosperm was achieved by using the tissue situated near the micropylar end of a young fruit. For initiation of callus, a high concentration of auxin (20 to 100 ppm) was added to the basal medium containing activated charcoal. Subcultured callus showed a 40-fold increase during culture of three months. Based on the analysis of fatty acid composition, the maturation of endosperm was characterized by an increase in short chain fatty acids (C8, C10, C12, C14)and a decrease in long chain fatty acids (C16, C18: 1, C18: 2). In developing endosperms, proportion of short chain fatty acids was higher in lipids of the antipodal than those of other regions. In the final stage of maturation, around 82% of total fatty acids was short chain fatty acids, while the proportion of long chain fatty acids decreased up to 16%. The fatty acid composition of callus subcultured for six months was comparable to that of the immature endosperm. Lipids were accumulated in callus as globular bodies.

Plant Regeneration from Callus Cultures of Allium trifoliatum subsp. hirsutum and Assessment of Genetic Stability by Isozyme Polymorphism

Plant regeneration from callus cultures of Allium trifoliatum subsp. hirsutum fertile accession F-370, was studied as a means for clonal multiplication and germplasm storage of Allium spp. Callus was induced on in votro-cultured basal leaf explants. Best proliferation was obtained on modified BDS medium supplemented with (mg/1): 0.75 picloram, 2.0 benzyl adenine, and 900 casein hydrolysate. Shoot and root organogenesis were obtained in 3 to 5 month old subcultured calli, on BDS or MS medium supplemented with (mg/1): either 0.03 picloram or no auxin, 2 BA or 2-isopentenyladenine, and 900 casein hydrolysate. Direct bulb formation, without shoot elongation, occurred on BDS medium with 10 mg/1 IBA. Under these conditions, callus formation and organogenesis were not obtained with A. trifoliatum subsp. hirsutum var. sterile, a male-sterile genotype. Most regenerants were phenotypically normal, but some abnormal shoots were also observed, i.e. shoots with vitrified or extremely broad leaves. Isozyme polymorphism analysis of seven proteins in the latter regenerants, and in several callus cultures, revealed significant deviation from the original pattern in esterase, 6-phosphogluconate dehydrogenase and superoxide dismutase. No such deviations were detected in normal regenerated plants.

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l-1 sucrose (1/2 MS) and supplemented with 2.2 μM BA, 5.4 μM NAA and 2.2–9.0 μM 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 μM BA+0.05 μM NAA+0.3 μM GA3+200−800 mg l-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 μM BA+0.3 μM GA3+24.7 μM adenine sulphate.