Callus proliferation from the protoplasts of embryogenic cells of Quercus serrata

Embryogenic culture was induced from the immature embryos of Quercus serrata using Marashige and Skoog's medium (MS) containing 0.1 M each of 2,4-d and BAP, and subcultured for seven months before isolation of protoplasts by using 1% Cellulase RS in 0.6 M mannitol solution. Efficient colony formation was obtained when protoplasts were cultured in a liquid MS medium containing 0.6 M mannitol, 3% sucrose and combination of 0.1 M or 1 M each of 2,4-d and BAP. Excluding ammonium nitrate from the MS medium resulted in the decrease of the percentage of colony formation. From colonies, both agar culture and liquid culture were sustained in the MS media without mannitol containing no plant growth regulator, or containing 0.1 M of BAP in combination with 0.1 M or 1 M of 2,4-d.Abbreviations BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium after Murashige & Skoog (1962).     

Cell regeneration and sustained division of protoplasts from cotton (Gossypium hirsutum L.)

Protoplasts were isolated from 12 day old subcultured phytohormone habituated callus tissue of Gossypium hirsutum L. (0.5% cellulysin-Calbiochem, 0.6% macerase-Calbiochem, 0.7M mannitol, and pH 5.0). After separation and purification (0.35M sucrose floatation medium), the protoplasts were cultured (K₃ media of Kao et al., 1974 with 0.9 M BAP, 5 M IAA and 0.35M sucrose) in both liquid and solid medium at a density of 5×10⁵ protoplasts/ml. Four weeks after isolation, cell regeneration and callus formation was observed.Abbreviations IAA indoleacetic acid - BAP 6-benzyl-adenine Arizona Experimental Station Publication No. 4373     

Direct Organogenesis from Mature Leaf and Petiole Explants of Eryngium Foetidum L.

Eryngium foetidum L. plants were regenerated from mature leaf and petiole explants through direct organogenesis without intervening callus phase. From leaf explants, adventitious multiple shoots raised on Murashige and Skoog (MS) medium supplemented with 4.43 M benzylaminopurine (BAP) and 0.57 M indole-3-acetic acid (IAA), whereas in petiole explants shoot regeneration occurred at 8.86 M BAP and 0.57 M IAAA. 80% of the leaf explants and 44% of petiole explants produced shoots after four weeks of culture. The regenerated plants were rooted on MS medium supplemented with 2.46 M indole-3-butyric acid and 2.88 M gibberellic acid. The plants were successfully established in the soil and showed 70.9% survival in the field.     

Isolation and culture of Lens culinaris Medik. cv. Eston epicotyl protoplasts to calli

Protoplasts of Lens culinaris Medik. cv. Eston were isolated from epicotyl tissues of seedlings grown on Murashige & Skoog basal medium. For isolating the protoplasts, epicotyl tissues were digested for 12–14 h at 25°C in an isolation mixture (pH 5.7) containing 1% Cellulase RS, 0.5% Driselase, 0.25% Pectolyase Y23, 0.2M calcium chloride, 10 mM mannitol and 10 mM MES. Protoplasts were purified by flotation over 20% sucrose and washed with 0.2 M calcium chloride solution supplemented with 10 mM mannitol. Purified protoplasts were cultured at a density of 10⁵ ml^-1 in agarose (Seaplaque, 0.6%) blocks which were suspended in an identical but liquid KM8P culture medium lacking amino acids, ammonium nitrate, and coconut water but containing 0.35 M glucose and a growth regulator complement of either 2.2 M 2,4-dichlorophenoxyacetic acid (2,4-D), 2.7 M naphthaleneacetic acid (NAA), 2.3 M N-(2-furanylmethyl)-1H-purine-6-amine (kinetin), 2.2 M benzylamino purine (BAP), 2.3 M 2-methyl-4-(1H-purine-6-ylamino)-2-buten-1-ol (zeatin), and 1.4 M gibberellic acid (GA₃), or 5.4 M NAA and 2.2 M each of 2,4-D and BAP. The osmotic potential of the liquid culture medium was gradually reduced over a period of 3 weeks by replacing the spent medium with a fresh medium containing 0.25, 0.1 and 0 M glucose at weekly intervals. About 6% of the dividing protoplasts developed into cell colonies after 3 weeks of culture at 25°C in diffuse light (10 E m^-2s^-1). In 35–42 days the microcolonies were about 1 mm in diameter and developed into calli on transfer to agar-solidified B5 medium supplemented with growth regulators used in the protoplast culture medium and 5 mM glutamine. Attempts to regenerate plants from protoplast-derived calli have so far been unsuccessful.Department of Applied Microbiology and Food Science, University of Saskatchewan     

Regeneration in Phaseolus vulgaris L. Promotive role of N6-benzylaminopurine in cultures from juvenile leaves

Plants were regenerated from cultured young leaves of Phaseolus vulgaris L. cv. Kinghorn. For inducing shoot regeneration the expiant had to consist of the petiole and a portion of the lamina, and N⁶-benzylaminopurine (BAP) had to be present in the culture medium. Furthermore, the frequency of shoot regeneration increased more than seven-fold if donor seedlings were raised on a medium containing 5 M BAP, followed by culture of the leaf explants on a medium containing 20 M BAP. Regenerated shoots developed roots on basal (hormone-free) medium and the resulting plantlets could be transplanted to soil.Abbreviations BAP N⁶-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium This research was supported by operating grants from the Research Board Grants Program of the University of Guelph and the Natural Sciences and Engineering Research Council of Canada to PKS. Technical and photographic assistance from Sangeeta Saxena and Jean Gerrath is gratefully acknowledged.     

Somatic Embryogenesis and Plant Regeneration in Pigeonpea

Somatic embryogenesis in pigeonpea [Cajanus cajan (L.) Millsp.] has been achieved using cotyledon segments of mature seeds as explants. A large number of globular somatic embryos were induced directly from cotyledons of genotypes T-15-15, GAUT-82-90 and GAUT-82-99 when cultured on EC6 basal medium supplemented with 2.22, 4.44, 13.32 or 22.2 M N⁶-benzylaminopurine (BAP) and 0.45, 1.36, 2.27, 4.54 and 13.62 M thidiazuron. Somatic embryos developed into cotyledonary stage when the globular embryos were transferred to Murashige and Skoog's (MS) basal medium containing 2.89 – 14.43 M gibberellic acid. Maturation of somatic embryos was achieved on half strength MS medium with 0.38 M abscisic acid. The mature somatic embryos were germinated on MS medium supplemented with 0.44 M BAP and the plantlets were hardened and transferred to soil.     

Adventitious shoot regeneration from leaf explants of tissue cultured and greenhouse-grown raspberry

Adventitious shoot regeneration was observed using leaf-petiole explants from shoot-proliferating cultures of Comet red raspberry (Rubus idaeus L.). A maximum regeneration rate of 70% (3.7 shoots/explant) was obtained using 4.5–9.1 M (1–2 mg l^–1) N-phenyl-N-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ) with 2.5–4.9 M (0.5–1 mg l^–1) 1H-indole-3-butanoic acid (IBA) or 2.3 M (0.5 mg l^–1) TDZ with 4.9 M (1 mg l^–1) IBA in modified Murashige-Skoog medium. TDZ was more effective than N-(phenylmethyl)-1H-purin-6-amine (BA) at promoting regeneration in combinations tested with IBA (maximum 50% regeneration rate; 1.8 shoots/explant). Variation in the agar concentration or incubation temperature, orientation or scoring of the leaf-petiole explants and use of separate leaf or petiole explants had no effect on shoot regeneration. Incubation in the dark for 1, 2 or 3 weeks prior to growth in the light did not influence the percent regeneration rate but depressed the number of adventitious shoots. Explant source, from micropropagated shoots or greenhouse-grown plants, had an effect on shoot regeneration that was genotype dependent. Only 8 of 22 (36%) raspberry cultivars were capable of regeneration from leaf explants derived from greenhouse-grown plants.     

Regeneration of plantlets from cell suspension culture derived callus of white poplar (Populus alba L.)

Friable calli derived from the stem tissues of Populus alba were used to establish cell suspension cultures which were characterized for in vitro growth and regeneration capacity. Suspended cells and callus recovered from these cells were maximal on a fresh weight basis using MS liquid medium containing 0.44 M BAP and 4.52 M 2,4-D. Shoot regeneration from the recovered callus was observed within 30 to 40 days of culture. The number of shoots was increased by subculturing the shoot-forming callus 2 to 3 times on MS medium supplemented with 19.7 M 2iP and 0.05 M IBA. Regenerated shoots were easily rooted on half-strength MS medium lacking growth regulators, and the plantlets were transferred to pots containing vermiculite for greenhouse growth.Abbreviations BAP 6-benzylaminopurine - 2iP 2-isopentenyladenine - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume - MS medium Murashige and Skoog medium (1962)     

Regeneration of Medicago truncatula from tissue culture: increased somatic embryogenesis using explants from regenerated plants

Plant regeneration has been achieved by somatic embryogenesis in Medicago truncatula Gaertn. (barrel medic) c.v. Jemalong, an annual legume species. Regenerated plants were obtained from cultured leaf tissue explants on a four-step modified B5 basal medium. Induction of embryo formation occurred on a medium containing 10 M NAA and 10 M BAP, and embryo maturation was promoted after transfer to a medium containing 1 M NAA and 10 M BAP. Shoot development, secondary somatic embryogenesis and occasional plantlet development occurred on a subsequent transfer to 0.1 M NAA and 1 M BAP. Plantlet formation could also be completed by transfer of well developed shoots to 0.05 M NAA. A high frequency of primary somatic embryos could only be obtained by using the same culture protocol with tissue from regenerated plants. Explants from regenerated plants showed a large increase in the number of primary embryos per callus and the number of calli producing embryos. Explants from plants derived from the seed of one regenerated plant also showed increased embryo formation. Although high embryo formation rates can be reproducibly obtained from this seed, embryo conversion rates to plants are currently low.Abbreviations BAP 6-benzylaminopurine - B5 medium of Gamborg et al. 1968 - 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium of Murashige and Skoog 1962 - NAA 1-naphthaleneacetic acid     

Plant regeneration from somatic embryos ofTaxus brevifolia

Summary Taxusbrevifolia is the source of paclitaxel (Taxol®), an anticancer drug. A method for regeneration ofTaxus brevifolia from immature zygotic embryos via somatic embryogenesis is described. Embryogenic callus tissues were obtained by culturing immature zygotic embryos on Lloyd and McCown medium (MCM) supplemented with 160 M 2,4-dichlorophenoxyacetic acid (2,4-D) + 5 M benzylaminopurine (BA) + 5 M naphthaleneacetic acid (NAA) for 4 weeks. Putative embryoids were obtained following transfer of cultures to MCM medium supplemented with 4 M BA + 5 M kinetin + 1 M NAA for 6 to 8 weeks. Conversion of embryos was obtained on MCM medium supplemented with 40 M abscisic acid (ABA) + 1% activated charcoal. Development of bipolar structures with recognizable shoot and root apices was observed in somatic embryos. Five percent of somatic embryos were regenerated into plantlets on half-strength growth regulator-free MCM medium.     

Tissue culture response of Beta germplasm: callus induction and plant regeneration

Callus cultures of 18 sugarbeet (Beta vulgaris) lines, two accessions of B. maritima and a B. macrocarpa accession were initiated from aseptically germinated seeds. Plant regeneration through organogenesis was obtained either on MS or B5 medium containing various concentrations and combinations of naphthaleneacetic acid (NAA), 6-benzylaminopurine (BAP), 2,3,5-triiodobenzoic acid (TIBA) and abscisic acid (ABA). Genotypes differed in their abilities of callus formation and regeneration: seven out of 18 sugarbeet lines, and an accession of B. maritima were capable of regenerating plantlets. Our data also indicated that 2 M TIBA promoted morphogenesis from callus culture in the presence of 5 M BAP.     

Direct regeneration of Drosera from leaf explants and shoot tips

An efficient protocol for the micropropagation of Drosera anglica, D. binata and D. cuneifolia is described. Proliferation was obtained from leaf segments and shoot tips, which served as initial explants. The regeneration capacity of explants was influenced by factors such as nutrient media, concentrations of growth regulators and the type of medium (liquid or solid). The highest number of plants regenerating from D. binata explants was obtained on the growth regulator-free Vacin and Went medium. In the case of D. anglica the highest proliferation rate was obtained on the Fast medium supplemented with 0.05 M 6-benzyladenine (BA) and 0.005 M -naphthaleneacetic acid (NAA), whereas for D. cuneifolia the optimal regeneration medium proved to be 1/2 MS with the growth regulator supplementation estimated at 0.2 M BA and 0.2 M NAA. Liquid media significantly increased the regeneration potential of D. anglica and D. binata explants.     

Regeneration of Elaeagnus angustifolia from leaf segments of in vitro-derived shoots

Shoot regeneration was achieved from in vitro-produced leaves of Elaeagnus angustifolia L. Half-leaf explants from the terminal part of the shoot produced more shoots than explants from the basal part of the in vitro-derived shoots on agar-solidified WPM medium supplemented with 1 M benzyladenine (BA). In liquid medium of the same formulation, compact shoots that did not elongate were formed on the explants. Leaf cross-section explants (1 mm thick) produced shoots both on solid and liquid medium with 1 M BA, whereas again compact shoots were formed with 10 M BA. Further shoot development on these explants was promoted by their transfer to fresh solid medium containing 1 M BA and 1 M gibberellic acid (GA₃).Abbreviations BA benzyladenine - GA₃ gibberellic acid - WPM woody plant medium     

Plant regeneration capacity of callus derived from leaf,stem, and root segments of Populus alba L. x P. grandidentata Michx.

Summary Expiants for establishing callus cultures originated from in vitro cultured hybrid poplar (Populus alba L. X P. grandidentata Michx.). Plant regeneration was achieved from established callus cultures derived from stem internodes (SI), leaf discs (LD), and root segments (RS). Shoot regeneration from callus occurred within 4 weeks of culture on most of the media tested. Frequency of shoot formation was greatly increased by subculturing the selected organogenic calli on regeneration media. The highest rate of multiple shoot formation (an average number of 7/SI, 11/LD, and 8/RS) was obtained by using 0.05 M IBA in combination with 22.5 M 2iP, 22.5 M zeatin, and 12.5 M 2iP, respectively. Regenerated shoots were easily rooted in polyterra peat plugs in transparent plastic boxes. The rooted plantlets were subsequently transferred to pots containing an artificial potting mix.Abbreviations BA 6-benzylaminopurine - 2iP N⁶-isopentenyladenine - Z zeatin - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - WPM Woody Plant Medium - PGR Plant Growth Regulator Journal Paper No. J-14119 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project No. 2210     

Plantlet regeneration from subculturable nodular callus of Pinus radiata

An efficient planlet regeneration system via nodular callus formation is described for Pinus radiata. Subculturable nodular callus was induced at its highest frequency (93%) on embryonic explants excised from seeds at an early stage of germination (radicle length 2–5 mm). The optimal medium for nodular callus tissue proliferation was LP basal medium that was modified by reducing the concentration of potassium nitrate to 500 mg l^–1 and supplemented with 22.2 M 6-benzyladenine (BAP) and 2.85 M indole-3-butyric acid (IBA). Bud differentiation from the nodules was achieved by reducing BAP and sucrose concentrations in the culture medium. The maximum frequency of adventitious bud formation occurred on LP basal medium containing 2% sucrose and 0.44 M BAP on which about 61% of the transferred nodules formed buds. During the next 6 weeks of culture on the same cytokinin-free medium multiple shoots elongated from the buds. These shoots were excised and transferred to root initiation medium (RIM2.1), consisting of full-stregth SH macro- and micro-salts, 1000 mg l^–1 myo-inositol, 0.4 mg l^–1 thiamine-HCl, 2% sucrose and a combination of naphthaleneacetic acid (NAA), IBA and BAP at concentrations of 2.69, 4.93 and 0.11 M, respectively. After 5–15 days, root meristems were initiated on the stem bases. The highest rooting frequency was achieved when shoots were treated for 10 days on RIM2.1 medium, before being transferred to half-strength Schenk and Hildebrandt medium with 1% sucrose and without growth regulators for root growth.     

Axillary shoot induction and plant regeneration in Plantago ovata Forssk

Axillary shoot induction and plant regeneration were obtained in Plantago ovata. The optimum medium for inducing axillary shoots was Murashige & Skoog (MS) medium [5] supplemented with 4.6 M kinetin and 0.05 M NAA. Rooting of shoots was best on half-strength MS medium containing 5.0 M IBA and 0.05 M kinetin. The regenerated plants were similar to the control plants in karyotypic and phenotypic details.     

Rapid plant regeneration from various explants of Jatropha integerrima

A simple, rapid and reproducible protocol for direct shoot regeneration from different explants of Jatropha integerrima was developed. Prolific adventitious shoot bud initiation was obtained using a combination of 2.2 or 4.4 M benzyladenine and 4.9 M indole-3-butyric acid (IBA). Reduction of IBA concentration (2.5 M) promoted further development of shoots. Regenerated shoots rooted readily on Murashige and Skoog (MS) medium lacking growth regulators. Plantlets were acclimatized and successfully transferred to pots.     

Cytoplasmic effects on the tissue culture response of callus from winter wheat mature embryos

A simple and reliable method was established for the maintenance of a permanent stock of several Medicago truncatula genotypes selected from a general seed stock by their in vitro culture amenability and embryogenic capacity. In the first step, multiple shoots were induced from the cotyledon axillary meristem meristem of pre-germinated seeds in Murashige and Skoog medium supplemented with cytokinins (9.3 M zeatin, 22.2 M benzylaminopurine or 4.5 M thidiazuron). In the second step, the induced shoots were allowed to develop in growth-regulator-free medium. Benzylaminopurine at 22.2 M supported the best combination of shoot quality and number of shoots produced. Rooting of microshoots depended on the cytokinin used for shoot induction and was faster for zeatin-treated shoots. In this work a propagation system was devised where the addition of growth regulators was restricted to the induction phase therefore reducing the risks of epigenetic and somaclonal variation.     

Transient gene expression in electroporated protoplasts of Eucalyptus citriodora Hook

Protoplasts isolated from cotyledons of Eucalyptus citriodora were electroporated using a rectangular pulse, with plasmid carrying the cat gene. The levels of transient expression and protoplast viability were influenced by the voltage and pulse duration. At a field strength of 800 V cm^-1 (1000 s), a protoplast viability of 57%, and 47% conversion of ¹⁴C-chloramphenicol to its acetylated forms, were obtained. Expression levels were improved by an increase in plasmid concentration (up to 60 g ml^-1), and also by the addition of carrier DNA. Gene expression was further enhanced by the addition of 40% (w/v) PEG, in the presence of the carrier DNA, to the protoplasts after electroporation.Abbreviations BAP 6-benzylaminopurine - CAT chloramphenicol acetyltransferase - CPW 13M CPW salts medium with 13% (w/v) mannitol - DC direct current - FDA fluorescein diacetate - f. wt fresh weight - GUS -glucuronidase - K Kao (1977) - MES 2-N-morpholinoethane sulfonic acid - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid - PVP-10 polyvinylpyrrolidone (Av MW 10,000) - TLC thin layer chromatography     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Bambusa balcooa</Emphasis> Roxb.: Factors affecting changes of morphogenetic competence in the axillary buds

Axillary buds from field-grown culms of Bambusa balcooa were used as explants to induce multiple shoots in liquid Murashige and Skoogs (MS) medium supplemented with 11.25 M of 6-benzylaminopurine (BAP) and 4.5 M kinetin (Kn). A clump of at least 3 shoots was used for root induction in half strength MS medium with 1.0 M of 3-indolebutyric acid (IBA). Morphogenetic competence of the axillary buds varied widely in different months of two consecutive calendar years. The highest morphogenetic potentials were observed in October. The major problem encountered was presence of systemic fungal contaminants. Perhaps, rainfall positively contributed to induce morphogenetic competence. A moderately high phenolic content of the nodal explant was also detrimental for in vitro morphogenesis. The morphogenetic competence of B. balcooa correlated with the season in which the explants were excised from the natural stands. To the best of our knowledge this is the first report on in vitro regeneration of B. balcooa from mature field-grown axillary buds.