In vitro plant regeneration via somatic embryogenesis from root culture of some rhizomatous irises

A method for plant regeneration of Iris via somatic embryogenesis is described. Root and leaf pieces from in vitro-grown plants of several genotypes of rhizomatous Iris sp. were cultured in vitro. Callus induction occurred only on root cultures incubated under low light intensity (35 mol m^-2 s^-1) on two induction media containing 2,4-D (4.5 or 22.5 M), NAA (5.4 M) and kinetin (0.5 M). Somatic embryos developed after transfer of callus onto four regeneration media containing 9 or 22 M BA, or 5 M kinetin and 2 M TIBA or 9 M BA and 4 M TIBA. Plantlets could be obtained from these somatic embryos. Genotypic differences were found both in callus induction and somatic embryo formation, with I. pseudacorus responding better than I. versicolor or I. setosa. Cytological analysis performed on root tips of 80 regenerated plants revealed that two of the I. pseudacorus regenerants were tetraploid.Abbreviations 2,4-D dichlorophenoxy acetic acid - NAA naphthaleneacetic acid - BA 6-benzyladenine - TIBA 2,3,5-triiodobenzoic acid - IBA indolebutyric acid     

Adventitious shoot formation and plant regeneration from tissues of tomatillo (Physalis ixocarpa Brot.)

Cultured hypocotyl explants of tomatillo (Physalis ixocarpa Brot.), were evaluated with regard to their morphogenic responses to combinations of benzyladenine (BA, 0–5 M) with either naphthaleneacetic acid (NAA, 0–50 M) or 2,4-dichlorophenoxyacetic acid (2,4-D, 0–50 M). The induction of shoots or roots was dependent on the cytokinin/auxin combination.Hypocotyl explants failed to form shoots when they were grown on media containing either a cytokinin or an auxin alone. The highest frequency of shoot formation was observed on media containing 12.5–25 M BA and 5 M NAA. Likewise the highest frequency of root formation was observed on media supplemented with 1 M BA and 1 M NAA. Complete plants were regenerated and transferred to soil, where they reached maturity.     

Micropropagation of parsley through axillary shoot proliferation

A micropropagation protocol of parsley,Petroselinum crispum (Mill.) Nyman (curled type) has been developed. Surface-sterilized axillary buds cultured on Murashige and Skoog medium supplemented with benzyladenine, kinetin, or thidiazuron developed axillary shoots (rosettes). Kinetin resulted in only a low proliferation rate. The concentrations of thidiazuron or benzyladenine that were optimal for shoot proliferation, resulted in shoots with a low capability to root. During the rooting treatment, these shoots showed wilting signs. Rooting was increased significantly by using a two-week inductive stage with 2.5 M naphthaleneacetic acid directly followed by acclimatization. Two proliferation media (5 M benzyladenine and 0.5 M naphthaleneacetic acid or 5 M kinetin and 2.5 M naphthaleneacetic acid) resulted in moderate proliferation but produced shoots that were easy-to-root. These media have been tested by repeated axillary proliferation on the same medium. The medium with 5 M benzyladenine and 0.5 M naphthaleneacetic acid was optimal.Abbreviations BA 6-Benzyladenine - IBA Indole-3-butyric acid - MS Murashige & Skoog - NAA -Naphthaleneacetic acid     

Micropropagation procedures for Leontochir ovallei

Leontochir ovallei Phil., an endangered Chilean species in the Alstroemericeae, was micropropagated on Murashige & Skoog medium supplemented with 4 M benzyladenine, 1 M indolebutyric acid and 146 mg l^-1 glutamine. Over 88% of the shoots rooted in vitro when treated with 10 M naphthaleneacetic acid and micropropagated plantlets were successfully transplanted into the greenhouse.Abbreviations BA benzyladenine - IBA indolebutyric acid - 2iP isopentenyladenine - NAA naphthaleneacetic acid - MS Murashige and Skoog (1962) medium     

Plant regeneration from cotyledons of niger

Callus initiation from seedling explants of niger (Guizotia abyssinica Cass) cv. Ootacamund was found to be better on LS medium containing kinetin (1.4 M) plus 2,4-dichlorophenoxyacetic acid (9 M) than its analogues. Embryoids were induced directly from cotyledons on LS medium supplemented with 2,4,5-trichlorophenoxyacetic acid and 2,4,5-trichlorophenoxypropionic acid. When cotyledon-derived callus was subcultured onto medium with 10.7 M naphthalene-acetic acid and 2.3 M kinetin, embryogenesis was observed. Multiple shoots were obtained from cotyledonary explants in presence of MS medium containing 4.4 M benzyladenine and 11.4 M indoleacetic acid. Regenerated plants that were transferred to pots and grown to maturity were morphologically normal and fertile.Abbreviations NAA naphthaleneacetic acid - IAA indoleacetic acid - BA benzyladenine - GA₃ gibberellic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxypropionic acid - 2,4,5-TP 2,4,5-trichlorophenoxypropionic acid - ABA abscisic acid     

Adventitions shoot formation on excised leaves of in vitro grown shoots of apple cultivars

Leaves taken from micropropagated shoots of several apple (Malus domestica Borkh.) cultivars were cultured in vitro on Linsmaier & Skoog (LS) medium or the rice anther culture medium of Chu et al. (N6) containing various concentrations of either benzyladenine (BA) or thidiazuron (TDZ) plus naphthaleneacetic acid (NAA). Of the TDZ concentrations tested, 10 M was most effective and it was equivalent to, or better than, 22 M BA for both the percentage of leaves regenerating shoots and number of shoots formed per regenerating leaf in almost every experiment. Lower concentrations of NAA (1.1 and 5.4 M) gave best results with both BA and TDZ. N6 medium gave consistently better results than LS. Lowering total salt concentration or total N concentration of LS to that of N6 did not improve the response nor did changing the NO₃:NH₄ ratio. The 3–4 leaves on the most distal part of the shoot were most responsive and tended to form the most adventitious shoots. Placing the leaf cultures in the dark for the first 2–3 weeks of the culture period produced the best results. Optimum results were obtained by culturing leaves from the distal part of the shoot in the dark for 2 weeks on N6 medium containing 10 M TDZ and 1.1 or 5.4 M NAA, then moving the cultures to 16 h daylight at a photon flux of 60 mol s^-1m^-2.     

Thidiazuron stimulates shoot organogenesis and somatic embryogenesis in white ash (Fraxinus americana L.)

Immature and mature nonstratified seeds of white ash (Fraxinus americana L.) were dissected transversely and 2/3 of each seed was placed onto agar-solidified Murashige and Skoog medium. Adventitious buds, shoots, and somatic embryos formed on callus, cotyledons, and hypocotyls of the resulting seedlings. Shoot organogenesis was induced on explants cultured on medium with 10 M thidiazuron but not on explants on media with benzyladenine (BA) or isopentenyladenine. Not all seed sources were equally capable of shoot organogenesis and embryogenesis. Atypical of adventitious regeneration of other woody plants, mature seed explants of white ash were more organogenic with shoots that elongated better than explants from immature seeds. Somatic embryogenesis was observed in cultures where mature seeds were first cultured for 4 weeks on a medium containing 10 M adenine 2,4-dichlorophenoxyacetic acid in combination with 0.1 and 1.0 M thidiazuron, followed by transfer to a medium containing 0.05 M 6-benzyladenine and 0.5 M naphthaleneacetic acid. Adventitious shoots and epicotyls from both seedlings and germinated somatic embryos were rooted under intermittent mist and acclimatized to the greenhouse.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - 2iP isopentenyladenine - NAA naphthaleneacetic acid - TDZ thidiazuron-N-phenyl-N-1,2,3-thiadiazol-5-ylurea - WPM woody plant medium     

Enhancement of shoot regeneration from cotyledon explants of Brassica rapa ssp oleifera through pretreatment with auxin and cytokinin and use of ethylene inhibitors

The presence of benzyladenine or naphthaleneacetic acid in seed germination medium markedly enhanced subsequent shoot regeneration from the base of the excised cotyledon explants of Brassica rapa cv. Horizon. Cotyledon explants from younger seedlings (3 or 4-day old) produced more shoots than those from older seedlings. Addition of the ethylene inhibitor aminoethoxyvinylglycine (1.0 M) to the regeneration medium improved shoot regeneration three fold.Abbreviations AVG aminoethoxyvinylglycine - BA benzyladenine - MGBG methylglyoxal-bisguanylhydrazone - MSBN ms (murashige & skoog 1962) medium supplemented with 4.4 m BA & 5.4 m NAA, 2% sucrose - NAA 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.     

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 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M 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 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Artemisia annua L.: dedifferentiated and differentiated cultures

Dedifferentiated and differentiated tissue cultures ofArtemisia annua L. for artemisinin production were carried out. The calluses were initiated on MS medium supplemented with sucrose (30 g l^-1), myoinositol (100 mg l^-1) and RT vitamins. The auxins used were naphtaleneacetic acid (NAA), indoleacetic acid (IAA), indolebutyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-d). These were added to the basal medium either singly or in combination. The best results were obtained with 2.4-d (4.5 M : 0.02 d^-1) and NAA (5.4 M : 0.06 d^-1). Cell suspensions were established on the same media without agar. Suspension cultures showed different morphological characteristics according to the plant growth regulator supplied. Organized cultures were initiated from callus obtained on 2,4-d (4.5 M) and from bud cultures. Medium containing 6-benzylaminepurine (BA) (8.9 M)+NAA (0.54 M); Zeatin (45.62 M)+NAA (5.37 M) or BA (8.9 M) stimulated both organogenesis in callus (frequency of induction =50%) and semi-organized tissue in shoot buds. BA (13.32 M)+NAA (1.08 M) or BA (13.32 M) only stimulated multiple shoot cultures (frequency of induction =80%). Regarding artemisinin content, while the values obtained were 1.13 and 0.78 mg gDW^-1 in primary callus, artemisinin was not detected in cell suspension and only traces of it were found in multiple shoot cultures.     

Plant regeneration from nucllar tissues of Aegle marmelos through organogenesis

A protocol for organogenesis from nucellar explants excised from fertilized ovules of immature fruits of Aegle marmelos Corr. was developed. Adventitious buds were initiated on Murashige and Skoog's (MS) medium containing various combinations of 6-benzyladenine (BA), -naphthalene-acetic acid (NAA), 3-indoleacetic acid and gibberellic acid. Medium containing 4.4 m BA and 2.7 M NAA produced the maximum number of adventitious buds per explant. Shoots were elongated by transferring explants with shoot buds to medium with a low concentration of BA (0.44 M). Rooting of in vitro-regenerated shoots was obtained in half-strength MS medium with 4.9 M indole-3-butyric acid. This is the first report of plant regeneration from nucellar explants of A. marmelos.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid - GA₃ gibberellic acid     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.     

In vitro regeneration through organogenesis of Meconopsis simplicifolia — An endangered ornamental species

Meconopsis simplicifolia (D.Don) Walp. could be propagated by induction of adventitious shoots from callus produced on hypocotyl, cotyledon and rosette leaf explants of 4-month-old seedlings. Callus was initiated on agar-solidified Murashige and Skoog medium supplemented with 1 M kinetin +10 M -naphthaleneacetic acid (NAA). Shoots formed when the callus was subcultured on medium supplemented with kinetin or benzyladenine (BA) in combination with NAA, indoleacetic acid, indolebutyric acid or gibberellic acid. Excised shoots were rooted on medium containing auxin with 10 M NAA producing the best rooting (55%).Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxy-acetic acid - FAA formalin-acetic acid-alcohol - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA -indolebutyric acid - NAA -naphthaleneacetic acid     

Micropropagation of Frazinus angustifolia from mature and juvenile plant material

Micropropagation of Fraxinus angustifolia Vahl has been successfully achieved both from mature and juvenile plant material using shoot tip and nodal explants. Several basal media supplemented with benzyladenine (BA) and indolebutyric acid (IBA) were tested for shoot proliferation. The most new explants per mature explant (5.3) was obtained on DKW medium plus 4.4 M BA+0.98 M IBA. The most new explants per juvenile explant (5.6) was produced on QL medium plus 8.9 M BA+0.49 M IBA. Rooting was achieved on WPM supplemented with 0.98–4.9 M IBA. Rooted plantlets were transferred to soil and acclimatized with 85% survival.Abbreviations BA benzyladenine - IBA indolebutyric acid - NAA 1-naphthaleneacetic acid     

Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

Shoot,root and flower morphogenesis on tomato inflorescence explants

Regeneration of de novo shoots, roots and flowers has been obtained on inflorescence explants of tomato (Lycopersicon esculentum Mill.). Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and -naphthaleneacetic acid (NAA) were added in a 3×3×3 factorial combination with kinetin, each at 0.001, 0.1 and 10 M concentrations. Direct shoot formation occurred on media with 10 M kinetin and 0.001 M IAA or NAA. Root formation was observed on media with 0.1–10 M IAA, IBA or NAA. Flower formation occurred on elongated shoots with several leaves on media with 10 M IAA and 0.1 M kinetin. Shoot organogenesis was increased by substituting 10 M zeatin or N⁶-benzyladenine (BA) for kinetin. Eleven tomato cultivars were tested for their ability to undergo de novo shoot regeneration on the improved medium. All tomato cultivars were capable of shoot morphogenesis with a mean number of shoots per explant that ranged from 1.3 (Red Alert) to 5.3 (Large Red Cherry). Histological studies revealed that active cell divisions occurred in subepidermal and cambial tisue during the first week of culture. Meristematic centers of dividing cells were evident by day 14, and well-developed shoot apices and leaf structures were observed on 50% of the explants 28 days after culture initiation.Abbreviations BA N⁶-benzyladenine - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - 2iP N⁶-[²-isopentyl]adenine - NAA -naphthaleneacetic acid - PGR plant growth regulator     

The in vitro production of an anthocyanin from callus cultures of Oxalis linearis

Callus growth and the production of anthocyanins were sustained on the salts and vitamins of Murashige and Skoog. Callus growth was stimulated at a concentration of 8–32 M -naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-d). Benzyladenine (BA) and zeatin at 8 M inhibited callus growth whereas isopentenyladenine (iP) stimulated callus growth. NAA repressed anthocyanin production with an increase in NAA from 8–32 M. Anthocyanin synthesis was promoted by an increase in 2,4-d from 0.5 to 2 M and decreased thereafter up to a concentration 32 M 2,4-d. A concentration of 8 M BA, thidiazuron and zeatin, respectively stimulated pigment production. Sucrose stimulated callus growth at 60 mM and pigment production at 120–360 mM.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - iP isopentenyladenine - TZ thidiazuron-N-phenyl-N-1,2,3-thiadiazol-5-yl-urea - Bu-HCl Butanol-2N HCl - BAW Butanol-acetic acid-water     

In vitro micropropagation of Arctostaphylos uva-ursi (L.) Sprengel.: Comparison between two methodologies

In vitro micropropagation of Arctostaphylos uva-ursi was performed to increase the number of ground cover species able to serve as substitute for members of the Rosaceae susceptible to fire blight. Explants (node segments) excised from plants growing in the greenhouse were established in vitro on a medium containing 10 M -naphthaleneacetic acid (NAA) and activated charcoal (2 g I^-1). Using in vitro grown shoots, two propagation procedures were used:- Culture of nodal fragments with 50 M NAA resulted in the growth of 6 to 7 nodes every 4 weeks, yielding 1 700 almost rootable shoots after 4 subcultures;- Development of axillary shoots obtained with media containing 25 M benzyladenine (BA) and 20 M indoleacetic acid (IAA) yielded almost 500 rootable shoots after 4 subcultures. The rate of propagation decreased after the 3^rd subculture.Percentage of in vitro rooted shoots reached 98% with diluted micronutrients and 10 M NAA but 31% of the plants died during acclimatization.Abbreviations BA benzyladenine - BM basal medium - HID high intensity discharge - IAA indoleacetic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid - PAR photosynthetic active radiation - 2iP 2-isopentenyladenine     

In vitro culture ofXanthium strumarium (Cocklebur)

Xanthium strumarium L. was micropropagated by rooting shoots proliferated from shoot-tip explants. The best shoot proliferation was obtained from explants growing on Murashige and Skoog medium supplemented with 4.4 to 8.9 M benzyladenine (BA) and 1.1 to 2.1 M naphthaleneacetic acid (NAA). The micropropagated plants were transferred to potting media and maintained under high humidity conditions in the greenhouse. The media that produced best shoot proliferation from shoot-tip explants also produced the most callus from hypotocotyl, cotyledon and shoot-tip explants, whereas more callus was produced on leaf explants with a lower BA concentration (1.1 M) and 1.1 M NAA.Abbreviations BA benzyladenine, 2 4-d-2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - MS Murashige and Skoog Technical contribution No. 3319 of the South Carolina Agricultural Experiment Station, Clemson University.