Developmental aspects of long-term callus culture ofVicia faba L.

Summary Vicia faba callus line (VFS 1), isolated from expiants of immature embryo, grew satisfactorily onMurashige andSkoog complete medium with 1.38 M 2,4-D, or with 0.92 M 2,4-D to which 1.0 M kinetin was added. It also grew well on the B 5 modified medium containing 2.3 M 2,4-D and 25.0 M kinetin. On the last of these media the cultures grew more uniformly and without necrosis. They also showed diminishing variation in polyploidy in favour of diploids and corresponding aneuploids (hypodiploids).After being cultured for nearly three years on MS containing 1.38 M 2,4-D, 8–33% of cultures of VFS 1 were able to regenerate roots when transferred to either MS half strength with 5.37 M NAA, or to a medium without 2,4-D, or else to media with the addition of kinetin only (in various concentrations).     

Somatic embryogenesis and plant regeneration in wild cotton (Gossypium klotzschianum)

A simple and efficient method for high frequency somatic embryogenesis and plant regeneration from hypocotyl-derived cultures and suspension cultures of Gossypium klotzschianum Anderss, a wild, diploid species of cotton is described here. Embryogenic cultures were induced from hypocotyl sections on MSB medium with 0.9 M 2,4-D and 2.32 M kinetin. MSB medium containing 0.045 M 2,4-D, 0.93 M kinetin, 2.46 M IBA promoted embryogenic culture proliferation and embryo development. Suspension cultures with 0.23 M 2,4-D and 0.93 M kinetin also produced many embryos. Somatic embryos cultured on MSB medium with PGRs produced secondary embryos, and embryos developed into normal plantlets on PGR-free MSB medium. Regenerated plantlets were transferred onto the quarter-strength MSB medium with 0.5% active charcoal to avoid recallusing. Hypocotyls were better than cotyledons for culture induction and plant regeneration. 2,4-D and kinetin were essential for culture induction and maintenance.     

Norway spruce somatic embryogenesis: high-frequency initiation from light-cultured mature embryos

Somatic embryos and rooted plantlets have been regenerated from light-initiated embryogenic callus derived from mature embryos of Picea abies. Under a 16 h photoperiod, mature zygotic embryos were cultured on a modified half-strength Murashige & Skoog medium without NH₄NO₃ and supplemented with 5 mM glutamine, 4.5 M N6-benzyladenine and 10.7 M naphthaleneacetic acid or 10 M 2,4-dichlorophenoxyacetic acid. White translucent embryogenic callus, proliferating from the callusing hypocotyl region after 3 weeks incubation, was isolated from the green non-embryogenic tissue and subcultured for over 12 months. Upon transfer of the embryogenic callus through a specific sequence of media, somatic embryos proceeded to mature, elongating and forming rings of cotyledonary leaves similar to those of zygotic embryos. Transferred to medium without growth regulators, the somatic embryos germinated and produced plantlets with green cotyledons, elongated hypocotyls and primary roots.     

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.     

Somatic embryogenesis and plant regeneration from immature embryos of saw palmetto,an important landscape and medicinal plant

Somatic embryogenesis and plant regeneration from immature zygotic embryos was achieved for saw palmetto (Serenoa repens (Bartr.) Small). Embryos, isolated from immature fruit of native-grown plants, were cultured on Murashige and Skoog medium plus 0.15% (w/v) activated charcoal and supplemented with 452 M 2,4-dichlorophenoxyacetic acid (2,4-D) and 14.7 M N⁶-(2-isopentenyl)adenine (2iP). Clusters of somatic embryos developed from all immature zygotic embryos 5 weeks after culture initiation. After 12 weeks, explants were transferred to the same medium with the amount of 2,4-D reduced to 90.4 M which resulted in somatic embryo proliferation. Somatic embryos were then transferred to the basal medium containing 0.9, 9 M thidiazuron (TDZ), or no growth regulator for conversion into plantlets. The 9 M TDZ treatment was ineffective for plant regeneration. However, 12% of the embryos subcultured on 0.9 M TDZ were able to produce complete plantlets. Shoot production was obtained from 35% of the embryos subcultured in the absence of growth regulators. Rooting (100%) was achieved when these shoots were transferred onto medium containing 22.2 M -naphthaleneacetic acid (NAA).     

Effect of thidiazuron on vegetative tissue-derived somatic embryogenesis and flowering of bamboo Bambusa edulis

Current research on somatic embryogenesis of bamboo uses reproductive tissue as explants. However, it was hard to obtain the explant. Shoots of a local accession (3–4 m high) were used for multiple shoot production. In order to obtain embryogenic callus, nodal and internodal tissues from in vitro plantlets were placed on Murashige and Skoog (MS) medium supplemented with 9.2 M kinetin (KN), 13.6 M 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1% (v/v) coconut milk, and 6% (w/v) sucrose. We studied the effects of sucrose and thidiazuron (TDZ) on callus proliferation. Optimal additives to the MS medium for embryogenic callus proliferation were 0.046 M TDZ, 13.6 M 2,4-D and 3% (w/v) sucrose. TDZ also promoted the germination of bamboo somatic embryos. The germination rate of the somatic embryos exceeded 80% on MS-based medium supplemented with 0.455M TDZ. Naphthaleneacetic acid (NAA) reduced germination. Well-developed plantlets were successfully transferred to soil. There was no albino mutant in subsequent culture. In vitro regenerants and potted plants flowered, but no seeds were produced.     

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).     

Tissue culture ofKarwinskia humboldtiana—a plant producing toxins with antitumoural effects

Isolated embryos ofKarwinskia humboldtiana were cultured in vitro. The growth of embryos and development to plantlets on woody plant medium supplemented with indole-3-acetic acid 6.10^-2 mol l^–1, gibberellic acid (GA₃) 3.10^-2 mol l^–1, and 6-benzylaminopurine (BA) 2 mol l^–1 was obtained. Multiplication of shoots and rooting of excised shoots has been achieved. Callus formation on modified Murashige-Skoog medium supplemented with 1-naphthaleneacetic acid 10 mol l^–1, GA₃ 14 mol l^–1, and kinetin 5 mol l^–1 on hypocotyls, or on root cultures on medium supplemented with 2.4-dichlorophenoxyacetic acid 10 mol l^–1 and BA 10 mol l^–1 was induced.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - TEM transmission electron microscopy     

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     

Direct somatic embryogenesis through thin cell layer culture in Panax ginseng

Direct somatic embryos were differentiated on cotyledon transverse Thin Cell Layers (tTCLs) of Panax ginseng after 9 weeks in the Murashige and Skoog basal (MS) medium containing 2,4-d (5M). When MS medium containing 2,4-d (5M) was used for seedling pretreatment and for tTCLs culture, somatic embryos were observed 2 weeks earlier, i.e. after 7 weeks of culture. On the tTCLs from seedlings pretreated with 2,4-d (5M) combined with benzyladenine and zeatin at 0.1 M (BZ), somatic embryos were observed after 6 weeks of culture and the percentage of embryogenesis was higher (62%) than when 2,4-d was used alone for pretreatment (40%). Similar results were also obtained from pretreatment with combinations of 2,4-d (5M) and thidiazuron (TDZ) (0.01, 0.1M). When a combination of 2,4-d (5M) and BZ (0.1M) was used both for seedling pretreatment and for tTCLs culture, both somatic embryos and shoots were observed after only 3 weeks. As the concentration of BZ increased, the percentage of somatic embryogenesis decreased but the percentage of organogenesis increased. Similar responses were obtained with a combination of 2,4-d (5M) and TDZ (0.01M). On the medium containing both NAA (0.3M) and BZ (1M), globular- and heart- stage embryos developed after 4 weeks of culture into cotyledonary-staged embryos which remained dormant after a short elongation of the embryo axis. The importance of seedling pretreatment by growth substances in enhancing somatic embryogenesis is reported.Abbreviations BA 6-benzyladenine - BZ combination of BA and zeatin - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium Murashige and Skoog basal medium - NAA a-naphthaleneacetic acid - TDZ thidiazuron - tTCLs transverse thin cell layers - TCL longitudinal thin cell layer     

TDZ-induced somatic embryogenesis in non-responsive caryopses of rice using a short treatment with 2,4-D

Caryopses cultures of rice on an auxin medium (2,4-D, 20 M) formed slow-growing tissues that failed to regenerate. The embryogenic tissue possibly lost its regeneration potential on the auxin medium. Reproducible regeneration, however, could be achieved by a short treatment with 20 M 2,4-D for 3 days. Transfer of these caryopses to the medium containing TDZ or BA at 10 M resulted in regeneration of somatic embryos and shoots in 30% of the cultures within 10–15 days. TDZ was better than BA for inducing shoot regeneration. Short treatment for 3 days at higher concentrations of auxin (40–80 M) and subsequent transfer to TDZ or BA medium resulted in an increased frequency (up to 50%) of regeneration.     

The initiation of somatic embryos and adventitious roots from developing zygotic embryo explants of Cercis canadensis L. cultured in vitro

Zygotic embryo explants of Cercis canadensis L. cultured in vitro responded to 1 and 5 M 2,4-dichlorophenoxyacetic acid (2,4-D) or 50 M -naphthaleneacetic acid (NAA) by initiating somatic embryos and adventitious roots. Somatic embryos and adventitous roots were formed from developing zygotic embryos, while fully developed embryos collected from mature seed initiated only adventitious roots. Following 2,4-D application, the number of somatic embryos decreased while adventitious roots increased with increasing developmental age of the explant. The greatest number of somatic embryos were initiated with a 5 to 20 day exposure to 5 M 2,4-D from zygotic embryos collected between 75 and 82 days post-anthesis in 1987. Somatic embryos formed directly from epidermal and subepidermal cells, while adventitious roots developed from interior cortex cells. Normal somatic embryos were recovered after a 20 day exposure to 5 M 2,4-D and acclimated to greenhouse conditions.     

In vitro somatic embryogenesis ofDalbergia sissoo Roxb. —a multipurpose timber-yielding tree

Somatic embryogenesis was achieved in callus cultures dervied from 40-day-old semimature zygotic embryos ofDalbergia sissoo on semi-solid Murashige and Skoog (MS) salts and vitamins supplemented with 0.46–1.16 M kinetin, 6.78–9.04 M 2,4-dichlorophenoxy acetic acid (2,4-D) and 30 g/1 sucrose. Somatic embryos proliferated rapidly by secondary somatic embryogenesis after transfer to half-strength basal MS medium supplemented with 0.46-1.16 M kinetin and 6.78–9.04 M 2,4-D with 2% (w/v) sucrose. The light-green somatic embryos germinated on half-strength MS salts and vitamins supplemented with 0.5 mg/1 abscisic acid and 2% (w/v) sucrose. The developmental stages of somatic embryogenesis were studied by light and scanning electron microscopy.Abbreviations ABA Abscisic acid - BA 6-benzyladenine - Kn kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - MS Murashige and Skoog (1962) basal medium     

Growth and rosmarinic acid accumulation in callus,cell suspension,and root cultures of wild Salvia fruticosa

The accumulation of rosmarinic acid (RA) in Salvia fruticosa callus, cell suspension, and root cultures was studied. For callus induction, leaves excised from microshoots were cultured on MS medium containing thidiazuron (TDZ) (0, 2.3, 4.6, 6.9, 9.2, or 11.5 M) and indole-3-acetic acid (IAA) (0 or 3 M). For root culture, hairy roots were cultured in B5 medium containing 2.7 M -naphthaleneacetic acid (NAA) and different concentrations of sucrose or phenylalanine. Induction of callus was completely inhibited in the absence of both TDZ and IAA and the largest callus (0.79 g) was obtained with a combination of 6.9 M TDZ and 3 M IAA. Culture duration of 5 weeks resulted in maximum callus growth and RA yield (2.12 mg/ 100 mg dry weight). Cell suspension growth and RA yield (5.1 mg/ 100 mg dry weight) were maximum after 20 days of culture. The highest root growth and RA yield (2.62 mg/ 100 mg dry weight) was obtained with 4% (w/ v) sucrose. Incorporation of 10 mg l^–1 phenylalanine in the medium increased RA yield in the roots to 4.68 mg/ 100 mg dry weight after 4 weeks of culture. Amounts of RA extracted from in vivo leaves and roots were 0.21 and 0.72 mg/ 100 mg dry weight, respectively.     

TIBA affects the induction of direct somatic embryogenesis from leaf explants of Oncidium

To study the effect of auxin on direct somatic embryogenesis from leaf cultures ofOncidium `Gower Ramsey', 1-cm-long explants have been cultured in vitro testing IAA, 2,4-, quercetin, TIBA and PCIB. On a modified MS medium devoid of plant growth regulators, leaf cells of three regions (leaf tips, adaxial sides and cut ends) formed somatic embryos. After 8 weeks in culture, the frequencies of embryo-forming explants were 55, 52.5 and 30 % on leaf tips, adaxial sides and cut ends, respectively, and the numbers of embryos per dish was 89.3. Except for TIBA, other growth regulators (IAA, 2,4-, quercetin, PCIB) and their combinations tested, all retarded direct embryo formation. In the presence of 0.1 and 0.5 M TIBA, leaf tip, adaxial sides and cuts end of explants gave almost the same embryogenic response as the control. However, 10 and 27.5 % of explants were induced to form embryos from abaxial sides, and these explants did not form embryos on cut ends. In addition, after 8weeks in culture, TIBA at 0.5M highly promoted the mean numbers of embryos per dish to 134.2.     

Clonal propagation of Callistemon by tissue culture techniques

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 M), nicotinic acid (20 M), pyridoxine hydrochloride (3 M), thiamine hydrochloride (2 M), riboflavin (10 M), cytokinins (5 M) and auxins (0.1 M). The presence of benzylaminopurine (5 M) and p-chlorophenoxyacetic acid (0.1 M) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 M. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 M.     

Somatic embryogenesis in cultured mature zygotic embryos of Abies balsamea

Embryo suspensor masses (ESMs) were induced by culture of isolated mature zygotic embryos of balsam fir [Abies balsamea (Mill.)] on media containing 10 M cytokinin [6-(--dimethylallylamino)purine (2iP), 6-benzyladenine (BA), or thidiazuron (TDZ)]. Once induced, ESMs proliferated on media containing 2iP, BA or TDZ (10 M) or on 4.5 M BA in combination with 10 M naphthyl-1-acetic acid. When ESMs were transferred to media containing 5–80 M abscisic acid, cotyledonary-stage embryos were formed. Embryos were readily germinated on medium lacking growth regulators.Abbreviations ABA abscisic acid - BA 6-benzyladenine - ESM embryo-suspensor mass - 2iP 6-(--dimethylallylamino)purine - NAA naphthyl-1-acetic acid - TDZ N-phenyl-N-1,2,3-thiadiazol-5-yl urea (thidiazuron)     

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.     

Plantlet regeneration from callus initiated from flower buds in the wild species Allium senescens var. minor

Callus regeneration was observed from flower buds of Allium senescens var. minor inoculated in BDS, MS or B5 medium supplemented with 4.4 M benzyladenine alone or in combination with 4.5 M 2,4-dichlorophenoxy-acetic acid (2,4-d), with 2,4-d and kinetin (4.5 M/4.6 M) or with 5.3 M naphthaleneacetic acid. Ovules enlarged initially but the embryogenic tissue degenerated as callus development progressed from the nectar regions of the petals. Shoot buds and leaf primordia developed from the meristematic protuberances that originated from the surface of the callus. BDS medium with 4.5 M 2,4-d and 13.3 M BA was most suitable for shoot multiplication. The regenerated shoots were rooted in respective liquid medium without any growth regulators and successfully transferred to soil with 90% survival rate.Abbreviations BA N⁶-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid     

A comparison of BA,zeatin and thidiazuron for adventitious bud formation from Picea glauca embryos and epicotyl explants

Picea glauca (white spruce) zygotic embryos and one-week-old-seedling epicotyl explants were placed on either Woody Plant Medium (WPM) or half-strength Schenk & Hildebrandt (1/2S&H) medium supplemented with varying levels of benzyladenine (BA) (0.1, 1.0, 10, 50, 100 M), zeatin (10, 50, 100 M) or thidiazuron (TDZ) (0.01, 0.1 M). In addition to differences in the number of buds induced at three months on the two media, buds induced on WPM were visually more uniform, less vitrified and elongated faster. On 1/2S&H supplemented with BA, maximum bud induction from embryos occurred on 1.0 M BA with 0.01 M TDZ with higher BA concentrations inhibitory to bud induction. In contrast, on WPM there was little difference in the number of buds induced from embryos placed on 10, 50 and 100 M BA with or without TDZ. One-week-old-seedling epicotyl explants required higher BA levels on 1/2S&H, as bud induction at three months was greatest at 10 M BA. On WPM, as with the embryos, there were only minor differences in the number of buds induced from epicotyl explants on the various BA levels. Zeatin was more effective at inducing buds than BA with both media. From embryos, bud induction was greatest on 50 or 100 M zeatin without TDZ and 50 or 100 M zeatin with or without TDZ on 1/2S&H and WPM respectively. From epicotyl explants on 1/2S&H, there was little difference in the number of buds induced with the zeatin concentrations used, while with WPM, 50 and 100 M zeatin induced the greatest number of buds. Interestingly, with BA, the epicotyl explants needed a higher level than the embryos for maximal response, while with zeatin, the level was the same for both embryos and epicotyl explants. Long-term (six month) survival was higher on WPM than with 1/2S&H. Additionally, embryos had a higher percentage of genotypes surviving at six-months when compared with epicotyl explants. For overall survival and development of the buds, 50 M zeatin with 0.01 M TDZ was the best treatment tested.Abbreviations BA benzyladenine, 1/2S&H-half-strength Schenk & Hildebrandt medium - TDZ thidiazuron - WPM woody plant medium