Somatic embryogenesis in cultured immature kernels of Pistachio,Pistacia vera L.

Embryogenic tissue was produced from kernels of immature fruits of Pistachio (Pistacia vera L.) cultured in liquid Murashige and Skoog media, supplemented with 200 mgl^–1 casein hydrolysate, 114 M 1-ascorbic acid, and benzylaminopurine. Compact embryogenic masses differentiated directly from the fruit explants after culture for 2 weeks in liquid medium with 8.9 M benzylaminopurine. After transfer of the embryogenic masses into the same medium, but with 4.4 M benzylaminopurine, somatic embryos appeared. Several stages of embryogenesis were present in the cultures. Adventive embryos were readily separated from the friable embryogenic masses by shaking. Separated somatic embryos, germinated on solidified Murashige & Skoog medium without growth regulators, developed into plantlets.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzylaminopurine (N⁶-benzyladenine) - EMS embryogenic mass - MS Murashige and Skoog medium (Sigma M-0404) - NAA -naphthalene acetic acid - PGR plant growth regulator - TDZ thidiazuron (1-phenyl-3-(1,2,3, thiadiazol-5-yl)urea) - WP McCown's Woody plant medium (Sigma M6774) - ABA abscisic acid     

Micropropagation of Uniola paniculata L.

Uniola paniculata L. is a major sand-dune stabilizing grass which is being utilized to prevent shoreline erosion. In vitro cultured caryopses of U. paniculata produced callus on MS medium supplemented with 22.5 M 2,4-D, 4.4 M BA and 87.6 mM sucrose. Shoot induction occurred after these calli were inoculated onto the same medium without 2,4-D. Rooting of in vitro-derived shoots occurred when transferred to a one-half strength MS medium containing 43.8 mM sucrose and 14.7 M IBA. Plantlets were planted after the roots reached a length greater than 20 mm.Abbreviations BA N-(phenyl-methyl)-lH-purine-6-amine - IBA lH-indole-3-butanoic acid - MS Murashige and Skoog (1962) - 2,4-D (2,4-dichlorophenoxy) acetic acid - TC agar tissue culture agar, (K.C. Biologicals, Lenexa, KS) - Subdue methaxyl:N-(2,6-dimethylphenyl)-N-(methyoxyacetyl) alanine methyl ester (CIBA- GEIGY)     

Effect of plant growth regulators on somatic embryogenesis in leaf cultures of Coffea canephora

The effects of plant growth regulators on somatic embryogenesis were studied in leaf cultures of Coffea canephora. The maximum number of somatic embryos were obtained on media that contained only cytokinin as a plant growth regulator. All of the auxins tested (NAA, IBA, IAA and 2, 4-D) inhibited the formation of embryos. The optimal concentration of each cytokinin (2-iP, BA and kinetin) for somatic embryogenesis was 5 M. Under optimal conditions, each explant formed more than 100 embryoids with little callus and few adventitious roots. Embryoids were formed only at the cut edges of the leaf discs. Cytokinins were absorbed only at the cut edges of leaf discs that were in contact with the medium, and were not transported to other parts of the explant.Abbreviations 2-iP iso-pentenyladenine - BA benzyladenine - NAA 1-naphthaleneacetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Plant regeneration through somatic embryogenesis from mature seed and young inflorescence of wild rice (Oryza perennis Moench)

Somatic embryogenesis in the wild rice species (Oryza perennis) was induced from cultured mature seeds and young inflorescences. Murashige and Skoog's (MS) medium supplemented with 2 mg/l 2,4-D and 0.2 mg/l BAP was used for induction of a compact, white nodular callus and somatic embryos. Plant regeneration occurred with the tranfer of the nodular callus to MS basal medium containing 0.5 mg/l IAA, 0.5 mg/l NAA, 4 mg/l BAP and 500 mg/l casein hydrolysate. The embryogenic nature of the callus from both explants was maintained over 10 subcultures for about 12 months. Plant regeneration with respect to the number of calli plated from the 6th to 10th passage varied from 80% to 60% for young inflorescence derived callus and from 75% to 69.8% for seed-derived callus.Abbreviations MS Murashige and Skoog medium - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthalene acetic acid - CH casein hydrolysate     

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)     

In vitro plant regeneration of leguminous trees (Albizia spp)

Hypocotyl explants of three leguminous forest tree species, Albizia amara, A. lucida and A. richardiana, have differentiated shoot buds on B₅ basal medium. Maximum number of shoots per explant developed on basal medium augmented with 2,4-D (0.1 M) in A. amara (2) and BA (10 M) for both A. lucida (2) and A. richardiana (1.6). Higher concentrations of auxins in the medium, in general, enhanced rooting and callusing but cytokinins promoted the growth of green calli. BA enchanced the differentiation of shoots in the three species. The in vitro grown shoots of A. amara and A. richardiana, after subculturing on B₅+1 M IAA developed roots (up to 30–40%). These plants have been successfully transferred to the field.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - BM Gamborg's B₅ medium with 0.9% agar+3% sucrose - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - Kn Kinetin - NAA -naphthaleneacetic acid     

Somatic embryos from callus of Sequoia sempervirens

Compact calli with a potential for somatic embryogenesis were obtained from complete or split mature zygotic embryos or from cotyledons and hypocotyls of in vitro grown seedlings of Sequoia sempervirens. Somatic embryos which showed a typical bipolar structure, were formed together with adventitious buds. When placed on filter paper supports they developed into complete plantlets. Of the various combinations tested, culture medium adapted from Murashige and Skoog mineral solution complemented with 6-benzylaminopurine (2 M), kinetin (2 M) and 2,4-dichlorophenoxyacetic acid (2.5 M) was established as the optimal for somatic embryo production.Abbreviations BM basic medium - 2,4-D dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - Kin Kinetin     

Propagation of Lirlodendron hybrids via somatic embryogenesis

Propagation of hybrids between the Chinese tuliptree (Liriodendron chinense) and the North American yellow-poplar (Liriodendron tulipiferea) could be greatly accelerated with a highly productive somatic embryogenesis system. Flowers were collected from a single Chinese tuliptree and the anthers used for controlled pollinations of 4 yellow-poplar mother trees. Aggregates of samaras resulting from the pollinations were harvested 8 weeks post-pollination. Following surface disinfestation, samaras were dissected and embryos and endosperm were cultured together on a semisolid induction medium containing 9.0 M 2,4-dichlorophenoxyacetic acid and 1.1 M benzyladenine. Following 2–3 months on induction medium, an average of 15.6 percent of the explants produced either somatic embryos or proembryogenic masses. Compared to pure yellow-poplar embryogenic cultures, putative hybrid cultures tended not to maintain growth as proembryogenic masses while exposed to auxin, instead proliferating via repetitive embryogenesis as globular-stage embryos. Four to six weeks following transfer of globular embryos to basal medium, mature embryos were produced from the putative hybrid lines. Mature embryos germinated following transfer to basal medium lacking casein hydrolysate. Plantlets survived transfer to potting mix and acclimatization to greenhouse conditions. Verification of the hybrid nature of the embryogenic lines and somatic embryo-derived plantlets was accomplished by Southern hybridization analysis with a species-specific DNA marker.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - PEM proembryogenic mass - CH casein hydrolysate - RFLP restricted fragment length polymorphism - CTAB hexadecyltrimethylammonium bromide - TBE Tris-borate-EDTA - SDS sodium dodecylsufate - SSC sodium citrate/chloride     

High efficiency adventive embryogenesis on somatic embryos of anther,filament and immature proembryo origin in horse-chestnut (Aesculus hippocastanum L.) tissue culture

Adventive embryogenesis was successfully induced in cultures of zygotic and somatic embryos on MS medium supplemented with BA and NAA. A procedure has been proved successful for the in vitro multiplication of somatic embryos regenerated at low frequencies from filament and callus cultures. The occurrence and rate of adventive embryogenesis did not depend on the origin of the primary embryos (zygotic and somatic), but did depend on the developmental stage. Primary embryos are capable of embryogenesis in each of the different phases of embryogenesis, though the rate is different. BA concentrations of 22–44 M increased the rate of adventive embryogenesis and accelerated the development of embryos. The highest proliferation rate (22–25x/5 weeks) was achieved at hormone concentrations of 44 M BA and 5.4 M NAA.Abbreviations BA benzyladenine - CH casein hydrolysate - CM coconut milk - 2,4-D dichlorophenoxyacetic acid - MS Murashige & Skoog medium - WPM woody plant medium - NAA 1-naphtaleneacetic acid     

In vitro propagation and low temperature storage ofSaussurea lappa C.B. Clarke — An endangered,medicinal plant

A procedure forin vitro multiplication ofSaussurea lappa (Asteraceae) is described. On Murashige and Skoog's medium (MS) containing benzylaminopurine and gibberellin 3.5-fold shoot multiplication occurred every three weeks. Shoots rooted on MS containing 0.5 M naphthaleneacetic acid with 90% efficiency. The shoot cultures stored at 5°C in the dark for 12 months without an intervening subculture survived with 100% viability. The shoots cold stored for 6 months or more showed higher rates of multiplication under culture room conditions than the untreated shoots.Abbreviations MS Murashige and Skoog 1962 - BAP Benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - IBA Indolebutyric acid - NAA Naphthaleneacetic acid - GA₃ Gibberellin     

Effect of thidiazuron on somatic embryogenesis of Cayratia japonica

Unpollinated ovary explants of Cayratia japonica (Thump.) Gagnep, were cultured on the revised Murashige & Skoog's medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) alone, or in combination with 0.009 M thidiazuron (TDZ) or 0.23 M kinetin for induction of embryogenic callus. The best results were obtained on medium containing 2.3 – 4.6 M 2,4-d and TDZ. When the calluses were subcultured on the basal medium (BM), somatic embryogenesis took place spontaneously at surfaces of the calluses, but only about 5% of the somatic embryos could develop to cotyledonary stage and most of the rest remained at the globular stage of development. If the calluses were transferred onto medium containing TDZ or TDZ combined with 0.27 M -napthaleneacetic acid, the number of cotyledonary somatic embryos increased up to 25%. When the somatic embryos of different stages were transferred onto fresh BM, only the cotyledonary embryos could convert into the plantlets. The results revealed that for the induction of embryogenic callus and somatic embryogenesis of Cayratia japonica, both cytokinin and auxin are required in the medium and the cytokinin activity of TDZ is much stronger than that of kinetin even when the concentration of TDZ used was only 4% of kinetin.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron (N-phenyl-1,2,3,-thi-diazol-5-ylurea)     

Somatic embryo induction in Eucalyptus dunnii

Somatic embryogenesis has been reported in three Eucalyptus species so far. Here we report the initial success on the somatic embryo induction of a fourth species, E. dunnii, which is one of two species of choice to be grown in southern Brazil. Induction was performed on three day-old seedlings by means of naphthaleneacetic acid (5.5 or 16.5 M) alone or in combination with 2,4-dichlorophenoxyacetic acid (4.5 M). Either 10% coconut milk (v/v) or 1 g l^-1 hydrolysed casein enriched auxin-free medium with was able to induce the development of the somatic embryos.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - B5 Gamborg et al. (1968) medium - MS Murashige & Skoog (1962) medium - NAA -naphthaleneacetic acid - BA benzyladenine     

The influence of auxins,light and cell differentiation on solasodine production bySolanum eleagnifolium Cav. calli

The effect of auxins, light and cellular production ofSolanum eleagnifolium Cav. calli were studied. 2,4-dichlorophenoxyacetic acid (4.5 M) was the plant growth regulator used for calli initiation and this produced the highest solasodine concentration. The solasodine concentration in darkness was significantly lower than that achieved under a photoperiod of 16 h. Differentiated tissue obtained by adequate hormonal balance (several ratios of 3-indolebutyric acid to 6-benzylaminopurine) produced higher yields of solasodine than non-differentiated tissue. 3-indolebutyric acid (2.5 M) and 6-benzylaminopurine (8.8 M) increased the productivity of solasodine by 100%.Abbreviations BAP 6-benzylaminopurine - KIN Kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA 3-indoleacetic acid - NAA 1-naphtaleneacetic acid - IBA 3-indolebutyric acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - DW dry weight - GI      

Hormonal regulation of berberine production in cell suspension cultures of Thalictrum minus

Effects of auxin and cytokinin on cell growth and alkaloid production in cell suspension cultures of Thalictrum minus were examined in an attempt to increase the productivity of a medicinal compound, berberine. In Linsmaier and Skoog medium containing auxin such as 2,4-D (1 M), the cultured cells grew rapidly, producing little berberine. On the other hand, the berberine-producing activity was remarkably enhanced by simultaneous administration of auxin and cytokinin, although cell growth was inferior. In particular, for the combination of NAA (60 M) and 6-benzylaminopurine (10 M), the yield of berberine was as high as 20 mg/30 ml medium after 2 weeks of culture. Furthermore, most of the berberine produced by the cells was released into the liquid medium, in which an excess of berberine crystallized. The results of the present experiments are suggestive of an advantage in adopting a two-stage culture method for the production of berberine in fermentor systems.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine     

The effect of in vitro pretreatments on subsequent shoot organogenesis from excised Rubus and Malus leaves

Shoot regeneration from Rubus leaves was obtained on a medium containing MS salts, vitamins and sugars, Staba vitamins, casein hydrolysate (100 mg l^–1) and 10 M thidiazuron. Shoot regeneration from Malus leaves was obtained on N⁶ rice anther medium with 5 M thidiazuron. In vitro pretreatment of source shoots with either colchicine or thidiazuron enhanced the organogenic potential of detached leaves of two Rubus hybrids. The response to colchicine was quadratic and occurred at non-mutagenic concentrations (75–250 M). The response to thidiazuron was exponential between 0 and 5 M. When applied as a pretreatment, the effectiveness of several different cytokinins (benzyladenine, thidiazuron, zeatin) at enhancing Malus and Rubus organogenesis was related to the shoot proliferation activity of the cytokinin and to treatment-induced variation in leaf and petiole size.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - MS Murashige & Skoog basal medium devoid of plant growth regulators - OI organogenesis-initiating subculture - PTI colchicine pretreatment subculture - PTII cytokinin pretreatment subculture - NAA naphthaleneacetic acid - TDZ thidiazuron - zeatin trans-zeatin     

Improvement of somatic embryogenesis in Medicago arborea

A number of medium constituents were evaluated in an attempt to improve somatic embryo production in Medicago arborea ssp. arborea, using cotyledons, petioles and leaves as explants. Two culture steps were applied: in the first stage (2 months), Murashige–Skoog (MS) medium was used, containing 2,4 dichlorophenoxyacetic acid (9M 2,4-D) and kinetin (9 M KIN) together with different nitrogen sources (alanine, glutamine, proline or tryptophan (2.5 and 5 mM); casein hydrolysate (100, 500 and 1000 mg l^–1; nitrate (4.69 and 9.39 mM) or casein hydrolysate (100 mg l^–1) and nitrate (4.69 mM)), polyalcohols (mannitol at 164 and 328 mM or sorbitol at 219 and 438 mM), sucrose (43.8 and 175.4 mM) or calcium (1.5 and 6 mM). In the second stage (3 months of cultivation), calli were transferred to a kinetin-free MS medium with 2,4-D (2.25 M) only. The inclusion of proline (2.5 mM) was the most effective treatment for the induction of somatic embryos, with the petiole being the best explant. Treatment with casein hydrolysate (100 mg l^–1) also improved the embryonic efficiency. The rest of the treatments neither affect nor inhibit the embryonic response.A special treatment with sorbitol (219 mM) in the second stage of cultivation produced a slight increase in embryogenesis, but less than that obtained with proline.     

Influence of dicamba and casein hydrolysate on somatic embryo number and culture quality in cell suspensions of Dactylis glomerata (Gramineae)

The effects of various concentrations and combinations of dicamba (3,6-dichloro-o-anisic acid) and casein hydrolysate on growth, mucilage accumulation, somatic embryo and root development in suspension cultures of Dactylis glomerata L. (orchardgrass) were examined. Fresh weight of culture tissue was increased with 20 M but not with 80 or 160 M dicamba in treatments with 1–4 g/l casein hydrolysate. Different casein hydrolysate concentrations did not alter the amount of mucilage (measured by viscosity) in the supernatant in the absence of dicamba. However, the addition of dicamba increased viscosity with 80 M giving the maximum response. Casein hydrolysate produced the greatest viscosity at 1–3 g/l in treatments where dicamba was present. Both dicamba and casein hydrolysate were required for development of somatic embryos. Dicamba at 40 M with 3–4 g/l casein hydrolysate produced approximately 2000 embryos/35 ml of suspension. Root development was inhibited by dicamba and stimulated by the presence of casein hydrolysate. The usefulness of medium component manipulations for influencing somatic embryogenesis and culture quality is discussed.     

Plant regeneration from leaf and seed-derived calli and suspension cultures of the African perennial wild rice,Oryza longistaminata

Plant regeneration from 2-month-old callus cultures derived from immature leaves of 7-day-old aseptic seedlings and mature embryos of the African wild rice Oryza longistaminata was achieved at 20% and 100% frequency, respectively. The morphogenic potential of the embryo-derived calluses dropped from 100% at the third subculture to 12.5 % at the 12th subculture. Five-month-old morphogenic calluses were used to establish a fast-growing suspension culture which, when plated onto semisolid medium, still retained its ability to regenerate plantlets 9 months after initiation. Histological analyses demonstrated that late plant regeneration from established callus and suspension cultures occured through organogenesis, although some embryogenesis events may have taken place during initiation of these cultures.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthalenacetic acid - BAP 6 benzylaminopurine - PAS Periodic acid shiff     

Somatic embryogenesis and plantlet regeneration in semul (<Emphasis Type="Italic">Bombax ceiba</Emphasis>)

Somatic embryogenesis in semul, Bombax ceiba L. (Bombacaceae) was achieved from immature zygotic embryo explants on MS medium. The cytokinin BA induced somatic embryogenesis at higher frequencies than the auxin 2,4-d. The rate of somatic embryogenesis was inversely related to the concentration of BA. A constant supply of 0.44 M BA was necessary for a high multiplication rate. Conversion of somatic embryos into plantlets is reported.     

Somatic embryogenesis and plant regeneration from cell suspension and tissue cultures of mature himalayan poplar (Populus ciliata)

Somatic embryogenesis and plantlet formation were obtained from callus and cell suspension cultures of 40-year- old Himalayan Poplar (Populus ciliata Wall ex Royle). Callus and cell suspensions were obtained by transfer of inoculum of semiorganized leaf cultures, which were maintained on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP), to MS with 2,4-dichlorophenoxyacetic acid (2,4-D). Reduction of 2,4-D concentration during subsequent subculture of cell suspensions resulted in the formation of embryoids. These embryoids developed further only after being transferred to agar-based MS medium supplemented with BAP and naphthalene acetic acid. Loss of embryogenic potential was observed in cell suspensions after 6 subcultures. However, callus cultures retained the embryogenic potential even after repeated subcultures for more than a year. Plantlets could be successfully hardened and grown in natural outdoor conditions.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthalene acetic acid - MS Murashige and Skoog (1962) medium