Changes in ovule protein profiles associated with somatic embryogenesis in Cercis canadensis (redbud)

Summary The somatic embryogenic potential of Cercis canadensis (redbud) ovules was compared to changes in ovule protein profiles over time. Ovules collected 82–159 dpa were cultured on a modified SH medium and evaluated after six weeks for the development of somatic embryos. Proteins were extracted from additional ovules and analyzed by SDS-PAGE. Ovules produced somatic embryos from 96–139 dpa and the maximum embryogenic response occurred at 107 dpa. Changes in the staining intensity of six protein bands were associated with changes in embryogenic potential. The intensity 32 and 36 kDa proteins decreased when ovules became competent to produce somatic embryos. The four remaining bands (18, 19, 56, and 94 kDA) increased in intensity from the middle to the end of the sampling period and these changes were associated with the loss of the somatic embryogenic potential.Abbreviations BSA Bovine Serum Albumin - dpa days post anthesis - 2,4-D 2,4-dichlorophenoxyacetic acid - kDa kilodalton - PVPP polyvinylpolyprrolidone - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - SH modified Schenk and Hildebrandt medium (1972)     

Somatic embryogenesis from immature embryos in meadowfoam (Limnanthes alba)

Developing embryos from immature seeds were excised and cultured. Optimal proliferation of differentiated secondary embryos occurred on Murashige-Skoog media containing 7% sucrose, 0.1 M 2,4-D, and 0.1–1.0 M zeatin. Higher levels of auxins inhibited embryo proliferation. Secondary embryos were subcultured to produce more embryos. The results indicate the feasibility of clonal propagation of meadowfoam.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-Benzyladenine     

Somatic embryogenesis from immature zygotic embryos ofGinkgo biloba

Immature zygotic embryos ofG. biloba were taken, at various developmental stages, from ovules harvested in November 1993. Zygotic embryos showing the beginning of the cotyledonary development cultured on modified Murashige & Tücker (1969) media proliferated intensely. In fact, 98.5% of the immature zygotic embryos produced embryogenic and undifferentiated tissues (calluses), in proportions varying depending on the hormonal composition of the induction media. After two weeks of culture, direct embryogenesis was observed on the hypertrophic cotyledons when benzyladenine 10 M was used as the sole plant growth regulator in the induction media. The addition of different concentrations of NAA (5–10–20 M) and of BA (5 M) to the induction media led to an indirect embryogenesis after two months, when the calluses were transferred to the development media without auxin. The highest frequency of embryogenic tissues (90–95%) and the highest number of somatic embryos per explant (9.6) were obtained with benzyladenine (10 M) as the sole exogenous growth factor. Some embryos isolated mechanically or in situ on the callus developed as far as the later cotyledonary stage.Abbreviations AUX Auxin - BA Benzyladenine - CYT Cytokinin - IZE Immature zygotic embryo - MT Murashige & Tücker (1969) medium - NAA Naphtaleneacetic acid     

Somatic embryogenesis from immature zygotic embryos of oil palm

Immature zygotic embryos at different developmental stages were used for callus induction and regeneration studies. Immature embryos excised from fruits 77, 91, 100, 114, 128, 140 and 193 days after pollination and mature embryos were cultured on modified Y3 medium containing 500 mgl^–1 cysteine, 0.5% (w/v) PVP-40, 500 M 2,4-d and 0.3% (w/v) charcoal. Compact embryogenic tissue began differentiating directly from embryo explants after 2 weeks of culture. The percentage of embryos forming compact embryogenic tissue ranged from 28.6% for 91-day-old embryos to 0% for 140-day-old and older embryos. Friable embryogenic tissue was observed in callus cultures derived from 100-day-old embryos. Although both compact and friable embryogenic tissues were successfully isolated, normal embryo and plantlet development was observed only from friable embryogenic tissue.Abbreviations ABA abscisic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - PVP polyvinylpyrollidone     

Micropropagation of Mexican redbud,Cercis canadensis var. mexicana

Mexican redbud (Cercis canadensis var. mexicana) shoot cultures were initiated from explants taken from both mature and juvenile stock plants. Culture conditions affecting shoot growth and proliferation and rooting of three clones were investigated. Shoot growth was best on media supplemented with 0.25% activated charcoal and solidified with 0.2% Gelrite. Four commercially available salt formulations (Anderson's rhododendron medium, WPM, MS, DKW) were tested for growth of shoot cultures, and Anderson's rhododendron basal salt mixture was superior. Axillary shoots grew from explants cultured media supplemented with a wide range of concentrations of benzyladenine and thidiazuron. Benzyladenine at 5.6–22.2 M supported the best combination of shoot quality and number. Rooting of microshoots in vitro was best on half-strength WPM containing 6.71 M naphthaleneacetic acid and 0.1% activated charcoal.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid - 2iP 6-(, -dimethylallylamino)purine - DKW Driver & Kuniyuki Walnut - kinetin 6-furfurlaminopurine - MS Murashige & Skoog - NAA -naphthaleneacetic acid - WPM Woody Plant Medium - TDZ thidiazuron - 1-phenyl-3 (1,2,3-thidiazol-5-yl)urea     

Somatic embryogenesis from immature zygotic embryos of Rosa bourboniana desp

Summary A protocol for the inducton of somatic embryogenesis from immature zygotic embryos of Rosa bourboniana, a scented rose species, was established. Somatic embryos were induced after 8wk of inoculation of zygotic embryos on MS medium supplemented with different concentrations of 2,4-dichlorophenoxy acetic acid (5–15 μM). In addition to 2,4-dichlorophenoxy acetic acid concentrations, somatic embryogenesis was also influenced by the month of collection of the explant and the stage of maturity of the hip. Maximum embryogenic response (16.6%) was observed using 2,4-dichlorophenoxy acetic acid (15 μM), from green hips in the month of September. The use of l-proline (800 mg l⁻¹) was found to be optimum for secondary embryogenesis. On periodic subculturing, the cultures formed somatic embryos sustainably over a period of 18 mo. For somatic embryo germination, 6-benzylaminopurine (5 μM) was found to be most suitable. Rooted plants were transferred successfully to soil and appear morphologically normal under greenhouse conditions. Transfer of plants for hardening was most suitable during the active growth period between June and September. IHBT Publication No: 0447     

Somatic embryogenesis and plant regeneration from immature embryos of western larch

Somatic embryogenesis was initiated from immature embryos of western larch (Larix occidentalis Nutt.) on media containing 2,4-dichlorophenoxyacetic acid and N6- benzyladenine. The effects of explant type and ammonium nitrate and glutamine concentrations on initiation were tested. Although 21–93% of explants rendered cultures in various experiments, only 3% yielded sustainable embryogenic lines. Excised embryos at the early cotyledonary stage were optimal for initiation. Maturation of somatic embryos was promoted by abscisic acid. Response to abscisic acid concentrations and duration of exposure to abscisic acid varied with genotype. Maximal results were obtained with 0.025 M abscisic acid for 1 to 2 weeks followed by individual culture on medium without growth regulators. Mature somatic embryos developed into shoots with roots. Plantlets have been established in peat.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA N⁶-benzyladenine - IBA indole-3-butyric acid - ABA abscisic acid     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of Japanese larch (Larix leptolepis)

Embryogenic tissue was initiated using LM, LP and MS media from open-pollinated immature embryos of Larix leptolepis. The initiation frequency varied with collection dates. The highest frequencies of embryogenic tissue initiation (60, 67 and 59% on LM, LP and MS media, respectively) were observed from cones collected on July 30. At this time, all the excised embryos were at the cotyledonary stage. ABA over a wide concentration and length of exposure range did not promote maturation, but was beneficial in reducing precocious germination. Of over 400 plants regenerated, 72 were transplanted into soil mixtures and to date, 69 of these (95%) have survived. This revised version was published online in June 2006 with corrections to the Cover Date.     

Multiple shoot formation from normal and malformed somatic embryo explants of Eastern redbud (Cercis canadensis L.)

A procedure for multiple shoot formation from somatic embryo explants of Eastern redbud (Cercis canadensis L.) cultured on DKW medium containing benzyladenine (BA) and thidiazuron (TDZ) was developed. TDZ in combination with BA produced more shoots than either treatment alone. The highest number of shoots (3.3 to 3.4 shoots per explant) was obtained from partially desiccated and wounded explants treated with a combination of 5 or 10 M BA and 0.5 or 1.0 M TDZ for 20 days before being transferred to the same medium without TDZ. The number of shoots formed was increased from 1.5 to 3.2 shoots per explant by cutting through the cotyledonary node prior to culture. In addition, the frequency of explants forming shoots was increased by desiccation of somatic embryo explants to approximately 50% moisture and by using somatic embryos with two well formed cotyledons as explants.Abbreviations ABA abscisic acid - BA benzyladenine - CRD Completely randomized design - DKW Driver and Kuniyuki medium - LSD Least significant differences - TDZ thidiazuron     

Somatic embryogenesis and bud formation from immature embryos of buckwheat (Fagopyrum esculentum Moench.)

Immature embryos of Fagopyrum esculentum cv. Pennquad were isolated from field-grown plants and cultured on media containing a high benzylaminopurine to indole-3-acetic acid ratio. Part of the embryos were grown in the presence of 2,4-dichlorophenoxyacetic acid and kinetin for the first 5 days, and then transferred to benzylaminopurine + indole-3-acetic acid medium. From callus tissues developed on hypocotyls and cotyledons, 3 types of tissue were selected in later subcultures: (a) callus tissue strains that produced buds, (b) embryogenic tissue, and (c) unorganized callus tissue, lacking any organogenic capacity. Pretreatment with 2,4-dichlorophenoxyacetic acid increased the number of explants which gave rise to bud forming and embryogenic tissue, but was not essential for morphogenesis. Somatic embryogenesis was confirmed by histological observation. Plantlets could be easily obtained by inducing adventitious roots on shoots, but spontaneous root development in somatic embryos was infrequent.Abbreviations BAP benzylaminopurine - IAA indole-3-acetic acid - 2,4-D dichlorophenoxyacetic acid - IBA indole-3-butyric acid     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.)

Summary Immature zygotic embryos from open-pollinated and selfed Carica papaya L. fruits, 90 to 114 days post-anthesis, produced 2 to 20 somatic embryos on apical domes, cotyledonary nodes, and radicle meristems after culture for three weeks on half-strength Murashige and Skoog (MS) medium supplemented with 0.1 to 25 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 400 mg l^–1 glutamine, and 6% sucrose. After six weeks of culture, about 40 to 50% of the zygotic embryos had become embryogenic, and each embryogenic embryo yielded hundreds of somatic embryos within five months of culture on media supplemented with 2,4-D. Somatic embryos matured on half-strength MS medium, germinated on MS medium containing 5 mg l^–1 kinetin, and grew large enough for greenhouse culture on MS medium. Shoots were rooted in vermiculite and grown in the greenhouse.Journal Series no. 3449 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Somatic embryogenesis and morphogenesis in callus derived from the epiblast of immature embryos of wheat (Triticum aestivum)

Factors affecting the formation of embryogenic callus from the epiblast of immature embryos of wheat (Triticum aestivum L.) are described. Embryos were incubated on a modifed Murashige and Skoog medium with the scutellum in contact with medium. Callus formation from the epiblast was affected by the type of cultivar used, the stage of embryo development, and the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) in the culture medium. The number of embryos forming embryogenic callus ranged from 10% to 72%, depending on the cultivar. Embryo development was classified into five distinct morphological stages and higher yields of callus were produced using embryos excised at stages II and III. Concentrations of 2,4-D higher than 1 μM were required for the formation of embryogenic callus. Epiblast callus gave rise to embryoids which differentiated into multiple plants at a high frequency when placed on hormone-free medium.     

Somatic embryogenesis and plant regeneration from immature embryos of five families of Quercus acutissima

Immature embryos of sawtooth oak (Quercus acutissima Carruth.) were obtained from five seed families and cultured on modified Murashige and Skoog nutrient medium containing 1 g/l l-glutamine and 5 mM proline and supplemented with 1.0 mg/l indole-3-butyric acid and 1.0 mg/l 6-benzylaminopurine. The frequency of somatic embryogenesis from immature embryos was a function of the collection date and seed family. The highest frequency of explants forming somatic embryos was obtained with seeds of family Chungnam 11, i.e. 7 weeks post-fertilization (90.9%) and 9 weeks post-fertilization (91.2%). No response was shown by families Chungnam 14, 15 and Jeonbook 29 (0%), at 10 weeks post fertilization. During germination, the highest frequency of epicotyl formation was obtained with Chungnam 11 (44.0%) or Chungnam 15 (43.5%), and the highest rate of radicle formation was shown by Chungnam 11 (26.1%). The most responsive seed family with respect to the formation of both epicotyl (43.5%) and radicle (26.1%) was Chungnam 11. Twenty plantlets were transplanted to a perlite:peatmoss:vermiculite (1:1:1) soil mixture, and 8 plants survived in the field. Received: 6 December 1996 / Revised version received: 18 April 1997 / Accepted: 10 May 1997     

Somatic embryogenesis and plant development from immature zygotic embryos of seedless grapes (Vitis vinifera L.)

Summary Somatic embryo formation occurred from immature zygotic embryos within ovules of stenospermocarpic seedless grapes (Vitis vinifera L.), when cultured for two months on liquid Emershad/Ramming medium. Somatic embryos continued to proliferate after excision and transfer to Emershad/Ramming medium supplemented with 1 M benzylaminopurine and 0.65% TC agar. Plant development from somatic embryos was influenced by genotype, medium, phase (liquid, agar), stage (torpedo, mature) and their interactions. Optimal plant development occurred on Woody Plant Medium supplemented with 1.5% sucrose + 1 M benzylaminopurine + 0.3% activated charcoal and 0.65% TC agar.Abbreviations ABA abscisic acid - BAP benzylaminopurine - ER Emershad/Ramming - GLM general linear model - MGLH multivariate general linear hypothesis - MS Murashige/Skoog - NaClO sodium hypochlorite - TC tissue culture - WP woody plant     

Somatic embryogenesis from immature inflorescences of oil palm

Summary Immature inflorescences of oil palm (Elaeis guineensis) var. Pisifera were inoculated onto modified MS medium containing 0.3% (w/v) activated charcoal and 475 M 2,4-D. After 2—3 months of culture, a hard yellow callus proliferated at the base of the shoot-like structures. The high incidence of phenolic oxidation required the use of increased levels of activated charcoal (0.5% w/v) and 2,4-D (500 M). Development of floral structures from inflorescence expiants was frequently observed during the culture period. After 81 weeks of culture, an embryogenic tissue characterized by compact consistency and pearly white color was observed in tissues derived from very young inflorescences. This compact embryogenic tissue differentiated into normal somatic embryos when transferred onto regeneration medium containing NAA (15 M) and ABA (2 M). Normal plantlets were recovered from these somatic embryos after 8 weeks on regeneration medium.Abbreviations 2, 4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - ABA abscisic acid - PVP-40 polyvinylpyrrolidone     

Somatic embryogenesis from peach palm zygotic embryos

The factors affecting the induction and development of somatic embryos and plantlet acclimatization of peach palm (Bactris gasipaes Kunth) were evaluated to establish an efficient regenerative protocol based on somatic embryogenesis. Mature zygotic embryos were cultured in Murashige and Skoog (MS) medium supplemented with 0–40 μM of picloram (4-amino-3,5,6-trichloropicolinic acid) and 0 or 5 μM of 2-isopentyladenine (6-dimethylaminopurine) (2-iP). After 5 mo. in culture embryogenic callus arose from primary calli. Picloram (10 μM) was effective in inducing embryogenic calli in 9.8% of the explants. The use of 1 μM of AgNO₃ enhanced embryogenic competence. Embryogenic calli showed an organized structure, a globular aspect, and were white to yellowish in color. Histological analyses showed that cell proliferation arose from subepidermal cells adjacent to vascular bundles, resulting in primary callus formed by a meristematic zone from which somatic embryos arose. Protein profile analyses revealed two high molecular mass bands in these embryogenic calli, but not in other tissues. Embryogenic calli were transferred to a culture medium containing 40 μM of 2,4-dichlorophenoxyacetic acid, 10 μM of 2-iP, plus 1 g l⁻¹ of glutamine, hydrolyzed 0.5 g l⁻¹ casein, and activated 1.5 g l⁻¹ of charcoal. Morphogenetic responses achieved in this medium were the development of somatic embryos, rooting, and loss of embryogenic capacity. Somatic embryos were converted to plantlets on MS medium plus 24.6 μM of 2-iP and 0.44 μM of naphthalene acetic acid. Plantlets were maintained in MS medium with activated charcoal (1.5 g l⁻¹) until they were 6 cm tall, and then acclimatized. After 16 wk, 84.2 ± 6.4% survival was observed. M. P. Guerra and C. R. Clement are Fellows of the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brasília, DF.     

Somatic embryogenesis from immature and mature embryos of a minor millet Paspalum scrobiculatum L.

Immature and mature zygotic embryos of Paspalum scrobiculatum L. cv. PSC 1 cultured on MS or N₆ nutrient medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), formed embryogenic callus. Induction of embryogenic callus and subsequent somatic embryogenesis was possible at a lower concentration of 2,4-D on N₆ than MS medium. Immature embryos were highly totipotent, forming somatic embryos at a higher frequency than mature embryos. Addition of amino acids (L-proline or L-tryptophan) to 2,4-D medium resulted in significant enhancement of embryogenesis on culture of mature embryos. Silver nitrate also supported an increased frequency of embryogenesis. Thus it is possible to have high frequency of somatic embryogenesis on culture of mature embryos, which are available in abundance and with ease than immature embryos. The somatic embryos readily germinated and formed plantlets on hormone-free regeneration medium. The regenerated plantlets were successful on transfer to soil and set seed.     

Habitat fragmentation influences genetic diversity and differentiation: Fine‐scale population structure of Cercis canadensis (eastern redbud)

Forest fragmentation may negatively affect plants through reduced genetic diversity and increased population structure due to habitat isolation, decreased population size, and disturbance of pollen‐seed dispersal mechanisms. However, in the case of tree species, effective pollen‐seed dispersal, mating system, and ecological dynamics may help the species overcome the negative effect of forest fragmentation. A fine‐scale population genetics study can shed light on the postfragmentation genetic diversity and structure of a species. Here, we present the genetic diversity and population structure of Cercis canadensis L. (eastern redbud) wild populations on a fine scale within fragmented areas centered around the borders of Georgia–Tennessee, USA. We hypothesized high genetic diversity among the collections of C. canadensis distributed across smaller geographical ranges. Fifteen microsatellite loci were used to genotype 172 individuals from 18 unmanaged and naturally occurring collection sites. Our results indicated presence of population structure, overall high genetic diversity (H_E = 0.63, H_O = 0.34), and moderate genetic differentiation (F_ST = 0.14) among the collection sites. Two major genetic clusters within the smaller geographical distribution were revealed by STRUCTURE. Our data suggest that native C. canadensis populations in the fragmented area around the Georgia–Tennessee border were able to maintain high levels of genetic diversity, despite the presence of considerable spatial genetic structure. As habitat isolation may negatively affect gene flow of outcrossing species across time, consequences of habitat fragmentation should be regularly monitored for this and other forest species. This study also has important implications for habitat management efforts and future breeding programs.     

Somatic embryogenesis from immature zygotic embryos and monitoring the genetic fidelity of regenerated plants in grapevine

Somatic embryogenesis and plant regeneration were successfully established on Nitsch and Nitsch (NN) medium from immature zygotic embryos of six genotypes of grapevine (Vitis vinifera). The optimum hormone combinations were 1.0 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction and 1.0 mg dm⁻³ α-naphthalene acetic acid (NAA) + 0.5 mg dm⁻³ 6-benzyladenine (BA) for embryos production and 0.03 mg dm⁻³ NAA + 0.5 mg dm⁻³ BA for embryos conversion and plant regeneration. The frequency of somatic embryogenesis varied from 10.5 to 37.5 % among six genotypes and 15.5–42.1 % of somatic embryos converted into normal plantlets. The analysis of DNA content determined by flow cytometry and chromosome counting of the regenerated plantlets clearly indicated that no ploidy changes were induced during somatic embryogenesis and plant regeneration, the nuclear DNA content and ploidy levels of the regenerated plants were stable and homogeneous to those of the donor plants. RAPD markers were also used to evaluate the genetic fidelity of plants regenerated from somatic embryos. All RAPD profiles from regenerated plants were monomorphic and similar to those of the field grown donor plants. We conclude that somaclonal variation is almost absent in our grapevine plant regeneration system.     

Somatic embryogenesis from pea embryos and shoot apices

Conditions were defined for plant regeneration via somatic embryogenesis in pea, using explants from immature zygotic embryos or from shoot apices. For the induction of somatic embryos, an auxin (picloram or 2,4-dichlorophenoxyacetic acid) was required. Embryogenic callus originated from embryonic axis tissue of immature embryos and from the axillary-bud region and the plumula of shoot apices. A clear effect of embryo size on somatic embryogenesis was shown. There were differences in frequency of somatic embryogenesis among the five genotypes used in the study. Additions of BA to auxin-containing medium reduced embryo production. Histological examinations confirmed the embryogenic nature of the immature embryo cultures and revealed that somatic embryos originated from the meristematic areas near the callus surface.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid