Initiation Of Embryogenic Tissue Research Articles

Plant regeneration by somatic embryogenesis in Pinus thunbergii resistant to the pine wood nematode

Pine wilt disease (PWD) is a severe threat to pine forests in East Asia. Screening and breeding of resistant varieties is a very effective way to prevent and control PWD; however, no reliable somatic embryogenesis system has yet been developed for the elite nematode-resistant Pinus thunbergii Parl. line. In this study, we studied the plant regeneration via somatic embryogenesis of nematode-resistant P. thunbergii. Initiation of embryogenic tissue was significantly affected by seed family (p = 0.017), immature zygotic embryo stage (p = 0.032), and initiation medium (p = 0.004). Seed family 37 was the most favorable female parent for initiation of P. thunbergii. Furthermore, the initiation rate increased from the pre-embryonic stage to the cleavage polyembryonic stage. The optimal medium was I2, containing 2,4-dichlorophenoxyacetic acid (9 μmol·L−1) and 6-benzyladenine (4.4 μmol·L−1). A statistically significant interaction between cell line and subculture time (24 months) was observed in the influence on proliferation rate, somatic embryo production, and percentage germination (p < 0.001). In this study, the highest somatic embryo production was achieved using cell line 37-1 (1983 somatic embryos per gram fresh mass), with approximately 83.5% of somatic embryos germinating after transferring to germination medium, of which 77.6% converted into plantlets.

Somatic Embryogenesis in Selected Conifer Trees Pinus nigra Arn. and Abies Hybrids.

Somatic embryogenesis was achieved in the conifers Pinus nigra Arn. and in the hybrids Abies alba ×A. cephalonica and Abies alba ×A. numidica. For initiation of embryogenic tissue in P. nigra, immature zygotic embryos enclosed in megagametophytes were used. The initiated embryogenic cultures were maintained and proliferated on solid culture medium DCR supplemented with 9 μM 2,4-D and 2.2 μM BA. Microscopic investigations revealed the presence of bipolar early somatic embryos in proliferating tissue. Suspension cultures have also been established by resuspending the embryogenic tissue in liquid culture medium. Experimentation with abscisic acid concentration resulted in successful somatic embryo maturation. Besides abscisic acid, the carbohydrate content or higher concentration of gelling agent in maturation medium were also important requirements for somatic embryo maturation. Germination of cotyledonary somatic embryos occurred on hormone-free medium and terminated in somatic seedlings regeneration. The regenerated somatic seedlings were transferred to soil and were capable of successful development. For initiation of embryogenic tissue in Abies hybrids juvenile explants as immature or mature zygotic embryos as well as cotyledons were used and 4.4 μM BA as sole plant growth regulator was sufficient. Medium of the same composition was also suitable for their long-term maintenance. Maturation of somatic embryos was achieved on solid DCR medium supplemented with 38 μM abscisic acid, polyethylene glycol (0, 5, 7.5, and 10% PEG-4000) and different carbohydrates such as maltose, sucrose and glucose (each 3%). PEG-4000 stimulated somatic embryo development depending on the carbohydrate source used. Cotyledonary somatic embryos germinated after desiccation treatment and the regenerated somatic seedlings were transferred to soil. Cryopreservation of embryogenic tissue could be an alternative method for long-term maintenance. For cryopreservation the slow-freezing method was used with success. Tissue regeneration in the post thaw period was relatively high and the regenerated tissue produced mature somatic embryos and subsequent plantlets. The embryogenic tissue was also used in experiments focused on genetic transformation either by biolistic (P. nigra) or Agrobacterium-mediated (Abies hybrids) methods. A proteomic study was performed to gain a deeper insight into the early stages of P. nigra somatic embryogenesis.

Simple and efficient protocols for the initiation and proliferation of embryogenic tissue of Douglas-fir

Key messageThis paper describes the micropropagation of Douglas-fir via somatic embryogenesis using media and methods available in the public domain.Douglas-fir is a conifer species with high and increasing interest for forest industries in both New Zealand and France. Delivery of the best trees to the forest from breeding programmes is currently constrained by an inability to effectively and reliably multiply selections through vegetative propagation. Somatic embryogenesis coupled with cryopreservation is essential biotechnology tool in conifers for scaling up variety design and production. The aim of this work was therefore to develop protocols for the initiation and proliferation of Douglas-fir embryogenic cell lines based on the modifications of previously published techniques and media available in the public domain, especially successful methods developed for P. radiata at Scion. Three years of initiation experiments have resulted in a simple and efficient protocol. Disinfection of whole cones (instead of seeds) was sufficient to prevent contamination. Immature zygotic embryos were excised from megagametophytes and placed onto a modified Litvay medium (Plant Cell Rep 4(6):325–328, 1985***). At optimal development stages of zygotic embryos, the average initiation percentage of embryogenic tissue was 69.3% when our most effective protocol was used. Proliferation of initiated cell lines on a Glitz formulation was challenging, with a high percentage of cell lines composed of a mixture of both embryonal masses and callus. 2,4-D at a lower concentration reduced the number of such mixed lines. Repeated liquid suspension and subculture of the embryogenic parts of the tissue was an efficient means to increase the number of embryogenic cell lines with sustained proliferation. Maltose added to the proliferation medium in place of sucrose improved fresh mass gain and consistently increased early somatic embryo patterning and growth. A sample of proliferating cell lines was successfully cryopreserved, thawed and somatic embryos were matured and germinated from these lines. The method may be of practical interest and provide new opportunities to realize increased genetic gain in this species through the clonal-assisted deployment of the best genetic material in both New Zealand and France.

Fraser fir somatic embryogenesis: high frequency initiation, maintenance, embryo development, germination and cryopreservation

Fraser fir (Abies fraseri [Pursh] Poir.) is a coniferous species native to the Southern Appalachian Mountains of the eastern United States. The species has high economic and recreational value but is vulnerable to extinction due to introduced pests and global warming. Somatic embryogenesis technology may assist in the clonal production of desired lines of Christmas trees and safekeeping of rare and valuable germplasm via cryopreservation. We have developed a highly effective medium for initiation of embryogenic tissue from immature or mature seeds of Fraser fir that contains AL salts (Kvaalen et al., in Can J For Res 35:1053–1060, 2005), brassinolide, paclobutrazol and abscisic acid. Using dominant embryos attached to the female gametophyte placed on medium, the highest initiation percentages occurred with precotyledonary stage 3 embryos. When tested with 11 high-value open-pollinated families over 5 years, initiation tests for medium containing brassinolide and paclobutrazol averaged 6–62 % initiation. A maintenance medium was developed that contained AL salts and 1.1 mg L−1 BAP and was able to capture approximately 50 % of the initiations. A maturation medium was developed containing AL salts, maltose, polyethylene glycol 8000 and abscisic acid that produced cotyledonary embryos capable of germination to produce a root and shoot. Culture cryopreservation and retrieval was also demonstrated.

Environmental conditions at the initial stages of Pinus radiata somatic embryogenesis affect the production of somatic embryos

Physicochemical conditions present during the initiation of somatic embryogenesis in Pinus radiata determine the rate of embryogenic cell lines generated as well as the final number of somatic embryos. The effect of physical and chemical conditions during the initiation and proliferation stages of somatic embryogenesis in radiata pine was investigated. The first objective was to assess if different temperatures and/or water availability during the initiation or proliferation of embryogenic tissue impacted the success rate during each stage of somatic embryogenesis. The second objective was to determine what stage has a greater influence on subsequent stages of development. Some treatments (18 °C, 4 g L−1 gellan gum) resulted in a higher percentage of initiation whereas others (28 °C, 2 g L−1 gellan gum) gave rise to lower initiation. Our results indicated that initiation at different temperatures affected the subsequent stages of somatic embryogenesis. Thus, these embryogenic tissues were influenced by the environmental conditions present at initiation. We were able to successfully regenerate somatic embryos from cell lines initiated under all the environmental conditions tested.

Somatic embryogenesis, plant regeneration, and cryopreservation for Torreya taxifolia, a highly endangered coniferous species

Torreya taxifolia Arn., an ancient evergreen tree, is on the brink of extinction from attack by a fungal disease, recently reported to be caused by a novel isolate of Fusarium. We report the development of a somatic embryogenesis tissue culture system that can be used for cryogenic storage of T. taxifolia cultures and subsequent plant regeneration. Initiation of embryogenic tissue from immature zygotic embryos occurred on a conifer tissue culture medium containing 0.25 % activated charcoal, 43.8 mM maltose, 0.5 mM 2,4-dichlorophenoxacetic acid, 0.2 mM 6-benzylaminopurine, 0.2 mM kinetin, 0.1 μM brassinolide, 3.8 μM abscisic acid, 20.5 μM biotin, 1.13 μM folic acid, 1.28 mM 2(n-morpholino)ethanesulfonic acid and 0.69 mM pyruvic acid. Embryo induction ranged from 60 to 100 % across six seed sources. Somatic embryo development occurred on a medium containing 43.8 mM maltose, 1 % activated charcoal, 37.8 μM abscisic acid, 20.5 μM biotin, 0.1 μM brassinolide, 0.205 mM folic acid, 1.28 mM 2(n-morpholino)ethanesulfonic acid and 0.69 mM pyruvic acid. Germination of somatic embryos ranged from 64 to 82 %. Embryogenic tissue cultures from 30 genotypes representing seed from six mother trees were cryopreserved, and culture recovery was demonstrated after freezing. In contrast to many other coniferous tree seeds, the measured water potential (−MPa) of T. taxifolia megagametophyte tissue rose greatly during seed after-ripening. Duplication of this rise in vitro allowed development of somatic embryos to the cotyledonary stage.

Somatic embryogenesis of Pinus rigida × P. taeda and the relationship between the initiation of embryogenic tissue and zygotic embryo development

The pitch-loblolly pine hybrid (Pinus rigida × P. taeda) has useful characteristics of the parents, but its exploitation is hindered by restrictions of conventional breeding and propagation methods. This study was undertaken to establish an effective in vitro system for propagating pitch-loblolly hybrid pine through somatic embryogenesis and to unravel the relationship between the efficiency of embryogenic tissue initiation and zygotic embryo development. To initiate embryogenic tissue, megagametophytes of developing seeds were used as explants. Seeds were collected weekly, examined, and tested during June and July 2004. The medium and seed collection date were the most important factors for the successful somatic embryogenesis of P. rigida × P. taeda. Five embryogenic lines were obtained using a modified P. taeda basal medium, and the highest initiation rate was 0.55%, for seeds collected in 2 weeks, between July 3 and 16. Histological observation revealed that zygotic embryos of those seeds were mostly at the proembryonic stage or in transition to precotyledonary stages. For the successful maturation of somatic embryos, abscisic acid and gellan gum were needed in the medium. The results show that, although further tests and development are required, somatic embryogenesis could provide a viable option for propagating P. rigida × P. taeda hybrids.

Somatic embryogenesis of selected spruce species (Picea abies, P. omorika, P. pungens 'Glauca' and P. breweriana)

Somatic embryogenesis was studied in four spruce species (<em>Picea abies</em>, <em>P. omorika</em>, <em>P. pungens</em> 'Glauca' and <em>P. brewenana</em>) to determine if this method can be used for in vitro propagation of coniferous trees. The highest frequency of initiation of embryogenic tissue was obtained when mature zygotic embryos were used as explants. It ranged then from 10.8% (<em>P. brewenana</em>) to 23.75% (<em>P. omorika</em> and <em>P. pungens</em> 'Glauca'). The frequency of embryogenic tissue initiation was strongly affected by medium composition, i.e. addition of appropriate auxins (2,4-D, NAA, Picloram) and sucrose concentration (10-20 g<sup>-1</sup>"1). A lower frequency was obtained in <em>Picea omorika</em> (10%) when megagametophytes (endosperms with immature zygotic embryos) were used as explants. No emryogenic tissue was produced from hypocotyls, cotyledons and needles. A satisfactory frequency was achieved with the use of somatic embryos of <em>Picea abies</em> (30%). The proliferation of embryogenic cell lines of spruces was affected by medium type. The experiments resulted in production of somatic plantlets of <em>P. abies</em> and <em>P. omorika</em>. This enables the application of this method of spruce micropropagation for genetic and breeding research or for nursery production.

Improved plantlet regeneration from open-pollinated families of Abies alba trees of Dobroč primeval forest and adjoing managed stand via somatic embryogenesis

AbstractPossibility to improve plantlet regeneration from Abies alba Mill. open-pollinated families of 4 trees in Dobroč primeval and 3 trees in managed forest was studied. Immature zygotic embryos were cultured in order to obtain initiation of embryogenic tissue. Totally, three from the families of the managed forest (57%) and two from the primeval families (50%) responded to initiation condition. Initiation frequencies among families ranged in managed forest: 4.5–56.2%, primeval: 5.4–16.8%. Maturation ability was shown by 77.3% of the primeval cell lines, 36.4% cell lines produced cotyledonary somatic embryos. In managed forest, in 62.5% of the cell lines embryo maturation was observed. Cotyledonary embryos developed only in 15% of cell lines. Regenerants were obtained from 9 cell lines of primeval and from 6 cell lines of managed forest. Biochemically, the mature somatic embryos were characterized by the variation in soluble and protein profiles. The corresponding profiles of insoluble proteins exhibited uniform pattern. The variation was characteristic for somatic embryos of individual cell lines rather than for the primeval and managed stands. Enzymatically, no indications were obtained supporting higher metabolic potential of somatic embryos derived from zygotic embryos of silver fir primeval stand than in somatic embryos originating from the trees of managed stand.

Douglas fir embryogenic tissue initiation

Somatic embryogenesis (SE) is expected to play an important role in the future of US forests by providing increased productivity, sustainability, and uniformity. For broad scale implementation to occur, SE technology must work with a variety of genetically diverse trees. Douglas fir (Pseudotsuga menziesii (Mirb) Franco) is the dominant tree in the Pacific Northwest and has great economic and recreational value. We have developed a highly effective medium for initiation of embryogenic tissue of Douglas fir that contains ABA, biotin, brassinolide, folic acid, MES, pyruvic acid and can be used as a gelled medium or in a gelled-liquid medium overlay system. When tested with many high-value crosses over 2 years, initiation tests averaged initiation in the range of 40–57%. Additionally, a time- and labor-saving tetrazolium chloride embryo staining technique was developed to evaluate seed health and screen out seed sources likely to perform poorly in the initiation process.

In vitro initiation and early maturation of embryogenic tissue of Quillaja saponaria.

Quillay ( Quillaja saponaria ) is an endemic tree that grows between IV and IX Region of Chile. The factors that affect the generation of somatic embryos were determined with the objective to study their clonal propagation. Hypocotyls of seeds and apexes of selected adult trees were used for the in vitro culture. The embryogenic induction was performed in 0.5xMS medium supplemented with 13.5 µM 2,4-D and 4.4 µM BAP, during 8 weeks. Later, the embryogenic masses accumulated reserves in medium with 15 mg·L -1 of ABA during 4 weeks. The addition of 4.4 µM BAP to the medium promoted the maturation of pro-embryos to embryos in globular state (Phase Maturation I). In addition, we determined that supplements of casein hidrolysate, magnesium sulphate and glutamine favored the development of the somatic embryos. However, the decrease in the concentration in the gelling agent and the variation in the concentration of oxidized and reduced sources of nitrogen did not significantly affect the maturation. These effects allow developing somatic embryos until globular state, from young and adult tissue of Q. saponaria. El quillay es un arbol endemico chileno que crece entre las regiones IV y IX. Con el objetivo de mejorar su propagacion clonal se determinaron los factores que afectan la generacion de embriones somaticos. Para el cultivo in vitro se utilizaron hipocotilos de semillas y apices de arboles adultos seleccionados de tres genotipos. La induccion embriogenica de los explantes se realizo en medio 0,5 x MS complementado con 13,5 µM de 2,4-D y 4,4 µM de BAP, durante 8 semanas. Posteriormente, los callos embriogenicos acumularon reservas al adicionar al medio 15 mg·L -1 de ABA durante 4 semanas. La remocion del 2,4-D y la mantencion de la concentracion de 4,4 µM de BAP en el medio de cultivo promovio la maduracion de pro-embriones a embriones en estado globular (Fase de Maduracion I). Ademas, se determino que los suplementos de hidrolizado de caseina, sulfato de magnesio y glutamina beneficiaron el desarrollo de los embriones somaticos. En cambio, la disminucion en la concentracion en el gelificante y la variacion en la concentracion de fuentes oxidadas y reducidas de nitrogeno no afectaron significativamente la maduracion. Los efectos estudiados permiten desarrollar embriones somaticos hasta estado globular tanto de tejido juvenil como de tejido adulto de Quillaja saponaria de tres genotipos.

Achieving desired plant growth regulator levels in liquid plant tissue culture media that include activated carbon

This paper is part of a series considering the impact of activated carbon (AC) on the composition of plant tissue culture media. Using liquid culture media for initiation of Norway spruce embryogenic tissue and eight different ACs, we present a method for achieving target plant growth regulator (PGR) levels in AC-containing medium based on sorption isotherms for individual PGRs. Linear relationships were found between PGR adsorption and specific BET (Brunauer, Emmett, Teller theory) surface area and specific total pore volume of AC. When using a new AC, this linear relationship allows one to achieve multiple PGR levels similar to historic levels through adjustment of the mass of AC based on its relative BET surface area or relative total pore volume. Target levels of PGRs and an initiation success similar to that in medium without AC were achieved with several different AC types when AC mass was adjusted on the basis of pore volume.

Somatic embryos can be induced from the vegetative shoot apex of mature Pinus patula trees

Embryogenic cultures were initiated and established and somatic embryos produced in Pinus patula using thin sections from the dome of the vegetative shoot apex. Factors affecting initiation, basal medium and plant growth regulators were investigated. We report for the first time the initiation of embryogenic tissue and embryos from shoot apical domes (zones) of 15-year-old Pinus patula trees. The growth of embryogenic lines were comparable to those obtained from zygotic embryos. Plantlets were regenerated.

Is there a negative genetic correlation between initiation of embryogenic tissue and fungus susceptibility in Norway spruce?

Genetic associations between initiation of embryogenic tissue (ET) and susceptibility to the phytopathogenic fungi Ceratocystis polonica (Siem.) C. Moreau and Heterobasidion annosum (Fr.) Bref. in Norway spruce have been studied by initiating ET from zygotic embryos of mature seeds collected from 19 clones tested for susceptibility to the pathogens in a clonal field trial. Initiation frequencies varied significantly among clones (families), ranging from 12 to 56%. The family variance component accounted for more than 40% of the total variance in initiation frequency of ET. The estimates of broad-sense heritability of fungus susceptibility of the clones ranged from 0.12 for length of phloem necrosis after low-density inoculation with H. annosum to 0.55 for blue-stained sapwood after mass inoculation with C. polonica. None of the susceptibility measures showed any phenotypic correlation with initiation of embryogenic tissue. Genetic correlations and their standard errors were estimated by bootstrapping. Two measures of fungal susceptibility correlated genetically with initiation of ET; estimated at –0.58 for lesion length after inoculation with C. polonica and –0.29 for H. annosum lesion length. A better measure of susceptibility, blue-stained sapwood following inoculation with C. polonica, was not correlated with initiation of ET.

Is there a negative genetic correlation between initiation of embryogenic tissue and fungus susceptibility in Norway spruce?

Is there a negative genetic correlation between initiation of embryogenic tissue and fungus susceptibility in Norway spruce?

Induction of somatic embryogenesis in loblolly pine (Pinus taeda L.)

Several factors that may affect induction of somatic embryogenesis in loblolly pine (Pinus taeda L.) were investigated in 1994 and 1995. Megagametophytes containing immature zygotic embryos were excised from seeds as explants. Potassium chloride, silver nitrate, myo-inositol, coconut water, or polyamine was added to the control media (U.S. patent no. 5,036,007) to determine the effects of each single ingredient or their combinations on the initiation of embryogenic tissue. Supplements of myo-inositol at 22.2 mM resulted in increases in frequencies of cell mass extrusion and proliferation compared with the control media in consecutive years. Addition of silver nitrate showed the potential to promote initiation of embryogenic culture. The combination of 10 mM potassium with 29.4 μM silver nitrate achieved the highest frequencies in both extrusion and proliferation of embryogenic tissue. The combination of silver nitrate at 29.4 μM with addition of myo-inositol at 11.1 or 22.2 mM achieved a higher conversion rate from extrusion to proliferation. Polyamine did not significantly affect the induction of somatic embryogenesis, but coconut water was inhibitory.

Effects of various partial pressures of oxygen and carbon dioxide on different stages of somatic embryogenesis in Picea abies

Effects of various partial pressures of oxygen (5, 20 and 45 kPa) and carbon dioxide (0.03 and 6 kPa) on initiation, proliferation and maturation of somatic embryos in Picea abies were studied. The pO2 had a significant effect on the initiation of embryogenic tissue from mature zygotic embryos. However, the effect of pO2 was dependent on the strength of the basal medium. Low pO2 stimulated the formation of embryogenic tissue when the zygotic embryos were incubated on full strength medium, but was inhibitory when half-strength medium was used. Proliferation of embryogenic tissue was stimulated by higher partial pressures of both CO2 and O2. The effect of the gas phase on maturation of somatic embryos varied between different cell lines. However, there was a general tendency for 5 kPa O2 and 6 kPa CO2 to stimulate maturation.

Plant regeneration from embryo-derived tissue cultures of soybeans

Routine regeneration of fertile plants from a tissue culture of soybean has been achieved. Serially propagated embryogenic cultures were initiated from immature embryos of many genotypes. Organized tissues developed only on the cotyledons of embryos. Genotypic variation in the frequency of initiation of embryogenic tissue was noted. However, embryogenic tissue cultures were generated from all genotypes tested. Embryogenic tissue was serially increased and underwent morphogenesis. Whole fertile plants were recovered. Cultures have been maintained for two years without loss of morphogenic competency.