Efficient plant regeneration from embryogenic suspension cultures of sweetpotato

Summary Using 15 Chinese and Japanese cultivars of sweetpotato, Ipomoea batatas (L.) Lam., we succeeded in developing an efficient plant regeneration system from embryogenic suspension cultures. The embryogenic callus derived from shoot apices of the 15 cultivars was used to initiate embryogenic suspension cultures in Murashige and Skoog (MS) medium containing 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Rapidly proliferating and well-dispersed embryogenic suspension cultures were established. Cell aggregates 0.7–1.1 mm in size from embryogenic suspension cultures were transferred to solid MS medium supplemented with 9.05 μM of 2,4-D and formed embryogenic callus with somatic embryos. The embryogenic callus with somatic embryos was further transferred to MS medium supplemented with 3.78 μM of abscisic acid, resulting in the germination of somatic embryos. Within 20 wk after the initiation, the frequencies of cell aggregates forming plantlets reached approximately 100% for the 15 tested cultivars. These plantlets, when transferred to soil, showed 100% survival. No morphological variations were observed.     

Plant regeneration from embryogenic suspension cultures of dune reed

Embryogenic callus, derived from mature seeds of dune reed (Phragmites communisTrinius) was used to establish suspension culture. Green shoot-forming type and albino shoot-forming type embryogenic callus of dune reed were selected carefully by the difference of shape and color of callus growing under light and mechanically dispersed before suspending in liquid MS medium supplemented with 1.0 mg l^–12,4-D. They were subcultured every 5 days to remove mucilaginous material in the early culture stage. Both fine albino and green shoot-forming cell suspension lines of dune reed were composed of rapidly growing small cell aggregates that were densely cytoplasmic and potentially embryogenic. Globular somatic embryos were continuously produced in each liquid medium containing 1.0 mg l^–1 2,4-D. The cell aggregates in fine albino cell suspension line (size below 300 m) were smaller than that of green shoot-forming cell suspension line (size between 300 and 800 m). Following transfer to a differentiation medium, both suspension cultures formed regenerating plants with normal roots and albinotic or green shoots, respectively.     

Differential gene expression in embryogenic and non-embryogenic clusters from cell suspension cultures ofCoffea arabica

Somatic embryogenesis (SE) is a very useful system for studying the differentiation process in plants and involves gene regulation at several levels. During SE induction in Coffea arabica cv. Catura Rojo two types of cell clusters, embryogenic (EC) and non-embryogenic (NEC), were observed. The goal of this work was to compare the most relevant characteristics between EC and NEC for a better understanding of the mechanism driving SE. Morphohistological observations indicated a correlation between the morphological features of clusters and their embryogenic competence. On the other hand, no variation at the DNA level, studied by AFLP, were found to explain the disparity in embryogenic competence of clusters, but gene expression, observed by RNA differential display, and SDS-PAGE showed differences that can explain that disparity. Our results lead us to propose that differential gene expression can modulate the embryogenic capacity of coffee cells and that the number of genes turned off in somatic cells to allow for the change from a somatic to an embryogenic state, is higher than those genes that are turned on.     

Effect of basal medium, growth regulators and Phytagel concentration on initiation of embryogenic cultures from immature zygotic embryos of loblolly pine (Pinus taeda L.)

Low initiation frequency is one of the main barriers in applying somatic embryogenesis to the clonal production of Pinus species. Factors affecting initiation, including basal medium, plant growth regulators, and Phytagel concentration, have been investigated in loblolly pine (Pinus taeda L.). BM₁ basal medium proved superior to DCR₁ and LP (LP basal salts plus BM₁ organic nutrients). No extrusion from megagametophytes was exhibited on LP medium. The combination of 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l 6-benzylaminopurine (BA) resulted in a higher extrusion frequency than that of 11 mg/l 2,4-D, 4.5 mg/l BA and 4.3 mg/l kinetin. Phytagel at 1 g/l resulted in the highest explant browning, but the lowest extrusion frequency, while 4 g/l Phytagel induced some dry embryogenic extrusions. Phytagel at 2 g/l was regarded as the best level for initiation of embryogenic cultures. Received: 23 December 1996 / Revision received: 22 July 1997 / Accepted: 2 September 1997     

Proliferation and maintenance of embryogenic capacity in elm embryogenic cultures

Summary This paper investigates maintenance and proliferation of somatic embryogenesis systems for Ulmus minor and U. glabra. Proliferation occurred with subculture of embryogenic calluses. The calluses were mainly formed by friable nodules composed of meristematic cells organized into proembryogenic cell masses (PEMs) and thin-walled vacuolated parenchymatic cells. Cotyledonary embryos, with procambial strands and differentiation of their vascular tissues as well as visible root meristems, were identifiable after 18d of culture on a proliferation medium with 0.44 μM benzyladenine (BA). The shoot meristem was only occasionally well developed. Somatic embryo multiplication from elm embryogenic calluses is a clearly asynchronic system, and PEMs as well as embryos at all stages of development are observed simultaneously at the end of subculture period. Factors affecting the proliferation of elm embryogenic callus, such as culture medium, carbon source and genotype, were studied. Basal medium (MS) or medium supplemented with 0.44 μM BA produced the highest number of somatic embryos. Somatic embryo production was higher with sucrose or glucose than with maltose, and significant differences were also found among the four embryogenic lines tested. The use of liquid medium with filter paper support is an essential step for the survival of isolated somatic embryos during the germination stage. The addition of 0.22 μM BA′ to liquid MS medium was the best treatment for germination and plantlet conversion of elm somatic embryos.     

Secretion of a chitinase-like protein in embryogenic suspension cultures of Dactylis glomerata L.

A chitinase-like 32 kDa acidic protein with a potential chitinase activity has been identified in the medium of embryogenic suspension cultures of Dactylis glomerata L. using an antiserum raised against endochitinase EP3 from Daucus carota L. The presence of this protein discriminates between Dactylis glomerata L. embryogenic and nonembryogenic suspension cultures and thus could be possibly used as a marker for embryogenic potential.     

Calcium increases the yield of somatic embryos in carrot embryogenic suspension cultures

An upward shift in the concentration of calcium present in the medium during somatic embryogenesis increased the number of embryos produced approximately two-fold. This was observed when embryogenic suspension cells grown in 2,4-D medium with the normal calcium concentration of 10^–3 M were transferred to hormone-free medium containing 10^–2 M calcium and when embryogenic suspension cells grown in 2,4-D medium containing 10^–4 M calcium were transferred to hormone-free medium with 10^–3 M calcium. At calcium concentrations between 6·10^–3 and 10^–2 M globular stage somatic embryos were found in cultures supplemented with 2·10^–6 M of 2,4-D indicating that elevated calcium counteracts the inhibitory effect of 2,4-D on somatic embryogenesis. No qualitative changes were found in the pattern of extracellular polypeptides as a result of growth and embryogenesis in media with different calcium concentrations.     

Genotype effects on plant regeneration in callus and suspension cultures of Avena

Regenerative potential of the calli of nineteen genotypes of Avena sativa, Avena nuda, Avena byzantina and one interspecific hybrid were compared over three successive cultures. Highly significant genotype and genotype × subculture interactions were observed. Among the highest plant regenerable genotypes were Corbit (first subculture); GAF/Park and 88Ab3073 (second subculture); and GAF/Park and 87Ab5932 (third subculture). These genotypes regenerated on an average 10 to 17 plants each from a 200 mg callus mass after a 30 to 45 proliferation period. GAF/Park, a progeny of an interspecific cross, regenerated plants at a significantly higher level (11.85 plants/rep), followed by the similarly performing A. sativa (6.23 plants) and A. nuda (5.06 plants) genotypes, which were significantly higher than the A. byzantina genotypes (2.07 plants). Four genotypes were tested for their adaptability to suspension culture and plant regeneration potential by separating their cells and cell clusters into two sizes: larger and smaller than 3 mm. Larger clusters yielded plants for three genotypes GAF/Park, 88Ab3073, and Tibor. The smaller clusters only regenerated plants for GAF/Park and 88Ab3073. From one gram of callus used to initiate suspensions of GAF/Park and 88Ab3073, 119.9 and 18.8 plants, respectively, were regenerated. The plants regenerated for various genotypes from agar-solidified or suspension culture experiments had normal growth and seed set. This study confirms high and sustained regenerative capabilities of GAF/Park, a restricted genotype due to the weedy Avena fatua genetic background and identifies alternative genotypes, especially 88Ab3073 for future tissue culture and transformation studies.     

High frequency plant regeneration from zygotic-embryo-derived embryogenic cell suspension cultures of watershield (Brasenia schreberi)

An improved protocol for high frequency plant regeneration via somatic embryogenesis from zygotic embryo-derived cell suspension cultures of watershield (Brasenia schreberi) was developed. Zygotic embryos formed pale-yellow globular structures and white friable callus at a frequency of 80% when cultured on half-strength MS medium supplemented with 0.3 mg l⁻¹ 2,4-D. However, the frequency of formation of pale-yellow globular structures and white friable callus decreased slightly with increasing concentrations of 2,4-D up to 3 mg l⁻¹, where the frequency reached ~50% of the control. Cell suspension cultures from zygotic embryo-derived white friable callus were established using half-strength MS medium supplemented with 0.3 mg l⁻¹ 2,4-D. Upon plating of cell aggregates on half-strength MS basal medium, approximately 8.3% gave rise to somatic embryos and developed into plantlets. However, the frequency of plantlet development from cell aggregates was sharply increased (by up to 55%) when activated charcoal and zeatin were applied. Regenerated plantlets were successfully transplanted to potting soil and grown to normal plants in a growth chamber. The distinctive feature of this study is the establishment of a high frequency plant regeneration system via somatic embryogenesis from zygotic embryo-derived cell suspension cultures of watershield, which has not been previously reported. The protocol for plant regeneration of watershield through somatic embryogenesis could be useful for the mass propagation and transformation of selected elite lines.     

Effect of culture period and cell density on regrowth following cryopreservation of embryogenic suspension cultures of Norway Spruce and Sitka spruce

Changes in cryotolerance, sedimented cell volume and dry weight were determined during a 21-day culture period for embryogenic suspension cultures of Norway spruce (Picea abies) and Sitka spruce (Picea sitchensis). Maximum cryotolerance was obtained for both species when the cultures were harvested in the phase of stationary growth in terms of dry weight. For P. abies, the culture period that resulted in maximum cryotolerance was similar to the culture period that resulted in maximum formation of mature embryos after 10 weeks of maturation. The initial cell density of the P. abies cultures is an important factor in affecting regrowth after cryopreservation and it was found that adjustment of the sedimented cell volume to 50% (v/v) resulted in maximum regrowth. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of gelling agents on culture medium gel strength, water availability, tissue water potential, and maturation response in embryogenic cultures of Pinus strobus L.

Summary Maturation of somatic embryos of Pinus strobus L. was evaluated on media containing various types (agars and gellan gum), brands and concentrations of gelling agents in the presence of 80 μM ABA and 0.09 M sucrose. The media were characterized with respect to gel strength, water potential and water availability. Embryogenic tissue and somatic embryos cultured on medium with various concentrations of gellan gum were used to determine their water potential (Ψ). Regardless of the type of gelling agent used, gel strength increased with gelling agent concentration and was critical to the maturation response. High gel strength was associated with reduced water availability from the medium to the cultures. The water potential of gelled maturation medium remained constant between 0.4 and 1.0% gellan gum. It is concluded that the embryogenic tissue was exposed to varying amounts of water at the onset of and during the culture period, and that the amount of water in the culture environment in turn influenced the maturation response. Cotyledonary somatic embryos derived from gellan gum medium of high gel strength had a lower Ψ than somatic embryos matured on medium of lower gel strength. Once somatic embryos developed to the cotyledonary stage on the maturation medium, they were transferred to the germination medium. The germination frequency and the number of morphologically normal germinants were higher for somatic embryos matured on medium of high gel strength. Raising the concentration of the gelling agent in the maturation medium may be an alternative to the use of solutes to restrict water available to the embryogenic cultures.     

Synthesis of extracellular proteins in embryogenic and non-embryogenic cell cultures of alfalfa

Extracellular proteins, released into the culture medium from alfalfa cells grown in embryogenic and non-embryogenic conditions, were ³⁵S-methionine labelled at different days of culture. SDS-PAGE analysis showed significant differences between the patterns of extracellular proteins secreted into the medium devoid of 2,4-d, in which cells formed somatic embryos, or in presence of 2,4-d, in which undifferentiated cell proliferation took place. Some proteins, evident in 2,4-d-supplied cultures, disappeared when cells were subcultured in the embryogenic conditions. Western analysis with antibodies against the carrot extracellular proteins EP1 and EP2 showed the presence of homologous alfalfa proteins. In 2,4-d depleted alfalfa cells, an EP1-like protein disappeared and another one was reduced, while the presence of the EP2-like protein was, in the same conditions, strongly enhanced.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - EP extracellular proteins - ns-LTP non specific lipid transfer protein - SDS-PAGE Sodium dodecyl sulphate polyacrilamide gel electrophoresis     

Interaction of plant growth regulators and organic C and N components in the formation and maturation of Abies alba somatic embryos

To promote SE maturation, the influence of different media components on different developmental stages was quantitatively evaluated. Advanced maturation was achieved with a sequence of culture media (prematuration medium and maturation medium) that contained various carbohydrates, organic nitrogen compounds and plant growth regulators. Application of lactose, BA, L-glutamine and casein hydrolysate in the prematuration medium enhanced the total number of SEs and promoted advanced differentiation. The highest number of late torpedo stage SEs was observed on maturation medium supplemented with 200 mM lactose and 29 mM sucrose. Lactose and sorbitol favoured SE maturation up to the early cotyledonary stage. With application of PEG and high ABA concentrations (20–40 M), only early torpedo stages were formed. The number of late torpedo stage SEs was significantly higher on hormone free media or with lower ABA concentrations (0–5 M). Formation of early and late cotyledonary SEs was significantly enhanced by adding BA in the maturation medium: neither Zeatin nor 2iP were effective. In addition, low sucrose concentrations in the proliferation medium (29 mM compared to 58 mM) also favoured the formation of cotyledonary SE in the maturation medium.     

Effect of bioreactor configuration on the growth and maturation of Picea sitchensis somatic embryo cultures

Somatic embryo suspension cultures of Picea sitchensis (Sitka spruce) derived from two cell lines, SS03 and SS10, were grown in shake flasks, air-lift, bubble, stirred tank and hanging stirrer bar bioreactors. Cell line SS03 yielded freely suspended and individual stage 1 embryos, while the embryos of SS10 were present in large aggregates. Compared to shake flasks, proliferation in bioreactors resulted in increased biomass; however, cell line morphology influenced the effect of different bioreactor configurations on growth and maturation of embryo cultures. Somatic embryos grown in shake flasks and bioreactors were matured on gelled solid medium and in submerged culture where gelled solid medium was covered with a layer of liquid medium. The number of stage 3 (mature) embryos produced from SS03 in the bubble bioreactor was significantly higher than those from stirred tank and hanging stirrer bar bioreactors with both solid medium and submerged culture. Submerged culture was unsuitable for SS10 embryo maturation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Multiple paternal genotypes in embryogenic tissue derived from individual immature loblolly pine seeds

Pine embryogenic tissue derived from immature zygotic embryos may consist of multiple genotypes due to simple polyembryony. To test this hypothesis, megagametophytes with intact zygotic embryos were cultured from immature loblolly pine (Pinus taeda L.) seeds of clone WV42 control pollinated with a 1:1:1 pollen mix of clones WV44, WV47, and WV48. Each pollen parent contained a marker allele at one or more of the following loci: aconitase, malic dehydrogenase, 6-phosphogluconate dehydrogenase, and shikimate dehydrogenase, allowing determination of the paternal parent. After two to four weeks in culture, embryogenic tissue derived from zygotic embryos extruded from megagametophytes was separated into individual embryos and sectors of embryogenic tissue. The paternal genotype of each resulting cell line was determined by starch gel electrophoresis. Three of thirty-six explants produced multiple cell lines with genotypic differences among the cell lines within each explant. Our results unequivocally show that it is possible to initiate embryogenic tissue from more than one zygotic embryo of a loblolly pine seed and that the resulting cell lines may be genetically different.Abbreviations ACO aconitase - MDH malic dehydrogenase - SKDH shikimate dehydrogenase - 6PGD 6-phosphogluconate dehydrogenase     

Efficient plant regeneration from protoplasts isolated from embryogenic suspension cultures of Cyclamen persicum Mill.

A protocol for plant regeneration from protoplasts has been developed, and then successfully applied to different genotypes of Cyclamen persicum Mill. Protoplasts were isolated from embryogenic suspension cultures by enzymatic digestion in 2% cellulase R10 and 0.5% macerozyme R10. Yields obtained varied between 1 and 5 × 10⁵ protoplasts per gram fresh mass depending on the genotype. Protoplasts were immobilized in alginate films, which promoted proper cell wall regeneration. The highest cell division frequencies were found in modified Kao and Michayluk (1975, Planta 126:105–110) medium containing the same types and concentrations of plant growth regulators that were applied for suspension culture (2.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid and 0.8 mg l⁻¹ 6-(γ,γ-dimethylallylamino)purine). Cell division was recorded for all 11 tested genotypes in frequencies of up to 12% and 18% after 7 and 14 days, respectively. However, cell division frequency varied strongly between different genotypes. After 4–6 weeks calluses were released from the alginate films and further cultured on hormone-containing medium for continued growth or transferred to hormone-free medium for regeneration of somatic embryos. Plant regeneration via somatic embryogenesis succeeded in 9 out of the 11 genotypes under investigation. Up to now protoplast-derived plants from four genotypes have been successfully transferred to soil.     

Effects of light on somatic embryo development and abscisic levels in carrot suspension cultures

Carrot cells were cultured under various light spectra and intensities at different times following the initiation of suspension cultures from callus. The highest intensity white and blue light treatments were inhibitory to growth and somatic embryogenesis. Red and green light were not different from dark treatments which produced the highest total number of embryoids. After extended time in culture, carrot cells in blue light produced secondary embryoids and anthocyanin. Cultures in red light had multiple cotyledons and orange-pigmented radicles. Leafy cotyledons occurred in all light treatments. Abscisic acid production peaked at the heart stage of embryogenesis and synthesis was most pronounced in blue light. Red light enhanced development to the heart stage. Both the red and blue light spectra may be used to manipulate carrot cell cultures to optimize growth.     

Induction and growth characteristics of embryogenic avocado cultures

Embryogenic cultures were induced from immature avocado zygotic embryos representing different botanical races and complex hybrids. The optimum induction medium consisted of B5 major salts, MS minor salts, 0.4 mg l⁻¹ thiamine HCl, 100 mg l⁻¹ myo-inositol, 30 g l⁻¹ sucrose, 0.41 μM picloram and 8 g l⁻¹ TC agar. Somatic embryogenesis occurred directly from the explants on induction medium, and secondary embryos and proembryonic masses proliferated in liquid and on semisolid maintenance medium. Embryogenic culture maintainance was optimized in liquid, filter-sterilized MS medium, supplemented with 30–50 mg l⁻¹ sucrose, 4 mg l⁻¹ thiamine HCl and 0.41 μM picloram. Two types of embryogenic cultures were recognized: –genotypes that proliferated as proembryonic masses in the presence of auxin (PEM-type) and; –genotypes in which the heart stage and later stages of somatic embryos developed in the presence of auxin(SE-type). Embryogenic suspension cultures became increasingly disorganized over time, and this was associated with progressive loss of embryogenic potential. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from zygotic embryo-derived embryogenic cell suspension cultures of Ranunculus kazusensis

Culture conditions for high frequency plant regeneration via somatic embryogenesis from cell suspension cultures of Ranunculus kazusensis are described. Zygotic embryos formed white nodular structures and pale-yellow calluses at a frequency of 84.9% when cultured on half-strength Schenk and Hildebrandt (SH) medium supplemented with 0.1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). However, the frequency of white nodular structure and off-white callus formation decreased with an increasing concentration of 2,4-D up to 10 mg l⁻¹, when the frequency reached 25%. Cell suspension cultures were established from zygotic embryo-derived pale-yellow calluses using half-strength SH medium supplemented with 0.1 mg l⁻¹ of 2,4-D. Upon plating onto half-strength SH basal medium, over 90% of cell aggregates gave rise to numerous somatic embryos and developed into plantlets. Regenerated plantlets were successfully transplanted to potting soil and grown to maturity at a survival rate of over 90% in a growth chamber. The plant regeneration system established in this study can be applied to mass propagation and conservation of this species.     

Explant region-specific embryogenic competence and plant recovery inCamellia japonica

Summary The culture conditions for direct, indirect, and repetitive embryogenesis were established forCamellia japonica cv. Elegans and cv. Ville de Nantes. Direct embryo production from leaves averaged 15.3 embryos per responsive leaf on Murashige and Skoog medium (MS) with 1.0 mg·liter^−1 N⁶-benzyladenine and 0.5 mg·liter^−1 2,4-dichlorophenoxyacetic acid. Plantlet production was 7.1 (±1.5) plantlets per leaf. Direct embryo production from stems averaged 5.7 embryos per shoot, and 2.7 embryos per stem portion, on MS medium supplemented with 1.0 mg·liter^−1 N⁶-benzyladenine and 0.1 mg·liter^−1 indolbutyric acid (MS28). Conversion was only obtained after repetitive embryogenesis. Embryogenesis from leaf-derived callus occurred in all callus after transfer to MS/2–25 medium (half strength MS medium with 25 g·liter^−1 D-glucose) (production stage). Plantlet production was 16.3 (±3.6) plantlets per callus. Repetitive embryogenesis increased embryo population by 2.3- to 3.6-fold every 4 wk. Conversion of secondary embryos was obtained on MS medium supplemented with 2.0 mg·liter^−1 N⁶-benzyladenine, 0.2 mg·liter^−1 indolbutyric acid, 5 mg·liter^−1 gibberellic acid (MS56). Direct embryo formation from leaves, stems, and cotyledons, and embryogenic callus formation from leaves were restricted to specific regions of the explant.