Morphogenic and genetic stability in longterm embryogenic cultures and somatic embryos of Norway spruce (Picea abies {L.} Karst)

Embryogenic cultures were initiated from mature zygotic embryos of Picea abies. The somatic embryos in the embryogenic cultures were first stimulated to mature and then either to develop further into plantlets or to differentiate new embryogenic cultures. The procedure was repeated three times during two years. The ability to give rise to new embryogenic cultures or to develop into plantlets was similar for all somatic embryos irrespective of how long they had been cultured in vitro. The nuclear DNA content, measured in a flow cytometer, was estimated at 32 pg/G1 nuclei in seedings developed from zygotic embryos. Nuclei isolated from embryogenic cultures and from plantlets regenerated from somatic embryos had the same DNA content as those isolated from seedlings.Abbreviations N⁶-benzyladenine BA - 2,4-dichlorophenoxyacetic acid 2,4-D - abscisic acid ABA     

Long-term cryopreservation of Greek fir embryogenic cell lines: recovery, maturation and genetic fidelity

In coniferous species, including Greek fir (Abies cephalonica Loud), the involvement of somatic embryo plants in breeding and reforestation programs is dependent on the success of long-term cryostorage of embryogenic cultures during clonal field testing. In the present study on Greek fir, we assayed the recovery, morphological characteristics and genetic fidelity of embryogenic cell lines 6 and 8 during proliferation and maturation after long-term cryostorage. Our results indicate successful recovery of both cell lines after 6 years in cryostorage. In the maturation phase, both cell lines were capable of producing somatic embryos although some differences were detected among experiments. However, these changes were more dependent on the differences in the components of the maturation media or in the experimental set-up than on the long-term cryostorage. During both proliferation and maturation phases, the morphological fidelity of the embryogenic cultures as well as of the somatic embryos were alike before and after cryopreservation. The genetic fidelity of the cryopreserved cell line 6 that was assayed by random amplified polymorphic DNA (i.e. RAPD) markers demonstrated some changes in the RAPD profiles. The results indicate possible genetic aberrations caused by long-term cryopreservation or somaclonal variation during the proliferation stage. However, in spite of these changes the embryogenic cultures did not lose their proliferation or maturation abilities.     

In vitro propagation and cryopreservation of Thuja koraiensis Nakai via somatic embryogenesis

Korean arbor vitae (KAV; Thuja koraiensis Nakai) is a critically endangered coniferous tree in Korea. Here, we report the somatic embryogenesis (SE) and cryopreservation system that can be used for micropropagation of KAV and long-term storage of KAV cultures. To induce SE in KAV, the influence of the developmental stage of zygotic embryos and the effect of basal medium on embryogenesis induction were examined. The developmental stage of zygotic embryos had a significant effect on the embryogenesis induction (P < 0.0001). The highest frequency of embryogenesis induction occurred in megagametophytes with zygotic embryos at precotyledonary (P) and late embryogeny (L1) stage (36%). The highest frequency of embryogenesis induction was obtained on initiation medium containing IM basal salts with 2.2 μM 6-benzylaminopurine and 4.5 μM 2,4-dichlorophenoxyacetic acid (35%). The effect of abscisic acid (ABA) on production of somatic embryos was tested. The highest number of somatic embryos per 50 mg of embryogenic tissue was achieved on maturation medium with levels of 100 μM ABA (24.0 ± 2.4). The effect of cryopreservation treatment to embryogenic tissues on the maturation capacity of somatic embryos was also tested. No significant differences between noncryopreservation and cryopreservation treatment were observed (P = 0.1896), and the highest mean number of somatic embryo per 50 mg of embryogenic tissues was obtained in noncryopreserved cell line (28.17 ± 5.66). Finally, the genetic identities of the plantlets regenerated from non- and cryopreserved embryogenic cell lines were verified and there was no genetic variation in the regenerated plantlets from cryostored embryogenic cell lines. This study is the first report on SE and the successful cryopreservation of embryogenic culture of the genus Thuja.

     

Mitochondrial DMA variation in somatic embryogenic cultures ofLarix

outhern hybridization analysis using wheat mitochondrial gene-specific probes indicates that changes in mitochondrial genomic organization and the relative representation of certain genomic regions occur during in vitro somatic embryogenic cell culture ofLarix species. We observed differences in the mitochondrial (mt)DNA hybridization patterns between somatic embryogenic cell cultures and trees grown from seed forLarix leptolepis,L. decidua, and the reciprocal hybrids of these twoLarix species. This is the first study to describe the correlation of molecular changes in a gymnosperm mitochondrial genome with in vitro somatic embryogenic cell culture. Quantitative differences in mtDNA hybridization signals were also observed among a 4-year-old somatic embryogenic cell culture ofLarix ×eurolepis trees regenerated from this culture, and the seed source tree from which the somatic embryogenic cell cultures were initiated.     

AFLP analysis of variation in pecan somatic embryos

Before somatic embryogenesis can be applied, the genetic fidelity of cultures needs to be determined. Problematic of tissue-cultured woody species is the extensive evaluation time needed for assessments. The development of methods whereby plants could be rapidly screened for potential tissue culture-derived genetic changes would be very valuable. We evaluated the applicability of AFLP (amplified fragment length polymorphism) analysis for the assessment of genetic variability in somatic embryos of pecan [Carya illinoinensis (Wangenh.) C. Koch] and made comparisons between and within embryogenic culture lines. AFLP readily detected differences between culture lines, with 368 polymorphic loci identified. Individual culture lines generally produced somatic embryos with similar overall banding patterns. Embryos derived from the same culture line generally grouped together in a phenogram generated by UPGMA (unweighted pair-group method, arithmetic average) analysis. However, a few somatic embryos exhibited higher levels of polymorphism and failed to group with others regenerated from the same line. The relation between the detected within-line differences and their contribution to phenotypic variation is yet to be determined. Received: 9 September 1998 / Revision received: 12 November 1998 / Accepted: 8 December 1998     

Somatic embryogenesis and plant regeneration from immature embryo cultures of onion (Allium cepa L.)

Somatic embryos were obtained and plants regenerated from immature embryos of onion following culture on embryogenic induction media. Highest rates of somatic embrogenesis resulted from 0.5- to 1.5-mm immature embryos cultured on media containing 5 mg/l of picloram. Somatic embryos formed either directly on the surface of embryos or developed from compact cultures. The production of somatic embryos was significantly affected by the addition of auxin, embryo size and cultivar. The potential of somatic embryogenic cultures for plantlet regeneration has been maintained for over 1 year in some lines. Three types of immature-embryo-derived cultures were characterized by histology. Some cultures were morphologically similar to immature-embryo-derived embryogenic cultures of other monocotyledonous species. Cultures such as these have proven to be useful target tissues in transformation studies. Received: 16 December 1997 / Revision received: 23 February 1998 / Accepted: 13 March 1998     

大豆体细胞胚系培养及其微卫星DNA稳定性研究(简报)

植物离体培养是植物基因操作中的重要一环,也是植物个体发育研究中基因表达研究的有益参考体系。在继代培养过程中发生的遗传变异有时会使再生植株丧失优良的性状而需加以控制和避免。为此,首先需要了解培养过程中的遗传变异情况。     

A key to totipotency: Wuschel-like homeobox 2a unlocks embryogenic culture response in maize (Zea mays L.)

The ability of plant somatic cells to dedifferentiate, form somatic embryos and regenerate whole plants in vitro has been harnessed for both clonal propagation and as a key component of plant genetic engineering systems. Embryogenic culture response is significantly limited, however, by plant genotype in most species. This impedes advancements in both plant transformation-based functional genomics research and crop improvement efforts. We utilized natural variation among maize inbred lines to genetically map somatic embryo generation potential in tissue culture and identify candidate genes underlying totipotency. Using a series of maize lines derived from crosses involving the culturable parent A188 and the non-responsive parent B73, we identified a region on chromosome 3 associated with embryogenic culture response and focused on three candidate genes within the region based on genetic position and expression pattern. Two candidate genes showed no effect when ectopically expressed in B73, but the gene Wox2a was found to induce somatic embryogenesis and embryogenic callus proliferation. Transgenic B73 cells with strong constitutive expression of the B73 and A188 coding sequences of Wox2a were found to produce somatic embryos at similar frequencies, demonstrating that sufficient expression of either allele could rescue the embryogenic culture phenotype. Transgenic B73 plants were regenerated from the somatic embryos without chemical selection and no pleiotropic effects were observed in the Wox2a overexpression lines in the regenerated T0 plants or in the two independent events which produced T1 progeny. In addition to linking natural variation in tissue culture response to Wox2a, our data support the utility of Wox2a in enabling transformation of recalcitrant genotypes.     

The Use of Single Somatic Embryo Culture in Propagating and Regenerating Lucerne (Medicago sativa L.)

Embryogenic cultures of lucerne (Medicago sativa L.) cv. Robothave been established and propagated on medium containing yeastextract. These cultures consisted of unorganized callus tissuebearing embryogenic centres which increased in size during subculture,yielding new regenerated somatic embryos at the end of each20-d subculture. A development in the propagation of the embryogenic cultureswas the establishment of single embryo culture in hormone-freemedium where, in selected cases, the process of recurrent somaticembryogenesis (RSE) took place on the hypocotyl of explantedembryos. The process was independent of supporting callus tissueand occurred on simple defined medium. Single embryos underwenteither plantlet development or continued RSE on the hypocotyl.One third of the regenerated plantlets showed RSE after thetwo to three trifoliate leaf stage. In these cases shoot developmentstopped and only somatic embryo production took place. In vitrocloning of regenerated plantlets allowed us to reproduce eachparticular genotype before transplantation into soil. Lucerne (alfalfa), Medicago sativa L., somatic embryogenesis, single embryo culture     

Cryopreservation and plant regeneration via somatic embryogenesis using shoot apical domes of mature Pinus roxburghii sarg, trees

Summary The present investigation reports optimized parameters for somatic embryogenesis and cryopreservation of embryogenic cultures using shoot apical domes from mature trees of Pinus roxburghii Sarg. Embryogenic tissue of P. roxburghii Sarg. was cryopreserved for 24 h, 10 d, and 8 wk using sorbitol and dimethylsulfoxide (DMSO) as cryoprotectants. Results indicate that 0.2M sorbitol and 5% DMSO had the best cryoprotecting effect. The recovered tissue showed luxuriant growth on maintenance medium (II). Partial desiccation of thawed embryogenic tissue for 24 h prior to transfer to maturation medium enhanced the maturation of somatic embryos. Maturation frequency increased from 1.3 to 18.3% after 12 h desiccation treatment, and from 18.3 to 61.8% after 24 h of desiccation. However, non-desiccated embryogenic tissue produced the least number of somatic embryos (1.3%) on the maturation medium with the same abscisic acid and Gellan gum concentration. All the three embryogenic lines produced plantlets and had the same appearance and normal growth as compared to unfrozen controls.     

Recovery of somatic embryos and plantlets from protoplast cultures of Larix × eurolepis

Summary Somatic embryos and plantlets were regenerated from protoplasts of hybrid larch (Larix × eurolepis) isolated from two embryogenic callus and cell suspension culture lines (L1 and L2). L2, which was highly embryogenic, consistently yielded protoplasts that gave rise to somatic embryos. Centrifugation on a discontinuous medium/Percoll density gradient resulted in accumulation of embryogenic protoplasts in one of the Percoll interfaces. First division frequencies were in the range of 28–39% in line 1 and 18–20% in line 2 in both liquid and agarose-solidified culture media. The critical factor in maintaining high viability of cultures was lowering of osmotic pressure by dilution of the initial medium. The first somatic embryos were detected in 23- to 28-day-old cultures. Some of these developed into plants that were transferred to soil.     

Efficient and stable regeneration from protoplasts of <Emphasis Type="Italic">Cyclamen coum</Emphasis> Miller via somatic embryogenesis

Embryogenic cultures of Cyclamen coum were established on solid media and in suspension, and their growth characteristics in response to different concentrations of plant growth regulators (PGRs) were evaluated. Embryogenic cultures exhibited a high regeneration capacity of 876 somatic embryos per gram fresh mass. Up to 4.24 × 10⁵ protoplasts per gram of fresh mass were isolated from somatic embryos and embryogenic suspension cultures. Protoplasts derived from both embryos and suspension cultures were successfully cultured in vitro and regenerated into plants via somatic embryogenesis. Phenotypic analyses and flow cytometric measurements revealed that some regenerated plants were tetraploid. About 20% of the protoplast-derived calluses used for regeneration were tetraploid, while tetraploidy was found in 0.9% of the plants regenerated from the embryogenic cultures.     

Evaluation of somaclonal variation during somatic embryogenesis of interior spruce (Picea glauca engelmannii complex) using culture morphology and isozyme analysis

Somaclonal variation during interior spruce (Picea glauca engelmannii complex) somatic embryogenesis was evaluated using culture morphology and isozyme analysis. Genotype-specific abscisic acid-dependent developmental profiles and isozyme patterns were similar for subclone and parent line embryogenic cultures and cotyledonary somatic embryos. Extensive analysis of fifteen hundred subclone embryos of one genotype revealed no isozyme pattern variation. Initiation of embryogenic cultures was dependent on the developmental stage of the explant although cultures derived from different stages were morphologically similar. The embryogenic cultures initiated from interior spruce embryos show a high degree of genetic stability in that the morphological behavior and isozyme phenotype were always consistent with that of the explant genotype. These results support the conclusion that this culture system is appropriate for clonal propagation of interior spruce.     

Occurrence of chromosomal variations and plant regeneration from long-term-cultured citrus callus

Summary Embryogenic callus of Anliucheng sweet orange (Citrus sinensis Osbeck) is theoretically diploid. However, significant chromosomal variations occurred when the calluses were subcultured and preserved for a long time. Cytological observation revealed a variety of mitotic irregularities underlying the occurrence of chromosomal variations. Despite the ubiquitous existence of chromosomal variations, long-term-cultured calluses were still capable of producing somatic embryos and plants. Interestingly, chromosomal variants were selected against when somatic embryos and plants regenerated from the embryogenic callus. Randomly amplified polymorphic DNA (RAPD) analysis was also carried out to detect DNA sequence variation in regenerated plants derived from the embryogenic callus. No difference in banding patterns was detected. It was clear that the plant regeneration from long-term-cultured callus was inclined to select against somaclonal variations.     

Somatic embryogenesis from stem and leaf explants of Quercus robur L.

Internodal and leaf segments from pedunculate oak (Quercus robur L.) seedlings were used as explant source to induce somatic embryogenesis. Auxin treatment influenced embryogenic response, which only occurred in explants initially cultured on media containing 4 mg/l naphthaleneacetic acid (NAA) and different benzyladenine (BA) concentrations. After 6 weeks of culture on induction medium, the explants were transferred to medium supplemented with 0.1 mg/l BA and 0.1 mg/l NAA, and 4 weeks later, they were subcultured in a growth-regulator-free medium, in which somatic embryos arose through indirect regeneration on the surface of a nodular callus. Somatic embryos were induced in explants of two out of four seedling provenances. The induction frequency ranged from 16% in leaf explants to 4% in internodal explants. Somatic embryos developed two cotyledons, which were translucent or opaque-white in appearance, but anomalous morphologies were also observed. Different embryogenic lines were established and maintained by repetitive embryogenesis in multiplication medium containing 0.1 mg/l BA plus 0.05 mg/l NAA. These results indicate that tissues from explants other than Q. robur zygotic embryos are able to produce embryogenic cultures. Received: 14 July 1998 / Revision received: 2 November 1998 / Accepted: 6 November 1998     

Evidence for in vitro induced mutation which improves somatic embryogenesis in Asparagus officinalis L.

Summary Somatic embryogenesis from different genotypes of Asparagus officinalis L. could be obtained by in vitro culture of shoot apices. Apices were first cultured on an auxin-rich inducing medium and then transferred onto a hormone-free development medium. All genotypes tested in this way produced a few somatic embryos. In some experiments, during the development phase, a new kind of friable highly embryogenic tissue appeared in a random manner. These tissues could be continuously subcultured on a hormone-free medium and were named embryogenic lines. Five of these embryogenic lines regenerated plants from somatic embryos. These regenerated plants exhibited an increased embryogenic response compared to the parent plants; e.g. apex culture produced somatic embryos without any auxin treatments. For one of the embryogenic lines, a genetic analysis showed that the improved embryogenic response of regenerated plants was controlled by a mendelian dominant monogenic mutation.Abbreviations LSEA low somatic embryogenesis ability - HSEA high somatic embryogenesis ability - NAA 1-naphthaleneacetic acid     

Screening of large numbers of seed families of Pinus strobus L. for somatic embryogenesis from immature and mature zygotic embryos

The somatic embryogenic process was investigated using immature and mature zygotic embryos originating from 13 open-pollinated seed families selected for their embryogenic capacity from over 128 seed families of Pinus strobus. In a first step, intact megagametophytes with precotyledonary embryos from these families were placed on modified Litvay medium. Embryogenic tissues (ETs) were obtained for 12 of them, with initiation rates varying from 2.6% to 23%. On average, 14% of the ETs (36/258) formed stable embryogenic cell lines (ECLs) after 4–6 months of subculture. Mature somatic embryos were produced for 30 out of 52 cell lines, and plants were regenerated. Later, initiation of ETs from mature zygotic embryos was also tested for the selected families. ECLs were obtained for five of them, with a maximum initiation rate of 2.7%, and plants were produced for four ECLs. Received: 19 March 1998 / Revision received: 22 May 1998 / Accepted: 5 June 1998     

Characterization of somatic embryogenesis in Pelargonium××hortorum mediated by a bacterium

Several cultivars of hybrid seed geranium (Pelargonium×hortorum Bailey), previously shown to be recalcitrant in culture, produced somatic embryos at high frequency when explants were co-cultivated with a morphogenesis promoting bacterium. This bacterium was isolated as an in vitro contaminant from cultures of geranium seedling explants and identified as belonging to the genus Bacillus and species circulans. Co-cultivation of hypocotyl explants with the bacterium promoted somatic embryo formation and improved both the frequency and quality of somatic embryos. In the cultivar Ringo Rose, the least responsive among the cultivars screened, the embryogenic response was more than four times that of axenic cultures. Nearly 70% of these embryos converted into plantlets, while the somatic embryos induced under axenic conditions developed poorly and plantlet formation was inconsistent. Among the different treatments of bacterial culture tested (autoclaved culture, culture filtrate, sonicated bacterial culture, sonication of bacterial culture followed by filtration, HPLC fractionation of crude bacterial lysate), only two HPLC fractions promoted embryogenesis to a marginal degree. Co-cultivation of the explants with bacterium during the first week of induction was crucial for obtaining high-frequency embryogenesis, indicating the role of bacterial stimuli during the induction process. Received: 23 June 1998 / Revision received: 20 August 1998 / Accepted: 27 October 1998     

Characterization of embryogenic mango cultures selected for resistance to Colletotrichum gloeosporioides culture filtrate and phytotoxin

 Embryogenic nucellar cultures of two polyembryonic mango cultivars, ‘Hindi’ and ‘Carabao’, were selected for resistance to the culture filtrate and phytotoxin of a virulent strain of Colletotrichum gloeosporioides Penz. that was isolated from mango leaves. The cultures were recurrently selected either with progressively increasing concentrations of culture filtrate or by continuous challenge with the same concentration of either culture filtrate phytotoxin. Mycelium growth was inhibited when the pathogen was cocultured with the selected, resistant embryogenic cultures. Conditioned plant growth medium containing macerated resistant embryogenic cultures did not inhibit mycelium growth, confirming that extracellular antifungal compounds were involved in the defense response. Enhanced secretion of chitinase and glucanase was observed in the plant growth medium in which resistant embryogenic cultures and regenerated somatic embryos were grown in comparison with the controls. Received: 6 February 1997 / Accepted: 4 November 1997     

Genome fidelity during short- and long-term tissue culture and differentially cryostored meristems of silver birch (<Emphasis Type="Italic">Betula pendula</Emphasis>)

Clonal trueness of micropropagated or cryopreserved material is essential, especially with long-living tree species. In this study, the growth rate and morphology of regenerated silver birch (Betula pendula Roth) plants growing in the nursery were evaluated after different treatments: short-term (14 months) and long-term (70 months) tissue culture periods, cryostorage of in vivo buds and cryopreservation of in vitro shoot apices using four different slow cooling cryopreservation protocols with PGD (10% PEG, 10% glucose, 10% DMSO) as cryoprotectant. Genetic fidelity of the regenerated plants compared to the original donor trees was evaluated using RAPD assays together with chromosome analysis. The regenerated plants showed no genetic or phenotypic changes, and can thus be considered as reliable material for any research, breeding or silvicultural activities.