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1.
The aim of the study was to develop an effective cryopreservation method for Scots pine (Pinus sylvestris L.) embryogenic
cultures. Altogether nine cell lines derived from three mother trees were cryopreserved after cold hardening using dimethylsulfoxide
or two different mixtures of polyethyleneglycol 6000, glucose and dimethylsulfoxide as cryoprotectants. Seventy-eight percent
of the cell lines remained viable after cryostorage, the best cryoprotectant treatment being 10% polyethyleneglycol 6000,
10% glucose, and 10% dimethylsulfoxide in water. This treatment resulted in significantly better regrowth of the embryogenic
cultures than with the other cryoprotectants or with the controls. According to microscopical observations, the cells that
retained their viability and regrowth ability after cryopreservation were the embryonal head cells, as well as some elliptic
suspensor cells close to the embryonal head cell area. When proliferation growth of the frozen cultures had started, their
morphological appearance was the same as the non-frozen cultures. In addition, the RAPD assays suggested that the cryostorage
treatment used here preserved the genetic fidelity of the Scots pine embryogenic cultures.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
2.
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. 相似文献
3.
Summary The usefulness of random amplified polymorphic DNA (RAPD) in assessing the genetic stability of somatic embryogenesis-derived populations of black spruce [ Picea mariana (Mill.) B.S.P.] was evaluated. Three arbitrary 11-mer primers were successfully used to amplify DNA from both in-vivo and in-vitro material. Twenty-five embryogenic cell lines, additional zygotic embryos and megagametophytes from three controlled crosses involving four selected genotypes of black spruce were used for the segregation analysis of RAPD variants. Ten markers were genetically characterized and used to evaluate the genetic stability of somatic embryos derived from three embryogenic cell lines (one cell line per cross, 30 somatic embryos per cell line). No variation was detected within clones. The utilization of RAPD markers both for the assessment of genetic stability of clonal materials and to certify genetic stability throughout the process of somatic embryogenesis is discussed. 相似文献
4.
Genetic controls for growth of embryogenic cultures, storage, maturation treatments, germination and cryopreservation in white spruce somatic embryogenesis (SE) were examined. These SE processes were under genetic control but less strongly so than the initiation phase. For all the SE characters examined, variance due to clones within families was significant and often the largest genetic component of variance. This was further partitioned using an additive-dominance-epistasis model. A relatively-large proportion of the total genetic variance was due to epistatic variance in the maturation and germination of somatic embryos. Embryogenic lines were cryopreserved easily without a distinct genetic influence being noticed. 相似文献
5.
Morphogenesis and maturation of somatic embryos, ploidy, and genotyping of cell lines (CL) of embryogenic cultures of Larix sibirica Ledeb. in vitro were investigated during 2–6 years. It was revealed that from 2000 to 11103 globular somatic embryos were formed in proliferating CL. However, the ability of somatic embryos to the maturation and germination decreased. Cytogenetic study of embryonal-suspensor masses (ESM) of Larix sibirica demonstrated that cells of long-term cultivated cultures remained diploid. According to microsatellite analysis, proliferating CL of Siberian larch were characterized by weak allelic variability, and cell line 6 and cloned seedlings of this line were genetically stable and corresponded to the donor tree. Embryogenic cell lines composed the collection bank, which will be successfully used for plantation forest growing. 相似文献
6.
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 0.2 M 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. 相似文献
7.
Somatic embryogenesis of Abies alba (Mill.) has significant potential to become an effective method for vegetative propagation of this species. To induce somatic embryogenesis in A. alba, the influence of the mother tree, sampling dates, and cold treatment storage of cones were examined. The initiation frequencies ranged from 1.7% to 16.6%. The sampling date and cone storage, but not the mother tree, had a significant effect on the initiation of embryogenic cultures. Storage of embryogenic cell lines was tested through cryopreservation for 6 yr. Four out of 12 cryostored embryogenic cell lines recovered, and the regeneration of cotyledonary embryos was obtained with two cell lines. The ability of embryogenic cell masses to produce somatic embryos and the mean number of cotyledonary embryos were higher when the maturation protocol was based on embryogenic suspensions dispersed on filter paper. The properly developed germinants were obtained only from maturation media where 32 μM abscisic acid was used, being 16.2% when polyethylene glycol (PEG) was not present and 1.8% when supplemented with 10% ( w/ v) PEG, respectively. The present study provides evidence that it is possible to cryopreserve A. alba embryogenic cultures while maintaining their maturing ability for the lengthy period (6 yr) needed for progeny testing of field-grown trees. Therefore, our findings are important for further studies and advanced breeding work of the species; however, the conversion of germinants into ex vitro conditions still remains a significant challenge. 相似文献
8.
The present study was conducted to improve the transition from proliferation to maturation in embryogenic cultures of Nordmanns fir. For that reason, chemicals reported to affect endogenous levels or activity of auxin were included in the growth media during maturation. The auxin antagonist PCIB reduced proliferation and promoted the development of numerous high-quality mature embryos in the tested cell lines. PCIB could not substitute for exogenously supplied ABA and the positive effect was only found when PCIB and ABA were used in combination. The effect of PCIB was dependent on the concentration and the application period. The auxin transport inhibitor TIBA also reduced proliferation, but had no positive effect on maturation. The auxin synergist phloroglucinol had the opposite effect of PCIB; proliferation was increased and no maturation was initiated. A lowered concentration of boron had no effect on proliferation but had some positive effect on maturation. The optimum protocol for PCIB application was strongly genotype dependent, and a general scheme that covered the tested cell lines could not be found. Overexposure to PCIB during maturation caused abnormal development of the mature embryos, which was revealed by a reduced number of cotyledons. These results suggest that endogenously produced auxin may be one reason for low or failing maturation of embryogenic cultures of Nordmanns fir, but also imply that auxin may play a critical role for proper development of cotyledons during the later stages of embryo maturation. 相似文献
9.
Experiments were performed to determine the influence of maturation medium carbohydrate content on the rates of germination and plantlet conversion (root and shoot growth) of somatic embryos from four embryogenic lines derived from leaf or internode explants of Quercus robur L. seedlings. The conversion rate was favoured by high carbohydrate content as long as the maturation medium contained at least 2% sucrose, which was necessary for healthy embryo development. Given this, sorbitol and mannitol favoured the conversion rate more efficiently than sucrose, the highest rate, 32%, being achieved by medium with 6% sorbitol and 3% sucrose. Maturation treatment did not affect the root or shoot lengths of converted embryos. In supplementary experiments, 2 weeks of gibberellic acid treatment between maturation and germination treatments did not improve germination rates, but did reduce root length and the number of leaves per regenerated plantlet. In the four embryogenic lines tested, plant recovery rate was enhanced by inclusion of benzyladenine into the germination medium following culture of the embryos on maturation medium with 6% sorbitol and 2-3% sucrose. In embryogenic systems it is important to assess the uniformity of the regenerants. Random amplified polymorphic DNA (RAPD) analysis using 32 arbitrary oligonucleotide primers was performed to study variability in DNA sequences within and between four embryogenic lines. No intraclonal nor interclonal polymorphism was detected between embryogenic lines originating from different types of explant from the same seedling, but every one of the primers detected enough polymorphism among clones originating from different plants to allow these three origins to be distinguished. No differences in DNA sequences between regenerated plantlets and their somatic embryos of origin were detected, but a nodular callus line that had lost its embryogenic capacity was found to be mutant with respect to three other clones originating from the same plantlet. This study shows that high carbohydrate levels in the maturation medium significantly increase plant conversion of oak somatic embryos, which exhibit no variation in DNA sequences when proliferated by secondary embryogenesis. 相似文献
10.
Platycladus orientalis is a widespread conifer, which is native in eastern Asia, and has recently attracted much attention due to its ornamental value for landscape and gardens. However, native P. orientalis populations have been in decline over the past century. Here, we established an in vitro propagation and cryopreservation system for P. orientalis via somatic embryogenesis (SE). Whole megagametophytes with four development stages (Early embryogeny: E1 and late embryogeny: L1, L2, and L3) of zygotic embryos from immature P. orientalis cones were used as initial explants and cultured on three different basal media such as initiation medium (IM), Litvay (LV), and Schenk and Hildebrandt (SH). Both the developmental stage of zygotic embryos and kind of basal medium had a significant effect on embryogenesis induction with IM ( P?<?0.001, respectively). The highest frequency of embryogenic callus induction was obtained in megagametophytes with zygotic embryos at L2 stage, which ranged as high as 30%. The maturation medium containing IM basal salts, vitamins and amino acids, 15 g l ?1 abscisic acid (ABA), 50 g l ?1 maltose, and 100 g l ?1 polyethylene glycol 4000 (PEG) was found to be the suitable medium for production of somatic embryos. The frequency of somatic embryo formation from both non-cryopreserved and cryopreserved cell lines was also tested. There were no statistical differences on the production of somatic embryos between non-cryopreserved and cryopreserved cells ( P?=?0.523). Genetic fidelity of the plantlets regenerated from non-cryopreserved and cryopreserved embryogenic cell lines was assessed by both random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis. There was no genetic instability in the regenerated plantlets from cryopreserved embryogenic cell lines. Both the SE protocol and cryopreservation protocols described here have the potential to contribute the conservation and clonal propagation of P. orientalis germplasm. 相似文献
11.
An approach combining morphological profiling and flow cytometric analysis was used to assess genetic stability during the several steps of somatic embryogenesis in Pinus pinaster. Embryogenic cell lines of P. pinaster were established from immature zygotic embryos excised from seeds obtained from open-pollinated trees. During the maturation stage, phenotype of somatic embryos was characterized as being either normal or abnormal. Based upon the prevalent morphological traits, different types of abnormal embryos underwent further classification and quantification. Nuclear DNA content of maritime pine using the zygotic embryos was estimated to be 57.04 pg/2C, using propidium iodide flow cytometry. According to the same methodology, no significant differences ( P ≤ 0.01) in DNA ploidy were detected among the most frequently observed abnormal phenotypes, embryogenic cell lines, zygotic and normal somatic embryos, and somatic embryogenesis-derived plantlets. Although the differences in DNA ploidy level do not exclude the occurrence of a low level of aneuploidy, the results obtained point to the absence of major changes in ploidy level during the somatic embryogenesis process of this economically important species. Therefore, our primary goal of true-to-typeness was assured at this level. 相似文献
12.
The aim of the present work was to study the effect of the developmental stage of the somatic embryos and of the genotype
on the genetic transformation of embryogenic lines of European chestnut ( Castanea sativa Mill.) and the cryopreservation of the embryogenic lines that are generated. As an initial source of explants in the transformation
experiments, it was found that the use of somatic embryos isolated in the globular stage or clumps of 2–3 embryos in globular/heart-shaped
stages was more effective (30%) than when embryos at the cotyledonary stage were used (6.7%). All of the seven genotypes tested
were transformed, and transformation efficiency was clearly genotype dependent. Three transgenic lines were successfully cryopreserved
using the vitrification procedure, and the stable integration of the uidA gene into the transgenic chestnut plants that were regenerated subsequent to cryopreservation was demonstrated. 相似文献
13.
Hardwood species are valuable biological resources that have an important role in the economy and ecology of ecosystems worldwide. Non-zygotic or somatic embryogenesis (SE) is a powerful tool in plant biotechnology as it is a form of clonal propagation, amenable to cryopreservation of valuable germplasm and genetic transformation including gene editing. The SE process involves five steps and includes somatic embryo induction, proliferation, maturation, plantlet conversion, and subsequent plant acclimatization. This review aims to provide a general overview of these steps in different SE systems developed for hardwood species. Factors that influence the induction stage such as the age of the donor plant, genotype and culture media are discussed. The role of different explant types, i.e. zygotic embryos and non-zygotic tissues, such as roots, flower tissues, nodes, internodes, leaves or shoot apices, in SE induction are especially emphasized. Histological studies of the origin of somatic embryos and the sequence of events leading to their development from initial explants are assessed. Maintenance of embryogenic capacity carried out by subculture of embryogenic inocula on semisolid or liquid media through cell suspension cultures or by temporary immersion systems is described. At present, the main concerns associated with the application of SE for large-scale propagation of elite hardwoods are related to the embryo maturation, germination, and plantlet conversion steps, and these are highlighted in this review. Finally, molecular aspects associated with somatic embryo induction and development are also described. Attempts to overcome the hurdles identified in the embryogenic process, and future lines of research are proposed. 相似文献
14.
Exogenous spermidine (Spd) and the ectomycorrhizal (ECM) fungus Pisolithus tinctorius (Pers.) Coker and Couch had a synergistic effect on the maturation of Scots pine (Pinus sylvestris L.) somatic embryos. Induced maturation was expressed as a higher number of cell masses able to form embryos and a greater number of embryos formed per cell mass. In contrast, treatment with P. tinctorius alone on the hormone-free medium resulted in the lowest embryo-forming capacity. Retarded proliferation growth appeared to be required for maturation, but did not explain the synergistic effect of the fungus and exogenous Spd. Simultaneous treatment did not result in lower concentrations of putrescine (Put), Spd or spermine (Spm) in the embryogenic cell masses relative to the separate treatments. Our study is the first report on the use of a specific ECM fungus to induce maturation of somatic embryos, and it indicates that P. tinctorius was able to modify the maturation media in a way that, together with exogenous Spd, positively affected embryogenic cultures of Scots pine. Our study also shows that it is possible to enhance plant development other than root formation by using specific ECM fungi. 相似文献
15.
The aim of this research was to establish a long-term somatic embryogenic cultures that could be used for cryopreservation.
For the induction of somatic embryogenesis, different levels of 2,4-D as well as the combination of 2,4-D and indole-3-acetyl- l-aspartic acid (IASP) were tested on cotyledons of zygotic embryos. The somatic embryogenic cultures were established and
maintained up to 2 years through frequent subculturing on a medium containing 2,4D + IASP. Light, activated charcoal, and
polyethylene glycol (PEG) were tested for the regeneration and maturation of somatic embryos, and the mature embryos were
germinated in JADS medium. The combination of light and PEG provided the highest number of mature embryos. The somatic embryos
obtained were smaller than zygotic embryos and lacked starch. There was an interaction between 2,4-D and IASP on the induction
and regeneration of somatic embryo in Myrciaria aureana. The combination of light and PEG increased the number of mature embryos; however, charcoal was detrimental to the process. 相似文献
16.
The effects of ectomycorrhizal (ECM) fungi, including Laccaria
bicolor (Maire) Orton, Laccaria laccata (Scop., Fr.) Berk. and Br., along with two strains of Pisolithus tinctorius (Pers.) Coker and Couch, on the proliferation and subsequent maturation of two embryogenic cell lines of Abies cephalonica Loud., designated lines 6 and 8, were investigated. In the presence of these ECM fungi, the proliferation of both embryogenic
cell lines was inhibited. L. bicolor and P. tinctorius strain 2 resulted in the highest inhibition rates. On the other hand, cultivation of embryogenic cultures along with ECM
fungi, termed a dual culture, increased radial growth of both P. tinctorius strains; whereas, L. bicolor and L. laccata did not grow as well in the presence of embryogenic cell masses. The dual culture during the proliferation period of embryogenic
cells, however, enhanced the subsequent embryo formation and maturation of A. cephalonica; i.e. the capability of embryogenic cell lines to form somatic embryos as well as increasing the mean number of somatic embryos
per 1 g fresh weight of embryogenic cell mass. However, levels of responses were highly dependent on the interaction between
the specific embryogenic cell line and fungal strain. 相似文献
17.
Strategies for genetic improvement programs of Pinus pinea L, an important tree species of the Mediterranean ecosystem, are focused on increasing pine nut yield. Somatic embryogenesis and cryopreservation of elite genotypes are emerging as key components of advanced forest breeding programs. This study was carried out with embryogenic lines of different ages obtained from selected half-sib families of the species. The effect of three cryoprotectant procedures on the recovery and maturation ability was tested in embryogenic lines that showed different growth rate, two of them at different ages. In general, cryopreservation drastically reduced growth rates of frozen and rewarmed tissues; however, the use of 5% PEG–sucrose–DMSO dramatically increased growth rates of rewarmed embryogenic cultures. Overall, embryogenic lines of stone pine were suitable for cryopreservation. Seven out of eight lines were recovered, although the initial growth rates were variable. Five of six lines including the three oldest ones were recovered using 5% PEG–sucrose–DMSO. No relation was observed between age and growth rate of embryogenic lines and their response to cryopreservation. The line 2F47 showed the most stable response after long-term subculture and recovery after cryopreservation, at different ages. On the contrary, younger embryogenic lines either recovered after cryopreservation or did not, depending on the applied procedure. Maturation of some of the older lines was restored or enhanced after cryopreservation. Somatic embryos were obtained in three out of five tested embryogenic lines recovered from cryopreservation. However, only a few plantlets from cryopreserved lines were regenerated indicating the process must be optimized further before it is a practical adjunct to breeding. 相似文献
18.
Trees were regenerated from six white spruce embryogenic clones after cryopreservation for 3 and 4 years, respectively. Genetic
stability was evaluated using randomly amplified polymorphic DNA (RAPD) fingerprints. Somaclonal variation was detected in
some in vitro embryogenic cultures 2 and 12 months after they were re-established following cryopreservation but not in the corresponding
regenerated trees. These results suggest that trees regenerated from cryopreserved cultures in subsequent years are primarily
genetically stable in the genomic regions tested and that variation observed due to the in vitro culture process infrequently affects trees regenerated from normally maturing and germinating somatic embryos. However, trees
regenerated from somatic embryos that matured or germinated abnormally in in vitro culture exhibited altered RAPD fragment patterns.
Received: 20 July 1998 / Revision received: 15 October 1998 / Accepted: 14 December 1998 相似文献
20.
Summary The present review summarizes the factors involved in controlling the process of oak somatic embryogenesis as a method for
vegetative plant propagation and includes also data on artificial seed production, cryopreservation and transformation. One
major limitation, the inability to initiate embryogenic cultures from mature trees, has been recently overcome. Leaves from
selected cork oak trees with an age of 50 yr and more have been used to initiale somatic embryogenesis (SE) with a frequency
of up to 20%. These findings offer encouraging prospects for cloning proven superior plant material and to integrate this
propagation system into tree improvement programs. Once the process of SE has been initiated, the multiplication cycle proceeds
via secondary embryogenesis, which can be maintained indefinitely. Problems are reported by the formation of anomalous embryos.
The mutability of somatic embryogenic cell lines of various oak species has been monitored by flow cytometry and molecular
markers. No somaclonal variation was detected applying random amplified polymorphic DNA (RAPD) or amplified fragment length
polymorphism (AFLP) markers, whereas DNA-content measurements via flow cytometry revealed tetraploidy in some cell lines after
several years of continuous subculture. Maturation and low germination frequencies are the main bottlenecks for a broader
use of this technique. Recently attention has been on embryo quality and parameters for conversion capacity such as high endogenous
cytokinin level and low abscisic acid (ABA) level. Although oak is probably the species that is the most well-developed system
for a broadleaved forest tree, data on growth performances of somatic embryo-derived plants are rare. 相似文献
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