| A protocol for somatic embryogenesis and development has been carried out,using young leaves and petioles from flowering plants as explants in Cyclamen persicum Mill. Another rapid and high-frequency somatic embryogenesis system has also been successfully achieved from globular embryo-explants. And an embryogenic suspension culture is further established by inoculating globular embryos into liquid medium. Meanwhile expression pattern of Cyclamen somatic embryogenesis receptor-like kinase (CpSERK) is investigated during acquisition of embryogenic competence of somatic cell from liquid and solid culture systems.The results show that embryogenic calli have been successfully induced and identified. The auxin 2,4-D (2-4mg.L-1) and 2iP (0.2-0.8mg.L-1) in half strength MS medium are important factors for the induction of somatic embryogenesis. The optimal sub-cultural media for embryogenic callus are supplemented with 1/2MS +2,4-D2mg.L-1 + 6-BA0.2mg.L-1 or 1/2MS+2,4-D2mg.L-1 + 2iP0.2mg.L-1. According to the color, friability, watery grades and morphological character et al., calli are classified and respectively proliferated after 3-4 sub-cultural periods on induction medium. Two types of embryogenic callus are verified and obtained, which one is white-brown and soft, and the other is similar to the former except for friability. Embryogenic competence of both calli can be retained for more than one year.Somatic embryos start to appear on the surface of both embryogenic calli on the same medium without growth regulators for about 14 days. Somatic embryos occur in clusters from soft embryogenic callus, and several scores of embryoids are observed in one cluster. However, somatic embryos from friable embryogenic callus occur in clusters in more cases, or occasionally in individual way, with a few or more than ten embryoids in one cluster. Somatic embryos developed asynchronously in the same cluster, containing different developmental stage of embryoids, such as globular embryos, heart-shaped embryos, torpedo-shaped embryos and even mature embryos. Majority of somatic embryos can be regenerated into whole plantlets for 60-90 days. Normal embryo frequency is 34.7% from embryogenic calli after 7-8 periods of subculture. Embryogenic competence and normal embryo frequency have decreased remarkably as subculture prolongs, being only below 7% of normal embryo frequency for more than one year.The observations of viable cells show that both embryogenic and non-embryogenic calli comprise different sizes and shapes of cells and cell aggregates. The number of embryogenic and non-embryogenic cells is obviously different in the two types of embryogenic calli. The soft embryogenic callus mostly comprises aggregates with more than several scores of embryogenic cells, but the friable embryogenic callus contains aggregates with several or more than ten embryogenic cells. And the number of embryogenic aggregates in the former callus is more than that of the latter callus in the same random scope. Embryogenic aggregates are derived from mother embryogenic cells by time-lapse track of viable cell. Embryogenic aggregates with several or more than ten cells are originated from an embryogenic mother cell by continuous division, but embryogenic aggregates with more than several scores of embryogenic cells could be from a few of mother embryogenic cells in groups.Histological and cytological analysis during somatic embryogenesis of cyclamen show that somatic embryo is originated from the single embryogenic cell, being developed into a whole plantlet through different stages, such as two-cell proembryo, four-cell proembryo, eight-cell proembryo, multicellular proembryo, globular, heart-shaped, torpedo-shaped embryo. The two pathways have been observed during cyclamen somatic embryo development and plant regeneration. The first pathway is that a whole plantlet is regenerated through different developmental stages, such as embryogenic cell, multicellular proembryos, globular embryo, heart-shaped embryo and torpedo-shaped embryo. The second pathway is that a tuber is firstly formed from globular embryo without torpedo-shaped embryo, then roots or buds appear, finally a whole plantlet is regenerated.Dynamic changes of starch metabolism are investigated during the development and regeneration of somatic embryos. Four accumulation peaks of starch grains respectively appear at the stage of embryogenic cells, globular embryos, the early torpedo-shaped embryos and the developing tuber of a plantlet, which may be closely related to somatic embryogenesis, development and plant regeneration for energy andsubstance. Embryogenic calli are successfully induced after 25-28 days from regenerated somatic embryos-explants on the above sub-cultural media(1/2MS+2,4-D 2mg.L-1 +6-BA 0.2 mg.L-1 and 1/2MS+2,4-D 2 mg.L-1 +2ip 0.2 mg.L-1). Primary callus percentages come to above 95%, except for small globular embryo-explants, using different types of somatic embryos regenerated from leaf-explants as explants. The initial calli have embryogenic capacity, which is optimal in embryogenic callus from large globular embryo-explants, secondly from small non-polar tuber-explants and small globular embryo-explants. The number of somatic embryos from the above three embryo-explants is respectively 734/g.FW, 423/g.FW and 342/g.FW and that of embryogenic calli is 118, 80 and 69 from single embryo-explants on free-growth-regulators medium for 30 days. Embryogenic competence shows significant difference among individuals from the same type of embryo-explants.Effects of sub-cultural periods on embryogenic competence are studied as large globular embryo explants. The results show that sub-cultural periods have no effects on embryogenesis percentage, but significant effects on embryogenic competence and normal somatic embryo frequency on the-free-growth-regulator medium for 50 days. Calli for the first sub-cultural period show the best embryogenic competence, and the somatic embryogenesis percentage, the total number of somatic embryos of per gram callus, the total number of somatic embryos from single embryo-explants and the frequency of normal somatic embryos are respectively 100%, 3304/g.FW, 1296/ g.FW and 8.54%.Effects of different concentration of sucrose (0, 10, 30, 60, 90, 120g.L-1) and ABA(0, 0.2, 0.5, 1.0, 2.0mg.L-1))have been evaluated on late development of somatic embryos. The frequency of normal somatic embryo has remarkably increased on the 1/2MS media supplemented with appropriate concentration of ABA. Higher concentration of ABA can clearly inhibit somatic embryogenesis, and the average number is only 292/g.FW on the medium with ABA 2.0 mg.L-1. ABA (0.5 mg.L-1) is the most effective for development of somatic embryos, which the average number is 648/g.FW and normal embryo frequency is 12.65%. The normal embryo frequency (4.09%)on the control medium was low as compared to the frequency of 9.92–12.65% on experimental medium supplemented with ABA. Appropriate concentration supplement of sucrose (30-60 g.L-1) is also helpful to somatic embryogenesis and development. The total number of somatic embryos and the normal embryo frequency are optimal on the medium with 60 g.L-1 sucrose, respectively 1384/ g.FW and 8.24%. Development of somatic embryos can be inhibited on the medium with less than 10g.L-1 or more than 120 g.L-1 of sucrose.Embryogenic suspension culture has been successfully established, using regenerated globular embryos as starting materials inoculated directly in liquid medium. Globular embryo-explants are dedifferentiated into embryogenic mother cells after initial suspension culture, from which embryogenic cell aggregates are derived for 2-3 periods of subculture. The optimal inoculated density is 5% PCV (packed cell volume) and the optimal period of subculture is 14 days in the suspension system. Somatic embryos have developed on solid or liquid media without growth regulators, and embryogenic competence in suspension culture can be retained for more than a year. The developmental pathway of somatic embryos belongs to the second pathway in liquid culture, but both developmental pathways occur on solid medium. It is further verified that yellow cells aggregates have completely lost embryogenic competence with better proliferation capacity.Specific expression of the related genes with somatic embryogenesis and development is investigated using the above different embryogenic systems as experimental materials. Two pairs of specific primers of SERK genes (SKF1/SKR1 或SKF2/SKR2)are designed using Primer Premier 5.0 software, according to the cDNA conserved sequences of cyclamen SERK genes which are BLAST searched against the GenBank database at the National Centre for Biotechnology Information (NCBI). Two putative SERK gene fragments are cloned and named CpSERKa and CpSERKb, respectively. The 464bp fragment is CpSERKa, coding 154 amino acids and the 768bp fragment is CpSERKb, coding 255 amino acids. The two CpSERK fragments are distinct but similar at both nucleotide level (89.2% similarity) and protein level (90.9% similarity) within the overlapped segment. The results from GenBank BLAST search and multiple sequence comparison reveal that CpSERKa is most similar to the SERK homologue CpSERK3 in the same species, with the similarities of 98.5% at nucleotide levels and 98.1% at protein levels. And CpSERKa is the partial sequence of CpSERK3, but CpSERKb is the other member of the same family. The results are confirmed by the phylogenic analysis in which CpSERKa, CpSERKb and CpSERK3 are tightly grouped together adjacent to AtSERK3. CpSERKa and CpSERKb are also highly homologous to SERK genes in Daucus carota, Solanum tuberosum and some other species.Expression of both CpSERKa and CpSERKb has successfully detected in tissues and cells of embryogenic competence from different embryogenic systems, but not in non-embryogenic callus and cell aggregates of losing embryogenic competence. The results show that SERK genes are also expressed in multicellular proembryos and globular embryos. The slight expression of CpSERKa is detected, but CpSERKb is not expressed at the stage of torpedo-shaped embryos.
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