Metabolite profiling reveals clear metabolic changes during somatic embryo development of Norway spruce (Picea abies)

Progress on industrial-scale propagation of conifers by somatic embryogenesis has been hampered by the differences in developmental capabilities between cell lines, which are limiting the capture of genetic gains from breeding programs. In this study, we investigated the metabolic events occurring during somatic embryo development in Norway spruce to establish a better understanding of the fundamental metabolic events required for somatic embryo development. Three embryogenic cell lines of Norway spruce (Picea abies (L.) Karst) with different developmental capabilities were studied during somatic embryo development from proliferation of proembryogenic masses to mature somatic embryos. The three different cell lines displayed normal, aberrant and blocked somatic embryo development. Metabolite profiles from four development stages in each of the cell lines were obtained using combined gas chromatography-mass spectrometry. Multivariate discriminant analyses of the metabolic data revealed significant metabolites (P ≤ 0.05) for each development stage and transition. The results suggest that endogenous auxin and sugar signaling affects initial stages of somatic embryo development. Furthermore, the results highlight the importance of a timed stress response and the presence of stimulatory metabolites during late stages of embryo development.     

High stability of nuclear microsatellite loci during the early stages of somatic embryogenesis in Norway spruce

Somatic embryos of Norway spruce (Picea abies (L.) Karst.) differentiate from proembryogenic masses (PEMs), which are subject to autodestruction through programmed cell death. In PEMs, somatic embryo formation and activation of programmed cell death are interrelated processes. We sought to determine if activation of programmed cell death in PEMs is caused by genetic aberrations during somatic embryogenesis. Based on the finding that withdrawal of auxin and cytokinin induces programmed cell death in PEMs, 1-week-old cell suspensions were cultured in medium either with or without auxin and cytokinin and then transferred to maturation medium containing abscisic acid. We analyzed the stability of three nuclear simple sequence repeat (SSR) microsatellite markers at successive stages of somatic embryogenesis in two cell lines. There were no mutations at the SSR loci at any of the successive developmental stages from PEMs to cotyledonary embryos, irrespective of whether or not the proliferation medium in which cell suspensions had been cultured contained auxin or cytokinin. The morphologies of plants regenerated from the cultures were similar, although withdrawal of auxin and cytokinin significantly stimulated the yield of both embryos and plants. We conclude, therefore, that the high genetic stability of somatic embryos in Norway spruce is unaffected by the induction of programmed cell death caused by withdrawal of auxin and cytokinin.     

Storage lipid dynamics in somatic embryos of Norway spruce (Picea abies): histochemical and quantitative analyses

Adequate storage compounds are a prerequisite for successful development during the later stages of somatic embryogenesis; however, the critical amount of reserves below which somatic embryos fail to mature and germinate has not been determined. We analyzed storage lipids during Norway spruce (Picea abies (L.) Karst.) somatic embryogenesis. As maturation progressed, lipids, which were stored as lipid bodies in the cytoplasm, were localized first in suspensor cells of the early embryos, and later in the embryonic root pole, superficial layers of the hypocotyl and in cotyledons. The concentration of total lipids exhibited marked variation, with values peaking during cotyledon development and then decreasing during maturation. Linoleic (18:2), oleic (18:1), palmitic (16:0) and 5,9-octadecenic (5,9-18:2) acids were the most abundant fatty acids in embryos. As embryos developed, linoleic acid concentration increased slightly, whereas oleic acid concentration decreased. Oleic acid was the most prominent component of the fatty acid spectrum in isolated dormant zygotic embryos and megagametophytes. Addition of 5% polyethylene glycol to the medium during somatic embryo maturation caused a shift in the fatty acid spectrum toward that of zygotic embryos. During maturation, changes in the exogenous carbohydrate supply had no significant effect on total lipid concentration in mature embryos. A marked decrease in lipid concentration was detected during desiccation, indicating the importance of adequate lipid reserves during this developmental stage. The lipid content of zygotic embryos differed considerably with harvest year and location, suggesting that zygotic embryo data cannot be an indicator of somatic embryo quality.     

黑松合子胚和体细胞胚发育阶段的形态特征比较

为掌握黑松合子胚和体细胞胚发育进程,以未成熟合子胚为外植体,建立了黑松体细胞胚胎发生体系。对合子胚和体细胞胚发育阶段的形态特征进行观察和比较,结果表明,合子胚和体细胞胚的发育进程均可划分为8个阶段,两者同一阶段的形态特征大致相同,只有第2阶段和第4阶段有细微差别。     

miR396在落叶松体细胞胚胎中的功能研究

[目的]探讨miR396在落叶松体细胞胚胎发生中的功能.以期该研究能为解决体胚发育中子叶畸形胚与生根率低的问题提供新的视角,同时也为进一步揭示体胚发育的分子调控网络做基础铺垫.[方法]构建pre-miR396超表达载体,通过农杆菌介导侵染,将pre-miR396转入落叶松胚性细胞中,筛选稳定抗性细胞系.在DNA与RNA水平上,对抗性细胞系进行的鉴定、检测,同时统计比对其与野生细胞系间发芽率、叶片数目以及生根率的差异.[结果]在抗性细胞系基因组中,扩增出HTP与miR396的特异性片段,且无农杆菌Virg基因片段.RNA表达水平上,抗性系pre-miR396与miR396表达皆增加,而靶基因LaGRFs的表达量下调.统计表明抗性系胚的发芽率、生根率、叶片数目畸形率分别为70.60%、0.60%、37%,而野生型分别为92.50%、10.55%、4%,抗性系胚的发芽率、生根率显著降低(Sig.=0.000),叶片数目畸形率显著增加(Sig.=0.000).[结论]miR396在落叶松体细胞胚胎发生过程中,可能通过负调节靶基因LaGRFs的表达或者通过LaGRFs影响与它相互作用的基因,参与体胚子叶原基细胞代谢,从而影响了子叶与叶片的发育;通过负调节LaGRFs或者其它与根部发育相关的靶基因,影响了根端分生区细胞的活动,参与调控根的发育.     

High-efficiency somatic embryogenesis and morphohistology and histochemistry of somatic embryo development in Larix leptolepis Gordon

A high-efficiency somatic embryogenesis protocol of Japanese larch (Larix leptolepis Gordon) has been established in our investigation. Calli were induced from immature zygotic embryos of female cones of L. leptolepis and then subcultured regularly on to a modified Gupta and Durzan (DCR) basal medium for 5 years. Embryogenic tissues showed distinct morphological changes dur-ing somatic embryo development when they were transferred to a maturation medium supplemented with abscisic acid (ABA) com-pared with the morphology in a medium lacking ABA. Histological observations indicated that polyembryony was a characteristic feature during early embryogeny and somatic embryos at later stages showed normal histodifferentiation. In addition, histochemical analysis revealed that abundant starch granules and proteins accumulated in mature embryos, indicating that they played important roles in the development and regeneration of normal plantlets from somatic embryos on hormone-free germination media     

Lodgepole pine: the first evidence of seed-based somatic embryogenesis and the expression of embryogenesis marker genes in shoot bud cultures of adult trees

Of the various alternatives for cloning elite conifers, somatic embryogenesis (SE) appears to be the best option. In recent years, significant areas of lodgepole pine (Pinus contorta) forest have been devastated by the mountain pine beetle (MPB) in Western Canada. In an attempt to establish an SE propagation system for MPB-resistant lodgepole pine, several families displaying varying levels of resistance were selected for experimentation involving shoot bud and immature seed explants. In bud cultures, eight embryogenic lines were induced from 2 of 15 genotypes following various treatments. Genotype had an important influence on embryogenic culture initiation, and this effect was consistent over time. These lines were identified by microscopic observation and genetic markers. Despite the abundance of early somatic embryos, the cultures have yet to develop into mature embryos. In contrast, immature zygotic embryos (ZEs) cultured from megagametophytes initiated SE at an early dominance stage via nodule-type callus in 1 of 10 genotypes. As part of the study, putative embryogenesis-specific genes, WOX2 (WUSCHELL homeobox 2) and HAP3A, were analyzed in cultures of both shoot bud explants and ZEs. On the basis of these analyses, we postulate that PcHAP3A was expressed mainly in callus and may be involved in cell division, whereas WOX2 was expressed mainly in embryonal mass (EM)-like tissues. The findings from this study, based on molecular assessment, suggest that the cell lines derived from bud cultures were truly EM. Moreover, these experimental observations suggest that PcWOX2 could be used as an early genetic marker to discriminate embryogenic cultures from callus.     

Storage protein changes during zygotic embryogenesis in interior spruce

The major storage proteins isolated from protein bodies of embryo tissues of interior spruce Picea glauca (Moench) Voss/Picea engelmanii Parry had apparent molecular weights of 41, 35, 33, 24 and 22 kD. Minor proteins of 30 and 27.5 kD were also observed. Based on their solubility characteristics, the 41 kD protein was identified as a water and buffer-soluble albumin, and the 35, 33, 24 and 22 kD proteins were characterized as buffer-insoluble, high salt-soluble globulins. Two-dimensional electrophoresis revealed each protein was composed of several isoelectric variants. Developmentally specific accumulation of storage proteins was observed during embryogenesis. The 41 kD protein only accumulated during the later stages of cotyledon maturation, whereas the other storage proteins began to accumulate during the early stages of embryo development. All storage proteins showed major accumulations during cotyledon maturation.     

Defence reactions of developing somatic embryos of Algerian fir (Abies numidica)

Defence reactions of embryonal suspensor mass (ESM), precotyledonary, cotyledonary and desiccated cotyledonary somatic embryos of Abies numidica were tested by dual cultures with Phaeolus schweinitzii. Defence reactions were expressed at a very early stage of somatic embryo development. Both ESM and early somatic embryos inhibited mycelial growth, but the strongest defence was shown by the precotyledonary somatic embryos. The cotyledonary and desiccated cotyledonary embryos also showed defence reactions but with less intensity. Eight major components of soluble proteins, already present in the ESM, increased in concentration during subsequent developmental stages. Synthesis of two low‐molecular components of 6 and 3 kDa appeared in desiccated embryos. Probable regulation of defence reactions by auxins in early somatic embryos, as well as by abscisic acid content and storage proteins in subsequent developmental stages, is discussed.     

Selection of culture conditions for callus induction and proliferation by somatic embryogenesis of Pinus koraiensis

The induction and proliferation of embryogenic callus are key steps for large-scale propagation of somatic embryogenesis pathway and long-term preservation of coniferous germplasm.Callus can be induced from immature embryos of Korean pine(Pinus koraiensis Sieb.et Zucc.;Pinaceae) as explants,but there are problems,such as low proliferation efficiency,loss of embryogenicity,poor vigor;thus,best conditions for proliferation and culture of immature embryos of Korean pine are not yet clear.To solve the problems with somatic embryogenesis of Korean pine and determine the best culture conditions for callus induction and proliferation,we varied hormone concentration,subculture cycle of proliferation and other plant growth regulators combinations in media to induce callus formation by megagametophytes of three Korean pine families at different developmental stages,then analyzed the effects on embryogenic callus retention and cell proliferation using a quadratic regression orthogonal rotation design.The results showed that the family origin and collection date of explants significantly affected callus induction(induction rate reached1.67%).Embryogenic maintenance and callus proliferation were best on DCR medium supplemented with 0.25 mg L~(-1)6-benzyl adenine,1 mg L~(-1) naphthaleneacetic acid,30 g L~(-1)sucrose,500 mg L~(-1),L-glutamine,500 mg L~(-1) casein hydrolysis and 6.5 g L~(-1) agar.In addition,the combination of 2,4-dichlorophenoxyacetic acid+6-benzyl adenine also had a better proliferative effect on callus.The effects of different combinations of growth regulators on callus proliferation efficiency were significantly different.Transfer to new medium every 13-15 days not only maintained robust callus vigor,but also yielded a larger proliferation coefficient.The techniques and conditions for embryogenic callus induction and proliferation of Korean determined here will serve as a foundation for establishing a large-scale system for somatic embryogenesis and propagation of Korean pine.     

Initiation of somatic embryogenesis from immature zygotic embryos of oocarpa pine (Pinus oocarpa Schiede ex Schlectendal)

The focus of the current project was to establish somatic embryogenesis protocols for the tropical pine species Pinus oocarpa using immature zygotic embryos (ZEs) as explants. Somatic embryogenesis is best supported by mimicking natural seed-embryo developmental conditions, through a tissue culture medium formulation based on the mineral content of the seed nutritive tissue [megagametophyte (MG)]. A novel culture medium (P. oocarpa medium, PO) was tested in combination with different plant growth regulator (PGR) concentrations and compared with standard Pinus taeda media for the initiation of somatic embryogenesis from immature ZEs of P. oocarpa. Immature MGs containing immature ZEs of two mother trees were used with 12 and 8% extrusion rates for mother tree genotypes 3 and 5, respectively. In both mother trees the percentage capture was 2%. Multiplication of two captured cell lines (T5C2S01 and T5C1S12) was improved by lowering the concentrations of PGRs to 2.5 μM each 2,4-dichlorophenoxyacetic acid and abscisic acid (ABA) plus 1.0 μM each 6-benzylaminopurine and kinetin. Mature somatic embryos formed on 40 μM ABA, 6% (w/v) maltose, 12% (w/v) PEG 8000 and 0.6% (w/v) Phytagel. While PO medium appeared suboptimal for somatic embryo induction, it did exhibit potential for enhanced culture proliferation and subsequent improved maturation with cell line T5C2S01, where microscopic analysis revealed better embryo morphology on PO medium than on 1250 medium. However, this enhancement was not observed with cell line T5C1S12. Germination was preceded by partial desiccation for a period of 2-3 weeks before transferring the embryos to germination medium. Germination was observed after 7 days under low light, and apical primordia slowly expanded after transfer to ex vitro conditions. To our knowledge, this is the first report on the production of somatic seedlings in P. oocarpa.     

水曲柳体细胞胚与合子胚发生的细胞学研究

水曲柳(Fraxinus mandshurica)属木犀科(Oleaceae)白蜡树属,是我国东北重要珍贵硬阔树种之一,主要分布于小兴安岭、长白山、辽宁东部山地等地区, 以材质优良而著称.由于长期不合理的采伐利用,目前可利用的资源急剧减少,已被列为国家三级保护植物(傅立国,1992).进行水曲柳体细胞胚胎发生的研究,在资源保护、树种快繁和基因工程育种上有其重要的现实意义.     

Fluorescein diacetate as a viability stain for tree roots and seeds

Fluorescein diacetate (FDA) was tested as a viability stain for roots of green ash as well as for seeds of green ash and 10 other tree species. The viability level indicated by FDA staining of green ash roots agreed well with root growth potential results, bud condition assessment, and foliage browning measurements. In seed viability experiments, the FDA staining intensity of embryos was related to germination in 9 out of 11 species tested using a 30 minute stain incubation period. In the other 2 species, eastern hemlock and Scotch pine, embryo FDA staining intensity and germination were also similar, provided an 18 h stain incubation period was used. When two seedlots of differing viability were tested in each of white spruce, Douglas-fir, and pitch pine, significantly higher germination was reflected in significantly higher embryo FDA staining intensity. In Sitka spruce seed that was heat treated to produce a range of viabilities, the semilog plot of germination (log scale) and FDA staining intensity of the embryo (linear scale) had an r²= 0.95. Based on these preliminary results, FDA shows promise as a rapid viability stain for tree roots and seeds.     

Variation in Elatobium abietinum Attack on Picea glauca and its Relation to Pissodes strobi Resistance

White spruce (Picea glauca (Moench) Voss) is host to several pests, including the white pine weevil (Pissodes strobi (Peck)) and the green spruce aphid (Elatobium abietinum (Walker)). The larvae of the white pine weevil damage spruce leaders by consuming the cortex while the green spruce aphid is a defoliator. White spruce emblings (seedlings produced by culturing tissues from seed embryos) from 18 families previously ranked for resistance to the white pine weevil were defoliated to varying degrees by the green spruce aphid in a natural outbreak that developed within a holding shadehouse. A strong relationship was shown between damage caused by the aphids and weevil resistance. Emblings ranked as highly weevil - resistant sustained significantly less aphid defoliation.     

Somatic embryogenesis and histological analysis from zygotic embryos in Vitis vinifera L. ''Moldova''

We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape 'Moldova' (Vitis vinifera L. 'Moldova'). Primary calli were initiated on Nitsch and Nitsch (NN) medium supplemented with 1.0mg'L-1 2,4-D and 0.5 mg'L-1 6-BA. Embryogenic calli were produced upon transfer to a NN medium with 0.5 mg-L-1 6-BA and 2 mg.L-1 NAA and somatic embryos were obtained on a half strength MS medium without plant growth regulators. During the somatic embryo germination, an addition of 1.0 mg.L-1 6-BA in the medium could accelerate somatic embryos to develop into normal plants and increase the conversion rate from 0 to 43.3%. Histological studies of embryogenic calli and somatic embryos demonstrated dy-namic changes of proteins and starch grains. The developmental processes of somatic embryos were similar to those of zygotic em-bryos, including typical epiderma, cotyledon primordium and vascular tissue.     

Somatic embryogenesis inCephalotaxus harringtonia embryo-megagametophyte co-culture

Somatic embryogenesis was initiated fromCephalotaxus harringtonia (Forbes) K. Koch embryo culture. Explants consisted of embryo and megagametophyte halves both cut longitudinally. They were removed aseptically from mature seeds and grown together on a solid Murashige and Skoog modified medium supplemented with 5 mg·l ⁻¹ 2,4-dichlorophenoxyacetic acid. Embryogenic cultures started from callus after three or more months on the primary medium. The embryogenic callus originated from the suspensor region of the embryo. All chromosome counts made in the cells of the embryonic structures demonstrated a diploid stage, which suggest that they originated from zygotic embryo tissue. The early stages of somatic embryogenic development were achieved,i.e., formation of small clusters consisting of an embryonal region made up of isodiametric meristematic cells. A more advanced stage was reached in some cultures in which the distal embryonal end of the embryo appeared smooth and opaque. The ultrastructural characteristics of the embryos, the two types of embryo cells, embryonal and suspensor cells, as well as their contents were similar to those already reported in the case of somatic embryogenesis of other conifers.