Efficiency Of Somatic Embryogenesis Research Articles

Insights into the developmental trajectories of zygotic embryo, embryogenic callus and somatic embryo in coconut by single-cell transcriptomic analysis

Comprehensive utilization of coconut tissue culture in propagation and transformation is limited by the low efficiency of somatic embryogenesis. Understanding the potential mechanisms and developmental regulators (DRs) will facilitate the process. Here, we performed single-cell RNA sequencing (scRNA-seq) using protoplasts isolated from the zygotic embryo (ZE), embryogenic callus (EC) and somatic embryo (SE) of ‘Hainan Tall’ coconut. Cell types were identified with the assistance of known cell marker genes and further validated by RNA in situ hybridization. Differentiation trajectories from the ZE to SE transition were then constructed and identified potential DRs in the process. Notably, developmental initiation cells for both ZE and SE were identified as quiescent center-like cells (QC-L), while it was the dividing cell (DC) in EC. CnGRF12 (Growth Regulating Factor) was preferentially expressed in these developmental initiation cells, and its co-expression with CnGIF1 (GRF-interacting Factor) by transient transformation suggested that CnGRF12 has potential as a DR to accelerate transformation. Our comprehensive cell atlas provides single-cell-level insights into the developmental trajectories of ZE, EC and SE cells in coconut, and potential DRs investigated in this study could be useful for the future genotype-independent transformation and gene editing of coconut.

Sucrose-induced stress and initial days after explant excision affect the pattern and efficiency of somatic embryogenesis in the tree fern Cyathea delgadii Sternb.

Sucrose-induced stress and initial days after explant excision affect the pattern and efficiency of somatic embryogenesis in the tree fern Cyathea delgadii Sternb.

H2O2 Significantly Affects Larix kaempferi × Larix olgensis Somatic Embryogenesis.

Larch is widely distributed throughout the world and is an important species for timber supply and the extraction of industrial raw materials. In recent years, the hybrid breeding of Larix kaempferi and Larix olgensis has shown obvious heterosis in quick-growth, stress resistance and wood properties. However, its growth and development cycle is too long to meet general production needs. In order to shorten the breeding cycle, we have for the first time successfully established and optimized a somatic embryogenesis system for Larix kaempferi × Larix olgensis. We found that the highest rate of embryonal-suspensor mass (ESM) induction was observed when late cotyledonary embryos were used as explants. The induced ESMs were subjected to stable proliferation, after which abscisic acid (ABA) and polyethylene glycol (PEG) were added to successfully induce somatic embryos. Treatment with PEG and ABA was of great importance to somatic embryo formation and complemented each other's effect. ABA assisted embryo growth, whereas PEG facilitated the formation of proembryo-like structures. On top of this, we studied in more detail the relationship between redox homeostasis and the efficiency of somatic embryogenesis (frequency of ESM induction). During subculture, we observed the gradual formation of three distinct types of ESM. The Type I ESM is readily able to form somatic embryos. In contrast to type I, the type III ESM suffers from severe browning, contains a higher level of hydrogen peroxide (H2O2) and demonstrates a decreased ability to form somatic embryos. External treatment with H2O2 decreased the somatic embryogenesis efficiency of Type I and type III ESMs, or the higher the exogenous H2O2 content, the lower the resulting somatic embryogenesis efficiency. We found that treatment with the H2O2 scavenger DMTU (dimethylthiourea) could significantly increase the somatic embryogenesis efficiency of the type III ESM, as a result of a decline in endogenous H2O2 content. Overall, these findings have contributed to setting up a successful somatic embryogenesis system for larch production.

Studies on Improving the Efficiency of Somatic Embryogenesis in Grapevine (Vitis vinifera L.) and Optimising Ethyl Methanesulfonate Treatment for Mutation Induction.

Somatic embryogenesis (SE) has many applications in grapevine biotechnology including micropropagation, eradicating viral infections from infected cultivars, mass production of hypocotyl explants for micrografting, as a continuous source for haploid and doubled haploid plants, and for germplasm conservation. It is so far the only pathway for the genetic modification of grapevines through transformation. The single-cell origin of somatic embryos makes them an ideal explant for mutation breeding as the resulting mutants will be chimera-free. In the present research, two combinations of plant growth regulators and different explants from flower buds at two stages of maturity were tested in regard to the efficiency of callusing and embryo formation from the callus produced in three white grape cultivars. Also, the treatment of somatic embryos with the chemical mutagen ethyl methanesulfonate (EMS) was optimised. Medium 2339 supplemented with β-naphthoxyacetic acid (5 μM) and 6-benzylaminopurine (BAP-9.0 μM) produced significantly more calluses than medium 2337 supplemented with 2,4-dichlorophenoxyacetic acid (4.5 µM) and BAP (8.9 µM) in all explants. The calluses produced on medium 2337 were harder and more granular and produced more SEs. Although the stage of the maturity of floral bud did not have a significant effect on the callusing of the explants, calluses produced from immature floral bud explants in the premeiotic stage produced significantly more SEs than those from more mature floral buds. Overall, immature ovaries and cut floral buds exposing the cut ends of filaments, style, etc., tested for the first time in grapevine SE, produced the highest percentage of embryogenic calluses. It is much more efficient to cut the floral bud and culture than previously reported explants such as anthers, ovaries, stigmas and styles during the short flowering period when the immature flower buds are available. When the somatic embryos of the three cultivars were incubated for one hour with 0.1% EMS, their germination was reduced by 50%; an ideal treatment considered to obtain a high frequency of mutations for screening. Our research findings will facilitate more efficient SE induction in grapevines and inducing mutations for improving individual traits without altering the genetic background of the cultivar.

Comparison of somatic embryogenesis efficiency from in vitro explant sources of Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.)

This study on the efficiency of embryogenesis from various explant sources of Ngoc Linh ginseng (Panax vietnamensis Ha et Grushv.) was conducted to select the appropriate materials and optimise embryogenesis in vitro. In this study, in vitroexplants from leaf fragments, petioles, roots, and embryos (globular, heart-shaped, and cotyledon) were cultured on MS medium supplemented individually or in combination with 2,4-D (2,4-dichlorophenoxyacetic acid: 0.2, 0.5, and 0.7 mg/l) and TDZ (Thidiazuron: 0.1, 0.3, and 0.5 mg/l). The results showed that all explants had embryogenesis after 6 weeks of culture (100%). The average number of embryos per leaf fragment (51.00 embryos) on the optimal medium was significantly higher than that of petioles (29.67 embryos) and root samples (18.00 embryos). Interestingly, the globular embryo explant was noted as the most suitable material source for embryogenesis (68.33 embryos/sample). The culture medium with the combination of 2,4-D and TDZ increased the ability to create secondary embryos in globular embryos, the highest result was achieved at the concentration of 0.7 mg/l 2,4-D combined with 0.1 mg/l TDZ in the medium containing 0.5 mg/l NAA - 1-Naphthaleneacetic acid (73.33 embryos/sample).

Transcriptome Landscape Analyses of the Regulatory Network for Zygotic Embryo Development in Paeonia ostii.

Paeonia ostii is a worldwide ornamental flower and an emerging oil crop. Zyotic embryogenesis is a critical process during seed development, and it can provide a basis for improving the efficiency of somatic embryogenesis (SE). In this study, transcriptome sequencing of embryo development was performed to investigate gene expression profiling in P. ostii and identified Differentially expressed genes (DEGs) related to transcription factors, plant hormones, and antioxidant enzymes. The results indicated that IAA (Indole-3-acetic acid), GA (Gibberellin), BR (Brassinosteroid) and ETH (Ethylene) were beneficial to early embryonic morphogenesis, while CTK (Cytokinin) and ABA (Abscisic Acid) promoted embryo morphogenesis and maturation. The antioxidant enzymes' activity was the highest in early embryos and an important participant in embryo formation. The high expression of the genes encoding fatty acid desaturase was beneficial to fast oil accumulation. Representative DEGs were selected and validated using qRT-PCR. Protein-protein interaction network (PPI) was predicted, and six central node proteins, including AUX1, PIN1, ARF6, LAX3, ABCB19, PIF3, and PIF4, were screened. Our results provided new insights into the formation of embryo development and even somatic embryo development in tree peonies.

Plant-Derived Smoke and Karrikin 1 in Seed Priming and Seed Biotechnology.

Plant-derived smoke and smoke water (SW) can stimulate seed germination in numerous plants from fire-prone and fire-free areas, including cultivated plants and agricultural weeds. Smoke contains thousands of compounds; only several stimulants and inhibitors have been isolated from smoke. Among the six karrikins present in smoke, karrikin 1 (KAR1) seems to be key for the stimulating effect of smoke. The discovery and activity of highly diluted SW and KAR1 at extremely low concentrations (even at ca. 10-9 M) inducing seed germination of a wide array of horticultural and agricultural plants have created tremendous opportunities for the use of these factors in pre-sowing seed treatment through smoke- or KAR1-priming. This review presents examples of effects exerted by the two types of priming on seed germination and seedling emergence, growth, and development, as well as on the content of some compounds and enzyme activity. Seed biotechnology may involve both SW and KAR1. Some examples demonstrate that SW and/or KAR1 increased the efficiency of somatic embryogenesis, somatic embryo germination and conversion to plantlets. It is also possible to stimulate in vitro seed germination by SW, which allows to use in orchid propagation.

Calcium dynamics and modulation in carrot somatic embryogenesis.

Free calcium (Ca2+) is a pivotal player in different in vivo and in vitro morphogenic processes. In the induction of somatic embryogenesis, its role has been demonstrated in different species. In carrot, however, this role has been more controversial. In this work, we developed carrot lines expressing cameleon Ca2+ sensors. With them, Ca2+ levels and distribution in the different embryogenic structures formed during the induction and development of somatic embryos were analyzed by FRET. We also used different chemicals to modulate intracellular Ca2+ levels (CaCl2, ionophore A23187, EGTA), to inhibit calmodulin (W-7) and to inhibit callose synthesis (2-deoxy-D-glucose) at different times, principally during the first stages of embryo induction. Our results showed that high Ca2+ levels and the development of a callose layer are markers of cells induced to embryogenesis, which are the precursors of somatic embryos. Disorganized calli and embryogenic masses have different Ca2+ patterns associated to their embryogenic competence, with higher levels in embryogenic cells than in callus cells. The efficiency of somatic embryogenesis in carrot can be effectively modulated by allowing, within a range, more Ca2+ to enter the cell to act as a second messenger to trigger embryogenesis induction. Once induced, Ca2+-calmodulin signaling seems related with the transcriptional remodeling needed for embryo progression, and alterations of Ca2+ or calmodulin levels negatively affect the efficiency of the process.

Efficient somatic embryogenesis and regeneration from leaf main vein and petiole of Actinidia chinensis planch. via thin cell layer culture technology

Somatic embryogenesis (SEMs) is an effective micropropagation method that has been successfully applied to many different plant species; however, little is known about the SE in Actinidia chinensis Planch. - A species with high economic value. In this study, the efficiency of SEMs from leaf main vein and petiole of A. chinensis (seedlings germinated from seeds sterilized with 200 ppm silver nanoparticles for 7 min) via thin cell layer (TCL) technique was performed and compared with that of whole leaf main vein and whole petiole explants. The leaf main vein (mv - 1 mm × 10 mm in size) and leaf petiole (p - 1 mm × 10 mm in size) explants of in vitro A. chinensis were cut through transverse and longitudinal TCL (mv-tTCL and mv-lTCL for mv explants or p-tTCL and p-lTCL for p explants) with different sizes (½, ¼, 1/6, 1/8 of mv-lTCL or p-lTCL and ½, 1/3, ¼ of mv-tTCL or p-tTCL, respectively). Somatic embryo (SE) was recorded in the wounds of mv and p explants after 8 weeks of culture. For mv explants which cultured on MS medium supplemented with 0.02 mg/L NAA, 0.5 mg/L TDZ, 30 g/L sucrose and 8 g/L agar – MSE medium, the ½ mv-tTCL explant (mv was cut into 2 tTCL explants) gave the highest SEMs (98.67%) and number of somatic embryos per explant (10.66 embryos); meanwhile, p explants cultured on MSE medium, the ½ p-tTCL explant (p was cut into 2 tTCL explants) obtained the highest number of somatic embryos per explant (8.66 embryos) after 8 weeks of culture. In addition, the growth correction factor of SEMs was optimal at ½ mv-tTCL explant (21.04) or 1/6 p-lTCL (p was cut into 6 lTCL explants) and 1/8 p-lTCL (p was cut into 8 lTCL explants) (31.36 and 32.00). The current study has shown that the procedure of leaf main vein and petiole TCL derived- SEMs of A. chinensis is the optimal one. Large amounts of somatic embryo induction were observed from mv-TCL and p-TCL with full shapes of the globular, heart, torpedo and cotyledon. The shoot regeneration from somatic embryo with the cotyledon shape was optimal on medium containing 1.0 mg/L BA in terms of total number of shoots, shoot height and number of shoots. In addition, plantlets derived from shoot cultured on the MS medium supplemented with 0.4 mg/L NAA and planted in plastic pots with the combination of humus, coconut fiber and biofertilizers substrates (with ratio of 2:3:1 in weight) showed higher acclimatization and growth than those from other treatments in the greenhouse.

Silver nanoparticles enhanced efficiency of explant surface disinfection and somatic embryogenesis in Begonia tuberous via thin cell layer culture

In vitro culture establishment is one of the most important stages in micropropagation. The disinfectant effectiveness depends on the type of surface disinfectant, concentration and the time treatment. In this initial study, silver nanoparticles (AgNPs) were used as a disinfectant for petioles, flower stalks and stems of Begonia tuberous. In addition, thin cell layer culture (TCL) technique has been applied for the purpose of somatic embryogenesis. The results showed that AgNPs were effective in eliminating infectious microorganisms on B. tuberous explants; which were identified included 4 species of fungi (Fusarium sp., Aspergillus aculeatus, Trichoderma sp. and Penicillium sp.) and 1 species of bacteria (Pseudomonas sp.). At concentrations of 200 ppm and 300 ppm, AgNPs were not only effective in disinfection but also increased the induction rate of somatic embryogenesis in flower stalk TCL explants (approximately 40.00%); a similar effect was observed in stem TCL explants at the same concentration. Meanwhile, for petiole TCL explants, the induction rate of somatic embryogenesis was optimal when using AgNPs at a concentration of 100 - 300 ppm to disinfected the explant. In contrast, at high (400 ppm) or low (50 ppm) concentrations of AgNPs did not play a disinfecting role and stimulated somatic embryogenesis. In addition, explants derived from AgNPs sterilization did not show any abnormalities in somatic embryogenesis with shapes such as globular, heart, torpedo, and cotyledon. AgNPs showed double efficacy in sterilization of explants and improved efficiency of somatic embryogenesis from TCL petioles, flower stalks and stems explants; thus increasing the efficiency micropropagation of B. tuberous.

HTS-Based Monitoring of the Efficiency of Somatic Embryogenesis and Meristem Cultures Used for Virus Elimination in Grapevine.

Meristem culture and somatic embryogenesis are effective tools for virus elimination of vegetatively propagated crops including grapevine (Vitis vinifera L.). While both have been shown to be useful to eliminate the main grapevine viruses, their efficiency differs depending on the virus and grapevine variety. In our work, we investigated the efficiency of these two virus elimination methods using small RNA high-throughput sequencing (HTS) and RT-PCR as virus diagnostics. Field grown mother plants of four clones representing three cultivars, infected with different viruses and viroids, were selected for elimination via somatic embryogenesis (SE) and meristem culture (ME). Our results show for the first time that using SE, elimination in mother plants was effective for all viruses, i.e., grapevine rupestris vein feathering virus (GRVFV), grapevine Syrah virus 1 (GSyV-1), Grapevine virus T (GVT) and grapevine Pinot gris virus (GPGV). This study also confirms previous studies showing that SE is a possible strategy for the elimination of GFkV, GRSPaV, HSVd, and GYSVd-1. Our results demonstrate that the efficacy of virus elimination via SE is relatively high while the purging of viroids is lower. Our work provides evidence that the efficiency of SE is comparable to that of the technically difficult ME technique, and that SE will offer a more effective strategy for the production of virus-free grapevine in the future.

Different Roles of Auxins in Somatic Embryogenesis Efficiency in Two Picea Species.

The effects of auxins 2,4-D (2,4-dichlorophenoxyacetic acid), NAA (1-naphthaleneacetic acid) or picloram (4-amino-3,5,6-trichloropicolinic acid; 9 µM) and cytokinin BA (benzyloadenine; 4.5 µM) applied in the early stages of somatic embryogenesis (SE) on specific stages of SE in Picea abies and P. omorika were investigated. The highest SE initiation frequency was obtained after 2,4-D application in P. omorika (22.00%) and picloram application in P. abies (10.48%). NAA treatment significantly promoted embryogenic tissue (ET) proliferation in P. abies, while 2,4-D treatment reduced it. This reduction was related to the oxidative stress level, which was lower with the presence of NAA in the proliferation medium and higher with the presence of 2,4-D. The reduced oxidative stress level after NAA treatment suggests that hydrogen peroxide (H2O2) acts as a signalling molecule and promotes ET proliferation. NAA and picloram in the proliferation medium decreased the further production and maturation of P. omorika somatic embryos compared with that under 2,4-D. The quality of the germinated P. abies embryos and their development into plantlets depended on the auxin type and were the highest in NAA-originated embryos. These results show that different auxin types can generate different physiological responses in plant materials during SE in both spruce species.

Exogenous Spermidine Promotes Somatic Embryogenesis of Cunninghamia lanceolata by Altering the Endogenous Phytohormone Content

In order to study how exogenous hormones in C. lanceolata (gymnosperm) regulate somatic embryogenesis, we measured the endogenous phytohormones of two genotypes with different somatic embryogenesis efficiency and found that an increase in endogenous concentrations of IAA and ABA may be correlated to more efficient somatic embryogenesis. By applying exogenous spermidine, we found that exogenous hormones may affect somatic embryogenesis efficiency through affecting the endogenous phytohormone content. Based on these results, further studies can be conducted whereby the concentration of exogenous hormones or the levels of endogenous phytohormones by molecular methods are regulated to promote somatic embryogenesis. Our research may benefit the long-term economic output of the forestry industry and lays the foundation to studying the molecular mechanism that controls somatic embryogenesis efficiency.

Progress in utilizing plant-derived smoke water and smoke-derived KAR1 in plant tissue culture

Plant-derived smoke, its water extract—the smoke water (SW) and karrikinolide (KAR1) isolated from smoke or synthesized from different substrates stimulate germination of dormant and non-dormant seeds of plants from fire-prone and fire-free areas, including horticultural crops, agricultural crops and weeds. Both SW and KAR1 can be successfully used in in vitro cultures. They are able to stimulate pollen germination and growth of tubes. Growth of callus, efficiency of somatic embryogenesis, germination of somatic embryos and its conversion to plantlets are also positively affected by SW or KAR1. SW can stimulate seed germination in vitro, promote extension of primary root section and increase frequency for initiation of lateral roots. SW and KAR1 can be used as elicitors to increase production of secondary metabolites. This article describes discovery of biological activity of SW and KAR1 and provides an overview research on use of above factors in tissue and organ culture. The similarity of signaling pathways of karrikins compared to strigolactones or gibberellins is also discussed.

Composition of the Reconstituted Cell Wall in Protoplast-Derived Cells of Daucus is Affected by Phytosulfokine (PSK).

Phytosulfokine-α (PSK), a peptidyl plant growth factor, has been recognized as a promising intercellular signaling molecule involved in cellular proliferation and dedifferentiation. It was shown that PSK stimulated and enhanced cell divisions in protoplast cultures of several species leading to callus and proembryogenic mass formation. Since PSK had been shown to cause an increase in efficiency of somatic embryogenesis, it was reasonable to check the distribution of selected chemical components of the cell walls during the protoplast regeneration process. So far, especially for the carrot, a model species for in vitro cultures, it has not been specified what pectic, arabinogalactan protein (AGP) and extensin epitopes are involved in the reconstruction of the wall in protoplast-derived cells. Even less is known about the correlation between wall regeneration and the presence of PSK during the protoplast culture. Three Daucus taxa, including the cultivated carrot, were analyzed during protoplast regeneration. Several antibodies directed against wall components (anti-pectin: LM19, LM20, anti-AGP: JIM4, JIM8, JIM13 and anti-extensin: JIM12) were used. The obtained results indicate a diverse response of the used Daucus taxa to PSK in terms of protoplast-derived cell development, and diversity in the chemical composition of the cell walls in the control and the PSK-treated cultures.

Identification of an Embryonic Cell-Specific Region within the Pineapple SERK1 Promoter.

Plant tissue culture methods, such as somatic embryogenesis, are attractive alternatives to traditional breeding methods for plant propagation. However, they often suffer from limited efficiency. Somatic embryogenesis receptor kinase (SERK)1 is a marker gene of early somatic embryogenesis in several plants, including pineapple. It can be selectively induced and promotes a key step in somatic embryogenesis. We investigated the embryonic cell-specific transcriptional regulation of AcSERK1 by constructing a series of vectors carrying the GUS (Beta-glucuronidase) reporter gene under the control of different candidate cis-regulatory sequences. These vectors were transfected into both embryonic and non-embryonic callus, and three immature embryo stages and the embryonic-specific activity of the promoter fragments was analyzed. We found that the activity of the regulatory sequence of AcSERK1 lacking −983 nt ~−880 nt, which included the transcription initiation site, was significantly reduced in the embryonic callus of pineapple, accompanied by the loss of embryonic cell-specific promoter activity. Thus, this fragment is an essential functional segment with highly specific promoter activity for embryonic cells, and it is active only from the early stages of somatic embryo development to the globular embryo stage. This study lays the foundation for identifying mechanisms that enhance the efficiency of somatic embryogenesis in pineapple and other plants.

Red light promotes cotton embryogenic callus formation by influencing endogenous hormones, polyamines and antioxidative enzyme activities

Genetic transformation through somatic embryogenesis is a major tool for improving cotton’s (Gossypium hirsutum L.) development and defenses. However, the low efficiency of somatic embryogenesis seriously limits cotton’s biotechnology-based breeding and gene functional analyses. In this study, the effects of different light qualities on callus morphology and embryogenic callus induction in cotton were evaluated. Embryogenic callus formation was affected by different light regimes during the induction and expression phases. Red light treatments during the induction phase significantly promoted the formation of embryogenic calli, reaching 94.0%, and the differentiation period was reduced to 30 days compared with more than 60 days under white light conditions. Calli were of a moderate size and had moderate peroxidase, superoxidase dismutase, and catalase activity levels under red light conditions, which may maintain the balance between callus growth and differentiation. The highest total polyamine, spermidine, and spermine contents, and the lowest putrescine content, were detected during red light treatments. Additionally, endogenous auxin levels were significantly greater in red light-treated calli, and the expression levels of the somatic embryogenesis marker genes AGAMOUS-like 15, LEAFY COTYLEDON1, LEAFY COTYLEDON2, BABY BOOM, and WUSCHEL were up-regulated under red light compared with white light conditions. Thus, red light applied in the induction phase favored embryogenic callus differentiation, possibly through physiological responses to light quality. A high-efficiency cotton somatic embryogenesis protocol using red light applications will help clarify the mechanism of somatic embryogenesis.

Chemical Compositions, Somatic Embryogenesis, and Somaclonal Variation in Cumin.

This is the first report evaluating the relationship between the chemical compositions of cumin seeds (based on the analysis of the content of catalase, ascorbate peroxidase, proline, protein, terpenic compounds, alcohol/phenols, aldehydes, and epoxides) and the induction efficiency of somatic embryogenesis in two Iranian superior cumin landraces (Golestan and North Khorasan). Cotyledons isolated from Golestan landrace seeds cultivated on MS medium supplemented with 0.1 mg/L kinetin proved to be the best primary explant for the induction of somatic embryogenesis as well as the regeneration of the whole plantlet. Results indicated that different developmental stages of somatic embryos were simultaneously observed on a callus with embryogenic potential. The high content of catalase, ascorbate peroxidase, proline, and terpenic hydrocarbons and low content of alcoholic and phenolic compositions had a stimulatory effect on somatic embryogenesis. Band patterns of RAPD markers in regenerated plants were different from those of the mother plants. This may be related to somaclonal variations or pollination system of cumin. Generally, measurement of chemical compositions can be used as a marker for evaluating the occurrence of somatic embryogenesis in cumin. Also, somaclonal variations of regenerated plants can be applied by the plant breeders in breeding programs.

Factors affecting somatic embryogenesis in eight Italian grapevine cultivars and the genetic stability of embryo-derived regenerants as assessed by molecular markers

Embryogenic cultures have been used in cryopreservation, genetic transformation, propagation, virus elimination, induced mutagenesis and in many other biotechnological applications, providing excellent opportunities for biotechnology advances in grapevine. Unfortunately the efficiency of somatic embryogenesis (SE) is genotype-dependent in addition to showing interaction with explant type used and the plant growth regulator (PGR) composition. In order to identify the interaction of these parameters in SE, we tested eight wine grapevine cultivars, three explant types (ovary, anther/filament and stigma/style) and four PGR combinations in a statistically designed experiment. The genotype was the major determining factor, with embryogenic response varying from 0.1 to 5.1% (about 50-fold difference). For PGR composition of the medium, embryogenesis ranged between 0.5 and 3.3% (a ≈7-fold difference). The explant type was the least important factor with embryogenesis ranging between 0.8% (anther/filament) and 2.3% (ovary)—only a ≈3-fold change. Anther/filament, that had generally been considered to be the most promising explant, surprisingly gave the lowest embryogenesis percentage. Genetic homogeneity of plants developed from in vitro SE was assessed in comparison to mother plants using six inter-simple sequence repeat and ten random amplified polymorphic DNA primers that produced reproducible and clear bands ranging from 150 to 3500bp. The amplification products were monomorphic across all the regenerated plants and their respective mother plants confirming the genetic homogeneity of the regenerants and demonstrating the suitability of SE for in vitro grapevine germplasm conservation and propagation.

Influence of seed provenance on the propagation of Picea abies (L.) Karst

Picea abies (L.) Karst. is the most common species cultivated in Italy for sale as Christmas trees. The aim of this study was to identify the best seed provenance to improve plant production. Three Italian provenances - Gran Bosco di Salbertrand, Pezzel e Fochino, and Val di Fiemme - were examined. A part of seeds were sown on benches in a greenhouse after cold stratification, and another part was used for induction of somatic embryogenesis. In the first experiment, two different amounts of organic matter (manure), as components of growing media, were evaluated, while in the second one different levels and combinations of growth regulators in the medium were tested. The seed provenance and composition of the growing medium influenced seed germination and seedling growth. The best performance on both growing media was achieved by the Val di Fiemme provenance, and in general seedling emergence and growth were the most favourable in the growing medium with a lower organic matter amount. Also, the in vitro cultures evidenced differences in the efficiency of somatic embryogenesis among the provenances.