Growth Of Somatic Embryos Research Articles

High-Efficiency Somatic Embryogenesis from Seedlings of Koelreuteria paniculata Laxm.

Research Highlights: In the current study, we established a method for plant regeneration via somatic embryogenesis (SE) in Koelreuteria paniculata Laxm. for the first time. Background and Objectives: K. paniculata is an important ornamental and medicinal plant in China. However, the plant has difficulty with asexual reproduction, which imposes a limitation on large-scale propagation. Materials and Methods: Embryogenic calluses were induced from stems of aseptic seedlings on induction media. The effects of different media types and concentrations of N6-benzyladenine (BA), α-naphthaleneacetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-D) on callus induction were examined. Embryogenic calluses were then transferred to Driver-Kuniyuki Walnut (DKW) media containing NAA (0.1–0.2 mg L−1) or 2,4-D (0.5–2.0 mg L−1) to develop somatic embryos. Cotyledon embryos were cultured on DKW media containing NAA (0.1–0.2 mg L−1) until maturation, and were then transferred to 1/2 DKW medium supplemented with 1.0 mg L−1 indole-3-butyric acid (IBA) to produce complete plants. The effects of IBA and NAA on rhizogenesis were then examined by clonal culture. Results: The maximum callus induction frequency (80.25%) was obtained on DKW medium supplemented by 0.5 mg L−1 BA, 0.25 mg L−1 NAA, and 1.5 mg L−1 2,4-D. NAA had a more pronounced effect on somatic embryo growth than did 2,4-D, with a maximum SE frequency (54.75%) observed with 0.1 mg L−1 NAA added to DKW medium. For clonal culture, the highest rooting rate (52%) was observed on 1/4 DKW medium containing 1.5 mg L−1 IBA. Histology studies confirmed the presence of embryogenic calluses and somatic embryos in different stages. Conclusions: This protocol provides a novel method for large-scale propagation of K. paniculata, and creates opportunities for genetic engineering in this species.

Overexpression of VpPR10.1 by an efficient transformation method enhances downy mildew resistance in V. vinifera.

Putrescine and spermidine increase the transformation efficiency of Vitis vinifera L. cv. Thompson seedless. Accumulation of VpPR10.1 in transgenic V. vinifera Thompson seedless, likely increases its resistance to downy mildew. A more efficient method is described for facilitating Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless somatic embryogenesis using polyamines (PAs). The efficacies of putrescine, spermidine and spermine are identified at a range of concentrations (10µM, 100µM and 1mM) added to the culture medium during somatic embryo growth. Putrescine (PUT) and spermidine (SPD) promote the recovery of proembryonic masses (PEM) and the development of somatic embryos (SE) after co-cultivation. Judging from the importance of the time-frame in genetic transformation, PAs added at the co-cultivation stage have a stronger effect than delayed selection treatments, which are superior to antibiotic treatments in the selection stage. Best embryogenic responses are with 1mM PUT and 100µM SPD added to the co-culture medium. Using the above method, a pathogenesis-related gene (VpPR10.1) from Chinese wild Vitis pseudoreticulata was transferred into Thompson Seedless for functional evaluation. The transgenic line, confirmed by western blot analysis, was inoculated with Plasmopara viticola to test for downy mildew resistance. Based on observed restrictions of hyphal growth and increases in H2O2 accumulation in the transgenic plants, the accumulation of VpPR10.1 likely enhanced the transgenic plants resistance to downy mildew.

Stimulation of somatic embryo growth and development in Picea spp. by polyethylene glycol

Stimulation of somatic embryo growth and development in Picea spp. by polyethylene glycol

The Effect of Gibberellin on Somatic Embryo Growth and Maturation and Plantlet Regeneration of Tangerine (Citrus reticulata Blanco.) var. Batu 55

The effect of gibberellin at multiplication stage on somatic embryo growth and maturation and plantlet regeneration of tangerine ( Citrus reticulata Blanco.) var. Batu 55 was assessed. Somatic embryo at globular phase was cultured on MT media + 30 gL -1 sucrose and various concentrations of gibberellin (0, 2, 4, 6, and 8 ppm). The somatic embryo was maturated on MT media + 500 ppm malt extract + 73 mM sorbitol + 73 mM galactose. Cotyledonary somatic embryo was regenerated into plantlet on MT media + 500 ppm malt extract + 30 gL -1 sucrose + 2 ppm GA 3 . The results showed that the addition of gibberellin in somatic embryo multiplication stage increased somatic embryo growth and maturation and plantlet regeneration of tangerine. Optimum concentration of gibberellin needed for somatic embryo growth was 4 ppm which yielded two-fold fresh weight compared to control. The percentage of maturation was very low below 5%. The addition of gibberellin in media at embryo multiplication stage slightly increased the percentage of maturation about 1-2%. Thirty percent of cotyledonary embryo was able to regenerate into plantlet. The addition of gibberellin in media at embryo multiplication stage increased the regeneration percentage, even the addition of 8 ppm gibberelline yielded regeneration percentage up to 70%. Keywords: gibberellin, growth, maturation, plantlet regeneration, somatic embryo

Conifer somatic embryogenesis: improvements by supplementation of medium with oxidation-reduction agents.

A major barrier to the commercialization of somatic embryogenesis technology in loblolly pine (Pinus taeda L.) is recalcitrance of some high-value crosses to initiate embryogenic tissue (ET) and continue early-stage somatic embryo growth. Developing initiation and multiplication media that resemble the seed environment has been shown to decrease this recalcitrance. Glutathione (GSH), glutathione disulfide (GSSG), ascorbic acid and dehydroascorbate analyses were performed weekly throughout the sequence of seed development for female gametophyte and zygotic embryo tissues to determine physiological concentrations. Major differences in stage-specific oxidation-reduction (redox) agents were observed. A simple bioassay was used to evaluate potential growth-promotion of natural and inorganic redox agents added to early-stage somatic embryo growth medium. Compounds showing statistically significant increases in early-stage embryo growth were then tested for the ability to increase initiation of loblolly pine. Low-cost reducing agents sodium dithionite and sodium thiosulfate increased ET initiation for loblolly pine and Douglas fir (Mirb) Franco. Germination medium supplementation with GSSG increased somatic embryo germination. Early-stage somatic embryos grown on medium with or without sodium thiosulfate did not differ in GSH or GSSG content, suggesting that sodium thiosulfate-mediated growth stimulation does not involve GSH or GSSG. We have developed information demonstrating that alteration of the redox environment in vitro can improve ET initiation, early-stage embryo development and somatic embryo germination in loblolly pine.

Pine somatic embryogenesis: analyses of seed tissue and medium to improve protocol development

The shift from vegetative to embryogenic growth requires tissue to enter a radically different program of development and can be studied in vitro through the development of somatic embryos. From an applied perspective somatic embryogenesis (SE) is expected to play an important role in increasing productivity, sustainability, and uniformity of future forests. For commercial use, SE technology must work with a variety of genetically diverse trees. Since the first reports of SE in Picea abies and Larix decidua in 1985, many different coniferous species have shown the ability to produce embryogenic tissue. However, initiation frequency is often low, many desired seed sources are recalcitrant, and culture survival is often poor, raising costs of somatic seedlings produced from successful genotypes. A number of tools are now available to improve embryogenic tissue initiation and somatic embryo development in vitro that have resulted from analytical studies of seed tissues, the seed environment and gene expression in megagametophyte, zygotic embryos and somatic embryos. Benefits have occurred from medium supplementation with hormones, plant growth regulators, hormone inhibitors and polyamines. Somatic embryo growth has been enhanced with medium supplementation of nutritional components including specific sugar types, vitamins, organic acids, and redox potential modifiers. Control of environmental factors including, water potential, pH, adsorption of medium components by activated carbon and liquid versus gelled medium have also led to SE protocol improvements. The use of analytical studies to duplicate the seed environment in vitro is improving protocol development resulting in increased initiation, improved yields and higher-quality somatic embryos.

Changes in ATP, glucose-6-phosphate and NAD(P)H cellular levels during the proliferation and maturation phases of Abies alba Mill. embryogenic cultures

The aim of the present study was to evaluate the adenosine triphospate (ATP), glucose-6-phosphate (glu-6P) and reduced form of nicotinamide adenine dinucleotide phosphate (NAD(P)H) cellular levels during the proliferation and maturation phases of Abies alba Mill. somatic embryos. For a better understanding of the dynamics of these parameters during the proliferation cycle, four embryonic cell lines were tested. During the maturation period, three independent experiments were conducted, focused on the effects of PEG-4000 (5 or 10% (w/v)) and abscisic acid (16, 32 or 64 μM) applied together (Experiments A and B) or with addition of gibberellic acid (Experiment C) on the dynamics of bio-energetic molecules and on the mean number of cotyledonary somatic embryos. Our results demonstrated that the cellular levels of bio-energetic molecules strongly depended on the composition of maturation media. Generally, the higher the number of cotyledonary embryos produced, the higher the level of ATP observed after a 2-week maturation period. The cellular level of ATP, glu-6P and NAD(P)H increased, particularly after the transition from the proliferation to the maturation phase when the differentiation and growth of somatic embryos occurred.

Changes in sugar levels during slow growth of Dendrocalamus hamiltonii somatic embryos due to liquid paraffin overlay

A liquid paraffin overlay (LPO) was used for storage of rapidly multiplying somatic embryos of Dendrocalamus hamiltonii under slow-growth conditions. Slow growth was associated with changes in sugar metabolism. In rapidly growing embryogenic tissues, a sharp decline in starch and non-reducing sugars indicated rapid utilization of starch. In contrast, under slow-growth conditions in somatic embryos stored under LPO, a gradual decline in starch indicated its slower utilization. As a result, growth of somatic embryos under LPO was suppressed and subculturing was not required. Following retrieval from growth under LPO after 30, 90, 180, 270, and 365 d of storage, the somatic embryos showed high recovery and germination (79.78%, 77.49%, 71.22%, 67.13%, and 59.99%, respectively) and were able to proliferate following transfer to Murashige and Skoog’s (Physiol. Plant. 15:473–497, 1962) medium containing 1 mgl−1 BAP and 2% sucrose. The study provides useful information on in vitro storage of embryogenic tissue of D. hamiltonii.

Myo‐inositol hexakisphosphate, isolated from female gametophyte tissue of loblolly pine, inhibits growth of early‐stage somatic embryos

• Myo-inositol hexakisphosphate (InsP(6)), abundant in animals and plants, is well known for its anticancer activity. However, many aspects of InsP(6) function in plants remain undefined. We now report the first evidence that InsP(6) can inhibit cellular proliferation in plants under growth conditions where phosphorus is not limited. • A highly anionic molecule inhibitory to early-stage somatic embryo growth of loblolly pine (LP) was purified chromatographically from late-stage LP female gametophytes (FGs), and then characterized structurally using mass spectrometry (MS) and nuclear magnetic resonance (NMR) analyses. • Exact mass and mass spectrometry-mass spectrometry (MS-MS) fragmentation identified the bioactive molecule as an inositol hexakisphosphate. It was then identified as the myo-isomer (i.e. InsP(6)) on the basis of (1)H-, (31)P- and (13)C-NMR, (1)H-(1)H correlation spectroscopy (COSY), (1)H-(31)P heteronuclear single quantum correlation (HSQC) and (1)H-(13)C HSQC. Topical application of InsP(6) to early-stage somatic embryos indeed inhibits embryonic growth. • Recently evidence has begun to emerge that InsP(6) may also play a regulatory role in plant cells. We anticipate that our findings will help to stimulate additional investigations aimed at elucidating the roles of inositol phosphates in cellular growth and development in plants.

HIGH FREQUENCY PLANT REGENERATION VIA IN VITRO SOMATIC EMBRYOGENESIS IN FIFTEEN CULTIVARS OF DIMOCARPUS LONGAN LOUR.

ISHS III International Symposium on Longan, Lychee, and other Fruit Trees in Sapindaceae Family HIGH FREQUENCY PLANT REGENERATION VIA IN VITRO SOMATIC EMBRYOGENESIS IN FIFTEEN CULTIVARS OF DIMOCARPUS LONGAN LOUR.

Non-antibiotic selection systems for soybean somatic embryos: the lysine analog aminoethyl-cysteine as a selection agent.

BackgroundIn soybean somatic embryo transformation, the standard selection agent currently used is hygromycin. It may be preferable to avoid use of antibiotic resistance genes in foods. The objective of these experiments was to develop a selection system for producing transgenic soybean somatic embryos without the use of antibiotics such as hygromycin.ResultsWhen tested against different alternate selection agents our studies show that 0.16 μg/mL glufosinate, 40 mg/L isopropylamine-glyphosate, 0.5 mg/mL (S-(2 aminoethyl)-L-cysteine) (AEC) and the acetolactate synthase (ALS) inhibitors Exceed® and Synchrony® both at 150 μg/mL inhibited soybean somatic embryo growth. Even at the concentration of 2 mg/mL, lysine+threonine (LT) were poor selection agents. The use of AEC may be preferable since it is a natural compound. Unlike the plant enzyme, dihydrodipicolinate synthase (DHPS) from E. coli is not feed-back inhibited by physiological concentrations of lysine. The dapA gene which codes for E. coli DHPS was expressed in soybean somatic embryos under the control of the CaMV 35S promoter. Following introduction of the construct into embryogenic tissue of soybean, transgenic events were recovered by incubating the tissue in liquid medium containing AEC at a concentration of 5 mM. Only transgenic soybeans were able to grow at this concentration of AEC; no escapes were observed.ConclusionGenetically engineered soybeans expressing a lysine insensitive DHPS gene can be selected with the non-antibiotic selection agent AEC. We also report here the inhibitory effects of glufosinate, (isopropylamine-glyphosate) (Roundup®), AEC and the ALS inhibitors Exceed® and Synchrony® against different tissues of soybean

Conifer embryogenic tissue initiation: improvements by supplementation of medium with D-xylose and D-chiro-inositol

A major barrier to the commercialization of somatic embryogenesis technology in loblolly pine (LP, Pinus taeda L.) is recalcitrance of some high-value crosses to initiate embryogenic tissue and to continue early-stage somatic embryo growth. Developing initiation and multiplication media that resemble the seed environment may decrease this recalcitrance. Sugar and sugar alcohol analyses were performed weekly throughout the sequence of seed development for female gametophyte and zygotic embryo tissues to determine physiologic concentrations (Pullman, G.S. and M. Buchanan. 2008. Identification and quantitative analysis of stage-specific carbohydrates in LP (Pinus taeda) zygotic embryo and female gametophyte tissues. Tree Physiol. 28:985-996). Major differences in stage-specific sugars were observed. A simple bioassay was used to evaluate the potential growth promotion of individual carbohydrates added to initiation or multiplication media at physiologic concentrations. Seventeen sugars were screened. Compounds showing statistically significant increases in early-stage embryo growth were then tested for the ability to increase the initiation of LP. d-xylose and d-chiro-inositol produced statistically significant increases in early-stage embryo growth. When tested for improved initiation in P. taeda, Pseudotsuga menziesii (mirb) Franco and Picea abies L., Karst., d-xylose increased the averages of initiation by 6.5%, 7.3% and 16.7%, respectively. d-chiro-inositol increased the initiation in P. taeda by 7.3% in one test but not in the other, whereas in P. menziesii the initiation increases averaged 8.4% in two tests. Analyses of sugars and sugar alcohols in the seed environment coupled with a bioassay to screen potential media supplements for protocol improvement resulted in statistically significant increases in embryogenic tissue initiation for several coniferous species.

Synthetic seed production from encapsulated somatic embryos of cork oak (Quercus suber L.) and automated growth monitoring

Cork oak (Quercus suber) somatic embryos were coated with alginate for the production of synthetic seeds and their storability for commercialization was investigated. Also, the automatic monitoring of somatic embryo growth with a digital system of image capture was tested. A power regression model was fitted between size and fresh weight (Adjusted R-squared = 0.96). This method permitted growth assessment without contamination risk and opens the possibility of an automated control of culture growth for the future up scaling of plant production. Conversion rate of synthetic seeds was higher on medium supplemented with mineral nutrients than on medium without nutrients. Also, when the somatic embryos were coated without mineral nutrients added to the capsule, conversion rate was significantly lower. The addition of sucrose to the capsule had no significant effect on the conversion rate. No differences were recorded between 50 and 100 mM CaCl2 for capsule complexation. Synthetic seeds were cold stored at 4°C for two months without significant loss of conversion capacity. The present study reports the first attempts to determine optimal storage time and conditions for conversion of encapsulated somatic embryos of cork oak.

Isolation and characterization of a molecule stimulatory to growth of somatic embryos from early stage female gametophyte tissue of loblolly pine

Loblolly pine (LP, Pinus taeda) is the primary commercial species in southern forests of the US. Somatic embryogenesis (SE) is an effective technique to implement clonal tree production of high-value genotypes from breeding and genetic engineering programs. Unlike angiosperm embryos with attached cotyledons as seed storage organs, the diploid conifer embryo is surrounded by the unattached haploid female gametophyte (FG). The FG is not present in culture. This presents a dilemma if the FG produces necessary or regulatory compounds for embryo growth, since in culture these important compounds would be missing and would have to be added as supplements. We report here the direct evidence that extracts from early-stage FG indeed stimulate early-stage somatic embryo (SME) growth and multiplication, whereas extracts from late-stage FG inhibit early-stage SME growth. Furthermore, we have now isolated this stimulatory substance from early-stage FG tissue, and identified this substance as citric acid on the basis of NMR and mass spectrometry. We then demonstrated that topical application of citric acid to SMEs stimulates embryo colony growth at P = 0.05. Moreover, we find that there is a good correlation between the amount of citric acid isolated from FG tissue (65 nmoles per stage 2-3 FG) and the amount of citric acid that stimulates colony growth (25-50 nmoles) when applied topically to SMEs. This approach of isolating and characterizing a molecule from plant tissue, and investigating its role on SE processes can provide valuable information leading to further applications of these molecules to improve LP SE protocols.

Ferula gummosa Boiss. Embryogenic Culture and Karyological Changes

Ferula gummosa Boiss. a highly valuable medicinal plant which naturally propagates in very limited areas of the Middle East with specific environmental conditions. The production of Ferula gummosa somatic embryos and the karyological analysis of somatic seedlings were the purpose of this study. High frequency indirect embryogenesis was induced in callus derived from zygotic embryonic axes. Embryogenesis was obtained when callus tissues were placed onto an agar induction Murashige and Skoog medium with 1-naphthalene acetic acid and after the transfer of the cultures in a thermoperiod regime of 16 h, 19 degrees C/8 h, 7 degrees C under photoperiod of 16 h light/8h dark. Embryogenic callus tissues were maintained by subculture on induction medium. Globular proliferation was achieved with suspension culture in the Murashige and Skoog medium added with 1-naphthalene acetic acid or 2,4-dichlorophenoxyacetic acid for two weeks. Maturation of embryos and development of plantlets arose on the induction agar medium, but was better after transfer into the hormone free Murashige and Skoog medium. However, the level of abnormal embryos was high. Direct embryogenesis was obtained from somatic seedlings. The best results were obtained from hypocotyl explants. Embryo induction was achieved by two week culture of the explants in 2,4-dichlorophenoxyacetic acid liquid medium; somatic embryo growth and maturation was recovered on the hormone free medium. High level of abnormalities was recorded in the culture. Karyological analysis showed a high incidence level of cytochimerism in somatic seedlings with chromosome stickiness, polypoidy and aneuploidy in metaphase cells of the same root tip. The frequency of these karyological changes varied with the type of somatic embryos with regard to morphological abnormalities. Normal and abnormal rooted somatic seedlings were able to grow until production of the first leaf and then entered dormancy in the same manner as zygotic plantlets.

Loblolly pine ( Pinus taeda L.) somatic embryogenesis: Improvements in embryogenic tissue initiation by supplementation of medium with organic acids, Vitamins B 12 and E

A major barrier to the commercialization of somatic embryogenesis technology in loblolly pine ( Pinus taeda L.) is recalcitrance of some high-value crosses to initiate embryogenic tissue and continue early-stage somatic embryo growth. Developing initiation and multiplication media that resemble the seed environment may decrease this recalcitrance. Organic acid analyses were performed weekly throughout the sequence of seed development for female gametophyte and zygotic embryo tissues to determine physiological concentrations present that may be beneficial if added to somatic embryogenesis media [14]. Major differences in stage-specific organic acids were observed. A simple bioassay was used to evaluate potential growth promotion of individual organic acids added to initiation or multiplication media at physiological concentrations. Nine vitamins and 25 organic acids were screened. Compounds showing statistically significant increases in early-stage embryo growth were then tested for initiation of loblolly pine. Two vitamins and five organic acids including Vitamins B 12 and E, α-ketoglutaric, pyruvic, succinic, oxalic, quinic, and ascorbic acids produced statistically significant increases in early-stage embryo growth. When tested for improved initiation, Vitamins B 12 and E, α-ketoglutaric, pyruvic, and succinic acids increased initiation when alone and when combined. Analyses of organic acids in the seed environment coupled with a bioassay to screen potential media supplements for protocol improvement resulted in statistically significant increases in the loblolly pine embryogenic tissue initiation protocol and saved much time and expense.

Somatic embryogenesis from embryogenic cell suspension cultures of california poppy, Eschscholzia californica Cham

A somatic embryogenesis protocol was developed for Eschscholzia californica Chan. (California poppy) using embryogenic cell suspensions and optimized media conditions. Rapidly-growing, finely-dispersed embryogenic cell suspension cultures were established from embryogenic callus and maintained in B5 liquid media supplemented with 0.5 mg 1−1 (2.26 μM) 2,4-dichlorophenoxyacetic acid. Culture conditions were optimized by investigating the effect of basal media composition, gyratory shaker speed, various carbon sources, different cytokinins, and AgNO3 on the efficiency of somatic embryogenesis. After 40 d in culture, the somatic embryos that formed were counted and their overall growth expressed as pecked cell volume. The selected media consisted of either Gamborg (B5) or Murashige and Skoog (MS) salts and vitamins supplemented with 40 g 1−1 (117 mM) sucrose, 0.05 mg 1−1 (0.22 μM) 6-benzylaminopurine, and 10 mg l−1 (58.8 μM) AgNO3. Somatic embryo production was substantially reduced at shaker speeds above 40 rpm. Glucose and snerose were the most effective carbon sources, whereas fructose, galactose, and maltose resulted in a reduced yield and growth of somatic embryos. The development of somatic embryos was promoted by AgNO3 at concentrations below 10 mg l−1 (58.8 μM). A semi-solid medium containing 1.5 g l−1 Gel-rite produced the highest frequency of somatic embryo conversion, and promoted the efficient growth of plantlets. Using the reported protocol, over 500 viable somatic embryos were produced per 25 ml of embryogenic cell suspension culture.

Effects of Tissue Support and Aeration on the Germination and Growth of Aralia cordata Somatic Embryos.

The effects of supporting materials and aeration treatment on the germination and growth of somatic embryos induced from suspension cells of Aralia cordata 'Aichi-bozu' were examined. 1. Germination and growth of somatic embryos differed depending on the kinds of supporting materials, e.g. the germination rate after a 4-week culture period was 75.0% for polyester, 56.3% for vermiculite, and 37.5% for agar. Fresh and dry weights of tops of plantlets after a 4-week culture period the polyester support was heavier than these on the others. 2. Seventy percent of the embryos of plot A, which were naturally aerated for the entire 5-week period germinated, whereas 69% of those in plot B, which were exposed to natural aeration for 2 weeks and forced aeration for another 3 weeks, germinated ; only 58% of the embryos germinated in plot C which were only forced aerated for 5 weeks. All somatic embryos which germinated developed into plantlets. The increment of plant height, fresh and dry weights of plantlets after a 5-week culture period was greater in plot A and B than that in plot C. All plantlets became swollen in plot A but not those in plot B and C. Nevertheless, many normal plantlets developed in plot B. Hence, we found that for the efficient production of the plantlets, polyester was the superior plant support and that high humidity in the first half of a culture period and low humidity in the second half yielded optimum results.

Light quality influences germination, root growth and hypocotyl elongation in somatic embryos but not in seedlings of norway spruce

We studied how light from different light sources influences germination and postgerminative growth of plants from somatic embryos and seeds of Norway spruce (Picea abies [L.] Karst). Somatic embryos of three spruce genotypes and seeds were subjected to light from commercially available light sources: Philips TLD Blue 18W/18 (BL), Osram Fluora (FL), Philips Cool White TL 50W/33 (CW), Osram Warm White 18W/30 (WW), Philips Yellow 36W/16 (YE) and Philips TLD Red 36W/15 (RE), 18 h a day, with a photon flux (PAR) at 30 µmol m−2 s−1. After 6 wk the germination frequencies of the somatic embryo-derived plantlets were 50% under BL and 98% under RE. The corresponding mean root lengths were 6.7 and 15.4 mm. In somatic embryo-derived plantlets cultured under BL, FL, CW and WW, both roots and hypocotyls turned brown, presumably due to production of phenolic substances. Browning was less severe in somatic embryo-derived plantlets cultured under RE and YE. Under RE, the epicotyl elongated in 37% of the plantlets after 6 wk, compared with 70% under the other light sources. Seed germination and postgerminative seedling growth was modestly influenced by light from these light sources. RE and WW initially delayed germination as compared with BL, FL and CW, but after 2 wk, more than 90% of the seeds had germinated under all light sources. In conclusion, germination and postgerminative growth of somatic embryos of spruce is sensitive to differences in light quality, whereas seed germination and seedling growth is not.

Influence of Medium Parameters on Somatic Embryogenesis from Hypocotyl Explants of Flax ( Linum usitatissimum L.) : Effect of carbon source, total inorganic nitrogen and balance between ionic forms and interaction between calcium and zeatin

Summary The effects of different carbon sources, total inorganic nitrogen concentration, nitrate to ammonium ratio and the interaction between calcium and zeatin (ZEA) levels on somatic embryogenesis from flax hypocotyl explants were studied in three independent factorial experiments. MS medium supplemented with the monosaccharides glucose or fructose at high concentrations (4 %) gave consistently highly embryogenic cultures, with higher somatic embryo frequencies and higher growth rates when compared with media supplemented with sucrose or maltose. Although media with maltose had performed well in a 1-4 % concentration range, media supplemented with sucrose at 4 %, appeared to inhibit the induction and development of somatic embryos. Independently of the effect of the nitrogen content, the balance between both ionic forms (N03- and NH4') played a dramatic role on the induction of somatic embryogenesis and somatic embryo growth. Nitrate is important for calli differentiation and growth, and a high ammonium content increased somatic embryo frequency. The embryogenic vs. undifferentiated cell growth commitment of flax explants was determined by an interaction between calcium and ZEA levels, a high calcium/low ZEA affording very low embryogenic potential and high calli biomass. A high ZEA concentration was essential for the normal development of somatic embryos.