Number Of Somatic Embryos Research Articles

Somatic embryogenesis in Alnus glutinosa (L.) Gaertn

Induction of somatic embryos and plant regeneration was demonstrated for the first time in Alnus glutinosa. Somatic embryos were initiated from zygotic embryos collected 1–3 weeks post-anthesis (WPA), i.e., when they were at globular or early cotyledonary stage and were 0.5–1 mm in length. Induction frequency (16.6 %) and the mean number of somatic embryos (4.5 embryos/explant) were highest after culture of zygotic embryos, collected at 3 WPA, on Murashige and Skoog medium (MS) supplemented with 0.9-μM 2,4-dichlorophenoxyacetic acid and 2.22-μM benzyladenine (BA). No embryogenic induction was observed on medium with BA alone. Initial somatic embryos differentiated indirectly from callus tissue formed at the surface of the zygotic embryos. Embryogenic competence was maintained by secondary embryogenesis, which was affected by explant type, plant growth regulators and genotype. Secondary embryogenesis was induced by culture of small groups of whole somatic embryos or isolated cotyledon explants on medium consisting of MS medium (half-strength macronutrients) supplemented with 0.44-μM BA. Histological study of isolated cotyledon explants revealed that secondary embryos developed directly from differentiated embryogenic tissue on the surface of cotyledons. Somatic embryos at successive stages of development, including cotyledonary-stage embryos with shoot and root meristems, were evident. For plantlet conversion, somatic embryos were transferred to maturation medium supplemented with 3 % maltose, followed by 6 weeks of culture in Woody Plant Medium supplemented with 0.44-μM BA and 0.46-μM Zeatin (Z). This novel protocol appears promising for mass propagation, conservation and genetic transformation of black alder.

Morphological and polyamine content changes in embryogenic and non-embryogenic callus of sugarcane

Differences in competence acquisition and subsequent embryo maturation in embryogenic and non-embryogenic callus of sugarcane var. SP79-1011 were evaluated using histomorphological analysis, growth curves, numbers of somatic embryos, and polyamine contents. Embryogenic callus was formed by cells with embryogenic characteristics such as a rounded shape, prominent nuclei, a high nucleus: cytoplasm ratio, small vacuoles and organized globular structures. However, non-embryogenic callus presented dispersed, elongated and vacuolated cells with a low nucleus: cytoplasm ratio; these characteristics did not allow for the development of somatic embryos even upon exposure to a maturation stimulus. These results suggest that non-embryogenic callus does not acquire embryogenic competence during induction and that maturation treatment is not sufficient to promote somatic embryo differentiation. The use of activated charcoal (AC; 1.5 g L−1) resulted in a higher somatic embryo maturation rate in embryogenic callus but did not yield success in non-embryogenic callus. Embryogenic callus incubated with control (10 μM 2,4-dichlorophenoxyacetic acid) and maturation (1.5 g L−1 AC) treatments for 28 days showed similar patterns of total free polyamines; these results differed from the results observed with non-embryogenic callus, suggesting that embryogenic callus already exhibits a characteristic pattern of endogenous polyamine levels. At 28 days of culture with maturation treatment, embryogenic callus exhibited significantly higher levels of free Spm than embryogenic callus incubated with control treatment and non-embryogenic callus incubated with both treatments. This result suggests that Spm could be important for the acquisition of embryogenic competence and somatic embryo maturation in sugarcane var. SP79-1011.

Efficacious somatic embryogenesis and fertile plant recovery from shoot apex explants of onion (Allium cepa. L.)

An efficient somatic embryogenesis and regeneration system was developed for the first time in onion using shoot apex explants. These explants were used to initiate callus in Murashige and Skoog (MS) medium supplemented with 4.0 mg l−1 2,4-dichlorophenoxyacetic acid. The induction frequency of primary callus in this medium was 85.3%. The primary calli were then transferred onto medium supplemented with 2.0 mg l−1 2,4-dichlorophenoxyacetic acid. Following two biweekly subcultures, embryogenic callus formed. Inclusion of a low concentration of 6-benzylaminopurine in the subculture medium promoted the formation of embryogenic callus. The addition of 2.0 mg l−1 glycine, 690 mg l−1 proline, and 1.0 g l−1 casein hydrolysate also increased the frequency of callus induction and embryogenic callus formation. The highest frequency of embryogenic callus (86.9%) and greatest number of somatic embryos (26.3 per callus) were obtained by the further addition of 8.0 mg l−1 silver nitrate. Somatic embryos formed plantlets on regeneration medium supplemented with 1.5 mg l−1 6-benzylaminopurine; addition of 2.0 mg l−1 glycine to the regeneration medium promoted a high frequency of regeneration (78.1%) and plantlet formation (28.7 plants per callus). The regenerated plantlets were transferred to half-strength MS medium supplemented with 1.5 mg l−1 indole-3-butyric acid for root development; the maximum frequency of root formation was 87.7% and the average number of roots was 7.6 per shoot. The regenerated plantlets were successfully grown to maturity after hardening in the soil. This is the first report of somatic embryogenesis and regeneration from shoot apex explants of onion.

An Improve Method for Somatic Embryogenesis of Schisandra chinensis (Turcz.) Baillon

A somatic embryogenesis protocol for plant regeneration of Schisandra chinensis (Turcz.) Baillon was established. Increased callus induction was obtained from mature zygotic embryos on Murashige and Skoog (MS) medium supplemented with 1.0 mg L(-1) N-phenyl-1,2,3-thidiazol-5-yl-urea (TDZ) or 2.0 mg L(-1) 2,4-dichlorophenoxyacetic acid (2,4-D). Addition of Zeatin (Zt) promoted the formation of embryogenic calli. To induce somatic embryogenesis, 2,4-D, TDZ and Zt were incorporated in the medium alone or in combination. Development of the maximum number of somatic embryos (81 globular, 37 heart, 52 torpedo and 37 cotyledon-stage) and germination of the highest number of embryos (50%) was observed on 1/2 MS medium supplemented with 1.0 mg L(-1) TDZ and 0.2 mg L(-1) Zt. Further development of somatic embryos into plantlets was completed in 1/2 MS medium free of plant growth regulators.

Influence of abscisic acid and sucrose on somatic embryogenesis in Cactus Copiapoa tenuissima Ritt. forma mostruosa.

Having produced the embryos of cactus Copiapoa tenuissima Ritt. forma monstruosa at the globular stage and callus, we investigated the effect of abscisic acid (ABA) in the following concentrations: 0, 0.1, 1, 10, and 100 μM on successive stages of direct (DSE) and indirect somatic embryogenesis (ISE). In the indirect somatic embryogenesis process we also investigated a combined effect of ABA (0, 0.1, 1 μM) and sucrose (1, 3, 5%). The results showed that a low concentration of ABA (0-1 μM) stimulates the elongation of embryos at the globular stage and the number of correct embryos in direct somatic embryogenesis, while a high ABA concentration (10–100 μM) results in growth inhibition and turgor pressure loss of somatic embryos. The indirect somatic embryogenesis study in this cactus suggests that lower ABA concentrations enhance the increase in calli fresh weight, while a high concentration of 10 μM ABA or more changes calli color and decreases its proliferation rate. However, in the case of indirect somatic embryogenesis, ABA had no effect on the number of somatic embryos and their maturation. Nevertheless, we found a positive effect of sucrose concentration for both the number of somatic embryos and the increase in calli fresh weight.

A comparative study between solid and liquid cultures relative to callus growth<br>and somatic embryo formation in date palm (Phoenix dactylifera L.) cv. Zaghlool

Tissue culture techniques enable mass propagation of elite cultivars of date palm (Phoenix dactylifera L.). The main limitations for date palm in vitro multiplication are the low rates achieved using solidified medium and the long period needed to produce acclimatized plantlets. This research focuses on the comparison between different culture types and plant growth regulator (PGR) combinations on callus growth and somatic embryo formation of cv. Zaghlool. For callus growth, 200 mg friable embryogenic callus dispensed in Rasotherm and Phytacon flasks containing 200 ml liquid (100 rotations per minute) and in temporary immersion system, RITA ® bioreactor (5 min immersion every 12 h), were compared with cultures grown on 200 ml solidified medium. For somatic embryo formation, 500 mg of friable embryogenic callus grown in Erlenmeyer flasks filled with 50 ml liquid or solid MS medium. The medium was supplemented with 0.1 mg l -1 Naphthaleneacetic acid (NAA), 1.5 g l -1 activated charcoal (AC) with or without 0.05 mg l -1 6-Benzyl amino purine (BAP) compared to PGR-free medium. Results proved that cell suspension cultures produced the highest callus fresh mass as compared to the other systems tested, and the callus fresh mass reached 4 g after 16 weeks. The temporary immersion system did not significantly enhance the fresh mass of callus compared to the solidified medium. For somatic embryo induction, the number of somatic embryos increased in cell suspensions 6-16 fold compared to the solidified medium. Using liquid MS medium enriched with 0.1 mg l -1 NAA and 1.5 g l -1 AC gave rise to the highest number of somatic embryos formed from 500 mg initial callus: 160 embryos. The number of somatic embryos was also affected by the callus source. The calli induced from leaflet segments excised from converted somatic embryos resulted in a lower somatic embryo number than those of shoot tip origin with about 60 somatic embryos per 500 mg callus. The formation of somatic embryos using liquid medium required only 6 weeks, thus considerably reducing the previously reported period of 18 weeks which is required for somatic embryo formation using solidified media.

Callose Deposition Is Required for Somatic Embryogenesis in Plasmolyzed Eleutherococcus senticosus Zygotic Embryos

Dynamic changes in callose content, which is deposited as a plant defense response to physiological changes, were analyzed during somatic embryogenesis in Eleutherococcus senticosus zygotic embryos plasmolyzed in 1.0 M mannitol. During plasmolysis, callose deposition was clearly observed inside the plasma membrane of zygotic embryo epidermal cells using confocal laser scanning microscopy. The callose content of zygotic embryos gradually increased between 0 and 12 h plasmolysis and remained stable after 24 h plasmolysis. During eight weeks induction of somatic embryogenesis, the callose content of explants plasmolyzed for 12 h was slightly higher than explants plasmolyzed for 6 or 24 h, with the largest differences observed after 6 weeks culture, which coincided with the maximum callose content and highest number of globular somatic embryos. The highest frequency of somatic embryo formation was observed in explants plasmolyzed for 12 h. The somatic embryo induction rate and number of somatic embryos per explant were markedly different in zygotic embryos pretreated with plasmolysis alone (78.0%, 43 embryos per explant) and those pretreated with plasmolysis and the callose synthase inhibitor 2-deoxy-d-glucose (11.5%, 8 embryos per explant). This study indicates that callose production is required for somatic embryogenesis in plasmolyzed explants.

In vitro germination and embryogenic competence acquisition of Euterpe edulis Martius immature zygotic embryos

This study evaluated different aspects of in vitro germination and embryogenic competence of immature zygotic embryos of E. edulis. The embryos germinated on full or half-strength MS (MS or MS/2) medium combined with sucrose (20, 30 and 40 g L-1). The effect of calcium chloride concentrations (0, 2, 4, 8, and 12 mM) on the induction of somatic embryogenesis was tested. The embryos were germinated on MS or MS/2. Germination of zygotic embryos and the number of roots per plantlet were not affected by the culture medium and sucrose concentration. Plantlet height and fresh weight were influenced by both; the difference was greatest in MS medium with 40 g L-1 sucrose. The induction of somatic embryogenesis was not influenced by doses of calcium chloride, whereas the number of somatic embryos formed was affected. The germination capacity of somatic embryos of heart-of-palm tree was not influenced by the media tested.

Somatic embryogenesis and plant regeneration through cell suspension in diploid (AB) banana cultivar Elakki Bale (syn Neypoovan)

An efficient protocol was developed using cell suspensions for somatic embryogenesis and plantlet regeneration in a most popular diploid AB banana (M.accuminata X M.bulbisiana hybrid) cv. Elakki Bale (syn Neypoovan) known for its taste and keeping quality in southern India. Floral primodia from position 8–16 of male inflorescence which were more responsive for embryogenesis were used as explants for the embryogenic callus production in MS media supplemented with different concentration of 2,4-D. A concentration of 18.1 μM 2, 4-D produced maximum embryogenic calli in 1 % of the explants inoculated. Embryogenic calli on repeated sub culturing on MA2 media produced good embryogenic cell suspensions (ECS). Microscopic examination of ECS showed globular, smaller with dense cytoplasm filled with starchy granules characteristic of embryogenic cells. Highest number of somatic embryos (189) was produced on modified MA3 media. A germination percentage of 31 % were observed in BAP 22.19 μM concentration. Regenerated plants with normal shoot and root were hardened in soilrite. Direct somatic embryogenesis and plant regeneration was also noticed in embryogenic calli which did not pass through the ECS stage. The protocol optimized for somatic embryogenesis through cell suspension and also direct embryogenesis leading to plantlet regeneration can be used for the micropropagation and genetic manipulation.

Impact of Osmotica and Abscisic Acid on Direct Somatic Embryogenesis in Tea

An in vitro protocol was developed to induce somatic embryos using mature cotyledons obtained from open pollinated flowers of cultivated Camellia species. Impact of abscisic acid (ABA) alone and its combination with osmotica was also evaluated on somatic embryogenesis. Various concentrations of osmoticum viz., polyethylene glycol (PEG) or mannitol or glycine betaine were tested where ABA 5 mg L-1 and PEG 3% increased the number of somatic embryo for- mation compared to the other osmoticum coupled with ABA. Results suggested that the use of osmoticum along with ABA was quite effective to induce somatic embryos in tea. But germination of embryos required a new combination of nutrient medium. Inclusion of 6-benzylaminopurine (BAP) and gibberellic acid (GA3) in the nutrient medium enhanced percent germination and its growth.

The micropropagation of chrysanthemums via axillary shoot proliferation and highly efficient plant regeneration by somatic embryogenesis

Protocols for axillary shoot proliferation and somatic embryogenesis were developed for Dendranthema × grandiflora (Ramat.) Kitamura cv. Palisade White. Shoot tips were cultured on a modified Murashige and Skoog (MS) media supplemented with benzyl aminopurine (BA) and gibberellic acid (GA 3 ) or BA, kinetin (Kin) and indole-3-acetic acid (IAA). The auxins indole -3-butyric acid (IBA) and IAA were used to induce rooting. Direct somatic embryogenesis was induced from leaf, internode’s stem and for the first time for chrysanthemums from petiole explants. Modified MS medium supplemented with 1 mg/L naphthalene acetic acid (NAA) or 2,4- dichlorophenoxyacetic acid (2,4-D), 0.1 mg/L BA, 200 mg/L casein hydrosylate (CH) and 290 mg/L proline was used for induction. Proliferation rate of 3.2 new microshoots per one inoculated was achieved when BA (0.1 mg/L) was used in combination with GA 3 (0.5 mg/L). The number of roots per shoot was higher using IBA (0.5 mg/L), but IAA (2 mg/L) promoted longer roots. A high percentage of embryogenesis was induced by both combinations of plant growth regulators (PGRs). Leaf explants were most responsive, demonstrating the highest percentage of embryogenesis (97.9%), followed by petiole and internode’s stem explants (56.3 and 35.1%, respectively). The number of somatic embryos per embryogenic explant was also the highest on leaf explants; however, the best conversion rate (53.8%) of somatic embryos to plantlets was observed from petiole explants. For this reason, petiole explants are the most suitable type of explants for plant regeneration of chrysanthemum cv. Palisade White through somatic embryogenesis. Key words : Chrysanthemum, Dendranthema × grandiflora (Ramat.) Kitamura cv. Palisade White, micropropagation, direct somatic embryogenesis, explant type.

Establishment of plant regeneration of Michelia champaca L. through cell suspension culture technique

Michelia champaca L. is a woody tree species, which has high economical value as a basic material for medicinal and fragrance products. However, there are several problems encountered in conventional propagation of M. champaca, which are generally low through seed germination, while vegetative layering technique produces limited planting materials. This experiment was conducted to establish high frequency M. champaca regeneration through cell suspension culture technique. Liquid Murashige and Skoog medium containing 2 mgL-1 α-naphthalene acetic acids induced high frequency of somatic embryos formation (47.67 ± 4.53 per ml). Meanwhile for germination of somatic embryos, the highest total number of somatic embryos germinated into plantlets obtained was 34% in hormone free Murashige and Skoog medium. Plant regeneration of M. champaca via cell suspension culture technique which was successfully established in this study could be used for mass production of planting materials of this tree. Key words: Michelia champaca, somatic embryogenesis, cell suspension culture technique.

Efficient somatic embryo production of Limau madu (Citrus suhuiensis Hort. ex Tanaka) in liquid culture

Effects of N6-benzylaminopurine (BAP) concentration, initial cell density and carbon sources and concentrations for producing cell suspension and somatic embryos of Limau madu ( Citrus suhuiensis Hort. ex Tanaka) were investigated using cell suspension culture. Cells were first inoculated into Murashige and Skoog (MS) medium (1962) supplemented with 4.4 to 13.3 μM BAP. Growth rate of cells was at its maximum (6.69 mg day -1 ) in media supplemented with a lower concentration of BAP (6.7 μM). Embryogenic cell at 2 mg ml -1 was found to be the most effective inoculum size for the highest growth rate (3.35 mg day -1 ) of cell proliferation within a period of 15 to 30 days after inoculation (DAI). This inoculum size resulted in 31.75% faster embryo growth than those with inoculum densities of 4 to 6 mg ml -1 . Sucrose (88, 117 and 146 mM), glycerol (16, 22 and 27 mM) and combinations of sorbitol and galactose (146:0, 110:36, 73:73, 36:110 and 0:146 mM) were tested for their effects on embryogenic cell proliferation and somatic embryo induction. Results indicates that sucrose at 146 mM induced cell proliferation (7.65 mg day -1 ) and produced a higher quantity of cells than glycerol at 27 mM (2.33 mg day -1 ) and a combination of sorbitol and galactose at 73:73 mM (4.64 mg day-1), but failed to induce somatic embryos. Glycerol in different concentrations was ineffective in cell proliferation and somatic embryo induction. At optimal BAP concentration (6.7 μM), a small amount of embryogenic cells (100 mg in 50 ml) can be multiplied profusely in sucrose-containing medium. A large number of somatic embryos (951) were induced in a medium containing 110 mM sorbitol and 36 mM galactose as the most effective carbon source for inducing somatic embryos without BAP. Key words : Limau madu, cell suspension, sucrose, sorbitol, galactose.

Induction of petal-bearing embryos from root-derived callus of Oncidium ‘Gower Ramsey’

A repeatable in vitro culture method was established and it could induce a series of abnormal embryos by which their scale leaves were substituted by petals. These petal-bearing embryos were derived from long-term root calli of an orchid cultivar, Oncidium ‘Gower Ramsey’. The calli were induced and subcultured on a modified 1/2MS medium supplemented with five combinations of TDZ and dicamba. When 1-year-old callus, which induced and subcultured at 3 mg/l TDZ and 5 mg/l dicamba (line 13 callus), was transferred onto 1/2MS medium supplemented with 0.1 ml/l NAA and 3 mg/l TDZ, it gave the highest number of petal-bearing embryos. However, line 13 root explants gave one of the lowest percentage of callus formation (12.5%) and the number of somatic embryos per callus was also one of the lowest (along with lines 10, 11 and 12). The efficiency of embryogenesis decreased with age of callus and the significant decrease was started from the third year of culturing. Flowering characteristics of plantlets from normal embryos and petal-bearing embryos were both evaluated after 3 years of culture in the greenhouse. Parameters including length of the longest inflorescence, numbers of flowers per plant, length of flowers and width of flowers were all not significantly different between abnormal embryo-derived plants and normal embryo-derived plants.

Hygromycin promotes somatic embryogenesis in spinach

Hygromycin (hyg) at low doses (0.5–1.0 mg l−1) promoted somatic embryogenesis from apical sections of spinach lateral roots. The highest promoting effect on both the frequency of regeneration and the mean number of somatic embryos (SE) per explant was achieved at 0.5 mg l−1 hyg. With increasing the concentration of hyg to 1 mg l−1, the regeneration frequency decreased, while the mean SE number remained significantly higher than in control (hyg-free medium). Complete inhibition of SE regeneration started at 7.5 mg l−1 hyg. Moreover, hyg efficiently promoted the process of secondary somatic embryogenesis. Compared to control, a 2.75-fold increase in the secondary somatic embryo (SSE) mean number was obtained at 0.5 mg l−1 hyg, and the increment was still discernible at 1.0 and 2.5 mg l−1 hyg. Both primary SE and SSE explants became completely necrotic at 12.5 mg l−1 hyg. Since attempts with direct selection at 20 mg l−1 hyg proved unsuccessful, the results obtained in this study suggest that a stepwise selection procedure is suitable, starting with selection at 0.5 mg l−1 hyg, to exploit the promoting effect of low hyg doses on SE regeneration from transformed cells, then gradually increasing the hyg concentration to 20 mg l−1 for final selection. Complete SE and SSE explant mortality at hyg above 12.5 mg l−1 guarantees a low possibility of escape during the selection process. This study will be useful for increasing the efficiency of transgenic plant regeneration following genetic transformation in spinach.

Plant Regeneration through Somatic Embryogenesis from Leaf Sheath Derived Callus of Sugarcane (Saccharum officinarum L.) var. Isd -16

Leaf sheath explants of an indigenous variety Isd-16 of sugarcane (Saccharum officinarum L.) produced light yellow friable callus after culturing on to MS with 2,4-D (2 - 4 mg/l) and NAA (3 - 5 mg/l) singly. Callus formation was the maximum on MS + 3 mg/l 2,4-D. Callus underwent embryogenesis producing huge number of somatic embryos when subcultured on MS with 15 - 30 mg/l L-proline, 3 mg/l 2,4-D + 5 - 10% coconut water (v/v) and 3 mg/l 2,4-D + 10% CW (v/v) + 300 - 500 mg/l CH. L-proline significantly enhanced somatic embryo-genesis and 25 mg/l L-proline in MS was the best culture medium formulation. Most of the somatic embryos germinated and developed plantlets after 1 - 2 weeks of incubation in proline-supplimented medium. On the other hand, maturation and germination of embryos were achieved on half-strength MS with or without 0.25 - 1.0 mg/l L-proline, and 5% coconut water (v/v). Somatic embryos derived plantlets were then successfully transferred to natural condition through successive phages of acclimation. Key words: Plant regeneration, Leaf sheath, Somatic embryogenesis, Sugarcane D. O. I. 10.3329/ptcb.v21i2.10237 Plant Tissue Cult. & Biotech. 21(2): 143-149, 2011 (December)

Effects of different ectomycorrhizal fungi on somatic embryogenesis of Abies cephalonica Loud

The effects of ectomycorrhizal (ECM) fungi, including Laccariabicolor (Maire) Orton, Laccaria laccata (Scop., Fr.) Berk. and Br., along with two strains of Pisolithus tinctorius (Pers.) Coker and Couch, on the proliferation and subsequent maturation of two embryogenic cell lines of Abies cephalonica Loud., designated lines 6 and 8, were investigated. In the presence of these ECM fungi, the proliferation of both embryogenic cell lines was inhibited. L. bicolor and P. tinctorius strain 2 resulted in the highest inhibition rates. On the other hand, cultivation of embryogenic cultures along with ECM fungi, termed a dual culture, increased radial growth of both P. tinctorius strains; whereas, L. bicolor and L. laccata did not grow as well in the presence of embryogenic cell masses. The dual culture during the proliferation period of embryogenic cells, however, enhanced the subsequent embryo formation and maturation of A. cephalonica; i.e. the capability of embryogenic cell lines to form somatic embryos as well as increasing the mean number of somatic embryos per 1 g fresh weight of embryogenic cell mass. However, levels of responses were highly dependent on the interaction between the specific embryogenic cell line and fungal strain.

Somatic embryogenesis and plant regeneration in avocado (Persea americana Mill.): influence of embryogenic culture type

Embryogenic cell lines of Persea americana were classified as SE- or PEM-type based on their morphology in maintenance medium. PEM-type cultures consisted of proembryonic masses (PEMs) with occasional development of proembryos and early-stage somatic embryos, while SE-type cultures consisted of somatic embryos from globular to cotyledonary stages with only a low frequency of proembryos and PEMs. Moreover, histological analysis revealed signs of cellular organisation, with a higher proportion of small cells in peripheral regions of the callus mass in the SE-type, but not in PEM-type cell lines. The effects of time in suspension, inoculum density and maintenance medium on the ability of avocado cell lines to produce mature somatic embryos were evaluated. Morphological differences observed during proliferation were correlated to the subsequent capacity of cultures to develop mature somatic embryos. The period of time in suspension and inoculum density were critical factors that influenced the ability of cultures to undergo maturation. Optimal conditions differed between SE-type cultures and PEM-type cultures. In SE-type cultures, the highest number of somatic embryos was observed in cultures grown in suspension for 9 days and 0.4 g inoculum; in PEM-type cultures a slight increase in mature somatic embryos production could be observed with 4 g inoculum size and 14 days in suspension. The presence of agar in the maintenance medium of SE-type cultures was essential for the maturation of somatic embryos.

Genotype and explant-type dependent morphogenesis and silicon response of common reed ( Phragmites australis) tissue cultures

The effects of silicon on the growth and development of Phragmites australis (Cav.) Trin. Ex Steud. (common reed) stem nodal and root embryogenic calli were investigated. Silicon is considered to be a beneficial or quasi-essential nutrient for several Gramineaceous plants, including reed. Seven callus lines of four geographical locations (genotypes 1–4) within Hungary were investigated. Callus lines 1A, 2A and 3A were produced from stem nodal explants, while lines 1B, 2B, 3B and 4 were produced from roots. For the assay of silicon-dependent growth of callus lines of identical genotype but originating from different explants, we measured the increase of fresh weight of lines 1A and 1B. The studied developmental parameters were the increase of the number of somatic embryos (for callus lines 1A and 1B) and plant or root production from somatic embryos (for all genotypes/callus lines). Silicon was added to the culture medium as sodium silicate. In control cultures, plant or root regeneration from embryogenic calli was strongly genotype- and explant type-dependent. Stem nodal explants developed plants on regeneration medium in case of callus lines 2A and 3A, while line 1A produced roots only. All root derived calli developed roots on regeneration medium. Silicon stimulated the growth of both stem nodal and root calli (callus lines 1A, B) however, the concentration optima were different. Somatic embryogenesis of root calli, but not of stem nodal calli, was stimulated by silicate at low concentrations. However, for both of these callus lines, root development was stimulated by silicon. It had genotype-dependent influences on plant regeneration: while stimulation was observed in case of callus line 2A, inhibition occurred for line 3A. Root morphogenesis on calli was significantly influenced by silicon and depended on the callus line studied. Root production was stimulated on callus lines 1A, B and 2B, while in case of callus line 3B, it was significantly inhibited. The morphogenetic effects of Si were similar for different explants of the same geographical origin, i.e. plant or root production was similarly stimulated or inhibited by this element. We can conclude that the effects of Si on plant or root development depend on reed genotype used for callus induction. Its effect on growth and somatic embryogenesis depends on the explant type used for callus production. This is the first detailed report on the role of silicon in plant vegetative development and morphogenesis of a Gramineaceous plant.

An efficient somatic embryogenesis based plant regeneration from the hypocotyl of Catharanthus roseus

An efficient and rapid somatic embryogenesis based plant regeneration from hypocotyls of three Catharanthus roseus cultivars, Pacifica cherry red (PCR), Heatwave mix color (HWMC), and Mediterranean Rose Red (MRR) was standardized. Hypocotyl-derived primary calluses (HPC) were formed on callus induction medium (MSCP1) after 10 days of culture. Embryogenic calluses (EC) with somatic embryos were induced from HPC on proliferation medium (MSCP2) within 2 weeks of culture. Later, EC were selected and maintained on plantlet formation medium (MSCP3) with periodic subculturing at an interval of 2 weeks. Somatic embryos were germinated well into plantlets on MSCP3 within 6 weeks of culture. As sources of nitrogen, casein hydrolysate (CH; acid free) and L-proline were necessary as standard addenda in the plant regeneration protocol of C. roseus . The promotive effects of CH and L-proline not only decreased the culturing time but also increased the number of somatic embryos and their derived plantlets. The percentage of EC and regenerated plant initiated from hypocotyls were higher than those previously reported.