Frequency Of Somatic Embryogenesis Research Articles

Somatic Embryogenesis Induction and Genetic Stability Assessment of Plants Regenerated from Immature Seeds of Akebia trifoliate (Thunb.) Koidz

Akebia trifoliata is a perennial woody plant with considerable potential in nutrition, food, and health, and the production of seedlings with high quality is critical for its economic utilization. Plant regeneration through somatic embryogenesis is a powerful alternative for propagating many plant species. In this study, a simple and practicable protocol was developed for plant regeneration from immature seeds of A. trifoliata via somatic embryogenesis, and the genetic stability of regenerated plants was also assessed. In the somatic embryo (SE) induction stage, the highest frequency of somatic embryogenesis (35.2%) was observed on the WPM medium containing 1.0 mg L−1 of thidiazuron (TDZ) and 1.0 mg L−1 of 6-benzyladenine (6-BA). The concentration of 6-BA was optimized at 1.0 mg L−1 for the proliferation and maturation of the induced SEs, and the combination of 2.0 mg L−1 of indole-3-butyric acid (IBA) and 0.5 mg L−1 of TDZ was the most responsive for root development and plant growth. The leaf morphological characteristics greatly varied among the established plants, and they could be grouped into three plant types, namely the normal type, Type Ι, and Type ΙΙ. Remarkable differences in the number, size, shape, and color of the leaflets were observed among the three types, while their ploidy level was the same via flow cytometry analysis. The Type ΙΙ and the Type Ι plants had the highest and the lowest net photosynthesis rate, transpiration rate, and stomatal conductance among the three groups, respectively, and both had a smaller size of stomatal guard cells than the normal type. Simple sequence repeat (SSR) analysis detected that 41 bands (43.62%) were different from those observed in the wild, indicating a high degree of polymorphism between the regenerants and their donor parent. The obtained plants might hold potential for future genetic improvement and breeding in A. trifoliata, and the established regeneration protocol might serve as a foundation for in vitro propagation and germplasm preservation of this crop.

Increasing the somatic embryogenesis frequency of Panax vietnamensis Ha et Grushv. by disinfection of leaf explant using nano silver and the addition of nano silver in culture medium

Somatic embryo is a developmental method for mass multiplication of valuable medicinal plants. In this study, leaf explants of Ngoc Linh ginseng were disinfected with nano silver at different concentrations and exposure times to eliminate infectious agents and induce embryogenic callus for the production of somatic embryos. The results show that the lowest contamination rate (20.00%) was observed when leaf explants were treated with 0.5 g/L nano silver for 15 minutes while the highest embryogenic callus induction rate (72.22%) and fresh weight (0.77 g) was determined at 0.2 g/L nano silver for 20 minutes. High frequency of somatic embryogenesis formation and germination were occurred on MS medium supplemented 1.0 mg/L 2,4-D; 0.5 mg/L NAA; 0.2 mg/L Kin and 1.6 mg/L nanosilver. After 8 weeks of culture, the number somatic embryos derived from nano silver treated-leaves was increased 2 times than non-treated explants. Addition of 1.0 mg/L NAA and 1.2 mg/L nano silver was showed the highest shoot and root length, root number, fresh and dry weight of plantlets. This research showed that pre-treatment and supplement of nano silver in culture medium is potentially useful for improving embryogenesis frequency, and plantlet formation of Ngoc Linh ginseng cultured in vitro.

ИЗМЕНЧИВОСТЬ И НАСЛЕДУЕМОСТЬ РЯДА ПРИЗНАКОВ У ПРОСТЫХ ГИБРИДОВ F1 КУКУРУЗЫ ПРИ ПОНИЖЕННОЙ ТЕМПЕРАТУРЕ И В ОБЫЧНЫХ УСЛОВИЯХ

The values of heritability coefficients of parental genotypes were determined for such characters as "frequency of callus formation", "frequency of somatic embryogenesis","frequency of plant regeneration" under normal and stressful in vitro conditions in simple maize F1 hybrids. The highest value of the coefficient of heritability of maternal genotypes in traits studied in vitro was found for “frequency of somatic embryogenesis” at low temperatures. The variability and heritability of characters during the initial stages of plant development under the actionof low temperatures, as well as quantitative characteristics of the plant and male gametophyte, were evaluated. Hybrids in which the inbred lines A239 and 092 were used as maternal genotypes showed under stress higher indices of the studied traits both invitro conditions and at the seed level. For the characters “number of grains per row” and “plant height”, the highest heritability coefficients of the interaction of maternal and paternal genotypes were noted. Hybrids A239xP502 and Co125xMK159 were selected by the higher values of the studied quantitative traits. Hybrid combinations with significantly high meanings of the most of the studied characters can be used to create cold-resistant and productive corn hybrids

Enhanced Secondary Somatic Embryogenesis in Suspension Culture of Four Diploid Banana Cultivars from Kerala

ABSTRACT Secondary somatic embryogenesis was observed in the suspension culture of bract-derived banana calli in the presence of liquid Murashige and Skoog medium with additives such as glutamine (0.68–34.21 µM), biotin (0.40–20.46 µM), or ascorbic acid (0.56–28.38 µM) along with malt extract (100 mg/l). After the initiation of homogenous cell suspension, fully developed somatic embryos were observed after fifth subculture in the four cultivars. Somatic embryos were successfully regenerated in basal MS medium and were transplanted after fifth week. After fifth subculture, the frequency of somatic embryos increased in each culture flask. Maximum number of somatic embryos were observed in cv. Sannachenkadali (46.12 ± 0.85a) when cultured in liquid MS medium supplemented with biotin (8.18 µM) along with malt extract (100 mg/l), in followed by cv. Matti (44.75 ± 1.19a) having (AA) genome. SEM analysis revealed the presence of secondary somatic embryos on the surface of primary somatic embryos in each culture and it attributed a high frequency of somatic embryogenesis. The addition of biotin along with malt extract increased the frequency of secondary somatic embryogenesis in the diploid banana cultivars.

Enhanced Secondary Somatic Embryogenesis in Suspension Culture of Four Diploid Banana Cultivars from Kerala

ABSTRACT Secondary somatic embryogenesis was observed in the suspension culture of bract-derived banana calli in the presence of liquid Murashige and Skoog medium with additives such as glutamine (0.68–34.21 µM), biotin (0.40–20.46 µM), or ascorbic acid (0.56–28.38 µM) along with malt extract (100 mg/l). After the initiation of homogenous cell suspension, fully developed somatic embryos were observed after 5th subculture in the four cultivars. Somatic embryos were successfully regenerated in basal MS medium and were transplanted after fifth week. After 5th subculture, the frequency of somatic embryos increased in each culture flask. Maximum number of somatic embryos were observed in cv. Sannachenkadali (46.12 ± 0.85a) when cultured in liquid MS medium supplemented with biotin (8.18 µM) along with malt extract (100 mg/l), in followed by cv. Matti (44.75 ± 1.19a) having (AA) genome. SEM analysis revealed the presence of secondary somatic embryos on the surface of primary somatic embryos in each culture and it attributed high frequency of somatic embryogenesis. Addition of biotin along with malt extract increased the frequency of secondary somatic embryogenesis in the diploid banana cultivars.

Peculiarities of induction of callusogenesis and morphogenesis in fennel depending on the age of seedlings

The influence of the age of seedlings (of which hypocotyl explants were isolated) and the composition of the culture medium on the callusogenesis and morphogenesis induction of fennel cultivars ‘Mertsishor’ and ‘Oksamit Kryma’ were studied. It was established that with the use of younger seedlings (7-day of age) callus induction began in a larger number of explants for a week of cultivation earlier compared to 14 and 21-day seedlings. The frequency of somatic embryogenesis in the callus obtained from 7-day seedling hypocotyl was almost twice higher than that of the callus from more mature seedling. It was revealed that the cultivar ‘Mertsishor’ had a higher morphogenetic potential compared to ‘Oksamit Kryma’. Improved methods of embryogenic callus induction and plant regeneration can be used for obtaining the initial breeding material of fennel.

Impact of Osmotica and Plant Growth Regulators on Somatic Embryogenesis of Date Palm

An efficient somatic embryogenesis system is reported for date palm cv. Al-Fayda, a genotype resistant to the bayoud disease. Callus induction was achieved from adventitious bud explants cultured for 6 months on semi-solid Murashige and Skoog (MS) medium containing 4.5 μM 6-(dimethylallylamino) purine (2iP) and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram. The highest somatic embryogenesis frequency (89%) was obtained on MS medium supplemented with 225 μM 2,4-D. Subsequently, embryogenic cultures were transferred to agitated liquid MS medium (maturation medium) containing various concentrations of mannitol, polyethylene glycol (PEG) or sorbitol. The highest rate of somatic embryo maturation (71.4 mature embryos per 100 mg callus) was achieved on the medium supplemented with 40 g l-1 PEG. Mature somatic embryos were then transferred to MS medium supplemented with gibberellic acid (GA3) or 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) at various concentrations. The highest frequency of germination and conversion (26%) was obtained on the medium containing 5 μM NAA and 5 μM BAP. The developed plants were then transferred to ex vitro conditions, where a survival rate of 77.02% was observed. The regeneration protocol established in the present investigation will be used for mass propagation of date palm cv. Al-Fayda.

Somatic embryogenesis and in vitro flowering in Hybanthus enneaspermus (L.) F. Muell. – A rare multipotent herb

Present study reports the various factors affecting somatic embryogenesis and in vitro flowering in Hybanthus enneaspermus (L.) F. Muell. The effect of the salts strength of Murashige and Skoog's (MS) medium, concentration of sucrose and plant growth regulators were analyzed for the induction of direct somatic embryogenesis using nodal segments as explants. High frequency of somatic embryogenesis was reported on full strength MS medium (with 3% sucrose) and additives supplemented with 0.5 mg/L 6-benzylaminopurine (BAP) and 0.25 mg/L indole-3-acetic acid (IAA). Maximum somatic embryos (207.0 ± 4.2) were germinated on 1/2 strength MS medium augmented with 0.5 mg/L BAP. Microscopic studies revealed the typical developmental patterns in somatic embryogenesis from globular to heart-shaped and followed by bipolar torpedo-shaped somatic embryos from nodal explants. The plantlets raised from the somatic embryos resulted in flowering on full strength MS medium augmented with 1.0 mg/L each of BAP and Kinetin (Kin) + 0.5 mg/L IAA at 50 μmol/(m2·s) SFPD for 13 h/d photoperiod. About 92% plantlets were successfully acclimatized in the greenhouse. Field transferred plants exhibit normal flowering and fruit setting. The study could be exploited for large scale propagation of true to type plants as conservation strategies of this rare and endemic medicinal plant.

Plant regeneration through somatic embryogenesis and shoot organogenesis from immature zygotic embryos of Sapium sebiferum Roxb.

Sapium sebiferium Roxb. is a highly valued ornamental plant species in tropical and subtropical regions. Here we present an efficient protocol for plant regeneration from immature zygotic embryos (IZEs) of S. sebiferium through direct somatic embryogenesis and shoot organogenesis for the first time. The results showed that mechanical damage, plant growth regulators (PGRs) and light regimes had great influence on in vitro morphogenesis. High frequency of somatic embryogenesis was obtained when the mechanical damaged IZEs were cultured on the Murashige and Skoog (MS) medium containing 1.0mgl−1 indole-3-acetic acid (IAA) under continuous darkness for two weeks followed by a light/dark photoperiod cycle for four weeks. The induction frequency of somatic embryogenesis was up to 90.0%. Somatic embryos (SEs) at early cotyledonary stage showed highest proliferation frequency (81.7%) and 80.0% of secondary somatic embryos (SSEs) germinated on plant growth regulators (PGRs)-free MS medium. For direct shoot induction pathway, the best shoot regeneration rate (63.3%) was obtained on the medium supplemented with 1.0mgl−1 6-benzyladenine (6-BA). The plantlets derived from two ways were successfully acclimatized in the greenhouse with more than 90% survival rate. This study will be beneficial for the rescue of immature embryo, inheritance of heterosis as well as the genetic improvement of S. sebiferium.

Somatic embryogenesis and genetic uniformity of regenerated cassava plants from low-temperature preserved secondary somatic cotyledons

Low plant regeneration and transformation frequencies in cassava have been attributed to recycling of cassava secondary somatic embryos for long periods. This study examined the suitability of low-temperature storage of dehydrated cassava secondary somatic embryos as an alternative short-term conservation approach to recycling. The study included two experiments: in the first one, somatic embryogenesis from cotyledons of dehydrated secondary somatic embryos stored at six temperatures for 4, 8 and 12 months was established; and in the second, plant regeneration via shoot organogenesis using somatic cotyledon explants obtained in the first experiment was examined. The growth, photosynthetic pigment contents and genetic stability of the regenerated plants were assessed. At each storage temperature, the survival of explants, somatic embryogenesis, shoot induction and elongation decreased with increases in storage period. At 4 months after storage (MAS), explant survival, frequency of somatic embryogenesis, shoot induction, elongation and rooting had the best responses at 16EC and 20EC. Similarly, the growth and photosynthetic pigment contents of plants regenerated from somatic embryos preserved at 16EC and 20EC recorded the best results at 4 MAS. However, at 8 and 12 MAS, the best explant survival, somatic embryogenesis, shoot induction and elongation were recorded at 16EC. Eight RAPD primers produced a total of 56 distinct and scorable bands, ranging from 250 bp to 3000 bp in size. An random amplified DNA (RAPD) analysis showed a uniform banding profile among regenerants and to that of the mother plant, indicating no genetic variation among regenerants and between the regenerants and the mother plant. The data presented here suggest that cassava secondary somatic embryos dehydrated to 50% moisture content should be preserved at 16-20EC for 4 months; for 8 and 12 months storage, the embryos should be stored at 16EC. No genetic alteration was detected among the regenerants. Therefore, the method is a suitable means for short-term conservation of cassava secondary somatic embryos.

Embryogenesis and synthetic seed production in Mondia whitei

An effective plant regeneration system via somatic embryogenesis and synthetic seeds was developed for Mondia whitei, an endangered medicinal plant. Friable embryogenic callus was induced by culturing leaf explants on solid Murashige and Skoog (MS) medium containing various concentrations and combinations of sucrose and plant growth regulators. The highest frequency of somatic embryogenesis (100 %) and production of all developmental stages of somatic embryos were obtained on MS medium with 40 g l−1 sucrose, 8 g l−1 agar, 20 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 μM thidiazuron. This was followed by establishment in MS medium with 20 g l−1 sucrose, 8 g l−1 agar, 0.5 μM meta-topolin riboside (mTR) and 0.25 μM indole-3-acetic acid (IAA). All the embryos germinated and produced healthy plantlets on the same medium. Somatic embryos at the heart, torpedo and cotyledonary-stages were collected from media (EDM) containing MS medium plus 20 g l−1 sucrose, 8 g l−1 agar, 0.5 μM mTR and 0.25 μM IAA. The embryos were encapsulated with liquid MS medium plus different concentrations of sodium alginate (SA) and calcium chloride (CaCl2·2H2O) with a 10 min exposure. A combination of 3 % SA and 100 mM CaCl2·2H2O provided higher survival (95.7 %) and germination (73 %) frequencies of synthetic seeds. Germination frequency of synthetic seeds was 51.6 % after 50 days of storage at 4 °C. Somatic embryos and synthetic seed-developed plantlets were successfully acclimatized in the greenhouse with 90 % survival ex vitro. Application of the protocol provides a relatively simple and rapid system for conservation of natural populations for germplasm conservation. Analysis of bioactive compounds and genetic transformation studies can also be performed.

English

An attempt was made to study the somatic embryogenesis and plant regeneration from the in vitro leaf explants of Rumex vesicarius L. a renowned medicinal plant, which belongs to polygonaceae family. Effective in vitro regeneration of R. vesicarius was achieved via young leaf derived somatic embryo cultures. Embryogenic callus was induced from leaf explants on Schenk and Hildebrandt (SH) medium supplemented with various concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D) (0.5 to 3.0 mg/l) along with Kinetin (Kn) (0.5 mg/l). High frequency of somatic embryogenesis was effective on SH medium with 2, 4-D (2.5 mg/l) + Kn (0.5 mg/l) from leaf explants. Secondary somatic embryogenesis was also observed when primary somatic embryos were subculture on the same somatic embryo induction medium. Well developed cotyledonary shaped embryos regenerate 80% of shoots on media containing 2,4-D 0.5 mg/l + 2.0 mg/l BA. The regenerated shoots transferred to rooting media containing Indole- 3- butyric acid (IBA). Efficient rooting of 90% was noted on SH media with 1.0 mg/l IBA. Finally, these in vitro regenerated plantlets were hardened, acclimatized and successfully transferred to the field. The post transplantation survival rate of these regenerated plants was 65 to 70%. The in vitro regenerated plants and flowers were similar to mother plants. This protocol will be useful for genetic transformation experiments in R. vesicarius L. Keywords: Rumex vesicarius L, 2,4-dichlorophenoxy acetic acid (2,4-D), kinetin (Kn), Benzyl adenine (BA), Indole- 3- butyric acid (IBA). African Journal of Biotechnology , Vol 13(45) 4268-4274

Establishment of somatic embryogenesis and podophyllotoxin production in liquid shake cultures of Podophyllum hexandrum Royle

The objective of this study was to establish a simple and efficient method for high frequency of somatic embryogenesis and plant regeneration of Podophyllum hexandrum and podophyllotoxin (PTOX) production. Embryogenic callus (100mg FW) cultured on 0.75 strength MS medium with 4% sucrose and 3g/l PVP produced maximum number of somatic embryos (1089) after five weeks of culture under dark condition. When these somatic embryos were subcultured to the same MS medium with 1mg/l ABA, the embryos matured after 2 weeks of culture. The MS medium supplemented with1.0mg/l GA3 led to high frequency germination (91.1%). The accumulation of PTOX varied in somatic embryos, matured embryos and germinated embryos. Highest PTOX accumulation (2.8mg/l) was recorded in somatic embryos cultured in 0.75 strength MS medium with 8% sucrose. Regenerated plantlets were successfully acclimatized in the growth chamber. The protocol established in this study will be helpful for large scale production of podophyllotoxin.

Plant regeneration through direct somatic embryogenesis from immature zygotic embryos of the medicinal plant, Paris polyphylla Sm.

A protocol for induction of direct somatic embryogenesis and subsequent plant regeneration for the medicinally important and endangered plant Paris polyphylla Sm. has been developed for the first time. Immature zygotic embryos (IZEs) were cultured on different media namely Gamborg (B5), ½ B5, Murashige and Skoog (MS), ½ MS, Chu et al. (N6), ½ N6, Schenk and Hildebrandt (SH) and ½ SH. Highest frequency of somatic embryogenesis (32.6 %) and mean number of somatic embryos (SEs) per explant (28.7 ± 1.7) were obtained on ½ MS medium directly without an intermediate callus phase. The frequency of SE induction was significantly increased to 40.7 % when ½ MS medium was solidified with gelrite compared to agar (32.6 %). Secondary somatic embryos (SSEs) appeared on the primary SEs in a repetitive way on plant growth regulator-free ½ MS medium but with a gradual decrease in embryogenic potential during subsequent subcultures. Plasmolyzing pre-treatment of SSEs with 1.0 M mannitol for 12 h effectively maintains its embryogenic capacity. Primary embryos at the elongated dimpled and early cotyledonary stage displayed the highest embryo forming capacity of 26.94 and 27.87, respectively. High frequency of SE germination (94.0 %) occurred on ½ MS medium with 0.5 mg/l gibberellic acid. Highest percentage of seedling to plantlet conversion was observed in the medium supplemented with 0.05 mg/l 6-benzylaminopurine and 0.1 mg/l α-naphthalene acetic acid. Regenerated plants displayed morphological characteristics similar to that of the wild plants. Flow cytometry analysis showed ploidy stability of the regenerated plants.

In vitro Plant Regeneration through Somatic Embryogenesis in Medicinally Important leaf explants of Coleus forskohlii Briq.

High-frequency somatic embryogenesis and complete plant regeneration from leaf explants of Coleus forskohlii Briq had been established. The influence of plant growth regulators (PGRs) on the frequency of somatic embryogenesis and plant regeneration were studied. Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or both with 6-benzylaminopurine (BAP), were superior for somatic embryogenesis. Primarily, embryogenic callus was initiated from leaf explants of young plant on MS medium supplemented with 2,4-D (1.0mgl -1 ), 3% sucrose and 0.9% agar in three weeks. Embryogenic callus transferred on 2,4-D and BAP-supplemented medium favored induction of embryos in advance as compared to that on 2,4-D alone supplemented medium. The highest frequency (80 %) of direct somatic embryogenesis was obtained in the combination of BAP (1.0 mg/l) and 2,4-D (0.5 mg/l). Eighty percent of the embryos underwent maturation and conversion to plantlets upon transfer to half-strength MS medium having α-naphthaleneacetic acid (NAA), with BAP. Embryo-derived plantlets were transferred onto specially made plastic cup containing soilrite followed by their transfer to the garden soil. Survival rate of the plantlets under ex vitro condition was 80%.

In vitro culture of Cucumis sativus L. VI. Histological analysis of leaf explants cultured on media with 2, 4-D or 2, 4, 5-T

The developmental sequence of callus initiation and somatic embryogenesis in leaf explants of <em>Cucumis sativus</em> cv. Borszczagowski was analysed and compared on media containing two different auxin phenoxy-derivatives (2,4-D and 2,4,5-T) and cytokinin (BAP or 2iP). During the first 20 days of culture on media with 2,4,5-T proliferation of parenchymatic tissue occurred mainly and only small meristematic centers were observed. There was an intensive detachment of parenchymatic cells and dissociation of their cell walls near vessels and in the lower part of the explant adjacent to the medium. These cells were strongly plasmolysed. On the 2,4-D containing medium mostly meristematic tissue developed, proliferating around vascular bundles and forming meristematic centers or promeristem-like structures. After 35-50 days of culture, secondary callus was formed by separation of meristematic cells from the meristem surface in explants cultured on the 2,4-D containing medium. On medium supplemented with 2, 4, 5-T the detachment of parenchymatic and meristematic cells occurred, along with formation of a gel-like substance. The gel-like callus contained multi-cellular aggregates, proembryoids and embryoids. This type of callus tissue was initiated more intensively on medium with 2, 4, 5-T, but the frequency of somatic embryogenesis was much lower. The periferial cells of aggregates, proembryoids and embryoids showed the tendency to separate from the surface of the tissue. Many embryoids formed adventitious embryos.

INDUCTION OF DIRECT SOMATIC EMBRYOGENESIS FROM IMMATURE EMBRYOS OF BRASSICA OLERACEA VAR. SABAUDA L.

An efficient plant propagation system through somatic embryogenesis by using immature zygotic embryos was established in Savoy cabbage (Brassica oleracea var. sabauda L.). The effects of zygotic embryo age, presence of 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) (B5-D) in the culture medium, and the pH (5.0 and 5.8) of the B5 plant growth regulator (PGR)-free (B5-0) induction media were investigated. It appeared that the developmental stage of the immature zygotic embryos used as explants was the most important factor for somatic embryogenesis. Highest frequency of somatic embryogenesis (86.67%) was achieved directly on zygotic embryos at cotyledonary stage (1.8 mm long) of development that cultured on B5-0 medium. Optimal pH of the medium was 5.0. Mean number of primary somatic embryos per explant on B5-0 pH 5.0 medium was 7.2. The inclusion of 2,4-D into the induction medium significantly reduced both frequency of somatic embryogenesis (to 53.34 %) and mean number of embryos per explant (to 2.62) in cotyledonary zygotic embryos. After transferring to PGR-free Murashige and Skoog (MS-0) medium 69.69% of the primary somatic embryos of the Savoy cabbage produced secondary somatic embryos (SSEs) Effective recurrent somatic embryogenesis may be an appropriate practical solution for clonal propagation and genetic modifications of Savoy cabbage.

High frequency regeneration via direct somatic embryogenesis and efficient Agrobacterium- mediated genetic transformation of tobacco

A direct somatic embryogenesis protocol was developed for four cultivars of Nicotiana species, by using leaf disc as an explant. Direct somatic embryogenesis of Nicotiana by using BAP and IAA has not been investigated so far. This method does not require formation of callus tissues which leads to somaclonal variations. The frequency of somatic embryogenesis was strongly influenced by the plant growth hormones. The somatic embryos developing directly from explant tissue were noticed after 6 d of culture. Somatic embryogenesis of a high frequency (87–96%) was observed in cultures of the all four genotypes (Nicotiana tabacum, N. benthamiyana, N. xanthi, N. t cv petihavana). The results showed that the best medium for direct somatic embryogenesis was MS supplemented with 2.5 mg/l, 0.2 mg/l IAA and 2% sucrose. Subculture of somatic embryos onto hormone free MS medium resulted in their conversion into plants for all genotypes. About 95% of the regenerated somatic embryos germinated into complete plantlets. The plants showed morphological and growth characteristics similar to those of seed-derived plants. Explants were transformed using Agrobacterium tumifacious LBA4404 plasmid pCAMBIA1301 harboring the GUS gene. The regenerated transgenic plants were confirmed by PCR analysis and histochemical GUS assay. The transformation efficiency obtained by using the Agrobacterium- mediated transformation was more than 95%. This method takes 6 wk to accomplish complete transgenic plants through direct somatic embryogenesis. The transgenic plantlets were acclimatized successfully with 98% survival in greenhouse and they showed normal morphological characteristics and were fertile. The regeneration and transformation method described herein is very simple, highly efficient and fast for the introduction of any foreign gene directly in tobacco through direct somatic embryogenesis.

Efficient callus induction and plantlets regeneration in durum wheat using mature embryos

The use of mature embryos as explants to initiate cultures is a best alternative to save time and costs, especially for producing somatic embryos for genetic transformation of durum wheat. However, plantlets regeneration from cultures derived from matured embryos is usually low. In this study, we tested matured embryos as explants from eight Moroccan durum wheat varieties (‘Irden’, ‘Marzak’, ‘Kyperounda’, ‘Isly’, ‘Amria’, ‘Karim’, ‘Marouane’ and ‘Tomouh’) to define suitable culture media for obtaining high frequencies of somatic embryogenesis and in vitro plantlets regeneration. For this purpose, we tested five induction and maintenance media (M1 to M5) based on MS media (macro and oligo-elements) which differed with respect to concentrations of plant hormones (2,4-D and BA), vitamins, sucrose, maltose, L-asparagine, and solidifying agents. All tested media induced embryogenic callus for the varieties and regenerate plantlets. However, a significant effect of variety, medium and variety × medium interaction were observed for callus induction and regeneration. Average callus growth as measured by relative fresh weight growth rate (RFWGR) across different media was the highest for ‘Amria’ (7215.4%) and the lowest for ‘Tomouh’ (2088.2%). M1 (2 mg/L 2,4-D) and M5 (3 mg/L 2,4-D) media gave highest RFWGR(6892.1% and 6332%, respectively) and M3 (1 mg/L 2,4-D) was the lowest (3708.8%), across different varieties. However, the embryogenic callus from M3 media regenerated the highest percentage of plantlet, upon transfer to regeneration medium, for most of the varieties. For the varieties ‘Marouane’, ‘Kyperounda’, ‘Marzak’, ‘Karim’, and ‘Tomouh’, the favourable medium was M3, whereas, for ‘Isly’, ‘Irden’ and ‘Amria’, both M2 (2.5 mg/L BA and 2.5 mg/L 2,4-D) and M3 were the favourable media for embryogenic callus induction. In this study, for the first time, favourable media for induction and regeneration from mature embryo of Moroccan durum wheat varieties were identified. These media will be used for callus induction and genetic transformation.

The positive effect of arabinogalactan on induction of somatic embryogenesis in Quercus bicolor followed by embryo maturation and plant regeneration

This is the first report on the successful induction of somatic embryogenesis in swamp white oak from leaf and shoot apex explants excised from in vitro shoot cultures derived from 6- to 7-year-old trees. We demonstrated that arabinogalactan from larch wood (2–4 mg/L) promoted embryogenesis in the three genotypes evaluated by increasing the frequency of somatic embryogenesis, the embryogenic sites per explant, and by speeding the onset of embryo initiation. The explants were cultured sequentially on three culture media consisting of Murashige and Skoog (MS) salts and vitamins supplemented with 500 mg/L casein hydrolysate and different concentrations of α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA). Somatic embryogenesis induction frequencies of up to 12.4, 4.5, and 0.7 % were obtained for the three genotypes. Clonal embryogenic lines were maintained by repetitive embryogenesis following culture on MS medium containing 0.44 μM BA with or without 0.27 μM NAA. Before germination, cotyledonary-stage embryos were cultured for 4 weeks in maturation medium (MS medium with half-strength macronutrients) containing 6 % sorbitol. Germination response was significantly improved by applying a 2-month cold storage as a post-maturation treatment. The mineral formulation and plant growth regulator content of the germination medium influenced the frequency of plantlet conversion with the best results achieved on Gresshoff and Doy medium with BA (0.25–0.44 μM). This procedure resulted in over 50–60 % of germinating embryos exhibiting continuous root growth and either epicotyl elongation or shoot development.