Efficient Somatic Embryogenesis Research Articles

Advancing Hemidesmus indicus propagation and conservation: Somatic embryogenesis, histology, metabolite assessment and genetic stability

Hemidesmus indicus L. R. Br. is a commonly traded ethnomedicinal plant, and a method has been developed to produce somatic embryos (SEs) from calli grown from the leaves of the plant. With a maximum callus induction frequency of 91.66 %, pink-red friable proembryogenic calli were generated on Murashige and Skoog (MS) and 5.0 µM 2, 4-dichlorophenoxyacetic acid (2, 4-D) from the leaf explant (LE). With the aim of achieving a high success rate of 95.00 % for the induction of SEs, with a yield of 91.33 SEs per 100 mg of callus, the proembryogenic calli were transferred to fresh culture medium containing MS and 5.0 µM 6-benzylaminopurine (BA). This induction medium allowed the embryos to develop to the torpedo stage, but their further growth into emblings required a different culture medium. ½ MS with 5.0 µM BA and 1.0 µM gibberellic acid (GA3) resulted in 99.16 % maturation and 39.66 emblings regeneration out of 40 immature embryoids, respectively. With a maximum survival rate of 91.67 %, healthy emblings were successfully acclimatized using Soilrite. DNA-based molecular markers (RAPD and ISSR) were used to confirm the genetic similarities of the in vitro grown plants. Histological examinations were performed to determine the ontogeny, morphology, and development of somatic embryoids. Gas chromatography and mass spectrometry (GCMS–) were performed for both the mother and the in vitro generated plant to analyse the root for secondary metabolites. In the physiological and biochemical studies, enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR) were found to increase in vitro derived H. indicus plants during different acclimatization periods (0, 7, 14, 21 and 28 days). With increasing acclimatization time, net photosynthetic rate (PN) and total chlorophyll show a decrease at first, then an increase. The protocol provides high callus induction rates, efficient somatic embryogenesis, successful maturation and regeneration, robust acclimatization, genetic fidelity, detailed insight into development, and screening of secondary metabolites.

Establishment of an Efficient Somatic Embryogenesis Protocol for Giant Reed (Arundo donax L.) and Multiplication of Obtained Shoots via Semi-Solid or Liquid Culture

This study developed an efficient protocol for the in vitro propagation of giant reed (Arundo donax L.) biomass, defining a complete cycle of the induction of somatic embryogenesis from immature inflorescences, followed by the maturation of somatic embryos and the subsequent multiplication of the derived shoots in liquid culture in a temporary immersion system (TIS). The best explants were found to be 30 cm long immature inflorescences, preferably collected in spring. Such an explant type was easy to decontaminate, and the spikelets isolated from it provided over 100 embryogenic callus lines. Among the callus induction media tested, gelled MS medium supplemented with 1.1 mg/L 2,4-D provided the highest percentage of responsive spikelets and the highest density of embryogenic callus. Maturation of the embryogenic callus was easily triggered on gelled MS medium devoid of plant growth regulators. The obtained shoots could be further multiplied on previously optimized gelled DKW medium supplemented with 30 g/L sucrose, 5 mg/L BA, 0.1 mg/L IBA, and 6.8 g/L plant agar. Subsequent high multiplication of the developed shoots was achieved in liquid culture in TIS using a Plantform™ bioreactor, with an immersion cycle of 12 min every 8 h.

Efficient Somatic Embryogenesis, Regeneration and Acclimatization of Panax ginseng Meyer: True-to-Type Conformity of Plantlets as Confirmed by ISSR Analysis

Panax ginseng Meyer grows in east Russia and Asia. There is a high demand for this crop due to its medicinal properties. However, its low reproductive efficiency has been a hindrance to the crop's widespread use. This study aims to establish an efficient regeneration and acclimatization system for the crop. The type of basal media and strength were evaluated for their effects on somatic embryogenesis, germination, and regeneration. The highest rate of somatic embryogenesis was achieved for the basal media MS, N6, and GD, with the optimal nitrogen content (≥35 mM) and NH4+/NO3- ratio (1:2 or 1:4). The full-strength MS medium was the best one for somatic embryo induction. However, the diluted MS medium had a more positive effect on embryo maturation. Additionally, the basal media affected shooting, rooting, and plantlet formation. The germination medium containing 1/2 MS facilitated good shoot development; however, the medium with 1/2 SH yielded outstanding root development. In vitro-grown roots were successfully transferred to soil, and they exhibited a high survival rate (86.3%). Finally, the ISSR marker analysis demonstrated that the regenerated plants were not different from the control. The obtained results provide valuable information for a more efficient micropropagation of various P. ginseng cultivars.

In Vitro Propagation by Axillary Shoot Culture and Somatic Embryogenesis of Daucus carota L. subsp. sativus, ‘Polignano’ Landrace, for Biodiversity Conservation Purposes

Carrots are a multi-purpose vegetable; hence, they have become a very popular consumers’ choice, also thanks to their nutritional value with health benefits. Several varieties and landraces exist of carrot (Daucus carota L. ssp. sativus). In Apulia (Italy), for example, a multicolored carrot landrace is present, the Polignano carrot (PC), threatened by the risk of genetic erosion. With the purpose of verifying the possibility of using in vitro culture for ex situ conservation purposes of PC, a rapid and reliable protocol for micropropagation and a highly efficient somatic embryogenesis protocol have been set up. For micropropagation studies, axillary buds from taproot were used as initial explants. Supplementation on basal medium (BM, a Murashige–Skoog modified medium) with low cytokinine (6-benzyladenine, BA, 0.22 μM) dosage was enough to obtain a good multiplication rate, also depending by the PC color. Rooting of proliferated shoots occurred easily in hormone-free medium and acclimatization was successfully reached in 6 weeks. Somatic embryos were formed from embryogenic callus induced by taproot sections placed on 2,4-dichlorophenoxyacetic acid (2,4-D)-added medium. After in vitro conversion in hormone-free medium, somatic embryos were also successfully acclimatized. Both microplants and somatic embryos produced true-to-type taproots (multicolored carrots), demonstrating the suitability of in vitro techniques for biodiversity conservation purposes.

Towards development of an efficient somatic embryogenesis protocol for the palm tree Euterpe precatoria (Mart.) from leaf tissues of adult plants

Towards development of an efficient somatic embryogenesis protocol for the palm tree Euterpe precatoria (Mart.) from leaf tissues of adult plants

Plant Regeneration of Malaysian Aromatic Rice MRQ88 Via Somatic Embryogenesis

Aromatic rice has become one of the most sought-after sources by today's consumers. As a result, by utilising mature seeds, an efficient somatic embryogenesis system for the MRQ88 variety has been constructed. Due to the highest percentage of callus initiation and no browning, MS-B5 medium supplemented with 1 mg/L 2,4-D and 10 mg/L NAA was the best medium for MRQ88 callus induction. Incubation of embryogenic callus on identical medium containing maltose 10 g/L and ABA 10 mg/L for 8 weeks resulted in the highest proportion of somatic embryo initiation (80%). The number of regenerated green plantlets on medium containing NAA and 2,4-D which was previously pre-treated with 5 to 10 mg/L ABA, 10 g/L maltose, showed good results, with 8-9 green plantlets developed.

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

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

Somatic Embryogenesis and Indirect In Vitro Plant Regeneration in Amorphophallus konjac K. Koch by One-Step Seedling Formation

Konjac (Amorphophallus konjac K. Koch) is a well-known tuberous vegetable belonging to the important medicinal family Araceae, and the plant grows from an underground tuber. Here, we used a “one-step seedling regeneration” tissue culture system to improve the plantlet regeneration efficiency of konjac using young leaves as an explant source. In the current study, we used several sterilization methods for tuber sterilization. Moreover, various plant growth regulator combinations were applied to achieve efficient somatic embryogenesis and plantlet regeneration. Our results showed that the optimal tuber sterilization was method C (75% alcohol for 15 s + 0.1% HgCl2 for 15 min + washing by double-sterilized water three times). Three types of embryogenic calli were induced on full-strength Murashige and Skoog (MS) basal medium supplemented with 0.5 mg/L of 6-benzylaminopurine (6-BA), 0.5 mg/L of naphthaleneacetic acid (NAA), 1.0 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D), and 30 g/L of sucrose. Of the three types of embryogenic calli, only type Ⅲ further regenerated plantlets, with a callus induction rate of 55.73% and a seedling induction rate of 92.73%. This suggests that the addition of the above hormones gives the optimal callus induction. The proliferation rate achieved was 38% on the MS basal medium containing 1.0 mg/L of 6-BA, 1.0 mg/L of indolebutyric acid (IBA), 0.2 mg/L of kinetin (KT), and 50 g/L of sucrose. The one-step seedling formation achieved in MS medium contained 2.0 mg/L of 6-BA, 0.5 mg/L of NAA, 0.1 mg/L of gibberellic acid (GA3), and 30 g/L of sucrose, and the number of regenerated shoots per explants was 6 ± 2. Therefore, we establish a one-step seedling regeneration system through indirect plant regeneration, which shortens the time for konjac in vitro regeneration, significantly increased the micropropagation efficiency, and decreased the cost of the konjac tissue culture.

Somatic embryogenesis of Eleusine coracana on inducted seed explants

Abstract. Gebremedhen H, Feyissa T. 2021. Somatic embryogenesis of Eleusine coracana on inducted seed explants. Cell Biol Dev 5: 26-31. Finger millet (Eleusine coracana (L.) Gaertn.) is one of the most important crops in Africa and India. E. coracana contains different nutrients and is a popular food among diabetic patients in different countries. E. coracana is an orphan crop in the developing world on which little research has been done. The improvement of this crop is crucial for abiotic and biotic stress tolerance. Tissue culture and transformation techniques are among the approaches to improve this crop. Therefore, the objective of this study is to develop an efficient somatic embryogenesis protocol for the regeneration of different E. coracana cultivars. Somatic embryogenesis protocol for regeneration of four cultivars of E. coracana is developed. For all cultivars, mature seeds were used for callus induction. After sterilization of the seeds, callus was induced efficiently in different concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D) in all cultivars. From 95 to100% callus induction were recorded on callus induction medium containing 1.0, 1.5, 2.0, 2.5 and 3.0 mg/L 2,4-D. MS-medium supplemented with 1 mg/L 2,4-D resulted in 45 to 58.3% somatic embryogenesis, and 1 mg/L KN in combination with 0.25 mg/L NAA resulted in the highest mean number (15.85±0.63) of shoots and 16.74±1.86 roots were recorded from AAUFM-19.

Maturation and Conversion of Somatic Embryos Derived from Seeds of Olive (Olea europaea L.) cv. Dahbia: Occurrence of Secondary Embryogenesis and Adventitious Bud Formation.

Maturation and conversion of somatic embryos are two crucial steps that hamper the development of efficient somatic embryogenesis systems in olive. Herein, a simple and efficient protocol for the maturation and conversion of olive somatic embryos is reported. Globular somatic embryos derived from seeds of cv. Dahbia were cultured on either half-strength olive (OM) or olive cyclic embryogenesis (ECO) media, with and without plant growth regulators (PGRs). The embryos reached the cotyledonary stage in 9 weeks, but those cultured on ECO medium containing 0.1 mg·L−1 6-(dimethylallylamino)purine (2iP), 0.1 mg·L−1 6-benzyladenine (BA) and 0.05 mg·L−1 indole-3-butyric acid (IBA) exhibited the largest sizes, with an average of 4.7 mm. Somatic embryo conversion into plantlets was evaluated using different culture media (half-strength OM or one-third strength Murashige and Skoog (MS)), light conditions (light or dark) and desiccation pretreatments. The highest rate of somatic embryo conversion (45%) was observed under a 16 h photoperiod on half strength OM medium containing 0.1 mg·L−1 gibberellic acid (GA3) and 0.1 mg·L−1 1-naphthalene acetic acid (NAA). The embryos that failed to germinate showed either necrosis, cotyledon greening with no further conversion, adventitious bud formation or secondary embryogenesis. The findings of this study will be beneficial for biotechnological applications in olive.

Somatic Embryogenesis from Mature Embryos of Olea europaea L. cv. 'Galega Vulgar' and Long-Term Management of Calli Morphogenic Capacity.

Several olive cultivars, characterized by high-quality olive oil show agronomical issues such as excessive vigor, high susceptibility to biotic and abiotic stresses, and low propagation ability. They are strong candidates for breeding based on new technologies to improve their performance in a short period of time. For this reason, the first step is developing efficient somatic embryogenesis (SE) protocols. Somatic embryogenesis in olive is highly genotype-dependent for both adult tissues and mature embryos as initial explants, requiring the development of specific protocols for each genotype. Trials using cotyledons and radicles as initial explants, isolated from ripe seeds from the Portuguese olive cv. ‘Galega vulgar’, gave more than 95% calli development. Radicles proved to be the most responsive tissue for SE induction, with an average of 2 embryos per callus after callus transfer to expression medium, and 14 embryos per callus after subculture on the olive cyclic embryogenesis medium (ECO). Embryogenic competence could be recovered after several subcultures on ECO medium that maintained cyclic embryogenesis for an indeterminate period of time. Embryo conversion and plant acclimatization were also attained with high success rates. Media management for cyclic embryogenesis maintenance is of general importance for SE protocols in any olive genotype. Somatic embryogenesis was thus attained for the first time in embryo-derived explants of cv. ‘Galega vulgar’.

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

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

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.

An Efficient Somatic Embryo Liquid Culture System for Potential Use in Large-Scale and Synchronic Production of Anthurium andraeanum Seedlings.

Anthurium andraeanum Lind. is the second most important tropical flower in the world flower market. Somatic embryogenesis and plant regeneration in Anthurium has been reported previously; however, a stable and effective method for its commercial use has not been available. In this study, an efficient somatic embryogenesis and liquid culture system for large-scale production of A. andraeanum seedlings was achieved. Building on previous research, this study investigated the main factors for proembryogenic mass (PEM) proliferation, somatic embryo (SE) development, and SE germination in Anthurium. The results showed that relatively low concentrations of plant growth regulators, mineral nutrition, and sucrose promoted PEM proliferation, SE formation, and germination in a liquid culture system. This system can be described as follows: PEMs were induced from leaf blade explants on Murashige & Skoog (MS) medium with half-strength MS macronutrients (1/2 MS) containing 2.0 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg L-1 kinetin (KT), and 3% sucrose and were proliferated in ½ MS liquid medium containing 1.0 mg L-1 2,4-D, 0.5 mg L-1 KT, and 3% sucrose. The highest proliferation coefficients were 5.11–5.16. PEMs were then transferred to MS medium with 1/8 MS macronutrients (1/8 MS) liquid medium containing 1% sucrose to develop into globular embryos and mature embryos. Finally, the mature embryos were placed on four layers of absorbent filter paper saturated with 1/8 MS liquid medium containing 1% sucrose for germination, and an average of 60 seedlings per gram SEs was obtained. This liquid culture system can be used in large-scale and synchronic production of Anthurium seedlings.

Identification and characterization of cell cultures with various embryogenic/regenerative potential in cotton based on morphological, cytochemical, and cytogenetical assessment

Somatic embryogenesis (SE) plays a vital role in genetic transformation and massive propagation of important agronomical and economical crops. Here, we conducted a systematic assessment of the morphological, cytochemical, and cytogenetical characteristics of six culture strains with various embryogenic/regenerative potential during SE process in cotton. Results indicated that the six cell culture strains had stable ploidy levels, and did not reveal any relationship between the cytogenetic state and their morphogenetic potential. Moreover, the six culture strains were compared via double staining with Evans blue and Acetocarmine to efficiently distinguish embryogenic and non-embryogenic cells and determine the embryogenic nature of the calli. In addition, the kind of auxins added in medium affected not only growth property, color, size of cell clumps but also ploidy level and regeneration ability. By combining analysis of morphological, cytochemical, and cytogenetical characteristics of the cell cultures, we are able to obtain and maintain homogeneous cell population with high morphogenic and regeneration ability and establish efficient somatic embryogenesis and regeneration system from short-term cell cultures in upland cotton, which highlight the application of biotechnological approaches in crop breeding, and above all, to better understand totipotency of cells in higher plants.

Transcriptome analysis of Lilium Oriental × Trumpet hybrid roots reveals auxin-related genes and stress-related genes involved in picloram-induced somatic embryogenesis induction

ABSTRACTHighly efficient somatic embryogenesis (SE) initiation systems are crucial for the clonal propagation and genetic transformation of Lilium Oriental × Trumpet hybrid. To elucidate the underlying mechanism of SE induction, we conducted de novo transcriptome sequencing of the roots at four different times in relation to SE initiation (0 days, control; 20 days, embryogenic callus induction (ECI) 1; 40 days, ECI2; 55 days, ECI3). A total of 4621, 7582, and 7427 differentially expressed genes (DEGs) were identified from the ECI1 vs. control, ECI2 vs. control, and ECI3 vs. control comparisons, respectively. The gene-enrichment analysis revealed 54 significant DEGs. Further analysis showed that the transcript levels of the auxin-related genes ARF9/15/16, the abscisic acid-related (ABA-related) genes PYL4/9, and stress-related transcription factors, including WRKY26, MYB36, NAC100, bHLH2/5/18/112, and HSP90, were increased at the ECI1 stage. ABI5 can influence the expression of stress-responsive genes as an ABA signalling regulator. The upregulated ABI5 could be caused by the increased ARF9/15/16, PYL4/9, WRKY26, MYB36, NAC100, bHLH2/5/18/112, and HSP90 at the ECI2 and ECI3 stages to regulate the ECI. These genes had the most abundant transcripts among the DEGs, indicating that auxin and stress-related signalling pathways are involved in SE initiation of Lilium spp.

Somatic embryogenesis of Limonium sinense: a study on the regeneration efficacy via direct and indirect approach followed by Agrobacterium-mediated genetic transformation

ABSTRACTAn efficient indirect somatic embryogenesis and Agrobacterium-mediated transformation protocol for Limonium sinense has been established, wherein neomycin phosphotransferase II (npt II) and β-glucuronidase (GUS) genes were used as selectable and screenable markers, respectively. The efficiency of plantlet regeneration from transformed tissue was compared between direct embryogenesis from leaf and indirect embryogenesis from callus. Embryogenic callus (EC) was initiated from leaf explants on MS medium supplemented with 6.7 μM 2,4-D and 2.22 μM BA. The somatic embryos were induced, matured, and germinated when ECs were transferred onto MS medium supplemented with 4.44 μM BA and 1.07 μM NAA. Agrobacterium tumefaciens strain LBA 4404 containing the vector pBI121 was used for the transformation. Transient GUS expression frequency was evaluated and putative transgenic plants were successfully grown on culture medium in presence of kanamycin (80–100 mg L−1). PCR analysis of putative transgenic plants confirmed the presence of GUS and nptII genes. The transformation efficiency obtained through indirect embryogenesis from calluses (4%) was much higher than through direct embryogenesis from leaf explants (0.9%).

Induction of Somatic Embryogenesis in Sengon (Falcataria moluccana) With Thidiazuron and Light Treatments

Falcataria moluccana is important for reforestation and afforestation in Indonesia. However, epidemic of gall rust disease in F. moluccana plantations decreases its productivity. Genetic engineering is an alternative solution to against gall rust disease. Somatic embryogenesis is an efficient in vitro plant regeneration for successful plant improvement through genetic engineering. The objective of this study was to investigate the effect of thidiazuron and light treatments on the induction of somatic embryogenesis of F. moluccana. The effects of thidiazuron concentration (5, 10 or 15 μM) and light (continuous light, 7 days of dark followed by light, or continuous dark) on the induction of somatic embryogenesis in leaf explants were assessed. The highest production of somatic embryos was obtained in 5 μM thidiazuron and dark treatments for 7 days followed by light in Murashige and Skoog medium supplemented with 1.2 g/L proline. Histological analysis in globular and cotyledon stages confirmed that cells had progressed to secondary somatic embryogenesis. This research needs more improvements to become a successful and efficient somatic embryogenesis method and as a potential method for successful plant improvement through genetic engineering in F. moluccana.

Somatic Embryogenesis of Immature Cunninghamia lanceolata (Lamb.) Hook Zygotic Embryos

Two efficient somatic embryogenesis systems were developed in Chinese fir, the most important conifer for industrial wood production in China. Three development stages (cleavage polyembryony, dominant embryo, and precotyledon) of immature embryos derived from 25 genotypes of open-pollinated mother trees were used as initial explants. Cleavage polyembryony-stage embryos with a 12.44% induction rate was the most embryogenic response stage. The highest frequency of embryogenic callus (13.86%) induction was obtained from DCR medium supplemented with 1.5 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.3 mg L−1 kinetin (KN). An average of 53.33 early somatic embryos were produced from approximately 0.2 g (fresh weight) embryogenic callus after 2 weeks of incubation on medium supplemented with 50 μmol L−1 abscisic acid (ABA) and 100 g L−1 polyethylene glycol (PEG) 6000. About 53% dominant embryos have an embryogenic response after a 6-week cultivation on medium supplemented with 1.0–2.0 mg L−1 benzyladenine (BA), 0.2 mg L−1 naphthylacetic acid (NAA) or 2,4-D, and 0.004 mg L−1 thidiazuron (TDZ). After three successive transfer cultures on medium supplemented with 1.5 mg L−1 BA, 0.2 mg L−1 NAA, and 0.004 mg L−1 TDZ, 4.49–16.51% of the embryos developed into somatic embryos.

High efficient somatic embryogenesis development from leaf cultures of Citrullus colocynthis (L.) Schrad for generating true type clones.

We report an efficient somatic embryogenesis and plant regeneration system using leaf cultures of Citrullus colocynthis (L.) and assessed the effect of plant growth regulators on the regeneration process. Initially leaf explants were cultured on Murashige and Skoog medium supplemented with different concentrations of auxins viz., 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid, gibberellic acid alone and along with combination of 6-benzylaminopurine. The different forms of calli such as compact, white friable, creamy friable, brownish nodular, green globular and green calli were induced from the leaf explants on MS medium containing different concentrations of auxins and gibberellins. Subsequently initial callus was subcultured at 1.5mgL(-1) BAP+1.0mgL(-1) 2,4-D which resulted in 25% somatic embryos from 85% nodular embryogenic nodular callus that is highest percentage. Similarly the lowest percentage of somatic embryos was recorded at 2.5mgL(-1) BAP+0.5mgL(-1) NAA from 55% embryogenic globular callus i.e., 16%. High frequency of embryo development takes place at intermittent light when compared with continuous light in the individual subcultures. The cotyledonary embryos were developed into complete platelets on MS medium. In vitro regenerated plantlets were washed to remove the traces of agar and then transferred to sterile vermiculite and sand (2:1) containing pot.