Proliferation Of Somatic Embryos Research Articles

Molecular Mechanism of ARF5-AHL15-Mediated Auxin-Induced Embryogenic Cell Formation in Apples.

The somatic embryo (SE) has bipolar characteristics, which is an ideal material for large-scale microproduction of woody plants represented by apples, and the somatic embryo is also an excellent receptor for genetic transformation. The formation of embryogenic cells is a prerequisite for somatic embryogenesis to occur. The embryogenic cells of apples cannot be obtained without induction of exogenous auxin, but how the auxin pathway regulates this process remains unknown. In this study, via RNA sequencing, MdARF5 and MdAHL15 were identified as differentially expressed genes involved in this process. Overexpression of MdARF5 and MdAHL15 induced the formation and proliferation of embryogenic cells and thus substantially shortened the induction cycle and improved the somatic embryo proliferation efficiency. A yeast one-hybrid assay showed that MdARF5 can directly bind to the promoter of MdAHL15. β-Glucuronidase (GUS) and dual-luciferase reporter assays revealed that MdARF5 activation of MdAHL15 transcription was substantial. In conclusion, our results suggest that MdAHL15 is induced by auxin and promotes the formation of embryogenic cells in early somatic embryogenesis via the positive regulation of MdARF5 in apples. The results will provide a theoretical basis for somatic embryogenesis-based development, reproduction, and transgenic breeding in apples.

DETEKSI VARIASI SOMAKLONAL PLANLET Jatropha curcas Linn. HASIL REGENERASI EMBRIO SOMATIK DENGAN MARKA MOLEKULAR ISSR

Physic nut (Jatropha curcas Linn.) has the potential as a source of sustainable biofuels. Somatic embryo proliferation of J. curcas may cause somaclonal variations. This research aimed to investigate somaclonal variations of J. curcas somatic embryo derived-plantlet using ISSR markers. Somatic embryos of J. curcas at the globular phase were cultured on liquid MS medium supplemented with 0, 0.5, 1.0, 1.5, and 2.0 mg L-1 of 2,4-D. Parameter observed were embryos weight, embryos volume, colour, and size of embryos. After proliferation, the embryos were cultured on a germination medium until the cotyledonary phase. The results showed that proliferation of J. curcas somatic embryos was optimal, with the highest weight and volume, at MS medium added with 1 mg L-1 2,4-D.

Effect of Forchlorfenuron on Somatic Embryo Proliferation and Plantlet Regeneration in Oil Palm SUP-PSU1

In vitro propagation of palm oil is still an obstacle for mass propagation due to the low plant regeneration frequency. The objective of this study was to investigate the effect of N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) on a somatic embryo (SE) proliferation and plant regeneration of oil palm SUP-PSU1. At an approximate size of 5 mm, SE from chopping with a sharp razor blade at 100 times was cultured on oil palm culture medium (OPCM) with 0.1 mg/L dicamba and different concentrations CPPU. The results showed that 0.3 mg/L CPPU gave the highest SE proliferation at 100 % and the number of SEs at 5.16 embryos/tube after 4 weeks of culture. SE was transferred to Murashige and Skoog (MS) medium supplemented with 0.2M sorbitol, a secondary somatic embryo (SSE), which was induced at 66.67 % with 21.2 embryos/tube after 8 weeks. Furthermore, cultured SSE on PGR-free MS medium for 8 weeks gave the highest germination rate at 65 %, with the number of shoots at 2.29 shoots. Thus, this study provides new information for improving the plantlet regeneration system through somatic embryogenesis in oil palm.

Efficient Transformation of Somatic Embryos and Regeneration of Cork Oak Plantlets with A Gene (CsTL1) Encoding a Chestnut Thaumatin-Like Protein.

We present a reproducible procedure for transforming somatic embryos of cork oak with the CsTL1 gene that codes for a thaumatin-like protein, in order to confer tolerance to Phytophthora cinnamomi. Different concentrations/combinations of the antibiotics carbenicillin and cefotaxime, as bacteriostatic agents, and kanamycin, as a selective agent, were tested. A lethal dose of 125 mg/L kanamycin was employed to select transgenic somatic embryos, and carbenicillin was used as a bacteriostatic agent at a concentration of 300 mg/L, which does not inhibit somatic embryo proliferation. The transformation efficiency was clearly genotype-dependent and was higher for the TGR3 genotype (17%) than for ALM80 (4.5%) and ALM6 (2%). Insertion of the transgenes in genomic DNA was confirmed by PCR analysis, whereas expression of the CsTL1 gene was evaluated by semi-quantitative real-time PCR (qPCR) analysis. A vitrification treatment successfully cryopreserved the transgenic lines generated. The antifungal activity of the thaumatin-like protein expressed by the gene CsTL1 was evaluated in an in vitro bioassay with the oomycete P. cinnamomi. Of the eight transgenic lines analyzed, seven survived for between one or two times longer than non-transgenic plantlets. Expression of the CsTL1 gene and plantlet survival days were correlated, and survival was generally greater in plantlets that strongly expressed the CsTL1 gene.

An efficient bioreactor platform for scaling up the proliferation of Nordmann fir’s (Abies nordmanniana) somatic embryos

Increased market demand for high-quality Nordmann fir (Abies nordmanniana) Christmas trees, achieving reduced production costs and improved pest resistance, necessitates the establishment of efficient mass micropropagation platforms for the species. Somatic embryogenesis is a powerful tool for clonal propagation of woody plants. Utilizing the advantages of liquid culture media over solid media, the scalability of Nordmann fir somatic embryo proliferation was investigated through developing a batch culture using the disposable WAVE bioreactor (DWB) system, without feedback controls for pH and dissolved oxygen. This study revealed that the inoculation of a 10-L cellbag with 1 g L−1 FW (fresh weight) of A. nordmanniana embryogenic callus can produce ~ 70,000–111,000 vigorous and synchronized somatic embryos. Such outcomes were achieved with the rock speeds of 25 and 35 rpm and rock angle of 6° for 25 days at 24 ± 1 °C, respectively. The gas mix was a combination of O2 (21%) and a stepwise reduction of CO2 ended by 2% (v/v), at a flow rate of 0.2 L min−1. The application of 500 mg L−1 of 2-(N-morpholino)ethanesulfonic acid (MES) effectively stabilized the pH between 5.3 and 5.4. Furthermore, DWB was found an efficient system for accelerating and scaling up the proliferation of A. nordmanniana somatic embryos, as it is a high-yielding, user-friendly, functional, and rapid alternative to conventional methods with solid media.

Somatic embryogenesis and shoot organogenesis from the hypocotyl slices and free radical scavenging activity of regenerants of collard greens (Brassica oleracea L. var. acephala)

Brassica oleracea var. acephala is an important leafy vegetable that has been widely consumed as a high-nutrient, low-calorie food. Because of the plant’s biennial and self-incompatibility nature, biotechnological approaches are alternative way for propagation and breeding improvements. Since tissue culture studies have been focused in other B. oleracea representatives, the aim of the present study was to achieve effective regeneration protocol distinctive for collard greens, and evaluate the total phenolic content and antioxidant activity of regenerants. The effect of 3 cytokinins [thidiazuron (TDZ), 6-benzyladenine (BA) and 6-furfuryladenine (kinetin, KIN)] at increasing concentrations (0, 5, 7.5, 10, 20 or 30 µM) in combination with tenfold lower concentration of 1-naphtaleneacetic acid (NAA) (0, 0.5, 0.75, 1, 2 or 3 µM, respectively) on the regeneration from hypocotyl slices was studied. Histological analysis revealed the two regeneration pathways, somatic embryogenesis and shoot organogenesis, simultaneously occurred in the same explant, regardless of the cytokinin/NAA combinations used. The regeneration frequency of 95.9%, with 7.5 morphogenic structures regenerated per explant, and the healthy appearance of regenerated plants indicated the optimal combination 20 µM TDZ + 2 µM NAA. TDZ at 5 µM provided the high somatic embryo proliferation rate by generation of secondary embryos (7.79) along with the lowest rate of their abnormalities. Embryo-developed plants were successfully acclimatised (above 90%). The plants regenerated and proliferated on TDZ-containing media had higher total phenolic content that correlated with the highest free radical scavenging activity (IC50 = 19.09 µg ml− 1). Somatic embryogenesis and shoot organogenesis from hypocotyl slices of the collard greens were established. The combination 20 µM TDZ + 2 µM NAA was optimal for regeneration providing higher total phenolic content.

Influence of auxin and its polar transport inhibitor on the development of somatic embryos in Digitalis trojana.

The present study reports the role of auxin and its transport inhibitor during the establishment of an efficient and optimized protocol for the somatic embryogenesis in Digitalis trojana Ivan. Hypocotyl segments (5mm long) were placed vertically in the Murashige and Skoog medium supplemented with three sets [indole-3-acetic acid (IAA) alone or 2,3,5-triiodobenzoic acid (TIBA) alone or IAA-TIBAcombination] of formulations of plant growth regulators, to assess their differential influence on induction and proliferation of somatic embryos (SEs). IAA alone was found to be the most effective, at a concentration of 0.5mg/l, inducing~10SEs per explant with 52% induction frequency. On the other hand, the combination of 0.5mg/l of IAA and 1mg/l of TIBA produced significantly fewer (~3.6SEs) and abnormal (enlarged, oblong, jar and cup-shaped) SEs per explant with 24% induction frequency in comparison to that in the IAA alone. The explants treated with IAA-TIBA exhibited a delayed response along with the formation of abnormal SEs. Our study revealed that IAA induces high-frequency SE formation when used singly, but the frequency gradually declines when IAA was coupled with increasing levels of TIBA. Eventually, our findings bring new insights into the roles of auxin and its polar transport in somatic embryogenesis of D. trojana.

EFFECTS OF EXPLANT TYPE, CULTURE MEDIA AND PICLORAM AND DICAMBA GROWTH REGULATORS ON INDUCTION AND PROLIFERATION OF SOMATIC EMBRYOS IN EUCALYPTUS GRANDIS X E. UROPHYLLA1

ABSTRACT The objective of the present study was to test the effects of explant type, auxin concentrations, culture media, and auxin concentrations on the induction and proliferation of somatic embryos of Eucalyptus grandis x E. urophylla. Seeds and cotyledons were used as explants and inoculated in culture media containing 1.13, 2.26, 3.39 and 4.52 µM dicamba or 4.14, 10.35, 20.71 and 31.06 µM picloram. Embryogenic calli induced in the picloram treatments were used as explants and inoculated in semisolid or liquid media containing 4.14, 10.35, 20.71 and 31.06 µM picloram and keeping the origin of the embryogenic callus (seeds or cotyledons) and the concentration of picloram in those who were in the induction phase. Statistical, descriptive and anatomical analyses were performed. Induction of somatic pro-embryos into the juvenile plant material of Eucalyptus grandis x E. urophylla was performed using seeds or cotyledons as the source of explants, with the addition of dicamba and picloram as growth regulators. The use of cotyledons as a source of explants and the concentration of 4.1 µM picloram added to the culture media resulted in a higher induction of somatic pro-embryos. Proliferation of secondary somatic embryos was achieved using liquid medium added with picloram.

Growth, lipid peroxidation, organic solutes, and anti-oxidative enzyme content in drought-stressed date palm embryogenic callus suspension induced by polyethylene glycol

Polyethylene glycol (PEG) can be used in somatic embryogenesis to enhance embryogenic development and improve the success of in vitro culture but PEG also causes osmotic stress in developing embryos. The effects of PEG on embryo growth and development in date palm cell suspension culture and associated antioxidant enzyme activities were evaluated. Callus maintained on MS basal media was transferred to regeneration liquid media supplemented with increasing levels (0–20%) of polyethylene glycol 6000 (PEG) to induce osmotic stress. The degree of embryogenic callus formation, its fresh weight, and the percentage of normal embryo callus shapes were increased with an increase in the level of PEG up to 10%. Total soluble protein (TSP), proline, glycine betaine (GB), total soluble phenol (TSPh), total sugars (TS), and total soluble organic acids (TOA) also increased whereas superoxide dismutase (SOD) activity decreased in response to PEG supplementation. Raising the PEG level increased malondialdehyde (MDA) concentration up to 10% PEG and thereafter decreased. Glutathione reductase (GR) and catalase (CAT) activities decreased at the highest levels of PEG. The proportion of normal embryo developmental shapes were about 50% compared with 20% abnormal shapes at optimum levels of PEG. Proliferation of somatic embryos was influenced by their developmental shapes. Cv. Samani accumulated more organic solutes compared with cv. Sewi in both control and stress inducing media. In contrast, lipid peroxidation, GR, SOD, and CAT activities were significantly higher in cv. Sewi than in cv. Samani indicating that the cv. Samani had the ability to tolerate a higher level of osmotic stress compared to cv. Sewi due to the enhanced osmotic re-balancing within its tissues.

Pengaruh 2,4-D terhadap Induksi Embrio Somatik Kopi Arabika

<p><strong>Abstract</strong></p><p>Direct induction of somatic embryos in Arabica coffee (Coffea arabica L.) using plant groth regulators (PGR's) has been successful. The concentration and combination of different kinds of PGR's can influence the response and success in embryo induction. An experiment was conducted to determine the optimal concentration of 2.4-D in combination with kinetin for direct induction and proliferation of somatic embryos. The plant material used was Arabica coffee var. Kartika-l originating from The Indonesian Coffee and Cacao Research Institute, Jember. Explants were taken from young leaves of reddish-green in color. Somatic embryos were induced directly on a Murashige-Skoog (MS) standard medium containing 30 g/l sucrose and supplemented with 0, 1, 2, 4, and 8 mg/l 2.4-D in combination with 0.1 mg/l kinetin each. The cultures were incubated in the dark at temperature 26oC and RH +60% for 6 weeks with 10 replications. The results showed that somatic embryogenesis in Arabica coffee was best induced in a culture medium wiyh 2.4-D at 4 mg/l, combination with 0.1 mg/l kinetin. Induction of somatic embryos was achieved at 100% 4 weeks after culture. Three morphological stages of embryo development were identified: globular, early heart, and middle heart. The embryos were of three distinct colors such as, yellowish, yellowish-white, and white. The highest rate of proliferation of somatic embryos was achieved at 2 mg/l, 2.4-D in combination with 0.1 mg/l kinetin averaging 68.53 embryos per explant 6 weeks after subculture.</p>

Indirect Somatic Embryogenesis from Mature Inflorescence Explants of Date Palm.

Due to the limitations associated with shoot tip explants in the micropropagation of date palm, inflorescence explants are an ideal alternative. This chapter focuses on the protocol for the induction of callus from inflorescence tissue, establishment for proliferation of somatic embryos, germination, elongation, rooting, and acclimatization. Female inflorescences, 30-40cm in length, cv. Shaishee, were used for culture initiation. After disinfection, the outer inflorescence cover (spathe) is cut open, and the spikelet explants, 1cm long, are cultured on modified Murashige and Skoog (MS) medium containing 100mg/L 2,4-D, 3mg/L kinetin, and 3mg/L 2ip and incubated at 25±2°C in the dark. Callus obtained after 6-8months of culturing is transferred to the culture medium to induce somatic embryogenesis and plant regeneration. Well-developed regenerated shoots are cultured on MS medium containing 0.2mg/L NAA for root induction and plantlets acclimatized in the greenhouse before transfer to the field.

Erratum to: Primary and secondary somatic embryogenesis in Chrysanthemum (Chrysanthemum morifolium) cv. ‘Baeksun’ and assessment of ploidy stability of somatic embryogenesis process by flow cytometry

We developed an efficient and simple system for inducing somatic embryogenesis and regenerating plantlets from petal explant of Chrysanthemum (Chrysanthemum morifolium) cv. ‘Baeksun’. Somatic embryogenesis was induced from petal explants on the Murashige and Skoog (MS) medium supplemented with 1.0 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 3.0 mg l−1 6-benzyladenine (BA), yielding the highest mean number of embryos (56.3) per explant after 5 weeks of culture. We evaluated the effects of basal medium and various concentrations of sucrose on the proliferation of secondary somatic embryos. MS medium was observed to be more effective in promoting the proliferation of somatic embryos than half-strength Murashige and Skoog (1/2MS). In addition, 1 % sucrose was also found to be the best in induction of secondary embryogenesis. The highest germination rate (70 %) of the somatic embryos was observed on the MS medium containing 0.2 mg l−1 α-naphthalene acetic acid and 1 g l−1 activated charcoal (AC). Shoots elongated rapidly and roots developed well on hormone-free MS medium with 1 g l−1 AC and successfully acclimated in the greenhouse. Flow cytometric analysis of the primary somatic embryos, secondary somatic embryos, and the somatic embryo-obtained plants along with the parent grown in the greenhouse showed that they all had same identical peaks, indicating that there was no variation of ploidy level during the regeneration process. We expect that our report would be useful for micropropagation and Agrobacterium-mediated genetic transformation studies of this cultivar.

Primary and secondary somatic embryogenesis in Chrysanthemum cv. Euro

Somatic embryogenesis was induced from in vivo grown leaf explants of Chrysanthemum cv. Euro incubated on Murashige and Skoog (MS) medium supplemented with 2.0 mg/L 2,4-dichlorophenoxyacetic acid and 2.0 mg/L Kinetin, yielding the highest mean number of embryos (42 ± 5.97) per explant after 5 weeks of culture. We evaluated the effects of basal medium, various concentrations of sucrose, and timentin on the proliferation of secondary somatic embryos. MS medium was observed to be the more effective in promoting the proliferation of somatic embryos than half-strength Murashige and Skoog (1/2MS). In addition, timentin was also more efficient in induction of secondary embryogenesis than sucrose. Whole plantlets were obtained by culturing of secondary embryos on hormone-free MS medium and successfully acclimated in the green house.

Differential responses to somatic embryogenesis of different genotypes of Brazilian oil palm (Elaeis guineensis Jacq.)

To assess the potential of different genotypes of Brazilian oil palm (Elaeis guineensis Jacq.) to somatic embryogenesis and somatic embryo proliferation, mature zygotic embryos of nine commercial genotypes of E. guineensis (BRSC2001, BRSC2328, BRSC2301, BRSC3701, BRSCM1115, BRSC7201, BRSC2528, BRSC2501, and BRSCN1637) were used. Explants were incubated on Murashige and Skoog (MS) supplemented with 450 μM picloram, 3.0 % sucrose, 500 mg l−1 glutamine, and 2.5 g l−1 activated charcoal, and gelled with 2.5 g l−1 Phytagel. After induction, for differentiation and maturation, the embryogenic calli (ECs) were transferred into fresh medium supplemented with 0.6 μM naphthaleneacetic acid (NAA) and 12.30 μM 2-isopentenyladenine (2iP) or 40 μM picloram in combination with 0.3 g l−1 activated charcoal, and 500 mg l−1 glutamine. Somatic embryos were converted into plants on MS medium with macro- and micro-nutrients at half strength, 2 % sucrose, and 2.5 g l−1 activated charcoal, and gelled with 2.5 g l−1 Phytagel. In general, zygotic embryos swelled after 14 days. Primary calli, which were observed in all the genotypes after 45–60 days of culture, eventually progressed to ECs at 90 days. At this time, scanning electron microscopy (SEM) analysis showed cellular differences between compact and friable calli. After 150 days in the induction phase, the ECs with proembryos that were transferred to the medium for differentiation and maturation, differentiated asynchronically into somatic embryos at globular and torpedo stages. The results showed that BRSC2328 and BRSCM1115 had the highest potential for EC formation (90–100 %) and somatic embryo differentiation (40.7 and 52.5 somatic embryos per callus, respectively) when compared to other genotypes. After approximately 90 days of culture on MS basal medium without growth regulators, protrusion of the leaf primordia was observed, characterizing the onset of germination of the somatic embryos into plants.

Primary and repetitive secondary somatic embryogenesis in Rosa hybrida ‘Samantha’

This study describes a plant regeneration system for Rosa hybrida ‘Samantha’, a mainstream commercial cultivar, via direct somatic embryogenesis. Somatic embryogenesis was induced from in vitro-derived leaf explants, achieving a frequency of 7.5% following 8 weeks of culture on a Murashige and Skoog (MS) medium supplemented with 3.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 30 g/L glucose. We evaluated the effects of various types and concentrations of carbohydrates and plant growth regulators (PGRs) on the proliferation and germination of secondary somatic embryos. MS medium containing 1.0 mg/L 2,4-D, 0.05 mg/L 6-benzyladenine (BA) and 60 g/L glucose was found to be the most effective in promoting the proliferation of somatic embryos. The highest germination rate (56.2%) of somatic embryos was observed on MS medium containing 1.0 mg/L Thidiazuron (TDZ) and 0.5 mg/L BA. Whole plantlets were obtained by culturing germinated shoots on rooting medium comprising 1/2-strength MS salts supplemented with 0.05 mg/L α-Naphthalene acetic acid (NAA). Plantlets grew vigorously with normal vegetative and flowering characteristics.

In Vitro Selection of a Salt-tolerant Embryogenic Line in Habanero Pepper (Capsicum chinense Jacq.)

The effect of NaCI salinity on growth and development of somatic embryos of Habanero pepper was examined. Addition of 75 and 100 mm NaCI into the medium greatly increased the growth and development of somatic embryos and both of these concentrations favored the proliferation of somatic embryos. However, supplementation of 200 and 300 mm NaCI to the medium showed a negative effect on the growth and development of somatic embryos. Concentration increases of NaCl provoked a significant reduction of the embryos survival rate with the average lethal dose (46%) being registered in the treatment of 100 mm. Furthermore, a lower tolerance to salt stress (NaCl) was observed in deformed somatic embryos. Concentrations of 200 and 300 mm NaCl significantly delayed development in the surviving embryos in both treatments. These embryos remained at the globular stage throughout culture time. At 75 mm NaCl, most of the embryos were observed in the torpedo stage. However, the embryos exposed to 100 mm NaCl were observed mainly in globular and cotiledonar stages. It is quite likely that the transition from one intermediate stage of development to another occurs rapidly. With the exception of the concentration at 300 mm NaCl, salt stress stimulated embryonic germination, particularly at 100 mm NaCl. The content of proline in somatic embryos increased substantially in response to salinization. The results suggest that somatic embryos of C. chinense can tolerate concentrations of NaCl up to 100 mm without their development being affected. Moreover, they have sufficient cellular mechanisms to tolerate salinity at relatively higher levels.

An efficient Agrobacterium-mediated transformation protocol for black pepper (Piper nigrum L.) using embryogenic mass as explant

A protocol was developed for an efficient Agrobactertium-mediated transformation of black pepper plants through somatic embryogenesis. Embryogenic mass derived from primary somatic embryos that were obtained from the micropylar region of mature germinating seeds of black pepper was found to be the ideal target tissue for transformation. Genetic fidelity test of embryogenic mass-derived plantlets by RAPD using 23 random primers revealed no genetic variation among the progenies and the parent plant. Among the antibiotics used for selection of transformants, cefotaxime at 100 μg mL−1 was found to be optimum to control Agrobacterium besides its ability to promote somatic embryo proliferation. In the case of kanamycin, a step-wise increase in concentration from 25 to 50 and then to 100 μg mL−1 were found to be optimum. Embryogenic mass co-cultivated with Agrobacterium carrying the β-glucuronidase (GUS) reporter gene were cultured on plant growth regulator-free Schenk and Hildebrandt (SH) medium and transformants were selected in selection medium containing cefotaxime and step-wise increase in kanamycin concentration. The transient GUS gene expression was determined histochemically. Transformants that survived in the selection medium were hardened in the greenhouse. An average of nine hardened putative plantlets was obtained per gram of embryogenic mass. The presence of transgene in these plantlets was assayed by PCR, dot blot, and Southern blot hybridization. Results presented demonstrated for the first time an efficient transformation and regeneration of black pepper without the use of growth regulators. This simple efficient procedure would allow transformation of black pepper with genes of desirable characters.

Endoreduplication in Phalaenopsis is affected by light quality from light-emitting diodes during somatic embryogenesis

Endoreduplication is a developmental process that is unique to plants and occurs in all plants. The present study aimed to assess endoreduplication in various explant tissues and regenerated somatic embryos of Doritaenopsis. We further investigated the effects of light quality on endoreduplication and somatic embryo proliferation. To this end, we studied endoreduplication in leaves and root tips from regenerated plantlets and somatic embryos and in developing somatic embryos under 4 types of lighting conditions: red light, red + far-red light, red + blue light, and white light. We found that the degree of endoreduplication varied in different explants, and that the choice of explants used also influenced the ploidy levels of the newly regenerated somatic embryos. The DNA content of the leaf (2C–8C) was less than that of the root tip (2C–16C) and somatic embryo (2C–64C). In terms of light quality, the combination of red and far-red light produced the highest number of somatic embryos, while maintaining a low degree of endoreduplication. The data obtained indicate that this light combination stimulates somatic embryogenesis in Doritaenopsis and may exert some control on endoreduplication during cell division. These findings can be applied to achieve a reduction in somaclonal variations for the purpose of mass proliferation and genetic improvement.

DEVELOPMENT, MATURATION AND GERMINATION OF HORSE CHESTNUT SOMATIC EMBRYOS

Somatic embryogenesis could represent a way to clonally propagate mature elite trees of horse chestnut. However, while a number of protocols describe successfully the induction and proliferation of somatic embryos a low frequency of embryos converted successfully into plants. The objective of this research was to identify a maturation medium that could contribute to the outcome of more efficient germination results. Therefore, in a first approach the effects of sucrose 3 and 6%, and maltose 3%, were evaluated on somatic embryo maturation, in terms of subsequent somatic embryo germination. Carbon source and concentration had a marked influence on maturation, and subsequent germination of horse chestnut somatic embryos, with sucrose 6% promoting the highest frequency of germinated embryos larger than 1 cm.

Growth regulators affect primary and secondary somatic embryogenesis in Madagaskar periwinkle (Catharanthus roseus (L.) G. Don) at morphological and biochemical levels

An efficient somatic embryogenesis system has been established in Catharanthus roseus (L.) G. Don in which primary and secondary embryogenic calluses were developed from hypocotyls and primary cotyledonary somatic embryos (PCSEs), respectively. Two types of calluses were different in morphology and growth behaviour. Hypocotyl-derived embryogenic callus (HEC) was friable and fast-growing, while secondary callus derived from PCSE was compact and slow-growing. HEC differentiated into somatic embryos which proliferated quickly on medium supplemented with NAA (1.0 mg l−1) and BA (1.5 mg l−1). Although differentiation and proliferation of somatic embryos were faster in primary HEC, maturation and germination efficiency were better in somatic embryos developed from primary cotyledonary somatic embryo-derived secondary embryogenic callus (PCSEC). At the biochemical level, two somatic embryogenesis systems were different. Both primary and secondary/adventive somatic embryogenesis and the role of plant growth regulators in two modes of somatic embryo formation have been discussed.