Mature Zygotic Embryos Research Articles

Plant regeneration through somatic embryogenesis from immature and mature zygotic embryos of Musa acuminata ssp. burmannica

A simple and efficient protocol has been developed for in vitro regeneration of M. acuminata ssp. burmannica (AA) plants. Somatic embryos were produced when immature and mature zygotic embryo explants were cultured on Murashige and Skoog medium supplemented with plant growth regulators 2,4-dichlorophenoxyacetic acid; (2,4-D), picloram or benzyl adenine and indole acetic acid. In general, immature embryos responded better than mature embryos. Callus proliferation was highest in medium supplemented with 2,4-D (4.5 μM). Subsequent transfer of callus to fresh medium produced rapidly proliferating embryogenic calli. Embryogenic calli were maintained in complete darkness for 15 d followed by cycles of 8 h dark and 16 h light, under white fluorescent lamps with a light intensity of 3,000 lm/m2 and at temperature of 28 ± 2°C. Regeneration of embryogenic calli into plantlets was higher for immature embryos (76.6%) than for mature embryos (50.6%). This plant regeneration protocol using mature or immature zygotic embryos, via somatic embryogenesis, has significant potential to improve germination efficiencies of hybrid progenies used in conventional breeding strategies. Furthermore, tests on seed storage showed that seed viability rapidly decline after harvesting and was negligible after 9 mo of storage. This indicates using freshly harvested seeds as explant material is necessary for maximizing the tissue culture response.

Changes in the protein profile of Habanero pepper (Capsicum chinense J.) somatic embryos during development

Protein profile was studied during the development of Capsicum chinense somatic embryos. The total protein content and profile of polypeptides (by sodium dodecyl sulfate polyacrylamide gel electrophoresis) of somatic embryos at different developmental stages (globular, heart-shaped, torpedo and cotyledonary stages) were analyzed. The protein profile of zygotic embryos included nine exclusive bands with molecular weights of 4.0, 5.2, 8.1, 13.7, 20.9, 23.7, 41, 50 and 69.3 kDa; these bands were not observed in the protein profile of somatic embryos. Coincidently, five of these bands possessed similar molecular weights to those reported for storage proteins in other plant species. Protein content showed a clear decreasing tendency with increasing somatic embryo development. The lowest protein content was detected in somatic embryos at the cotyledonary stage (0.436 µg/mg fresh weight), and the highest content was found in somatic embryos at the globular stage (2.98 µg/mg fresh weight). Total proteins two-dimensional electrophoresis (2-DE) analysis of mature zygotic embryo (prior to the desiccation) and cotyledonal somatic embryo, showed significant differences in the protein profile of both types of embryos. Zygotic embryo showed the proteins expression of isoelectric point between 4 to 7 and 7 to 10, and molecular weights between 25 to 36 KDa, which were not expressed in the cotyledonal somatic embryo. The low protein content during the development of the somatic embryos, particularly at the cotyledonary stage, is a factor that could be related with the low rate of conversion to plantlets and the high frequency of deformed somatic embryos of C. chinense . Key words : Recalcitrance, maturation, germination, conversion.

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.

Testing of Algerian fir zygotic and somatic embryos on defence reactions in vitro

Defence reactions of desiccated cotyledonary somatic embryos and mature zygotic embryos of Abies numidica were tested by dual cultures with tester, fungus Phaeolus schweinitzii. Both types of embryos expressed defence reactions. Mutual comparisons of zygotic and somatic embryos showed important differences between defence reactions against mycelial growth towards these embryos. Greater defence reactions were observed in zygotic embryos relative to defence found in somatic embryos.  

Comparative Proteomic Analysis of Auxin-Induced Embryogenic and Nonembryogenic Tissues of the Solanaceous Tree Cyphomandra betacea (Tamarillo)

Cyphomandra betacea (tamarillo) is a tree that produces edible, highly nutritional fruits. In tamarillo, somatic embryogenesis (SE) is achieved through a two-step process starting with the formation of an embryogenic tissue on an auxin-rich medium and further development of embryos, following tissue transfer to an auxin-free medium. During the induction stage, both embryogenic (EC) and nonembryogenic calli (NEC) arise from the same explant (immature leaves or mature zygotic embryos) in the presence of either picloram or 2,4-dichlorophenoxyacetic acid. In an attempt to find somatic embryogenic-specific proteins, a comparative analysis of the proteome of tamarillo's EC and NEC was performed. Analysis of 2-DE gels revealed ca. 150 differentially expressed proteins, from which 22 have been identified by LC-MS/MS. Proteins exclusively or predominantly expressed in EC included metabolism-related proteins, such as enolases or treonine synthases, and also heat-shock and ribosomal proteins. Pathogenesis-related proteins were found mainly in NEC. A number of additional differentially expressed proteins involved in various functional categories were also identified. A quantitative real time PCR (qPCR) analysis revealed no significant differences at the mRNA level for 11 differentially expressed proteins, with exception of the pathogenesis-related proteins that were up-regulated in NEC. This seems to indicate that a posttranscriptional control might be responsible for the proteomic differences detected.

Micropropagation of Romulea sabulosa Schltr. ex Beg. - A potential ornamental plant

Romulea sabulosa Schltr. ex Beg. is an endemic, attractive and rare flowering species in South Africa with horticultural potential. There is a great need to develop in vitro propagation techniques for this species for the purpose of conservation and commercialization. In this regard, the suitability of the intact seeds and embryos as explants was examined. Excised mature zygotic embryos were selected as the explant source due to a low percentage of seed germination (4.5%). Excised embryos were placed on Murashige and Skoog (MS) media supplemented with different concentrations of kinetin and 2,4-dichlorophenoxyacetic acid (2,4-D). Shoot clusters developed on MS medium supplemented with kinetin without 2,4-D. Multiplication was best on a MS medium supplemented with 2.5μM mTR [6-(3-hydroxybenzylamino)-9-β-d-ribofuranosylpurine]. Shoots were multiplied from selected explants on a MS medium with 23.2μM kinetin and were then used for corm induction experiments. Shoots were placed on MS media supplemented with 3, 6, or 9% sucrose, 5mgl−1 activated charcoal or 23.2μM kinetin. Corm formation was observed for all treatments placed at 10, 15, and 20°C. Corm induction was significantly higher at 10 and 20°C. The heaviest corms were produced at 15°C on a MS medium supplemented with 6% sucrose. In some cases multiple corms formed from a single shoot. The findings of this study will assist in propagating R. sabulosa for commercial and conservation purposes.

Assessing the viability of cryopreserved coconut zygotic embryos by electrolytic conductivity and potassium leaching

The objective of this work was to adapt the application of electrolytic conductivity and potassium leaching tests to assess the viability of cryopreserved embryos of 'Anão Verde do Brasil de Jiqui' (AVeJBr) coconut. The zygotic embryos were excised, sterilized and subjected to four cryoprotectant treatments combined with three incubation times (12, 16 and 20 hours), totaling 12 treatments. The pre-treatment of mature zygotic embryos of AVeJBr coconut using a cryoprotectant with 1.75 mol L-1 of sucrose + 15% glycerol for 12 and 16 hours promoted lower embryo humidity and increased viability in electrolytic conductivity and potassium leaching tests. Samples with ten embryos are sufficient for electrolytic conductivity analysis in cryopreserved or non-cryopreserved AVeJBr coconut zygotic embryos. The 4 to 8 hour imbibition period of the embryos is promising for the electrolytic conductivity analysis of non-cryopreserved mature zygotic embryos of AVeJBr coconut.

In vitro zygotic embryo culture of Pinus peuce Gris.: Optimization of culture conditions affecting germination and early seedling growth

This study reports a protocol for the germination and early seedling growth of Pinus peuce Gris. using zygotic embryo culture. In order to overcome seed dormancy and optimize organogenesis, the effect of nutritional, plant growth regulatory and physical factors on in vitro germination and growth of isolated mature zygotic embryos of P. peuce were investigated.

Somatic embryogenesis and bulblet regeneration in snakehead fritillary (Fritillaria meleagris L.)

Induction of in vitro morphogenesis of mature zygotic embryos of Fritillaria meleagrisL. was investigated. Somatic embryogenesis and whole plant regeneration were achieved. Isolated zygotic embryos were cultured on MS medium that contained 3% sucrose, 0.7% agar, 250.0 mg/l casein hydrolysate, 250.0 mg/l L-proline, 80 mg/l adenine sulfate and 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) or thidiazuron (TDZ). Embryogenic callus was derived from mature zygotic embryos after 4 weeks on TDZ containing medium. Somatic embryos at the early stages of development arose from the surface of the embryogenic callus. Multiplication of somatic embryos, formation of bulblets and shoot development were observed on the same medium. Scale sections prepared from the formed bulblets were cultured on MS media supplemented with 2,4-D (0.1 to 10.0 mg/l) and TDZ (0.05 to 2.0 mg/l). Somatic embryos and bulblets were frequently produced from the scale sections. This is a successful report of plant regeneration through somatic embryogenesis for this very important medicinal and horticultural plant. Histological observation revealed that plants of F. meleagris L. were regenerated via somatic embryogenesis. Key words: Bulbous plant, medicinal plant, snakehead fritillary, somatic embryos.

Somatic embryogenesis in Jarilla heterophylla (Caricaceae)

It is reported for the first time an efficient protocol for indirect somatic embryogenesis and plant regeneration for Jarilla heterophylla (Caricaceae). Initial explants were mature zygotic embryos obtained from seeds collected in two regions of the state of Jalisco, Mexico (San Luis Soyatlan and Zacoalco de Torres). Two protocols for somatic embryogenesis used previously for Carica papaya were evaluated. The addition of 2,4-dichlorophenoxyacetic acid (2,4-D) to induction medium A1 without the addition of adenine hemisulphate yielded the best production of embryogenic callus with the higher total of somatic embryos per explant. With this protocol the plant regeneration process took about five months.

Somatic embryogenesis of a wild passion fruit species Passiflora cincinnata Masters: histocytological and histochemical evidences

The characterization of cellular changes that occur during somatic embryogenesis is essential for understanding the factors involved in the transition of somatic cells into embryogenically competent cells and determination of cells and/or tissues involved. The present study describes the anatomical and ultrastructural events that lead to the formation of somatic embryos in the model system of the wild passion fruit (Passiflora cincinnata). Mature zygotic embryos were inoculated in Murashige and Skoog induction media supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Zygotic embryo explants at different development stages were collected and processed by conventional methods for studies using light, scanning, and transmission electron microscopy (TEM). Histochemical tests were used to examine the mobilization of reserves. The differentiation of the somatic embryos began in the abaxial side of the cotyledon region. Protuberances were formed from the meristematic proliferation of the epidermal and mesophyll cells. These cells had large nuclei, dense cytoplasm with a predominance of mitochondria, and a few reserve compounds. The protuberances extended throughout the abaxial surface of the cotyledons. The ongoing differentiation of peripheral cells of these structures led to the formation of proembryogenic zones, which, in turn, dedifferentiated into somatic embryos of multicellular origin. In the initial stages of embryogenesis, the epidermal and mesophyll cells showed starch grains and less lipids and protein reserves than the starting explant. These results provide detailed information on anatomical and ultrastructural changes involved in the acquisition of embryogenic competence and embryo differentiation that has been lacking so far in Passiflora.

Somatic embryogenesis of the hybrid Abies cilicica × Abies cephalonica

The interspecific hybrid fir Abies cilicica × Abies cephalonica is based on native species Abies cephalonica (Loud.) and Abies cilicica (Ant. et Kotschy) Carr. Many variants of nutrient media have been tested. The medium BAP at a concentration of 1 mg·l<sup>–1</sup>, 1,000 mg·l<sup>–1</sup> myo-inositol, 500 mg·l<sup>–1</sup> glutamine, and 1,000 mg·l<sup>–1</sup> casein was found to be the most suitable initiation medium for somatic embryogenesis of this hybrid. Embryogenic suspensor mass was produced from immature and mature primary explants. The initiation frequency of ESM was dependent on the composition of nutrient medium, plant growth regulators, and time of seeds collection. The ESM initiation from immature seeds was higher in comparison with mature zygotic embryos, but the results were not significantly different. As the most suitable maturation medium was evaluated the medium based on Murashige and Skoog medium which contained 10 mg·l<sup>–1</sup> abscisic acid, 100 g·l<sup>–1</sup> PEG and 40 g·l<sup>–1</sup> maltose (MM 2 medium).  

In vitro plant regeneration and de novo differentiation of secretory trichomes in Passiflora foetida L. (Passifloraceae)

The only species in the genus Passiflora (Passifloraceae) known to produce resin glands is P. foetida. These glands are secretory trichomes mainly present on the floral bracts and leaf stipules. The secretion produced by these glands has received attention recently due to the presence of substances with pharmacological properties. Attempts to apply in vitro cell culture methods for the large scale production of highly valuable metabolites has been rather limited due to the fact that these compounds are produced by highly differentiated secretory cells in trichomes which are seldom obtained or because differentiation is inhibited by in vitro conditions. Here we describe the in vitro plant regeneration of P. foetida obtained via organogenesis, using mature zygotic embryos as explants. Differentiated plantlets and, more important, the de novo differentiation of secretory trichomes in vitro could be observed in less than 30 days. There was a clear effect of the concentration of 2,4-dichlorophenoxyacetic acid in the culture media on the regeneration of plants and on the differentiation of glandular trichomes. Our results should be useful for the micropropagation of P. foetida, as well as for studies of the process of secretory trichome differentiation and the implemention of biotechnological methodologies for in vitro mass production of passifloricin and/or other substances present in the P. foetida resin.

Somatic embryogenesis from mature zygotic embryos of commercial passionfruit (Passiflora edulis Sims) genotypes

Mature zygotic embryos of three genotypes of Passiflora edulis Sims, including ‘FB-100’, ‘FB-200’, and ‘FB-300’ were incubated on a Murashige and Skoog (MS) (1962) medium supplemented with different concentrations (18.1–114.8 μM) of 2,4-diclorophenoxyacetic acid (2,4-D) and 4.4 μM of 6-benzyladenine (BA). MS basal medium and MS with BA induced germination of P. edulis embryos. The highest frequencies of embryogenic calli were observed when explants were incubated on MS medium supplemented with 72.4 μM 2,4-D and 4.4 μM BA for ‘FB-200’, which showed the highest potential for embryogenic callus formation. Cytological and histological analyses of pro-embryogenic callus revealed two distinct cell types: thin-walled, small, isodiametric cells with large nuclei and dense cytoplasm, typical of intense metabolic activity; and elongated and vacuolated cells, with small nuclei and less dense cytoplasm. Differentiation of somatic embryos was promoted on MS medium supplemented with activated charcoal and indole-3-acetyl-l-aspartic acid (IAA-Asp) either with or without 2,4-D. However, no conversion of somatic embryos into plantlets was observed.

Effect of Growth Regulators on In Vitro Morphogenic Response of Boscia senegalensis (Pers.) Lam. Poir. Using Mature Zygotic Embryos Explants

The percent study describes the in vitro responses of mature zygotic embryos of Boscia senegalensis to different concentrations (0.0–5.0 mg/L) of 6-benzyladnine (BA), Thidiazuron (TDZ), α-Naphthalene acetic acid (NAA), and 2, 4-Dichlorophenoxyacetic acid (2, 4-D) supplemented on Murashige and Skoog medium (MS). The plant growth regulators (PGRs) were considerably affected the morphogenetic responses. BA produced adventitious shoots through two ways: direct organogenesis and auxiliary shoot formation. Both 2, 4-D and TDZ tend to produce callus, whereas NAA improve the development of embryos to seedlings. Maximum number of shoots/explant (14.8 ± 0.6) was obtained on MS medium supplemented with 3.0 mg/L BA. 67.0% of excised shoots were rooted either on 1/2 MS medium augmented with or without 0.25 mg/L IBA. The highest number of roots (1.2 ± 0.4) and root length (0.5 ± 0.2 cm) was produced on 0.25 mg/L IBA-containing medium. Regenerated plants were successfully acclimatized and transferred to the green house with 70% survival rate. All the plants appeared morphologically uniform with normal growth pattern. A rapid (30 days), efficient and without subculturing protocol for in vitro regeneration of B. senegalensis was developed.

Somatic embryogenesis of selected coniferous tree species of the genera Picea, Abies and Larix

Experiments on somatic embryogenesis in selected spruce, fir and larch species were performed to determine if this method of micropropagation enables production of quality seedlings for forest nurseries. High frequencies of embryogenic callus in spruce (23-31%) and fir (29%) were achieved when mature zygotic embryos were used as explants, while in larches (36%) only when megagametophytes (endosperms with immature embryos) were used. The possibility of somatic embryogenesis initiation also from somatic embryos (resulting in secondary and third generation of embryogenic callus) indicate high efficiency of this method of micropropagation. The best results at all stages of somatic embryogenesis (good proliferation of embryogenic callus, high rate of embryo regeneration and their survival) were obtained for Norway spruce, European larch and some hybrid larches.

Somatic embryogenesis of selected spruce species (Picea abies, P. omorika, P. pungens 'Glauca' and P. breweriana)

Somatic embryogenesis was studied in four spruce species (<em>Picea abies</em>, <em>P. omorika</em>, <em>P. pungens</em> 'Glauca' and <em>P. brewenana</em>) to determine if this method can be used for in vitro propagation of coniferous trees. The highest frequency of initiation of embryogenic tissue was obtained when mature zygotic embryos were used as explants. It ranged then from 10.8% (<em>P. brewenana</em>) to 23.75% (<em>P. omorika</em> and <em>P. pungens</em> 'Glauca'). The frequency of embryogenic tissue initiation was strongly affected by medium composition, i.e. addition of appropriate auxins (2,4-D, NAA, Picloram) and sucrose concentration (10-20 g<sup>-1</sup>"1). A lower frequency was obtained in <em>Picea omorika</em> (10%) when megagametophytes (endosperms with immature zygotic embryos) were used as explants. No emryogenic tissue was produced from hypocotyls, cotyledons and needles. A satisfactory frequency was achieved with the use of somatic embryos of <em>Picea abies</em> (30%). The proliferation of embryogenic cell lines of spruces was affected by medium type. The experiments resulted in production of somatic plantlets of <em>P. abies</em> and <em>P. omorika</em>. This enables the application of this method of spruce micropropagation for genetic and breeding research or for nursery production.

A Comparison of Soluble Sugar Accumulation in Zygotic and Somatic Pea Embryos

This study compares the soluble sugar content of zygotic and somatic pea embryos. It was noted that mature somatic embryos differed from zygotic embryos with respect to carbohydrate composition. Mature zygotic pea embryos contained glucose, myo-inositol, sucrose, maltose, galactinol, galactosyl-cyclitols, raffinose, stachyose and verbascose. The presence of maltose, galactosyl-cyclitols, stachyose and verbascose was not determined in somatic embryos, and their total soluble sugar content was below that of zygotic embryos. High sucrose levels in somatic embryos most probably resulted from the presence of sucrose in the growth medium. Monocotyledonous and irregular somatic embryos were characterized by a different sugar profile than regularly shaped somatic embryos and seeds.

Direct Organogenesis and Plantlet Regeneration from Mature Zygotic Embryos of Chinese Pine(Pinus tabulaeformis Carr.)

Mature zygotic embryos of Chinese pine were cultured as initial explants to investigate the process of direct organogenesis.Adventitious buds were initiated on DCR and MS medium supplemented with 1~5 mg·L-1 benzyladenine(BA) with or without 1~10 mg·L-1 2,4-dichlorophenoxyacetic acid(2,4-D).The highest induction frequency of adventitious buds(90%) was obtained on DCR medium containing 1 mg·L-1 BA.Subsequent transfer of buds to plant growth regulator(PGR)-free medium in time was advantageous for elongation of adventitious buds.1/2DCR medium with 1 mg·L-1 indole-3-butyric acid(IBA) was suitable for adventitious root initiation,on which average rooting frequency was 47.1%.Plantlets were successfully acclimatized in vermiculite and transferred to greenhouse,where they had a survival rate of 86.4% and no morphological abnormalities.

Effect of plant growth regulator, light and media formulation on initiation and growth of Parkia biglobosa (Jacq.) R. Br. Ex Don callus cultures from cotyledons and mature zygotic embryos

Cotyledons from 14-days old seedlings and mature zygotic embryos (MZE) of Parkia biglobosa were cultured on either Murashige and Skoog (1962) medium (MS) or woody plant medium (WPM) supplemented with a range of 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations and incubated in either 24-hour darkness or 16-hour daylight to induce callus formation. Cotyledon explants exhibited 100% callus formation while MZE gave 66.3%. 0.55mg l-1 2,4-D gave the highest size and weight of callus formed in both explants. WPM favored callogenesis better than did MS, and incubation in the dark promoted callus formation better than a 16-hour light regime. Analysis of variance revealed varying degrees of interaction between the tested factors on callus induction (p< 0.05). Induced calluses were sub-cultured on callus proliferation medium consisting WPM supplemented with 0.55mgl-1 2,4-D and 0.1mg l-1 benzylaminopurine (BAP) singly or in combination for growth. Callus growth after 3 weeks was highest (9.2mg) in media supplemented with 2,4-D only. Results of the present study are novel because callus induction in Parkia biglobosa was quantified in terms of size and weight for the first time. It was also found that WPM may be the medium of choice (rather than MS which has hitherto been used) for initiating callus P. biglobosa callus cultures using 2,4-D. These findings will enhance efficient production of P. biglobosa callus for somatic embryogenesis, callus-mediated regeneration, genetic transformation, and as raw material for secondary metabolite (thioproline) production from cell cultures.Keywords Parkia biglobosa, Leguminosae, callus induction, woody plant medium, plant genetic resources.