Immature Zygotic Embryos Research Articles

In vitro Culture and Plant Regeneration of Sorghum Genotypes Using Immature Zygotic Embryos as Explant Source

In vitro Culture and Plant Regeneration of Sorghum Genotypes Using Immature Zygotic Embryos as Explant Source

Regeneration of tropical maize lines ( Zea mays l .) from mature zygotic embryo through callus initiation

The use of immature zygotic embryos as an explant for maize regeneration has been hampered by the strictly limited suitable duration of immature embryos for culture. In contrast, mature zygotic embryos harvested from dry seeds are ubiquitous. However, generally mature embryos and especially tropical maize genotypes have been considered as the most recalcitrant for tissue culture work. Consequently tropical maize regeneration from mature embryos has not been reported so far. Here, we report successful regeneration of one inbred and one open pollinated tropical maize line from mature zygotic embryos using split seed technique. The maximum average callus induction recorded using LS basal salts and B5 vitamins supplemented with 3 mg l-1 2,4-D alone was 90% and 52.5% when same level of 2,4-D was combined with Kinetin. A maximum of 75.6% Type II and 62.3% Type I callus was produced after maintaining calli on media composed of LS basal salts and B5 itamins supplemented with 2 mg l-1 of 2,4-D. The frequency of regenerable calli induced was 21.14% for CIMMYT maize line 216 and 16.51% for Katumani. The number of shoots regenerated per callus induced on single split seed ranged from 1-5.

Synthetic seed production from somatic embryos of Pinus radiata

Pinus radiata is one of the most important forestry species in the southern hemisphere. This work describes the regeneration of this plant via somatic embryogenesis from immature zygotic embryos. To improve this process, somatic embryogenic cell suspensions were established in liquid media for the generation of material for embryo maturation. Each developmental stage of these suspensions was characterized by microscopy and their growth phases quantified. An alginate-containing medium was used as an encapsulation method for the somatic embryos that were then germinated as artificial seeds in vitro. The protocols described in this work are both useful and reliable for industrial purposes.

English

  Dwarf chickling (Lathyrus cicera L) is an important forage legume crop. However, due to the presence of neurotoxin amino acid (b-N-oxalyl-L-a,b-diaminopropionic acid) in the dwarf chickling, its cultivation is limited. There is need to carry out studies for the development of varieties with reduced or none of this neurotoxin for beneficial and prolong consumption by animals. The study reports tissue culture approaches for multiplication of the plant using thidiazuron (TDZ), 6-benzylaminopurine (BAP), and α-naphthalene acetic acid (NAA) from immature zygotic embryos of the plant. The highest number (16.25) of shoots per explant was recorded on MS medium supplemented with 0.45 mg/l TDZ and 0.4 mg/l ascorbic acid. Much reduced shoot regeneration was recorded on MS medium containing different combinations BAP+NAA+ascorbic acid. The TDZ-regenerated shoots were rooted by pulse treatment with 50 mg/l IBA for 7 min. The plantlets were successfully acclimatized under the greenhouse conditions.    Key words: Immature zygotic embryos, dwarf chickling, Lathyrus cicera L., axillary shoot regeneration, rooting.

Improved germination of somatic embryos and plant recovery of European chestnut

For the mass production of chestnut trees with selected, hybrid, or genetically engineered genotypes, one potentially desirable propagation strategy is based on somatic embryogenesis. Although methods exist for the initiation of embryogenic cultures of Castanea sativa from immature zygotic embryos or leaf explants, the embryos produced have had low rates of conversion into plantlets. This study explored the possible benefits for somatic embryos that have already undergone maturation and cold treatments, of (a) partial slow or fast desiccation, and (b) of the addition of plant growth regulators or glutamine to the germination medium. Germination response was evaluated in terms of both conversions to plantlets and through embryos developing only shoots (shoot germination) that could be rooted following the micropropagation protocols developed for chestnut. Two or 3 wk slow desiccation in sealed empty Petri dishes resulted in a slight reduction in water content that nevertheless increased total potential plant recovery, shoot length, and the number of leaves per plantlet. However, best results were achieved by 2 h fast drying in a laminar flow hood, which reduced embryo moisture content to 57–58% and enhanced the potential plant recovery and quality of regenerated plantlets. Plant yield was also promoted by addition of 0.44 μM benzyladenine and 200–438 mg/l of glutamine to the germination medium, and plantlet quality (as evidenced by root, shoot, and leaf growth) by the further addition of 0.49 μM indole-3-butyric acid.

English

  Triticale is an important cereal crop grown throughout the world. The study reports somatic embryogenesis from immature zygotic embryos of 5 Turkish triticale genotypes. The explants were initially cultured on MS medium supplemented with 2 mg dm-3  2,4-D, 500 mg dm-3 glutamine, 100 mg dm-3casein hydrolysate, 2% sucrose and 7% agar for two weeks in the dark. The temperature was maintained at 24±2ºC. Thereafter, the developing embryogenic calli were transferred to MS medium without 2,4-D to achieve embryogenesis under light intensity of 30 000 lux in 16 h light 8 h dark photoperiod at 24±2ºC for 2 weeks. The developing somatic embryos were then transferred to MS medium to mature them and obtain plantlets. The highest number of 9.63 somatic embryos per explant were recorded for genotype LAD388 from 14 - 16 days old explants. The experiment clearly established that 14 - 16 days old explants were superior compared to 17 - 19 days old explants in terms of number of somatic embryos per explant. The matured plantlets were acclimatised in the greenhouse on organic matter rich soil mix contained in pots.   Key words: Triticale, immature zygotic embryos, somatic embryogenesis, age.

Somatic embryogenesis from immature zygotic embryos and monitoring the genetic fidelity of regenerated plants in grapevine

Somatic embryogenesis and plant regeneration were successfully established on Nitsch and Nitsch (NN) medium from immature zygotic embryos of six genotypes of grapevine (Vitis vinifera). The optimum hormone combinations were 1.0 mg dm-3 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction and 1.0 mg dm-3 α-naphthalene acetic acid (NAA) + 0.5 mg dm-3 6-benzyladenine (BA) for embryos production and 0.03 mg dm-3 NAA + 0.5 mg dm-3 BA for embryos conversion and plant regeneration. The frequency of somatic embryogenesis varied from 10.5 to 37.5 % among six genotypes and 15.5-42.1 % of somatic embryos converted into normal plantlets. The analysis of DNA content determined by flow cytometry and chromosome counting of the regenerated plantlets clearly indicated that no ploidy changes were induced during somatic embryogenesis and plant regeneration, the nuclear DNA content and ploidy levels of the regenerated plants were stable and homogeneous to those of the donor plants. RAPD markers were also used to evaluate the genetic fidelity of plants regenerated from somatic embryos. All RAPD profiles from regenerated plants were monomorphic and similar to those of the field grown donor plants. We conclude that somaclonal variation is almost absent in our grapevine plant regeneration system.

Efficient somatic embryogenesis in sugar beet (Beta vulgaris L.) breeding lines

Efficient regeneration via somatic embryogenesis (SE) would be a valuable system for the micropropagation and genetic transformation of sugar beet. This study evaluated the effects of basic culture media (MS and PGo), plant growth regulators, sugars and the starting plant material on somatic embryogenesis in nine sugar beet breeding lines. Somatic embryos were induced from seedlings of several genotypes via an intervening callus phase on PGo medium containing N6-benzylaminopurine (BAP). Calli were mainly induced from cotyledons. Maltose was more effective for the induction of somatic embryogenesis than was sucrose. There were significant differences between genotypes. HB 526 and SDM 3, which produced embryogenic calli at frequencies of 25–50%, performed better than SDM 2, 8, 9 and 11. The embryogenic calli and embryos produced by this method were multiplied by repeated subculture. Histological analysis of embryogenic callus cultures indicated that somatic embryos were derived from single- or a small number of cells. 2,4-dichlorophenoxyacetic acid (2,4-D) was ineffective for the induction of somatic embryogenesis from seedlings but induced direct somatic embryogenesis from immature zygotic embryos (IEs). Somatic embryos were mainly initiated from hypocotyls derived from the cultured IEs in line HB 526. Rapid and efficient regeneration of plants via somatic embryogenesis may provide a system for studying the molecular mechanism of SE and a route for the genetic transformation of sugar beet.

Patterns of somatic embryo formation in Siberian larch: Embryological aspects

Somatic embryogenesis was induced in Siberian larch by in vitro culturing zygotic embryos at different developmental stages. Cultures were grown in modified Murashige and Skoog medium supplemented with hormones 2,4-dichlorophenoxyacetic acid (2 mg/l) and 6-benzylaminopurine (0.5-1 mg/l). The success of somatic embryogenesis in this species depended on the tree genotype and developmental stage of embryos used for culturing. Somatic embryogenesis from immature zygotic embryos at the stage of cotyledon initiation was most active. After 5-10 days, such embryos formed the embryogenic tissue including two cell types--elongated highly vacuolated embryonic tubes and small embryonic cells. Somatic embryos were isolated from proliferating embryogenic tissues after 2 months of culture.

Somatic embryogenesis in Araucaria angustifolia

Immature and mature zygotic embryos were used as source of explants for induction of somatic embryogenesis in Araucaria angustifolia. Embryogenic cultures (EC) were only obtained from immature zygotic embryos. Basic medium, carbon source, and genotype showed a significant influence on the formation of stage I somatic embryos (SE). When EC were submitted to maturation conditions, SE continued their individual development until stage II, but mature embryos were not obtained. Proteins secreted by embryogenic cultures were, to a certain degree, genotype specific and included an extracellular class IV chitinase and β-1-3-glucanase.

Optimization of particle bombardment parameters for the genetic transformation of Brazilian maize inbred lines

The objective of this work was to develop a genetic transformation system for tropical maize genotypes via particle bombardment of immature zygotic embryos. Particle bombardment was carried out using a genetic construct with bar and uidA genes under control of CaMV35S promoter. The best conditions to transform maize tropical inbred lines L3 and L1345 were obtained when immature embryos were cultivated, prior to the bombardment, in higher osmolarity during 4 hours and bombarded at an acceleration helium gas pressure of 1,100 psi, two shots per plate, and a microcarrier flying distance of 6.6 cm. Transformation frequencies obtained using these conditions ranged from 0.9 to 2.31%. Integration of foreign genes into the genome of maize plants was confirmed by Southern blot analysis as well as bar and uidA gene expressions. The maize genetic transformation protocol developed in this work will possibly improve the efficiency to produce new transgenic tropical maize lines expressing desirable agronomic characteristics.

Growth regulators influence the fatty acid profiles of in vitro induced jojoba somatic embryos

Inducing somatic embryogensis from jojoba [Simmondsia chinensis (Link) Schneider] explants to produce artificial seeds in the laboratory (in vitro) may prove highly profitable, as the seeds contain a characteristic liquid wax of economic importance in industry, nutrition and medicine. Thus, there is a need to examine the effect of the factors involved in the in vitro process on the quality and quantity of the synthesized fatty acids in comparison with those naturally produced in vivo. Immature zygotic embryos and mature leaf explants were cultured on Murashige and Skoog basal medium (MS) supplemented with various levels of 2,4-D, BA and sucrose. Embryogenic calluses developed from the zygotic embryos and leaf explants over a period of 2–4 weeks with the highest response at 0.4 μM 2,4-D, 2.2/4.4 μM BA and 117 mM sucrose (4%). Following induction, the zygotic embryo derived somatic embryos developed to the globular, heart, torpedo, and cotyledon stages. Direct somatic embryogenesis was observed with some of the zygotic embryo explants. Leaf-derived embryogenic calluses did not mature on any of the maturation/germination media examined up to 4 weeks of culture. Analysis of fatty acids indicated that the mature seeds are characterized with long chain saturated fatty acids C22:0 behenic Acid. The zygotic embryo-derived somatic embryos (SE-Z) and leaf-derived somatic embryos (SE-L) are characterized with the induction of the essential polyunsaturated fatty acid C18:2 (omega-6) linoleic acid, (omega-3) alpha-linolenic acid (ALA), with higher values of long chain saturated fatty acids C16:0 palmitic acid and monounsaturated fatty acid C18:1 oleic acid. These results indicate that manipulating the growth regulators in the induction media influenced the fatty acids synthesis and hence the fatty acids profile in jojoba somatic embryos.

Developing mutant sunflower lines (Helianthus annuus L.) through induced mutagenesis

SUMMARY Immature zygotic embryos of sunflower fertility restorer line 147 R were treated with ultrasound before plating to embryo culture medium. Some mutant plants were isolated and self-pollinated for several generations. New sunflower forms with inherited morphological and biochemical changes were obtained through selection and self-pollination. Genetic changes that occurred during the regeneration procedure included fifteen morphological and biochemical characters. In this study, negative genetic changes were registered for most of the indices. Positive changes were registered for leaf petiole length and number of leaves. Plant height was the least stable of all characters under study. Mutation for resistance to the local population of Orobanche cumana (race A-E) was obtained from the susceptible Bulgarian control line 147 R. All five investigated mutant restorer lines possessed 100% resistance to Orobanche and stable inheritance in subsequent generations. Our results showed that mutagenesis in sunflower can be successfully used to develop new lines useful for heterosis breeding. RESUMEN Se trataron embriones cigóticos inmaduros de la línea restauradora de girasol 147 R con ultrasonido antes de su traspaso a un medio de cultivo de embriones. Algunas plantas mutantes se aislaron y autofecundaron durante varias generaciones. Se obtuvieron nuevas variantes de girasol con cambios morfológicos y bioquímicos heredables a través de selección y autofecundación. Los cambios genéticos ocurridos durante la regeneración incluyeron quince caracteres morfológicos y bioquímicos. Como resultado de nuestro estudio, se registraron cambios genéticos negativos para la mayoría de los índices. Los cambios positivos opuestos incluyen los índices de largo de pecíolo y número de hojas. El índice altura de planta fue el menos estable de todos los caracteres estudiados. Se obtuvo una mutación para resistencia a la población local de Orobanche cumana (razas A-E) a partir de la línea búlgara susceptible 147 R. Las cinco líneas restauradoras mutantes investigadas poseyeron 100% de resistencia a Orobanche , cuya herencia fue estable en las generaciones siguientes. Nuestros resultados muestran que se puede utilizar exitosamente la mutagénesis en girasol para desarrollar nuevas líneas útiles para el mejoramiento basado en la heterosis. RÉSUMÉ Les embryons immatures de tournesol de la lignée restauratrice de fertilité 147 R ont été traité avec des ultrasons avant de les semer. Certaines plantes mutantes ont été isolées et autofécondées sur plusieurs générations. Les nouvelles formes de tournesol avec les changements morphologiques et biochimique dont elles ont hérité ont été obtenues au travers d’une sélection et d‘une autofécondation. Les changements génétiques qui se sont produits durant la procédure de régénération ont inclus 15 caractères morphologiques et biochimiques. Notre étude montre des changements génétiques négatifs ont été enregistrés pour la plupart des indices. A l’opposé, les changements positifs concernent les indices de longueur du pétiole de la feuille ainsi que le nombre de feuilles. La hauteur de la plante a été le moins stable de touts les caractères étudiés. La mutation pour la résistance à la population locale de Orobanche cumana (race A-E) a été obtenue à partir de la lignée bulgare 147 R de témoin de sensibilité. La totalité des 5 lignées restauratrices mutantes étudiées possèdent 100% de résistance à l’ Orobanche et ceci est fixé dans les générations suivantes. Nos résultats montrent que la mutagénèse du tournesol peut être utilisée avec succès pour développer de nouvelles lignées utiles à la sélection pour l’heterosis.

Characterization of leafy cotyledon1-like during embryogenesis in Theobroma cacao L.

Theobroma cacao L., an economically important crop for developing countries, can be experimentally propagated by somatic embryogenesis. Because of their potential roles in embryogenesis, a gene candidate strategy was initiated to find gene homologues of the members of the leafy cotyledon family of transcription factors. A homologue of the leafy cotyledon1-like gene, that encodes the HAP 3 subunit of the CCAAT box-binding factor, was found in the cocoa genome (TcL1L). The translated peptide shared a high amino acid sequence identity with the homologous genes of Arabidopsis thaliana, Phaseolus coccineus and Helianthus annuus. TcL1L transcripts mainly accumulated in young and immature zygotic embryos, and, to a lesser extent, in young and immature somatic embryos. In situ hybridization specified the localization of the transcripts as being mainly in embryonic cells of young embryos, the meristematic cells of the shoot and root apex of immature embryos, and in the protoderm and epidermis of young and immature embryos, either zygotic or somatic. Non-embryogenic explants did not show TcL1L expression. Ectopic expression of the TcL1L gene could partially rescue the Arabidopsis lec1 mutant phenotype, suggesting a similarity of function in zygotic embryogenesis.

Recovering polyploid papaya in vitro regenerants as screened by flow cytometry

Several protocols have been proposed for in vitro propagation of papaya, either based on somatic embryogenesis or shoot organogenesis. It is well-known that tissue culture-based approaches are frequently associated with somaclonal variation. Whether on the one hand this phenomenon can preclude further stages of in vitro culture, on the other hand it can generate useful genetic variability for crop improvement. However, somaclonal variation analyses are limited in papaya tissue culture. The DNA ploidy level of 250 papaya somatic embryogenesis-derived plantlets from immature zygotic embryos was analyzed by flow cytometry. In vitro-grown and greenhouse seed-derived plantlets were used as diploid standards. Flow cytometry unambiguously evidenced euploid (diploid, mixoploid, triploid and tetraploid) and aneuploid papaya plantlets, indicating that in vitro culture conditions can lead the occurrence of somaclonal variation. Additionally, the two subsequent flow cytometry analyses showed that the DNA ploidy level remained stable in all cloned papaya plantlets during the successive subcultures in the multiplication medium.

Effect of plant growth regulators on direct somatic embryogenesis in camphor tree (Cinnamomum camphora L.) from immature zygotic embryos and embryogenic calli induction

A description of a successful direct somatic embryogenesis induction from immature zygotic embryos of a camphor tree (Cinnamomum camphora L.) is presented. After a subculture of 2–3 years, embryogenic calli could be derived from primary somatic embryos. Immature zygotic embryos were cultured on a Murashige and Skoog (MS) basal medium supplemented with a range of combinations of cytokinins (BA) and auxins (2,4-D or NAA) for somatic embryo induction. Primary somatic embryos could be induced directly in almost all PGR combinations. A positive effect of 2,4-D on somatic embryogenesis from immature zygotic embryos of camphor tree was obtained. BA at appropriate concentrations ( 5 mg·L−1) of BA had the effect of restraining somatic embryo induction. NAA had a less positive effect on somatic embryogenesis than 2,4-D.

Improvement of Agrobacterium-mediated transformation in Hi-II maize (Zea mays) using standard binary vectors

High-frequency transformation of maize (Zea mays L.) using standard binary vectors is advantageous for functional genomics and other genetic engineering studies. Recent advances in Agrobacterium tumefaciens-mediated transformation of maize have made it possible for the public to transform maize using standard binary vectors without a need of the superbinary vector. While maize Hi-II has been a preferred maize genotype to use in various maize transformation efforts, there is still potential and need in further improving its transformation frequency. Here we report the enhanced Agrobacterium-mediated transformation of immature zygotic embryos of maize Hi-II using standard binary vectors. This improved transformation process employs low-salt media in combined use with antioxidant L-cysteine alone or L-cysteine and dithiothreitol (DTT) during the Agrobacterium infection stage. Three levels of N6 medium salts, 10, 50, and 100%, were tested. Both 10 and 50% salts were found to enhance the T-DNA transfer in Hi-II. Addition of DTT to the cocultivation medium also improves the T-DNA transformation. About 12% overall and the highest average of 18% transformation frequencies were achieved from a large number of experiments using immature embryos grown in various seasons. The enhanced transformation protocol established here will be advantageous for maize genetic engineering studies including transformation-based functional genomics.

Induction, development and maturation of somatic embryos in Bunge’s pine (Pinus bungeana Zucc. ex Endl.)

Embryogenic tissue was induced from developing immature zygotic embryos in Bunge’s pine (Pinus bungeana Zucc. ex Endl.). Induction rate reached 84.4% with our best treatment. Zygotic embryos were dissected from megagametophytes and inoculated on different induction media, DCR1 (Gupta PK, Durzan DJ (1985) Plant Cell Rep 4:177–179), BM1 (Gupta PK, Pullman G (1991) U.S. Patent No. 5,036,00) and MSG (Becwar MR, et al. (1988) Somatic cell genetic of woody plants. Kluwer Academic Publishers, Dordrecht, pp. 1–18), supplemented with 2,4-dichlorophenoxyacetic acid (2, 4-D) and 6-benzylaminopurine (BA). DCR1 was the best medium for initiating embryogenic tissue. Induction rates were affected significantly by developmental stages of explants. The highest induction rate was obtained with embryos collected on either June 20 or June 30 with 10 mg l−1 2, 4-D and 4 mg l−1 BA. Embryogenic tissue was subcultured monthly on DCR1 medium supplemented with 0.3 mg l−1 2, 4-D and 0.2 mg l−1α-naphthaleneacetic acid. In order to enhance embryo maturation, embryogenic tissue was transferred onto DCR1 medium for two weeks, in which 1,000 mg l−1 myo-inositol was included and all plant growth regulators were eliminated. This pretreated tissue was then transferred onto a maturation medium that was DCR1 medium containing 50 g l−1 sucrose and 0.1 mg l−1 indolebutyric acid. In this study, benefits of embryo maturation were not observed when abscisic acid and polyethylene glycol were applied in the culture.

High-efficiency somatic embryogenesis and morphohistology and histochemistry of somatic embryo development in Larix leptolepis Gordon

A high-efficiency somatic embryogenesis protocol of Japanese larch (Larix leptolepis Gordon) has been established in our investigation. Calli were induced from immature zygotic embryos of female cones of L. leptolepis and then subcultured regularly on to a modified Gupta and Durzan (DCR) basal medium for 5 years. Embryogenic tissues showed distinct morphological changes during somatic embryo development when they were transferred to a maturation medium supplemented with abscisic acid (ABA) compared with the morphology in a medium lacking ABA. Histological observations indicated that polyembryony was a characteristic feature during early embryogeny and somatic embryos at later stages showed normal histodifferentiation. In addition, histochemical analysis revealed that abundant starch granules and proteins accumulated in mature embryos, indicating that they played important roles in the development and regeneration of normal plantlets from somatic embryos on hormone-free germination media

Histological analysis of direct somatic embryogenesis in Arabidopsis thaliana (L.) Heynh

In Arabidopsis the in vitro culture of immature zygotic embryos (IZEs) at a late stage of development, on the solid medium containing synthetic auxin, leads to formation of somatic embryos via direct somatic embryogenesis (DSE). The presented results provide evidence that in IZE cells competent for DSE are located in the protodermis and subprotodermis of the adaxial side of cotyledons and somatic embryos displayed a single- or multicellular origin. Transgenic Arabidopsis lines expressing the GUS reporter gene, driven by the DR5 and LEC2 promoters, were used to analyse the distribution of auxin to mark embryogenic cells in cultured explants and develop somatic embryos. The analysis showed that at the start of the culture auxin was accumulated in all explant tissues, but from the fourth day onwards its location shifted to the protodermis and subprotodermis of the explant cotyledons. In globular somatic embryos auxin was detected in all cells, with a higher concentration in the protodermis, and in the heart stage its activity was mainly displayed in the shoot, root pole and cotyledon primordia. The embryogenic nature of dividing protodermal and subprotodermal cells accumulating auxin was confirmed by high expression of promoter activity of LEC2 in these cells. Analysis of symplasmic tracer (CFDA) distribution indicated symplasmic isolation between tissues engaged in DSE and other parts of an explant. Symplasmic isolation of somatic embryos from the explant was also detected.