Initiation Of Embryogenic Cultures Research Articles

Use of Plant Antimicrobial Peptides in in vitro Embryogenic Cultures of Larix sibirica

The effect of plant antimicrobial peptides on the initiation of callus and embryonal suspensor masses, formation of somatic embryos, and germination of regenerants of Siberian larch has been studied. Protein/peptide extracts isolated from Amarantus retroflexus (seeds), Nigella sativa (seeds), and Elytrigia elongata (spikelets) have been used as objects of plant origin. Peptides have been introduced into the nutrient media at the stage of initiation of embryogenic cultures and somatic embryo germination. The stimulating effect of peptides on the formation of embryogenic cultures of Siberian larch has been found. No other differences in the dynamics of growth in the control and experimental regenerants have been observed. This study is supposed to contribute to enhancing the immunity of the clonal planting stock of Siberian larch.

Degeneration pattern in somatic embryos of Pinus sylvestris L.

Somatic embryos can be used for propagating forest trees vegetatively, which is of great importance for capturing the genetic gain in breeding programs. However, many economically important Pinus species are difficult or impossible to propagate via somatic embryogenesis. In order to get a better understanding of the difficulties to propagate Pinus species via somatic embryogenesis, we are studying the developmental pathway of somatic embryos in different cell lines. In a previous study, we showed that the morphology of early somatic embryos in Scots pine (Pinus sylvestris) differs between cell lines giving rise to normal or abnormal cotyledonary embryos. In this study, we have compared the proliferation and degeneration pattern of early and late embryos in a normal and abnormal cell line. In both cell lines, a high frequency of the embryos degenerated. Among the degenerating embryos, two main degeneration patterns could be distinguished. In the normal cell line, the embryos degenerated similar to how the subordinate embryos are degraded in the seed. In the abnormal cell line, the degeneration of the embryos resulted in a continuous loop of embryo degeneration and differentiation of new embryos. We observed a similar degeneration pattern when embryogenic tissue was initiated from megagametophytes containing zygotic embryos at the stage of cleavage polyembryony. Based on our results, we suggest that the degeneration pattern in abnormal cell lines starts during initiation of embryogenic cultures.

Overexpression of PaHAP3A stimulates differentiation of ectopic embryos from maturing somatic embryos of Norway spruce

Somatic embryogenesis can be used for large-scale propagation of plants. In most conifers, it is only possible to establish embryogenic cultures from zygotic embryos or young seedlings. There is, however, a great interest to propagate selected trees with valuable traits via somatic embryos. To be able to establish embryogenic cultures from adult tissues, more knowledge about the molecular regulation of totipotency and embryogenic potential is needed. In Arabidopsis (Arabidopsis thaliana), LEAFY COTYLEDON1 (LEC1) is required for somatic embryogenesis, and overexpression of LEC1 can stimulate the formation of embryo-like structures from vegetative tissues. We have previously characterized a conifer LEC1-type gene, PaHAP3A, which is preferentially active during embryo development in Norway spruce (Picea abies). In this work, we show, by using histochemical GUS assays, that PaHAP3A is expressed in germinated embryos at presumptive sites from which embryogenic tissue differentiate during initiation of embryogenic cultures. Furthermore, we have overexpressed PaHAP3A, using both constitutive and inducible promoters, in order to elucidate whether elevated transcript levels of PaHAP3A are sufficient to induce embryonic properties after germination. In contrast to its angiosperm counterpart, PaHAP3A does not stimulate embryonic features in vegetative tissues. However, overexpression of PaHAP3A during the maturation stage leads to the differentiation of ectopic embryos from maturing somatic embryos. Our results not only indicate at least partial conservation between the conifer and angiosperm LEC1-type genes but also suggest that PaHAP3A is dependent of the cellular status to confer its presumptive role as an important factor influencing the competence of the tissue to initiate somatic embryos.

Whole transcriptome profiling of maize during early somatic embryogenesis reveals altered expression of stress factors and embryogenesis-related genes.

Embryogenic tissue culture systems are utilized in propagation and genetic engineering of crop plants, but applications are limited by genotype-dependent culture response. To date, few genes necessary for embryogenic callus formation have been identified or characterized. The goal of this research was to enhance our understanding of gene expression during maize embryogenic tissue culture initiation. In this study, we highlight the expression of candidate genes that have been previously regarded in the literature as having important roles in somatic embryogenesis. We utilized RNA based sequencing (RNA-seq) to characterize the transcriptome of immature embryo explants of the highly embryogenic and regenerable maize genotype A188 at 0, 24, 36, 48, and 72 hours after placement of explants on tissue culture initiation medium. Genes annotated as functioning in stress response, such as glutathione-S-transferases and germin-like proteins, and genes involved with hormone transport, such as PINFORMED, increased in expression over 8-fold in the study. Maize genes with high sequence similarity to genes previously described in the initiation of embryogenic cultures, such as transcription factors BABY BOOM, LEAFY COTYLEDON, and AGAMOUS, and important receptor-like kinases such as SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASES and CLAVATA, were also expressed in this time course study. By combining results from whole genome transcriptome analysis with an in depth review of key genes that play a role in the onset of embryogenesis, we propose a model of coordinated expression of somatic embryogenesis-related genes, providing an improved understanding of genomic factors involved in the early steps of embryogenic culture initiation in maize and other plant species.

Effect of various sterilization procedures on the in vitro germination of cotton seeds

In vitro manipulations of cotton often require high-quality sterile seedlings as a source of hypocotyl and cotyledon explants for initiation of embryogenic cultures or embryo apexes for shoot production. Unfortunately, in vitro seed germination is often hindered if cotton seeds were collected from the open field and stored under improper conditions. In our case a limited supply of field-grown cotton seeds was received, which necessitated the development of a more effective surface sterilization protocol. Seeds from two accessions, designated as I and II, with very high contamination levels and lowered germination rate were used in this study. These seeds were treated with the most commonly used sterilizing agents, which included commercial bleach, chlorine gas and hydrogen peroxide. Additional steps such as soap-water washes (SW), 70 % ethanol (ETH) and plant preservative mixture rinses were also included in the sterilization procedures to improve the efficiency of tested protocols. Surface sterilized seeds were germinated on Linsmaier and Skoog medium and the percentage of contamination free and well-germinated seeds were recorded for each treatment. Seeds treated with hydrogen peroxide showed significant improvement in germination rate and level of contamination when compared to identical seeds treated with chlorine gas and commercial bleach. The most effective sterilization protocol for all genotypes tested consisted of SW wash followed by ETH rinse and H2O2 sterilization for 7 h. This protocol was successfully used to sterilize seeds of 55 cotton lines.

Progress towards initiation of somatic embryogenesis from differentiated tissues of radiata pine (Pinus radiata D. Don) using cotyledonary embryos

Green cones of radiata pine were collected from two open-pollinated, elite families in two successive years at weekly intervals, and initiation of embryogenic cultures was investigated as a function of sampling date, initiation medium, explant type, and developmental stage. A combination of dissected embryos and a modified Litvay medium, Glitz, was best. This combination gave the highest rate of initiation, and it was possible to initiate somatic embryogenesis (SE) from differentiated cells in the epicotyledonary region of post-cotyledonary zygotic embryos from the two tested families with an average initiation rate of approximately 24% and 7% from stage five and six embryos, respectively. This is different from established initiation protocols of embryogenic cultures in radiata pine, which has traditionally been based on embryo rescue and continued proliferation of immature zygotic embryos. A further implication of initiation of SE from excised post-cotyledonary embryos was that the period of initiation of embryogenic cultures was extended from 4 to 12 wk.

Initiation, long-term cryopreservation, and recovery of Abies alba Mill. embryogenic cell lines

Somatic embryogenesis of Abies alba (Mill.) has significant potential to become an effective method for vegetative propagation of this species. To induce somatic embryogenesis in A. alba, the influence of the mother tree, sampling dates, and cold treatment storage of cones were examined. The initiation frequencies ranged from 1.7% to 16.6%. The sampling date and cone storage, but not the mother tree, had a significant effect on the initiation of embryogenic cultures. Storage of embryogenic cell lines was tested through cryopreservation for 6 yr. Four out of 12 cryostored embryogenic cell lines recovered, and the regeneration of cotyledonary embryos was obtained with two cell lines. The ability of embryogenic cell masses to produce somatic embryos and the mean number of cotyledonary embryos were higher when the maturation protocol was based on embryogenic suspensions dispersed on filter paper. The properly developed germinants were obtained only from maturation media where 32 μM abscisic acid was used, being 16.2% when polyethylene glycol (PEG) was not present and 1.8% when supplemented with 10% (w/v) PEG, respectively. The present study provides evidence that it is possible to cryopreserve A. alba embryogenic cultures while maintaining their maturing ability for the lengthy period (6 yr) needed for progeny testing of field-grown trees. Therefore, our findings are important for further studies and advanced breeding work of the species; however, the conversion of germinants into ex vitro conditions still remains a significant challenge.

Lodgepole pine: the first evidence of seed-based somatic embryogenesis and the expression of embryogenesis marker genes in shoot bud cultures of adult trees

Of the various alternatives for cloning elite conifers, somatic embryogenesis (SE) appears to be the best option. In recent years, significant areas of lodgepole pine (Pinus contorta) forest have been devastated by the mountain pine beetle (MPB) in Western Canada. In an attempt to establish an SE propagation system for MPB-resistant lodgepole pine, several families displaying varying levels of resistance were selected for experimentation involving shoot bud and immature seed explants. In bud cultures, eight embryogenic lines were induced from 2 of 15 genotypes following various treatments. Genotype had an important influence on embryogenic culture initiation, and this effect was consistent over time. These lines were identified by microscopic observation and genetic markers. Despite the abundance of early somatic embryos, the cultures have yet to develop into mature embryos. In contrast, immature zygotic embryos (ZEs) cultured from megagametophytes initiated SE at an early dominance stage via nodule-type callus in 1 of 10 genotypes. As part of the study, putative embryogenesis-specific genes, WOX2 (WUSCHELL homeobox 2) and HAP3A, were analyzed in cultures of both shoot bud explants and ZEs. On the basis of these analyses, we postulate that PcHAP3A was expressed mainly in callus and may be involved in cell division, whereas WOX2 was expressed mainly in embryonal mass (EM)-like tissues. The findings from this study, based on molecular assessment, suggest that the cell lines derived from bud cultures were truly EM. Moreover, these experimental observations suggest that PcWOX2 could be used as an early genetic marker to discriminate embryogenic cultures from callus.

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.

Somatic embryogenesis in Greek fir

To induce somatic embryogenesis of Greek fir, Abies cephalonica Loud., the influence of different cultivation media, sampling dates, and cold treatment storage were examined. The initiation frequencies ranged from 1% to 25%. The sampling date had a significant effect on the initiation of embryogenic cultures and a high frequency was observed within a 5 week window. The effect of cone storage, composition of initiation media, and the medium by storage interaction also remained nonsignificant after 3 and 10 months of proliferation growth. At the maturation phase, the highest tested concentrations of abscisic acid (ABA), 16 and 32 µmol/L, increased both the capability of embryogenic cell masses (ECMs) to produce embryos and the mean number of embryos. Of the two tested carbohydrates, maltose-containing media produced more embryos altogether (1943, on the average of 22.3 embryos per gram fresh mass) and 76% of the ECMs were able to produce embryos when compared with the corresponding values (152, on the average 3.0 embryos per gram fresh mass) and 15.3%, respectively, on sucrose-containing medium. However, 93.5% of embryos on maltose-containing medium died during the germination phase, whereas the corresponding percentage on sucrose-containing medium was 78.2%.

Role of antioxidants and amino acids on somatic embryogenesis of Pinus patula

This study investigated the role of antioxidants and amino acids on somatic embryogenesis using the vegetative shoot apices of three genotypes of mature Pinus patula trees. Apart from dithiothreitol at 0.1%, pretreatment of explants or incorporation of antioxidants in the basal nutrient medium had a negative effect on the initiation of embryogenic cultures, somatic embryo production, and plantlet recovery. Inclusion of an amino acid solution mixture, at a concentration of 10.0 mM, in the maturation medium tended to increase somatic embryo production.

Forest Biotechnology '99—A Joint Meeting of the International Wood Biotechnology Symposium and the Iufro Working Party for Molecular Genetics of Trees, 11–16 July, 1999 Oxford, UK

Forest Biotechnology '99—A Joint Meeting of the International Wood Biotechnology Symposium and the Iufro Working Party for Molecular Genetics of Trees, 11–16 July, 1999 Oxford, UK

Enhancement of embryogenic culture initiation from tissues of mature sweetgum trees.

Male inflorescences, female inflorescences, and leaves collected from dormant buds of three sweetgum (Liquidambar styraciflua) trees were tested for induction of somatic embryogenesis following treatment with thidiazuron, naphthaleneacetic acid (NAA) or different combinations of the two. Explants were placed into culture either within a few days after collection or following 2 months of storage at -15 °C. Although embryogenic cultures were obtained from all three trees, embryogenesis induction was strongly affected by genotype (source tree), with 100% of the staminate inflorescence explants from one tree producing embryogenic cultures in one experiment. Embryogenesis induction was also influenced by explant type, with staminate inflorescences up to five times more likely to produce an embryogenic culture than female inflorescences. No embryogenic cultures were obtained from leaf explants. While treatment with plant growth regulators was not required for embryogenesis induction from inflorescence explants, culture on medium with NAA alone resulted in the highest production of repetitively embryogenic cultures and cultures producing proembryogenic masses. Dormant buds stored for 2 months at -15 °C were still able to produce embryogenic cultures, although frozen storage decreased this ability by over one-half for staminate inflorescences.

Effect of basal medium, growth regulators and Phytagel concentration on initiation of embryogenic cultures from immature zygotic embryos of loblolly pine (Pinus taeda L.).

Low initiation frequency is one of the main barriers in applying somatic embryogenesis to the clonal production of Pinus species. Factors affecting initiation, including basal medium, plant growth regulators, and Phytagel concentration, have been investigated in loblolly pine (Pinus taeda L.). BM1 basal medium proved superior to DCR1 and LP (LP basal salts plus BM1 organic nutrients). No extrusion from megagametophytes was exhibited on LP medium. The combination of 3 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l 6-benzylaminopurine (BA) resulted in a higher extrusion frequency than that of 11 mg/l 2,4-D, 4.5 mg/l BA and 4.3 mg/l kinetin. Phytagel at 1 g/l resulted in the highest explant browning, but the lowest extrusion frequency, while 4 g/l Phytagel induced some dry embryogenic extrusions. Phytagel at 2 g/l was regarded as the best level for initiation of embryogenic cultures.

Embryogenic culture initiation and somatic embryo development in hybrid firs (Abies alba×Abies cephalonica, and Abies alba×Abies numidica)

Embryogenic culture initiation and somatic embryo development in hybrid firs (Abies alba×Abies cephalonica, and Abies alba×Abies numidica)

Embryogenic culture initiation and somatic embryo development in hybrid firs (Abies alba x Abies cephalonica, and Abies alba x Abies numidica)

Embryogenic cultures were established from silver fir (Abies alba Mill.) female megagametliophytes with developing embryos and from excised mature embryos after pollination with Abies cephalonica Lond. or Abies numidica DeLann pollea The frequency of embryogenic callus formation was dependent on genotype, collection time, medium and explants used. The embryogenic callus initiation potential of megagamethophytes with developing embryos in both hybrids was higher in early July and dropped as the zygotic embryos matured. Excised cotyledonary embryos were less suitable for induction of embryogenic cultures. SH medium supplemented with 1mg/l BAP was the most efficient for callus induction and maintenance. Cultures were composed of early somatic embryos with an embryonal mass formed of highly cytoplasmic cells, rich in cell organelles and a suspensor built up by vacuolated, strongly elongated cells. Maturation of embryos was detected with the formation of bipolar structures with shoot and root apices. Nutrition reserves were observed in cells of embryos cultured on DCR medium containing 1 or 10 mg/l ABA. Cotyledon formation, hypocotyl elongation and low frequency germination occured following transfer of the embryos to the same medium without ABA.

Improved tissue culture response of an elite maize inbred through backcross breeding, and identification of chromosomal regions important for regeneration by RFLP analysis

The frequency of initiation of friable, embryogenic callus from immature embryos of the elite maize inbred line B73 was increased dramatically by introgression of chromosomal segments from the inbred line A188 through classical backcross breeding. Less than 0.2% of the immature B73 embryos tested (5 of 3,710) formed embryogenic callus. The breeding scheme consisted of six generations of backcrossing to B73 with selection at each generation for high frequency initiation of embryogenic cultures. BC6 individuals were selfed for four generations to select homozygous lines. The average embryogenic culture initiation frequency increased to 46% (256/561). Nearly all (91%) of the embryos from one BC6 S4 plant formed embryogenic cultures. RFLP analysis was used to determine the locations and effects of the introgressed A188 chromosomal segments. Five segments were retained through at least the fifth backcross generation. The hypothesis that one or more of these five regions contains genes controlling somatic embryogenesis in maize was tested using an F2 population of the cross A188 X Mo17. A set of five DNA markers (three of them linked) explained 82% of the observed phenotypic variance for percentage of immature embryos forming embryognic callus. Four of the five markers were located in or near introgressed A188 chromosome segments.The region marked by probe c595 on the long arm of chromosome 9 was highly associated with several measures of in vitro culture response (percent embryogenic embryos, plants per embryo, and plants per embryogenic embryo). We propose that there is a major gene (or genes) in this region in A188 that promotes embryogenic callus initiation and plant regeneration in B73, Mo17, and probably many other recalcitrant inbred lines of maize.

Evaluation of somaclonal variation during somatic embryogenesis of interior spruce (Picea glauca engelmannii complex) using culture morphology and isozyme analysis

Somaclonal variation during interior spruce (Picea glauca engelmannii complex) somatic embryogenesis was evaluated using culture morphology and isozyme analysis. Genotype-specific abscisic acid-dependent developmental profiles and isozyme patterns were similar for subclone and parent line embryogenic cultures and cotyledonary somatic embryos. Extensive analysis of fifteen hundred subclone embryos of one genotype revealed no isozyme pattern variation. Initiation of embryogenic cultures was dependent on the developmental stage of the explant although cultures derived from different stages were morphologically similar. The embryogenic cultures initiated from interior spruce embryos show a high degree of genetic stability in that the morphological behavior and isozyme phenotype were always consistent with that of the explant genotype. These results support the conclusion that this culture system is appropriate for clonal propagation of interior spruce.

Relation of the developmental stage of zygotic embryos of yellow-poplar to their somatic embryogenic potential

The goal of the study was to characterize the optimal developmental stage of zygotic embryo expiants of the hardwood forest tree species yellow-poplar (Liriodendron tulipifera L.) for the initiation of embryogenic cultures, using morphological measurements and polypeptide profiles of the embryos. Developing zygotic embryos from seeds of six full-sib families, collected every two weeks from 4 weeks postpollination until seed maturity (18 weeks postpollination) were divided into 2 subsamples for each collection date. One group was used to initiate tissue cultures. Embryos in the other group were measured (total length, cotyledon length and hypocotyl thickness) and soluble polypeptide profiles of the embryos were analyzed by SDS-polyacrylamide gel electrophoresis. Potential of an expiant to produce an embryogenic culture peaked during the eighth week following pollination, with an average of 28% of the expiants producing proembryogenic masses, and declined to near zero for mature zygotic embryos. The maximum embryogenic potential corresponded to the globular stage of embryo developmet. Soluble protein profiles of zygotic embryos from 5 sampling dates indicated that decline in embryogenic potential appeared to parallel an increase in the level of a polypeptide of approximately 55 kDa, possibly a storage protein.

Factors Affecting in Vitro Embryogenesis from Undeveloped Ovules of Mature Citrus Fruit

Abstract The effects of genotype and growth additives on the induction of embryogenesis from undeveloped ovules from mature fruit of Citrus were examined. Embryos were produced in cultures of 15 of the 17 representative polyembryonic cultivars examined. Cultured ovules of 5 monoembryonic cultivars did not become embryogenic. Percentages of ovules producing embryos in the responsive polyembryonic cultivars ranged from 5 to 20 after 56 days in culture. Mean numbers of embryos per responsive ovule ranged from 1.9 to 13.2. Effects of growth additives on the production of embryos from undeveloped ovules from mature fruit of ‘Marsh’ grapefruit were tested. A low concentration (0.01 mg/liter) of butanedioic acid mono (2, 2-dimethylhydrazide) (daminozide) was most effective at increasing the induction of somatic embryogenesis over the control treatment of 500 mg/liter malt extract. The presence of abscisic acid or a higher concentration of malt extract (100 mg/liter) in the medium also increased embryo induction, but germination of embryos obtained on the malt extract medium was poor. The use of undeveloped ovules from mature fruits allows the initiation of embryogenic cultures from a wide range of Citrus species.