Embryogenic Masses Research Articles

Histological analysis of somatic embryogenesis and organogenesis induced from mature zygotic embryo-derived leaflets of peanut ( Arachis hypogaea L.)

The initiation and development of somatic embryos and organogenic buds in peanut ( Arachis hypogaea L.) obtained from mature zygotic embryo-derived leaflets (MZELs) was studied histologically. The MZELs were cultured on Murashige and Skoog (MS) basal medium supplemented with 20 mg l −1 2,4-dichlorophenoxyacetic acid (2,4-D) for embryogenic mass induction. Somatic embryos developed from these masses following transfer to a medium containing 3 mg l −1 2,4-D and were germinated on hormone-free medium. A combination of 4 mg l −1 α-naphthaleneacetic acid (NAA) and 5 mg l −1 6-benzylamino purine (BAP) was optimum to induce organogenic buds which developed into shoots once placed on a medium containing 0.5 mg l −1 BAP and 0.5 mg l −1 kinetin. Histological studies of explants at various developmental stages of somatic embryogenesis and organogenesis revealed that both somatic embryos and organogenic buds developed directly from the mesophyll layers of MZELs, and both were of multicellular origin. Initially, MZELs underwent periclinal division when cultured on MS medium containing 20 mg l −1 2,4-D giving rise to somatic embryos. Interestingly, MZELs underwent anticlinal division when cultured on MS medium containing 4 mg l −1 NAA and 5 mg l −1 BAP that resulted in organogenesis. Initial failure of somatic embryos to convert into plantlets was due to malformation of the shoot meristem. However, transfer to medium containing 2 mg l −1 BAP and 3 mg l −1 kinetin, or 5 mg l −1 thidiazuron (TDZ) resulted in the appearance of broad shoot apices which could be induced to produce plantlets.

Exogenous abscisic acid fate during maturation of hybrid larch (Larix ×leptoeuropaea ) somatic embryos

The maturation of somatic embryogenesis of hybrid larch is an essential step for plantlet production. ABA controls not only synchronicity of maturation, but has important consequences on eventual viability of the plantlet. To gain understanding of the role of this plant growth regulator during the maturation process of hybrid larch somatic embryos, we studied the incorporation of [ 3 H]-( ± )-abscisic acid [tritiated ( ± )-ABA] over 6 weeks of culture. Results showed a rapid incorporation of label into the tissues directly in contact with the culture medium. Accumulation of tritiated ( ± )-ABA occurred mainly in the maturing somatic embryos found at the periphery of the embryogenic mass but not in direct contact with the culture medium. Tritiated ( ± )-ABA was mainly conjugated as a glucose ester form. Rates of tritium incorporation indicated a significant build-up in tritiated ( ± )-ABA metabolization at the third week of culture. The weekly measurement of labelling in the culture medium over 6 weeks showed no localized exhaustion of tritiated ( ± )-ABA in positions where the embryogenic masses were placed in Petri dishes. The calculated ABA internal content of the maturing somatic embryos was similar to published ELISA measurements of ABA. This result suggests an absence of endogenous ABA synthesis by somatic embryos of hybrid larch during maturation.

Induction of somatic embryogenesis in cashewnut (Anacardium occidentale L.)

Somatic embryogenesis was induced in callus cultures derived from nucellar tissue of cashewnut (Anacardium occidentale L.). Callus was obtained from nucellar tissue after 3 wk of culture on semisolid Murashige and Skoog (MS) basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 5 μM)+gibberellic acid (GA3, 15 μM)+N6-benzyladenine (BA, 5 μM). This callus gave rise to an embryogenic mass after 9 wk on maintenance medium containing 2,4-D (10 μM)+GA3 (15 μM)+4% sucrose +0.5% activated charcoal +10% coconut water (CW) +0.05% casein hydrolysate (CH). The embryogenic mass, after transfer to medium supplemented with 2,4-D (5 μM)+GA3 (30 μM)+4% sucrose +0.5% activated charcoal +10% CW +0.05% CH, gave rise to somatic embryos. The developmental stages of somatic embryos were observed using light and stereo microscopes. Histological study of somatic embryo development was also carried out. The present study would be useful for clonal propagation, and variety improvement in cashewnut, which is essential due to its increasing demand and export potential.

Analysis of the effects of maturation treatments on the probabilities of somatic embryo germination and plantlet regeneration in pistachio using a linear logistic method

Embryogenic masses (EMSes) of pistachio (Pistacia vera L.) were proliferated in liquid Murashige and Skoog (MS) medium without growth regulators. To determine the effects of benzylaminopurine (BAP), racemic (±) abscisic acid (ABA) and sucrose treatments during maturation on the subsequent germination and plantlet regeneration, clusters of mature somatic embryos were transferred from maturation medium onto the surface of 0.7% agar-solidified Murashige and Skoog medium. Neither germination nor plantlet development medium contained BAP or ABA. Germination studies were carried out using 80 somatic embryos at every combination of four sucrose concentrations, three maturation periods and either five concentrations of BAP or four of ABA, and the numbers germinating were recorded after four durations of culture. A similar experimental plan was used to study plantlet regeneration. The number of germinated somatic embryos increased markedly with duration of the culture on germination medium, and was influenced by the concentrations of BAP or ABA in the maturation medium; the concentration of sucrose in this medium had little influence. Plantlet regeneration also increased with culture duration and was reduced at the highest levels of BAP or ABA; with ABA, the probability of plantlet regeneration was lower for longer maturation periods. ABA and BAP have similar effects on somatic embryo germination (except at the highest levels used), but BAP is superior to ABA for promoting subsequent plantlet regeneration. Linear logistic models were used to investigate the significance of the treatments, and to estimate the optimum conditions for germination and plantlet regeneration.

Somatic embryogenesis in Clematis integrifolia × C. viticella

Nodal sections of Clematis integrifolia × C. viticellawere cultured at 24 °C in darkness on a medium containing the salts and vitamins of Murashige and Skoog (1962) supplemented with sucrose (30 g l−1), 2 μM 6-benzylaminopurine (BAP) and 0.5 μM 4-indole-3-yl-butyric acid (IBA). These explants produced a white friable callus on their surfaces. Somatic embryos were first observed on the surface of the callus after eighteen months in culture. Thereafter, pieces (125 mm3) of the embryogenic mass were subcultured every 4 weeks and continued to produce somatic embryos over the two-year period of observation. A mean (± SE) of 64±4 cotyledonary stage embryos were observed per 125 mm3 callus four weeks after transfer to fresh medium. Comparison of the effect of growth regulators on conversion showed that the highest frequency of unipolar and bipolar conversions occurred on medium containing 10 μM kinetin and 1 μM BAP. Shoots were excised from embryos and inserted in Sorbarods saturated with liquid medium containing 0.05 μM IBA and 0.05 μM 1-naphthalenacetic acid. After four weeks 88% had formed root and survived transfer to compost in a greenhouse.

Regeneration and Possibility for Genetic Transformation of Rose

ABSTRACTThe important stage of genetic transformation is development effective and successful system for plant regeneration. As a major cut flower crop, it have been focused the attention according to this problems of many research groups from all over the world during the last 10 years. The research investigation concerns a development of new varieties to satisfy the continuously changeable requirements of numerous clients and admirers of this flower crop, and varieties more resistant to biotic and abiotic stress. In our study were used leaf explants from Bulgarian novel selected c.v. Anny (Rosa hybrida L.), using two pathways for plant regeneration- somatic embryogenesis and adventituos organogenesis. Several variants were tested for somatic embryogenesis. Some of the best results were achieved in liquid induction medium B5IV (Denchev P. et al., 1991) for a period of 2 weeks, induced explants were replaced on solidified medium for development B53MT (basal B5 supplemented with TDZ-0.2 mg/l and 3 % maltose) for 5 days. The maturation of somatic embryos was carried out on B5reg (B5 basal medium with 0.5 mg/l BAP and 0.01 mg/l NAA) resulting with around 50 % regeneration. This study attempted to achieve regeneration via adventituos organogenesis from leaf explants. The best results were showed on solidified MSN nutrient medium (basal MS supplemented with BAP-5 mg/l, TDz-1mg/1 and NAA-1 mg/l), cultivated first two weeks on dark, after that in conditions of growth room for 30 days. These results were reliable to carry out experiments of direct gene transformation with leaf disks and emryogenic mass (c.v. Anny). As a plasmid DNA was used binary vector pBECKS BIN400, containing selective mark genes GUS (under promoter 35S) and NPT II gene with promoter NOS for kanamycin resistance. Transient transformation was confirmed by glucuronidase activity of plant tissue using a fluorometric analysis of Jefferson et al., (1987) and putative stable-transformed rose embryogenic mass has been isolated. Several factors that have a significant affect on transformation efficiency were examined in an effort to optimize the biolistic process for gene transfer in roses. Some of the best results were achieved with leaf disks experiments.

Influence of boric acid on somatic embryogenesis of a cytosterile line of indica rice

The effect of boric acid on somatic embryo induction in rice was investigated using mature seeds of a widely used cytoplasmic male sterile line of indica rice IR-58025. Boric acid was added at a concentration of 100.00, 161.29, 241.93, 322.58 and 403.22 μM to the embryo induction medium containing basal salts, with 9.84 μM 2,4-dichlorophenoxyacetic acid. Boric acid was found to exert different effects on scutellum, coleoptile and root tissues. Increased growth of embryogenic masses with increasing boric acid concentrations was observed on coleoptile tissues of mature seed. Embryogenic response was observed at 161.29 μM boric acid on coleoptile and 241.93 μM boric acid on root tissue. Scutellum tissue showed embryonic response in 161.29 and 241.93 μM boric acid. The results indicate that the boric acid used in standard Murashige and Skoog medium is not sufficient to support induction of somatic embryogenesis in IR-58025 A.

Genotypic control of peanut somatic embryogenesis.

The protocol for obtaining a high frequency of plant development via somatic embryogenesis from mature zygotic embryo-derived leaflets of peanut (Arachis hypogaea L.) involves multiple stages; these include the induction of embryogenic masses, development of embryos, radicle emergence/conversion of embryos and the development of plants from rooted abnormal embryos. Sixteen genotypes were subjected to this protocol by exposing mature zygotic embryo-derived leaflets to the common media sequence and comparing responses. Although the protocol was effective for all the genotypes, variation in frequency of response at each stage of development indicated that, with the exception of root meristem differentiation and subsequent radicle emergence, the whole process of somatic embryogenesis depended on the genotypic constitution of the original plant. The failure of somatic embryos to undergo conversion to plantlets could be a genotype-dependent characteristic.

Somatic embryogenesis and somaclonal variation in Norway spruce: morphogenetic, cytogenetic and molecular approaches

Four embryogenic clones of Norway spruce have been subcultivated and observed over several years to determine the evolution of production of mature embryos and to assess the quality of the embryos produced. A wide range of intraclonal quantitative and qualitative variability has been observed within this production. Certain morphologic deviations appeared at the immature stage and after maturation, such as immature embryos with a diffuse organization, complete or part albino mature embryos or acclimated somatic seedlings comparable to dwarf mutants. All of these phenotypic variations could be the result of a modification of the genome itself or of only the expression of the genome. Two approaches, chromosome counting and RAPD (random amplified polymorphic DNA), were chosen for their capacity to detect genotypic variations: respectively, genomic and chromosomic or genic mutations. The cytogenetic approach revealed, for the first time in this species, three cases of mutated acclimated somatic plants: one totally trisomic and two chimeras with trisomic buds and diploid roots. Other cases of 5-year-old trisomic, double trisomic, tetraploid or mixoploid embryogenic masses were also detected. The molecular approach (RAPD) revealed no somaclonal variation despite the large sample of DNA and primers used and the important interclonal variation observed.

Histology of somatic embryo initiation and organogenesis from rhizome explants of Musa spp.

The origin of somatic embryos derived from rhizome explants of triploid Musa cv. Grand Nain was the subject of histological studies during different phases of ontogenetic development. The investigation revealed that the majority of somatic embryos showed normal root formation and consisted of highly vacuolated cells in the poorly structured shoot apex. The embryogenic mass and somatic embryos were mostly derived from several morphologically competent cells. Single cell origin depended on the presence of organogenetically functional vascular cells of rhizome explants and occurred infrequently. The implications of these findings for genetic improvement of banana and plantain by in vitro mutation breeding and gene technology are discussed.

An Improved Method for Embling Production in Pistachio, Pistacia vera L. Using

Benzylaminopurine (BAP) and racemic (±) abscisic acid (ABA) together with sucrose were tested for their ability to affect the maturation, germination and embling development of somatic embryos of the pistachio. The number of matured somatic embryos was found to be influenced significantly by concentrations of BAP , ABA and sucrose in the Iiquid maturation medium. The maturation of somatic embryos (SEs) was best achieved with a iow concentration (i.e. 0.5 mg 1{^-1}) of ABA, BAP, and plant-growth regulator-free Iiquid Murashige and Skoog (MS) medium. The duration of the embryo maturation treatment, the duration of the culture for germination and embling development, and the concentrations of ABA and BAP during germination and embling development also produced significant effects. Germination probabilities varied from 0.59 to 0.33 and 0.50 to 0.26 with BAP and ABA, respectively. The frequency of germination (0.62) was highest with the plant-growth regulator-free (PGR-free) control treatment and Iowest (0.26) with 2 mg 1{^-1} ABA. Under the same germination conditions, embling probabilities varied from 0.45 to 0.27 and 0.24 to 0.13 with BAP and ABA, respectively . The frequency of developed emblings was also highest (0.52) in the PGR-free control treatments and Iowest (0.13) with 2 mg 1{^-1} ABA. Abbreviations: ABA - abscisic acid; BAP - 6-benzylaminopurine (N^6-benzyladenine); EMS(es)embryogenic mass(es); MS - Murashige & Skoog; P-fitted probability; PGR(s) - plant - growth regulator(s); RH - relative humidity; SE(s) - somatic embryo(s)

Characterization of somatic embryogenesis-related cDNAs from alfalfa (Medicago sativa L.).

Messenger RNAs from cultures of embryogenic and non-embryogenic alfalfa (Medicago sativa L.) genotypes were used to differentially screen a cDNA library prepared from embryogenic cell masses of somatic embryo cultures to identify early-stage embryo transcripts. The three alfalfa somatic embryogenesis-specific transcripts cDNAs (ASET1, ASET2 and ASET3) identified by this screen were enriched in RNA samples from embryogenic tissues of the embryogenic genotype but were absent from petioles or mature embryos of an embryogenic genotype as well from tissue cultures of a nonembryogenic genotype. The ASET clones did not cross-hybridize and showed different patterns of expression in northerns of RNA from various fractions of alfalfa somatic embryo cultures. The ASET clones did not hybridize with the soybean embryogenesis-specific clone (Sbh1) which was shown to be expressed in embryogenic and non-embryogenic alfalfa tissue cultures. Sequencing showed ASET1 to be a partial transcript 595 nucleotides long. ASET2 was a complete transcript of 1193 nucleotides. From a comparison of the predicted open reading frame with the GenBank protein database it was concluded that ASET2 was a novel transcript. The protein predicted by the ASET2 sequence has several potential membrane-spanning domains and a potential phosphorylation site. In addition, the ASET2 cDNA had a long 5' region that contained two upstream reading frames (URFs) which could potentially code for 30 and 6 amino acid polypeptides.

Plant regeneration from encapsulated embryoids and an embryogenic mass of pistachio, Pistacia vera L.

Pieces of an embryogenic mass (EMS) induced in culture from immature fruits of pistachio, Pistacia vera L., were encapsulated into calcium alginate beads. Somatic embryos were also encapsulated individually into calcium alginate beads to produce synthetic seeds. The viability of the encapsulated EMS and somatic embryos was investigated immediately following encapsulation, and after storage for 60 days at 4°C. The encapsulated-stored EMS fragments recovered their original proliferative capacity after two months storage following two sub-cultures, but non-encapsulated-stored EMS failed to recover. The conversion frequency of synthetic seeds to seedling plants was 14% after storage for 60 days at 4°C, from which it may be concluded that encapsulation is a practical procedure for short-term storage of embryogenic pistachio tissue, and may be applicable to the preservation of desirable elite genotypes.

A comparison of normal callus culture and habituated ESM of silver fir (Abies albamill.) for defense reactions to the fungusPhaeolus schweinitzii

Normal callus culture and habituated embryogenic suspensor mass (ESM) of silver fir (Abies alba Mill.) were compared enzymatically using three enzyme systems. Significant reduction of peroxidase activity, more intensive glutamate dehydrogenase activity and higher number of isoenzymes of non‐specific esterase were found in habituated ESM as compared with the normal calli. Defense reactions of normal callus culture and habituated ESM were tested by a simple method of dual cultures. Habituated ESM showed strengthened defense reactions against tester Phaeolus schweinitzii. Reasons of this phenomenon are discussed.

Polyamine biosynthesis during somatic embryogenesis in interior spruce (Picea glauca x Picea engelmannii complex)

Putrescine, spermidine, and spermine levels during somatic embryogenesis of interior spruce (Picea glauca x Picea engelmannii complex) were quantified On abscisic acid supplemented growth medium putrescine and spermidine levels increased two-fold coinciding with maturation of the early somatic embryos to globular embryos. Polyclonal antibodies raised against Escherichia coli arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), following affinity purification specifically recognized spruce ADC and ODC, which corresponded to 85kD and 65kD bands on western blots of total protein extracts from embryogenic masses, Immunoassays using these antibodies showed increased ADC levels corresponding to embryo maturation while ODC levels remained the same. From these results it is concluded that polyamines are involved in the maturation of somatic embryos of interior spruce.

Somatic embryogenesis in cultured immature kernels of Pistachio, Pistacia vera L.

Embryogenic tissue was produced from kernels of immature fruits of Pistachio (Pistacia vera L.) cultured in liquid Murashige and Skoog media, supplemented with 200 mgl(-1) casein hydrolysate, 114 μM 1-ascorbic acid, and benzylaminopurine. Compact embryogenic masses differentiated directly from the fruit explants after culture for 2 weeks in liquid medium with 8.9 μM benzylaminopurine. After transfer of the embryogenic masses into the same medium, but with 4.4 μM benzylaminopurine, somatic embryos appeared. Several stages of embryogenesis were present in the cultures. Adventive embryos were readily separated from the friable embryogenic masses by shaking. Separated somatic embryos, germinated on solidified Murashige & Skoog medium without growth regulators, developed into plantlets.

In vitro organogenesis from diploid tissues of Cycas revoluta Thunb.

Zygotic embryos, stratified and collected at 8, 16 and 24 weeks and cotyledonary and epicotyl explants from 30 and 45 day old Cycas revoluta Thumb. seedlings were cultured in vitro to determine their morphogenic potential. Depending upon the duration of stratification and growth regulator type, zygotic embryos exhibited a different morphogenic response. The youngest embryos rarely survived, while larger ones formed a callus, depending on the growth regulator in the medium, and the oldest ones produced putative embryogenic masses. Cytokinin levels double the auxin concentrations resulted in shoot regeneration in almost mature embryos. In addition, in vitro culture of younger cotyledonary explants gave shoot regeneration, while older ones formed adventitious roots. The effectiveness of BA in inducing shoot regeneration from seedling explants was confirmed. NAA in combination with cytokinin in the induction medium inhibited bud induction. These results show that the morphogenic potential of tissues is linked to their developmental stage.

In vitro regulation of morphogenesis in peanut ( Arachis hypogaea L.)

Callusing, caulogenesis, in vitro flowering and somatic embryogenesis were induced from the base of leaflets derived from mature embryos of peanut ( Arachis hypogaea) by altering the hormonal composition of the Murashige and Skoog's (MS) basal medium. A combination of 4 mg/l alpha napththaleneacetic acid (NAA) and 5 mg/l 6-benylaminopurine (BAP) was optimum for inducing caulogenic buds. The caulogenic buds proliferated in medium with 3 mg/l BAP. Differentiation of these buds to shoots was achieved in MS basal medium with 0.5 mg/l each of BAP and kinetin (KN). Shoot buds and flower buds were produced when caulogenic buds were cultured on medium containing 1 mg/l BAP and 1 mg/l KN, prior to elongation. Clonally propagated plantlets derived from axillary buds elongated, formed roots and were grown to maturity in soil. Embryogenic mass formation was induced from the leaf base in the presence of 20 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos developed upon reducing 2,4-D to 3 mg/l.

Development and germination of pecan somatic embryos derived from liquid culture

Aggregates of globular and pre-globular stage somatic embryos from suspension cultures of pecan (Carya illinoensis Koch) were cultured on solidified media for embryo development. Embryo aggregates and pre-globular stage embryo masses were given various treatments to further ontologic development. A 2- to 4-wk mild dehydration of the embryo aggregates suppressed recurrent embryogenesis, promoted development of globular embryos into cotyledonary stage embryos, and enhanced plant development beyond germination. Fine embryogenic tissue masses filtered from suspension formed cotyledonary-staged embryos when the collection filters were plated on solified medium. The embryogenic capacity of preglobular stage embryo masses was compared between media supplemented with varying concentrations of polyethylene glycol (molecular weight 8 000) vs. filter overlays. The filter paper overlays were not necessary for embryo development. An inverse relationship was found between the number of embryos that developed and the concentration of polyethylene glycol in the medium. However, this relationship was reversed for ability of embryos to germinate and develop into a plant.

Somatic embryogenesis of Prunus subhirtella autumno rosa and regeneration of transgenic plants after Agrobacterium-mediated transformation

Embryogenic lines of Prunus subhirtella autumno rosa were established on a modified MS medium supplemented with 1 mg/l NAA, 0.06 mg/l IBA and 0.04 mg/l BA from petioles of axenically grown shoots of adult origin. To induce normal development of plantlets we compared a range of approaches on solid culture media as well as in suspension cultures including treatments with ABA, GA3, zeatin, darkness, and cold. A series of experiments were conducted to follow the temporal pattern of somatic embryo development.Separation of embryos at different stages of development was carried out by sieving the suspension cultures through nylon nets. While the embryogenic masses were used for further subcultures, well formed embryos were used for germination experiments.Transformed Prunus subhirtella plants were regenerated from somatic embryos by inoculating an embryogenic callus with Agrobacterium strain LBA 4404 containing the ß-glucuronidase (GUS) gene on plasmid pBinGUSint. Several putative transformed embryogenic calli were selected for continued proliferation on kanamycin containing media. Finally transgenic plants were regenerated on shoot multiplication medium containing kanamycin. Embryos and plants were shown to express the GUS gene by histochemical assays and northern blot analysis. With a yield of 110 transgenic lines from a single transformation experiment this approach appears ideal for the study of the influence on level of expression caused by different copy number, site of insertion etc. This will be helpful in establishing parameters according to which the best transgenic line for a chosen purpose should be selected.