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.     

Detection of somaclonal variations in tissue cultured date palm (Phoenix dactylifera L.) using transposable element-based markers

Plant Cell, Tissue and Organ Culture (PCTOC) - In vitro regeneration of date palm (Phoenix dactylifera L.) plants through somatic embryogenesis leads to the generation of somaclonal variants. The...     

Induction of somatic embryogenesis in Brassica juncea L. and analysis of regenerants using ISSR-PCR and flow cytometer

A new and simple protocol has been developed and standardized for direct somatic embryogenesis and plant regeneration from aseptic seedlings derived from immature Brassica juncea seeds. Depending on the age of immature seeds and nutrient media, in vitro occurrence of embryogenesis and the number of embryos from each seedling have varied greatly. The largest number of somatic embryos, producing 12.7 embryos per seedlings, have been developed by seedlings obtained from immature seeds collected after 21 days of pollination (DAP). Effect of different nutrient media [Gamborg (B5), Murashige and Skoog (MS) and Linsmaier and Skoog (SH)] and carbon sources (fructose, glucose, maltose and sucrose) were assessed to induce somatic embryos and the maximum response were achieved on Nitsch culture medium fortified with sucrose (3% w/v) followed by fructose and maltose. The somatic embryo converted into complete plantlets within 04-weeks of culture on Nitsch medium containing half-strength of micro and macro salts. The regenerated plantlets were successfully established in soil with 90% survival rate. The acclimated plants were subsequently transferred to field condition where they grew normally without any phenotypic differences. Genetic stability of B. juncea plants regenerated from somatic embryos were confirmed by inter-simple sequence repeat (ISSR)-PCR analysis and flow cytometry. No significant difference in ploidy level and ISSR banding pattern were documented between somatic embryo’s plants and control plants grown ex vitro.     

Detection of somaclonal variation of cotton (Gossypium hirsutum) using cytogenetics, flow cytometry and molecular markers

Two protocols of plant regeneration for cotton were adopted in this study, namely, 2, 4-D and kinetin hormone combination and IBA and kinetin hormone combination. Twenty-eight embryogenic cell lines via somatic embryogenesis and 67 regenerated plants from these embryogenic calli were selected and used for random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), chromosomal number counting, and flow cytometric analysis. The roles of RAPD and SSR markers in detecting somaclonal variation of cotton (Gossypium hirsutum L.) were evaluated. Two cluster analyses were performed to express, in the form of dendrograms, the relationships among the hormone combinations and the genetic variability. Both DNA-based techniques were able to amplify all of the cell clones and regenerated plantlets genomes and relative higher genetic variation could be detected in the culture type with 2, 4-D and kinetin hormone combination. The result suggested that 2, 4-D and kinetin hormone combination could induce relative high somaclonal variation and RAPD and SSR markers are useful in detecting somaclonal variation of regenerated cotton plants via somatic embryogenesis. Chromosome number counting and flow cytometry analysis revealed that the number of chromosomes and ploidy levels were nearly stable in all regenerated plants except two regenerated plantlets (lost 4 and 5 chromosomes, respectively) which meant that cytological changes were not correlated with the frequency of RAPD and SSR polymorphisms. This result also might mean that the cell lines with variation of chromosome numbers were difficult to regenerate plants.     

Selection of a super-growing legume root culture that permits controlled switching between root cloning and direct embryogenesis

 Root cultures, displaying vigorous growth and high embryogenic capacity, were established in the legume forage species Lotus corniculatus (bird’s-foot trefoil). Root cloning as well as plant regeneration was achieved on hormone-free medium, in agitated culture in the dark or under stationary conditions in the light, respectively. These qualities of vigorous growth and regeneration faded with time in hormone-free culture, with slow-growing roots turning brown in color. Addition of the synthetic cytokinin-like hormone benzylaminopurine to the culture medium, however, re-established the aging tissue’s capacity for somatic embryogenesis and plant formation. During continuous initiation of new cultures, it was possible to obtain one root culture (selected from 11 960 seeds at a 65% germination rate) which did not show the typical decline of qualities after prolonged proliferation but distinguished itself by displaying even faster growth and more vigorous embryogenic plant production on hormone-free medium. There was no decline since its initiation 9 months earlier. This super-growing root culture produces plants that show no morphological differences as compared to wild-type regenerants or seedlings. Roots, dissected from plantlets derived from super-root embryogenesis, expressed all the super-root qualities again when cultured in vitro. This is the first report on somatic embryogenesis from sustained root cultures without exogenous hormone application. Such a hormone-free, continuous root culture should provide a superior experimental system for genetic or developmental studies that might be sensitive to exogenous hormones, such as somaclonal variation in transgenesis or, since introduced in a legume species, nodulation in vitro. Received: 22 September 1997 / Accepted: 21 October 1997     

An efficient in vitro system for somatic embryogenesis and podophyllotoxin production in Podophyllum hexandrum Royle

Podophyllum hexandrum Royle known as Indian mayapple is an important medicinal plant found only in higher altitudes (2,700 to 4,200 m) of the Himalayas. The highly valued anticancer drug Podophyllotoxin is obtained from the roots of this plant. Due to over exploitation, this endemic plant species is on the verge of extinction. In vitro culture for efficient regeneration and the production of podophyllotoxin is an important research priority for this plant. Hence, in the present study, an efficient plant regeneration system for mass multiplication through somatic embryogenesis was developed. We have screened P. hexandrum seeds collected from three different regions in the Himalayas to find their regenerative potentials. These variants showed variation in germination percentage as well as somatic embryogenic frequency. The seeds collected from the Milam area of Pithoragarh district showed better germination response (99.3 %) on Murashige and Skoog (MS) medium fortified with Gibberellic acid (GA₃ [5 mg/l]) and higher direct somatic embryogenic frequency (89.6 %). Maximum production of embryogenic callus (1.2 g fresh weight [FW]) was obtained when cotyledons containing the direct somatic embryo clusters were cultured in MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D [1.5 mg/l]) after 4 week of culture in complete darkness. In the present investigation, somatic embryogenesis was accomplished either by direct organogenesis or callus mediated pathways. The latter method resulted in a higher frequency of somatic embryo induction in hormone-free MS medium yielding 47.7 embryos/50 mg of embryogenic callus and subsequent germination in MS medium supplemented with GA₃ (5 mg/l). Seventy-nine percent of embryos attained complete maturity and germinated into normal plants with well-developed roots. Systematic histological analysis revealed the origin of somatic embryo and their ontogenesis. The higher level of podophyllotoxin (1.8 mg/g dry weight [DW]) was recorded in germinated somatic embryos when compared to field grown plants. The present system can be widely used for mass propagation, transgenic recovery, and podophyllotoxin production for commercial utilization.     

Detection of somaclonal variants in somatic embryogenesis-regenerated plants of <Emphasis Type="Italic">Vitis vinifera</Emphasis> by flow cytometry and microsatellite markers

Flow cytometry and microsatellite analyses were used to evaluate the trueness-to-type of somatic embryogenesis-regenerated plants from six important Spanish grapevine (Vitis vinifera L.) cultivars. Tetraploid plants were regenerated through somatic embryogenesis from all of the cultivars tested with the exception of ‘Merenzao’. In addition, an octoploid plant was obtained in the cv. ‘Albariño’, and two mixoploids in ‘Torrontés’. The most probable origin of these ploidy variations is somaclonal variation. The cv. ‘Brancellao’ presented significantly more polyploids (28.57%) than any other cultivar, but it must be noted that 50% of the adult field-grown ‘Brancellao’ mother plants analysed were mixoploid. Hence, it is probable that these polyploids originated either from somaclonal variation or by separation of genotypically different cell layers through somatic embryogenesis. Microsatellite analysis of somatic embryogenesis-regenerated plants showed true-to-type varietal genotypes for all plants except six ‘Torrontés’ plants, which showed a mutant allele (231) instead of the normal one (237) at the locus VVMD5. There was not a clear relationship between the occurrence of the observed mutant regenerated plants and the callus induction media composition, the developmental stage of the inflorescences, the type of explant used for starting the cultures or the type of germination (precocious in differentiation medium or normal in germination medium) in any of the cultivars tested, except ‘Torrontés’. The mutant plants described herein have been transplanted to soil for future evaluation of putative phenotypic traits of interest. These mutants can be useful both for breeding programs and for functional genomic approaches aimed at increasing knowledge of the biology of grapevine.     

A simple method for the recovery of multiple fertile plants from individual somatic embryos of soybean [Glycine max (L.) merrill]

Summary We describe a method for obtaining and proliferating multiple, fertile plants from somatic embryos of several experimental and commercial soybean varieties. Shoot-bud cultures were initiated by placing cotyledonary and torpedo-stage somatic embryos derived from immature seedling cotyledons onto Cheng’s basal medium (CBO) containing 0.5 to 2.5 mg/liter 6-benzyladenine (6-BA). Prolific masses of adventitious shoots were produced within 6 to 18 wk. These cultures can be propagated indefinitely with regular subcultures to CBO containing 0.5 mg/liter 6-BA. Individual shoots were separated from the clusters and were rooted on CBO medium without exogenous growth regulators. By this method any number of plants can be produced from individual somatic embryos. The risk of losing valuable genotypes (e.g., derived from in vitro selection or transformation) due to inefficient embryo germination and embryo-to-plant conversion is thus greatly reduced. Plants were established in the greenhouse and progenies were field tested. Progenies from shoot-bud culture-derived plants showed no somaclonal variation for the seven recessive marker traits or quantitative agronomic characters evaluated under field conditions.     

A rapid protocol for somatic embryogenesis from immature leaflets of groundnut (Arachis hypogaea L.)

Summary Plant regeneration via somatic embryogenesis was developed in two groundnut varieties. Somatic embryogenesis was induced from immature leaflets on MS medium with different concentrations of the auxins 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) in combination with 0.5 mg/l of the cytokinin BA. The highest frequency of somatic embryo formation occurred on MS medium fortified with 20 mg 2,4-D per l. Of the two auxins tested individually 2,4-D was more effective for induction of embryogenesis as well as production of embryos. Embryo development and maturation was achieved on MS medium supplemented with N⁶-benzyladenine (BA) (0.5–2.0 mg/l) and 2,4-D (0.5 mg/l). Plant conversion frequency from somatic embryos was highest in presence of 2.0 mg BA per l and 0.5 mg NAA per l. The frequency of embryogenesis and plant regeneration was higher in the VRI-2 cultivar than in the other cultivar tested. Regenerated plants were transferred to soil, grown to maturity, and produced viable seeds.     

Analysis of genomic DNA methylation patterns in regenerated and control plants of rye (Secale cereale L.)

Rye (Secale cereale L.) is a species that has shown high rates of somaclonal variation when plants obtained by in vitro culture were analysed using different techniques. In this study, using methylation-sensitive amplified polymorphism (MSAP) markers, we analysed the cytosine methylation status at genomic level of regenerated plants of rye that were obtained by somatic embryogenesis. Such plants were originated from three different cell lines and the results were compared with the data obtained from the control plants grown from seeds of the same cultivar and lot. A similar total number of MSAP markers was observed in the regenerated (937) and control plants (1,022), while the mean number detected per plant was significantly higher in regenerated (554.43) than in control plants (356.00). The analysis indicated conservation of the number of partially-methylated CCGG/GGCC sites for all type of plants. However the mean number of non-methylated sites was near twofold in the regenerated plants (442.48) than in control plants (248.19). Methylation changes have been detected in all the regenerated plants when compared within cell lines, with an average frequency of 9.01 % of the detected markers. We also observed that regenerated plants from one or several cell lines shared methylation changes at the same locus pointing to a non-random behaviour of the changes in genomic methylation.     

Chromosomal location of genes controlling short-term and long-term somatic embryogenesis in wheat revealed by immature embryo culture of aneuploid lines

The expression of essential genes during somatic embryogenesis can be analysed by inducing aneuploid cells to undergo embryogenesis during immature embryo culture and then determining whether defects occur. Triticum aestivum disomic and aneuploid stocks, including 36 ditelosomics and 7 nullitetrasomic Chinese Spring wheats, were compared for their ability to undergo somatic embryogenesis after 2 months of in vitro immature embryo culture. Their regeneration capacity was observed after 4 and 14 months of in vitro culture to determine which chromosome arms influence the process. The large range of variation found among the tested aneuploids suggested that genetic control of the somatic tissue culture ability is polygenic. Our results indicate that genes affecting somatic embryo-genesis and regeneration are located in all of the homoeologous chromosome groups. The lack of chromosome arms 1AL (DT 1AS) and 3DL (DT 3DS) practically suppresses somatic embryogenesis, demonstrating that major genes on wheat chromosome arms 1AL and 3DL control regeneration capacity. Results suggest that plants were mainly produced from somatic embryo development. Although the control of somatic embryogenesis and regeneration is polygenic, the genes located on the long arms of homoeologous group 3 chromosomes have a major effect. We also have evidence of chromosome arms that determine the time required for regeneration.     

Developmental and hormonal regulation of direct shoot organogenesis and somatic embryogenesis in sugarcane (Saccharum spp. interspecific hybrids) leaf culture

Rapid and efficient in vitro regeneration methods that minimise somaclonal variation are critical for the genetic transformation and mass propagation of commercial varieties. Using a transverse thin cell layer culture system, we have identified some of the developmental and physiological constraints that limit high-frequency regeneration in sugarcane leaf tissue. Tissue polarity and consequently the orientation of the explant in culture, size and developmental phase of explant, and auxin concentration play a significant role in determining the organogenic potential of leaf tissue in culture. Both adventitious shoot production and somatic embryogenesis occurred on the proximal cut surface of the explant, and a regeneration gradient, decreasing gradually from the basal to the distal end, exists in the leaf roll. Importantly, auxin, when added to the culture medium, reduced this spatial developmental constraint, as well as the effect of genotype on plant regeneration. Transverse sections (1-2 mm thick) obtained from young leaf spindle rolls and orienting explants with its distal end facing the medium (directly in contact with medium) are critical for maximum regeneration. Shoot regeneration was observed as early as 3 weeks on MS medium supplemented with alpha-naphthalenencetic acid (NAA) and 6-benzyladenine, while somatic embryogenesis or both adventitious shoot organogenesis and somatic embryogenesis occurred on medium with NAA and chlorophenoxyacetic acid. Twenty shoots or more could be generated from a single transverse section explant. These shoots regenerated roots and successfully established after transplanted to pots. Large numbers of plantlets can be regenerated directly and rapidly using this system. SmartSett, the registered name for this process and the plants produced, will have significant practical applications for the mass propagation of new cultivars and in genetic modification programs. The SmartSett system has already been used commercially to produce substantial numbers of plants of orange rust-resistant and new cultivars in Australia.     

In vitro manipulation and propagation of medicinal plants

Well developed techniques are currently available to help growers meet the demand of the pharmaceutical industry in the next century. These protocols are designed to provide optimal levels of carbohydrates, organic compounds (vitamins), mineral nutrients, environmental factors (e.g. light, gaseous environment, temperature, and humidity) and growth regulators required to obtain high regeneration rates of many plant species in vitro and thereby facilitate commercially viable micropropagation. Well-defined cell culture methods have also been developed for the production of several important secondary products. An overview of the regeneration of medicinal plants by direct and indirect organogenesis and by somatic embryogenesis from various types of explants is presented, and the use of these techniques combined with other biotechnological approaches to improve medicinal plants through somaclonal variation and genetic transformation is reviewed.     

Influence of antibiotic cefotaxime on somatic embryogenesis and plant regeneration in indica rice

An antibiotic, cefotaxime (Omnatax) has been found to promote somatic embryogenesis and subsequent plant regeneration in vitro in indica-type basmati rice cultures. Response was highly genotype specific. The number, mass and morphology of the calli formed on the scutellar tissues were dependent on the growth medium (with or without cefotaxime). The embryogenic nature of nodular calli was confirmed through histological analysis and their plant regeneration ability. The calli of variety Pusa basmati 1 grown on medium supplemented with cefotaxime (100 mg/L) exhibited up to 70.5% plant regeneration as compared to control (51.51%). Plants regenerated from emryogenic calli were phenotypically normal and identical to seed-derived plants and exhibited normal fertility. A limited humidity and an optimal aeration of the culture tubes further enhanced the frequency of somatic embryogenesis and plant regeneration.     

High frequency regeneration via direct somatic embryogenesis and efficient Agrobacterium- mediated genetic transformation of tobacco

A direct somatic embryogenesis protocol was developed for four cultivars of Nicotiana species, by using leaf disc as an explant. Direct somatic embryogenesis of Nicotiana by using BAP and IAA has not been investigated so far. This method does not require formation of callus tissues which leads to somaclonal variations. The frequency of somatic embryogenesis was strongly influenced by the plant growth hormones. The somatic embryos developing directly from explant tissue were noticed after 6 d of culture. Somatic embryogenesis of a high frequency (87–96%) was observed in cultures of the all four genotypes (Nicotiana tabacum, N. benthamiyana, N. xanthi, N. t cv petihavana). The results showed that the best medium for direct somatic embryogenesis was MS supplemented with 2.5 mg/l, 0.2 mg/l IAA and 2% sucrose. Subculture of somatic embryos onto hormone free MS medium resulted in their conversion into plants for all genotypes. About 95% of the regenerated somatic embryos germinated into complete plantlets. The plants showed morphological and growth characteristics similar to those of seed-derived plants. Explants were transformed using Agrobacterium tumifacious LBA4404 plasmid pCAMBIA1301 harboring the GUS gene. The regenerated transgenic plants were confirmed by PCR analysis and histochemical GUS assay. The transformation efficiency obtained by using the Agrobacterium- mediated transformation was more than 95%. This method takes 6 wk to accomplish complete transgenic plants through direct somatic embryogenesis. The transgenic plantlets were acclimatized successfully with 98% survival in greenhouse and they showed normal morphological characteristics and were fertile. The regeneration and transformation method described herein is very simple, highly efficient and fast for the introduction of any foreign gene directly in tobacco through direct somatic embryogenesis.     

Regeneration by somatic embryogenesis of triploid plants from endosperm of walnut,Juglans regia L. cv Manregian

Plants were regenerated by somatic embryogenesis from endosperm tissue of open-pollinated seeds of Juglans regia L. cv Manregian. These plants were obtained by growing endosperm tissue on media similar to those used for plant regeneration from walnut cotyledons (Tulecke and McGranahan 1985). The plants appear morphologically uniform and have a triploid chromosome number of 3n=48. Nine plants have been grown to a young sapling stage in soil. This embryogenic line from endosperm has been maintained in culture for two years by the process of repetitive somatic embryogenesis.     

Analysis of lipid composition and morphological characteristics in soybean regenerants

A study was conducted to examine the extent of somaclonal variation of soybean plants, Glycine max (L.) Merrill cv. McCall, regenerated via somatic embryogenesis from cultured immature cotyledons using two different protocols. The sexual progeny of regenerants were compared with normal, seed-derived populations for morphological characteristics and fatty acid composition of seeds. First generation progeny of regenerants showed greater phenotypic variation than the control population, but this variation was not observed in the second generation. No stable somaclonal variants for fatty acid composition of the seed oil or morphological characteristics were observed, indicating that this somatic embryogenesis system should be adaptable for transformation with minimal generation of unwanted variation.Abbreviations NAA 1-naphtaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid Contribution from the Department of Agronomy, University of Kentucky, Lexington, KY 40546. The research, was supported by the American Soybean Association and the Lubrizol Genetics Company, published with the approval of the director of the Kentucky Agricultural Experiment Station as Journal Article Number 88-3-264. Offprint requests to: D.F. Hildebrand     

Somatic embryogenesis of the banana hybrid cultivar FHIA-18 (AAAB) in liquid medium and scaled-up in a bioreactor

The development of somatic embryos in liquid culture medium has a number of advantages for large-scale propagation of plants. This paper describes an improved system for the mass propagation via somatic embryogenesis of the banana hybrid cultivar FHIA-18 (AAAB). Explants from immature male flowers were used to form high frequency embryogenic tissue, this tissue was then used to establish embryogenic cell suspensions in a basic MS medium plus 1.0 mg l^–1 biotin, 100 mg l^–1 glutamine, 100 mg l^–1 malt extract (Sigma), 1.0 mg l^–1 2,4-D and 45 g l^–1 sucrose. Secondary multiplication of somatic embryos was achieved in liquid media in rotary shaker and in bioreactors. The number of embryos per litre obtained with 80.0% DO₂ and effects of pH were also studied. A high regeneration percentage of plants were obtained (89.3%) in only 1 month of culture, somatic embryos were then placed to germinate in temporary immersion systems and field testing of somaclonal variation.     

Factors Influencing Somatic Embryogenesis Induction and Plant Regeneration with Particular Reference to Arabidopsis thaliana (L.) Heynh

The broad applications of somatic embryogenesis, both in basic and applied research, have stimulated studies on the determination of in vitro conditions for the induction of somatic embryos and their conversion into plants. As a result, efficient protocols on SE induction and plant regeneration have recently become available for many plant species, including Arabidopsis thaliana (L.) Heynh., a model plant in genetics and embryogenesis.Studies on factors controlling in vitro plant morphogenesis are highly desirable not only for the development of improved regeneration systems, but also for the analysis of molecular mechanisms underlying plant embryogenesis. This review focuses on the conditions influencing the induction of embryogenic potential in in vitro cultured plant cells. The roles of explant type, endo- and exogenous plant growth regulators and stress factors in the induction of somatic embryogenesis are especially emphasized. Possible mechanisms by which different factors induce or modify embryogenic competence in cultured plant cells are also discussed. Since the production of genetically solid and true-to-type plants is desired, especially for transformation and micropropagation practice, the problem of the genetic characteristics of regenerants, in terms of their chimerism and somaclonal variation, is discussed in some detail.Special consideration is given to A. thaliana– a major model plant species for classical genetics and genomics. Recent availability of efficient embryogenic cultures in this organism makes it possible to benefit from advanced genomic research of Arabidopsis to study plant embryogenesis on the molecular level.     

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.