Embryogenic Response Research Articles

First stages of microspore reprogramming to embryogenesis through anther culture in Prunus armeniaca L.

Prunus armeniaca L. is a worldwide known species, very important particularly in the Mediterranean basin. Microspore embryogenesis through in vitro anther culture is a widely used method to obtain haploid and doubled haploid (DHs) plants which are being routinely used in breeding programmes for new superior cultivar development in many crops. Haploid–diploidization through gametic embryogenesis allows single-step development of complete homozygous lines from heterozygous parents. In the case of fruit crops, with long reproductive cycle, a high degree of heterozygosity, large size, and, often, self-incompatibility, there is no way to obtain haploidization through conventional methods. Induction of microspore embryogenesis in vitro is switched by a stress treatment. In many species, heat or cold stress has been reported to trigger pollen embryogenesis, the response being genotype dependent. In the present work we analyzed whether microspore reprogramming could be induced in apricot cultivars by cold stress through anther culture. We report the development of an in vitro anther culture protocol in P. armeniaca L. and analyse the response of several cultivars to stress treatments and culture media for inducing pollen embryogenesis. Results showed the formation of multicellular pollen and proembryos. The effect of two culture media in the embryogenic response was also analyzed, being the responses genotype-dependent. Monitoring of the cellular changes on the microspores was performed by structural and confocal microscopy analyses. Results indicated that the reprogramming of the microspore and the first steps of the embryogenic pathway have been achieved in different varieties of P. armeniaca, which constitutes a crucial step in the design of protocols for the regeneration of microspore-derived embryos and DH plants, for future potential applications in breeding programmes of this economically important fruit tree.

Factors influencing induction and maintenance of Vitis rotundifolia Michx. embryogenic cultures

Unopened leaves, petioles and fully opened leaves from micropropagation cultures of five Vitis rotundifolia Michx. varieties were cultured on induction medium to study their embryogenic response. Among the various explants tested, the maximum number of varieties produced embryogenic cultures from unopened leaves followed by fully opened leaves and petioles. Based on morphological differences, two types of embryogenic cultures were identified. Friable cultures typically arose as proembryonic masses (PEM) on induction medium, whereas somatic embryo production without an intervening PEM stage was observed in compact cultures. Of the five varieties tested, the highest frequency of embryogenic response was observed from fully opened leaves of ‘Supreme’ and unopened leaves and petioles of ‘Delicious’. Attempts to initiate suspension cultures from varieties resulted in proliferation and maintenance of ‘Alachua’ and ‘Carlos’ cultures in liquid medium for 16 weeks. Embryogenic potential of varieties was studied on cultures growing on embryo development medium. The maximum number of cotyledonary stage somatic embryos from 0.2 g proembryonic masses were observed in ‘Carlos’ (379.3) followed by ‘Alachua’ (350.0) and ‘Delicious’ (305.0). Cotyledonary stage somatic embryos germinated when cultured on Murashige and Skoog medium containing 1 μM Benzyladenine (BA). Although high embryo germination rates (80–100%) were observed in the varieties tested, plant recovery from germinated somatic embryos ranged from 6–47%. Embryogenic cultures could be maintained on X6 medium and used in genetic engineering studies.

Haploids and doubled haploids in Brassica spp. for genetic and genomic research

The availability of a highly efficient and reliable microspore culture protocol for many Brassica species makes this system useful for studying basic and applied research questions. Microspores and microspore-derived embryos are ideal targets for modification by mutagenesis and transformation. Regenerated doubled haploid plants are widely used in breeding programs and in genetic studies. Furthermore, the Brassica microspore culture system allows the identification of genomic regions and genes involved in the microspore embryogenic response, spontaneous diploidization and direct embryo to plant conversion. This review summarizes current achievements and discusses future perspectives.

DEVELOPMENT OF AN EFFICIENT REGENERATION SYSTEM FOR EGYPTIAN CULTIVAR(S) OF BARLEY (Hordeum vulgare L.)

Cultivated barley (Hordeum vulgare L.) belongs to the Triticeae in the grass family, Poaceae. Barley ranks as the world’s fourth most important cereal crop after wheat, maize and rice. It is grown over a wide environmental range than any other cereal crop. It is more tolerant to drought, summer frosts, saline and alkaline soils than other cereals; the grain is used for human food, animal feed and malt (FAO, 1998). Although barley is an important member of the cereal crops, it lags behind in gene transfer technology with regard to the introduction of elite agronomic traits such as disease and herbicide resistance. Non-sexual transfer of DNA to cereal species such as Hordeum vulgare L. has historically been problematic, chiefly because of problems encountered during attempts to regenerate cultured transgenic cells. Frequently, few or no plants can be recovered following selection for transformed cells, or only albino can be recovered (Lemaux et al., 1999). The recovery of fertile plants from transgenic callus is a critical component of many cereal transformation systems (Bregitzer et al., 1998; Hussein et al., 2004; Assem et al., 2008). Efficient regeneration of plants from transformed embryogenic callus is often limited to specific genotypes that exhibit vigorous plant regeneration (Bregitzer et al., 1998). Improved plant regeneration from elite barley cultivars will facilitate their genetic transformation. The media composition is an important key factor influencing the regeneration ability of different genotypes. Different media compositions have been applied by Bregitzer (1992) and Hussein et al. (2004). Therefore, it is necessary to optimize the regeneration conditions for the Egyptian cultivars. The objectives of the present investigation were: to establish an efficient regeneration system for some Egyptian barley genotypes using immature embryos as explants, and to evaluate the effect of different media composition on the embryogenic response and regeneration ability of the barley genotypes.

Embryogenic responses of Beta vulgaris L. callus induced from transgenic hairy roots

Agrobacterium rhizogenes A4M70GUS-mediated transformation of two local breeding lines of sugar beet was obtained using 4-week-old seedlings. Root formation efficiency was 61.54% for SBa genotype and 36.36% for SBb genotype. Five highly proliferated hairy root lines have been established in liquid hormone-free MS medium. Transgenic nature of the hairy root clones was evaluated by GUS assay, PCR and RT-PCR analyses. Hairy root-derived calli were induced using different plant growth regulators (PGRs): auxin, auxin/cytokinin and cytokinin. The best callus induction response was achieved on MS medium containing both 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg/l thidiazuron (TDZ). Globular embryo-like structures were observed in friable callus after its prolonged cultivation on MS medium supplemented with TDZ and giberellic acid (GA3) at 1 mg/l each, followed by growth on MS medium containing 1% glucose and 0.5 mg/l 2,3,5-triiodobenzoic acid (TIBA). Histological analysis revealed somatic embryos at different stages of development in hairy root-derived callus of sugar beet.

Influence of genotype and age of explant source on the capacity for somatic embryogenesis of two Cavendish banana cultivars (Musa acuminata Colla, AAA)

The embryogenic capacities of flower explants from one- and two-week-old male inflorescence buds from Musa acuminata Cavendish, AAA, genotypes ‘Williams’ and ‘Grand Naine’ were investigated. Explants of hands with immature flowers were excised and induced for embryogenesis. Highly significant differences were found in the embryogenic response, depending on the genotype and the developmental stage of the buds from which the explants were excised. After four months of induction, the total percentage of callus formed ranged from 97.81% in explants of two-week-old ‘Williams’ buds to 52.11% in explants of two-week-old ‘Grand Naine’ buds. Embryogenic callus formation was, on average, higher in the two-week-old ‘Williams’ explants (10.01%) than in the one-week-old explants (0.78%). The opposite was true for ‘Grand Naine’, with 7.51% embryogenic callus produced in explants of one-week-old buds compared to 2.49% in two-week-old buds. Selected embryogenic calluses that were successfully established on proliferation medium led to embryogenic cell suspensions with good regeneration capacities. Fifteen to thirty-five percent of the embryos germinated, demonstrated high plant-conversion capacity (99%). The effect of the interaction between the developmental age of explant and the genotype on the embryogenesis response is discussed.

Nitrogen compounds in embryogenic and non-embryogenic calluses of Medicago arborea L.

Nitrogen (N) metabolism during embryogenesis may be fundamental in the embryogenic response. We used different explants of Medicago arborea L. subsp. arborea seedlings: cotyledons, petioles and leaves, which form calluses with different embryogenic responses. The endogenous contents of total nitrogen, nitrate, nitrite and ammonia and nitrate reductase activity were determined in embryogenic and non-embryogenic calluses induced from the different explants. The endogenous total N content decreased in the calluses as the culture time progressed, this decrease being more pronounced in the more embryogenic calluses obtained from petioles with the H8 and F0 media. Inorganic N decreased during embryogenesis, coinciding with an increase in organic N. Thus, N metabolism somehow seems to be essential in embryogenesis. The N detected in calluses, at the start of culture, was mainly metabolised to nitrite. This metabolism was very pronounced; especially in embryogenic calluses obtained from cotyledons and petioles. That is, the metabolism of N seemed to be more marked in the calluses in which embryogenesis was greater. The nitrite content decreased in all the calluses, the contents being lower, especially in the last months of culture, in the more embryogenic calluses obtained from petioles. In many calluses, ammonia levels did not follow any general pattern. Neither was it possible to detect changes in ammonia levels between the embryogenic and non-embryogenic calluses. Regarding nitrate reductase activity, no clear differences between embryogenic and non-embryogenic calluses were found.

Regeneration of soybean (Glycine max L. Merrill) through direct somatic embryogenesis from the immature embryonic shoot tip

We describe here a simple and efficient system of soybean (Glycine max L. Merrill) regeneration through direct somatic embryogenesis by using immature embryonic shoot tips (IEST) as explants. The cultivar Kaohsiung 10 (cv. K10) used in this study did not show embryogenic response either from mature seed-derived explants (cotyledon, embryonic tip, leaf, shoot and root) or immature cotyledons. However, it showed a high percentage (55.8%) of somatic embryo (SEm) formation from the IEST excised 2–3 wk after flowering, thus indicating the crucial roles of type and age of explants. The IEST put forth primary SEm after 2 mo of culturing on Murashige and Skoog (MS) medium supplemented with 6% sucrose, 164.8 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 5 mM asparagine and 684 µM glutamine. Subsequently, secondary SEm were developed 1 mo after culturing on MS medium containing 123.6 µM 2,4-D and 3% sucrose. Cotyledonary embryos were induced on MS medium supplemented with 0.5% activated charcoal after 1 mo. The embryos were desiccated for 72–96 h on sterile Petri dishes and regenerated on hormone-free MS medium. Plantlets with well-developed shoots and roots were obtained within 5–6 mo of culturing of IEST. The SEm-derived plants were morphologically normal and fertile. Various parameters thought to be responsible for efficient regeneration of soybean through somatic embryogenesis are discussed. To our knowledge, this is the first report to employ IEST as explants for successful direct somatic embryogenesis in soybean.

Development of embryo-like structures in the suspension cultures of flax coincides with secretion of chitinase-like proteins

Flax suspension cultures have been established from the callus induced from the cotyledons, hypocotyls, and immature zygotic embryos (iZE). The growth of flax suspension culture (expressed as a sedimented cell volume) was compared in both conditioned (by liquid from embryogenic Pinus nigra suspension culture) and non-conditioned media. Conditioning of media significantly increased the growth of the cell lines of hypocotyl and iZE origin; however, it had no promotive effect on embryogenic response of these flax liquid cultures. Formation of embryo-like structures (ELS), confirmed also histologically, has only been found in the cell line derived from iZE and cultivated in non-conditioned MS medium supplemented with 1 mg l−1 2,4-D. The process of ELS formation in this cell line was accompanied by the expression of the protein(s) with chitinolytic activity and molecular weight approximately of 25 kDa. The relationship between the formation of ELS and secretion of chitinase(s) is discussed.

Somatic embryogenesis and plant regeneration in zygotic embryos of Trifolium nigrescens (Viv.)

This study developed a plant regeneration protocol for Trifolium nigrescens (Viv.) via somatic embryogenesis (SE). Immature zygotic embryos at tor- pedo (TsE) and cotyledonary (CsE) stage were cultured on media with different auxins and cytokinins at different concentrations. The cultural requirements for SE differed between the explants used: the addition of 6-furfurylam- inopurine (kinetin) or N 6 -(2-isopentenyl)-adenine (2iP) along with 2,4-dihydrophenoxyacetic acid (2,4-D) or 1-naphthaleneacetic acid (NAA) was needed to elicit the embryogenic response of CsE, but an exogenous cytoki- nin totally inhibited 2,4-D-induced SE from TsE. When applied alone, neither the cytokinin nor NAA induced SE in TsE or CsE. In all effective cultures the first somatic embryos appeared directly from the upper part of the hypocotyl (TsE and CsE) and from the margin of coty- ledons (TsE) on day 7. Embryogenic callus occurred on CsE after 10 days. At comparable concentrations 2,4-D was a more potent SE inducer than NAA, but most of the embryoids induced on media with 2,4-D displayed mor- phological abnormalities, whereas those produced in the presence of NAA generally resembled zygotic embryos. Plant regeneration was achieved after transfer of somatic embryos or embryo-derived first shoots to medium without plant growth regulators (PGRs). The frequency of plant recovery was about 30% for embryoids obtained on media containing 2,4-D, and for material from media with NAA the recovery rates were 44-68% (somatic embryos) and 72-100% (embryoid-derived shoots). Regenerants appeared identical to each other and to wild plants; they produced flowers and had the chromosome complement typical for the species, 2n = 16, in root tip cells.

LEAFY COTYLEDON2 gene expression and auxin treatment in relation to embryogenic capacity of Arabidopsis somatic cells

The expression pattern of the LEC2 gene during somatic embryogenesis (SE) in Arabidopsis explants (immature zygotic embryos) induced in vitro was followed, using real-time quantitative PCR (qRT-PCR). The analysis revealed differential expression of LEC2 transcripts within a 30 days time course of somatic embryo development. A significant auxin-dependent upregulation of the LEC2 gene was found to be associated with the induction phase of SE. In contrast to embryogenic culture the level of LEC2 expression was noticeably lower in non-embryogenic callus of Col-0 and hormonal mutants (cbp20 and axr4-1) with low SE-efficiency. The study with 35S::LEC2-GR transgenic plants showed that overexpression of LEC2 can compensate for the auxin requirement, and that transgenic explants formed somatic embryos when cultured in vitro under auxin-free conditions. However, unlike in auxin-induced SE, intense callus formation preceded the embryogenic response triggered via LEC2 overexpression, suggesting an indirect pathway of morphogenesis. Moreover, a negative interaction between auxin treatment and LEC2 overexpression in terms of SE efficiency was observed, as transgenic explants cultured on auxin medium displayed a significantly reduced level of embryogenic potential. The study provides further experimental evidence that in the determination of the embryogenic response in Arabidopsis somatic cells, a close link exists between auxin and the LEC2 activity.

Optimizing Initiation and Maintenance of Vitis Embryogenic Cultures

Stamens and pistils from mature grapevines and leaves from in vitro micropropagation cultures were used to optimize parameters influencing somatic embryogenesis in Vitis. Embryogenic competence was dependent on species/variety, explant type and developmental stage, medium composition, and growth regulator concentration. Of varieties evaluated, a greater number produced embryogenic cultures from stamens and pistils (26) compared with leaves (six). Among the different stamen and pistil stages, Stage II and III explants produced the maximum embryogenic response regardless of genotype and medium composition. Of seven culture media tested, the highest embryogenic response was recorded from varieties cultured on MSI (18) and PIV (16) media. Experiments annually repeated over 3 to 10 years demonstrated reproducible results. Highly reliable protocols for somatic embryogenesis were obtained for 29 Vitis species and varieties, including 18 Vitis vinifera varieties, Vitis riparia, Vitis rupestris, Vitis champinii, and eight Vitis hybrids. Embryogenic cultures were maintained on X6 medium for a period of 6 months to 2 years depending on the variety and used in studies involving genetic transformation and transgenic plant regeneration.

Direct somatic embryogenesis in Myristica malabarica Lam., an endemic, threatened medicinal species of Southern India and detection of phytochemicals of potential medicinal value.

Direct somatic embryogenesis was obtained from intact and fragmented zygotic embryos of Myristica malabarica , an endemic, threatened medicinal species of Western Ghats of Southern India while cultured in Murashige and Skoog medium containing activated charcoal. In the absence of activated charcoal there was no embryogenic response but only callus formation in zygotic embryos and their fragments. The addition of gibberellic acid resulted in the emergence of shoot from the somatic embryos. The various developmental stages of somatic embryos were examined using scanning electron microscope. Thin layer chromatography revealed the presence of compounds similar to lignans in the embryogenic mass. GC-MS analysis of the embryogenic mass revealed the presence of several compounds of potential clinical value including malabaricone A , α- spinasterol and γ- sitosterol. The spent medium showed strong anti-bacterial activity against Pseudomonas aeruginosa . The results are significant since this is the first report of tissue culture and induction of somatic embryogenesis in Myristica malabarica . The embryogenic culture system is potentially useful for efficient and consistent production of bioactive compounds and also has strong implications for conservation of its valuable germplasm.

Direct somatic embryogenesis in Myristica malabarica Lam., an endemic, threatened medicinal species of Southern India and detection of phytochemicals of potential medicinal value.

Direct somatic embryogenesis was obtained from intact and fragmented zygotic embryos of Myristica malabarica, an endemic, threatened medicinal species of Western Ghats of Southern India while cultured in Murashige and Skoog medium containing activated charcoal. In the absence of activated charcoal there was no embryogenic response but only callus formation in zygotic embryos and their fragments. The addition of gibberellic acid resulted in the emergence of shoot from the somatic embryos. The various developmental stages of somatic embryos were examined using scanning electron microscope. Thin layer chromatography revealed the presence of compounds similar to lignans in the embryogenic mass. GC-MS analysis of the embryogenic mass revealed the presence of several compounds of potential clinical value including malabaricone A , αspinasterol and γ- sitosterol. The spent medium showed strong anti-bacterial activity against Pseudomonas aeruginosa. The results are significant since this is the first report of tissue culture and induction of somatic embryogenesis in Myristica malabarica. The embryogenic culture system is potentially useful for efficient and consistent production of bioactive compounds and also has strong implications for conservation of its valuable germplasm. Keywords: Myristica malabarica, activated charcoal, somatic embryogenesis, lignan.

Overexpression of WUSCHEL in C. chinense causes ectopic morphogenesis

Capsicum chinense is a recalcitrant species for in vitro morphogenesis, and up to date there is no efficient system for genetic transformation and regeneration of this species via somatic embryogenesis. Here, we carried out an in vitro transformation of C. chinense via Agrobacterium tumefaciens co-cultivation with a system that expresses the heterologous gene WUSCHEL from Arabidopsis thaliana. WUSCHEL has been shown to promote the transition from vegetative to embryogenic state when overexpressed. We tested if the expression of WUSCHEL in C. chinense would promote an embryogenic response in this species. After 15 days of induction, the segments of transformed stems begun to form globular structures, suggesting that heterologus WUSCHEL was active and involved in the process of morphogenesis.

Effects of genotype, light regime, explant position and orientation on direct somatic embryogenesis from leaf explants of Phalaenopsis orchids

The influence of light regime, explant position and orientation on direct embryo formation from leaf explants of two Phalaenopsis, P. amabilis and P. Nebula, were investigated to optimize the protocol for regenerating of this orchid. When explants were cultured in light, direct embryogenesis was retarded in both species. Embryos showed whitish to pale green in color and larger size than those cultured in darkness. Furthermore, light regime induced explant browning, embryo necrosis and eventually low plantlet conversion rate. Sixty days of culture in darkness is the most suitable duration for direct embryo induction. Explant orientation also significantly affected direct embryo formation, and explants placed adaxial-side-up on culture medium had higher embryogenic response than abaxial-side-up orientation. In both species, the cut end had highest embryogenic competence than other parts of the explant. Moreover, when the leaf explant was cut transversely into two segments, the leaf basal segment had higher embryogenic competence than the leaf tip segment.

Callus induction and plant regeneration by Brazilian new elite wheat genotypes

The distinction of genotypes responsive to tissue culture and the development of an efficient regeneration system are the first steps towards transgenic plant production. Nine Brazilian wheat (Triticum aestivum L.) genotypes were cultivated in vitro to evaluate the embryogenetic capacity. The explants (immature zygotic embryos) were tested in two different culture media, MS (Murashige and Skoog 1962) and modified MS - MMS (Zhou et al. 1995) with decreasing dosages of hormone regulators. Three distinct phases were observed in each medium: induction, maintenance and regeneration. After induction, the somatic embryogenesis of calli was evaluated every 21 days. Genotypes responded differently to the different culture media. The embryogenic response of genotype CD104 was best in both culture media tested. On MMS, the values of callus induction, plant regeneration and ratio of regenerated plantlets per rescued embryo of this genotype were 100%, 99.5% and 1.1%, respectively. Genotypes CD104, CD200126 and CDFAPA 2001129 were most responsive on MS (regeneration capacity of 37.5%, 33.5% and 33% respectively), and therefore interesting for genetic transformation in plant breeding programs that develop new elite cultivars with a commercial purpose.

Somatic embryogenesis and plant regeneration from pistil transverse thin cell layers of lemon (Citrus limon)

Callus induction, somatic embryogenesis and plant regeneration were obtained in Citrus limon (L.) Burm. (cv. Femminello) from cultures of pistil transverse thin cell layer explants [(t)TCLs]. Explants were cultured on two different media, based on Murashige and Skoog salts and vitamins, supplemented with 500 mg l−1 malt extract (MSI), or 500 mg l−1 malt extract and 13.3 μM 6-benzylaminopurine (MSII). Sucrose (146 mM) was used as carbon source. Somatic embryos appeared 3 months after culture initiation from stigma and style (t)TCLs; they were observed at the surface of the (t)TCL-derived callus. Although ovary (t)TCLs showed the highest callus formation, they never differentiated somatic embryos. Percentages of embryo formation from (t)TCLs incubated on MSI (13% and 2% for stigma and style, respectively) were lower than those from (t)TCLs incubated on MSII (36% and 7% for stigma and style, respectively). The embryogenic response of stigma (t)TCLs was usually higher than that of style (t)TCLs. After about 3 months, somatic embryos developed into plantlets at high frequencies (57% and 62% for stigma- and style-derived somatic embryos, respectively).

Activated charcoal induced high frequency microspore embryogenesis and efficient doubled haploid production in Brassica juncea

Three Indian Brassica juncea cultivars were studied for embryogenic response of microspores, microspore embryo regeneration, ploidy assessment of microspore-derived plants and their diploidization. Genotype dependence for microspore totipotency was observed and a significant effect of genotype by bud size selection was established. The addition of activated charcoal in NLN medium containing 13% (w/v) sucrose and 10 μM silver nitrate resulted in a fourfold increase in microspore embryogenesis, ranging from 100 to 405 embryos per Petri dish corresponding to 2,700–10,935 embryos per 100 buds. Conversion/germination of embryos produced in presence or absence of activated charcoal was similar but air-drying of microspore embryos was essential. Incubation of microspore embryos at 4 ± 1°C for 10 days in dark resulted in 82.3% conversion. The majority of plants produced from these embryos was haploid. Treating microspore-derived plants at the 3–4 leaf growth stage with 0.34% colchicine for 2–3 h resulted in greatest survival (70%) and chromosome doubling (75%) frequencies. Doubled haploid plants were self-pollinated and grown to maturity under field conditions.

Biosynthesis of Dibenzyl Trisulfide (DTS) from somatic embryos and rhizogenous/embryogenic callus derived from Guinea hen weed (Petiveria alliacea L.) leaf explants

ABSTRACT A procedure for producing somatic embryos enriched with dibenzyl trisulfide (DTS) using a hormone-dependent culture system is reported for Petiveria alliacea L. (Guinea hen weed). Leaf explants were cultured on a Murashige and Skoog medium supplemented with a range of naphthaleneacetic acid (NAA) concentrations and a fixed concentration of benzyladenine (BAP) at 11.0 μM and sucrose or glucose at 30 g l−1. Leaf explants cultured on all media types started to form callus at the cut surfaces of the discs 10–14 d after initiation. The type of sugar used influenced average fresh weight, the propensity to form roots, as well as the embryogenic response. The highest mean fresh weight (337.7±26.18 mg) and mean root number (23.7±1.69) was produced on media enriched with sucrose and supplemented with 53.7 μM NAA and 11.0 μM BAP. An ethanol extract of rhizogenic/embryogenic callus or somatic embryos was subjected to high-performance liquid chromatography analysis, which revealed the presence of DTS in both ...