Somatic embryogenesis in Araujia sericifera

This work presents the preliminary results of in vitro studies with Araujia sericifera, which is cultivated for ornamental purposes. Immature seeds from wild plants were used to start the cultures. Somatic embryos and friable embryogenic calluses were obtained from white cotyledons in media containing naphthaleneacetic acid and benzyladenine or 2,4-dichlorophenoxyacetic acid. Plants were regenerated from these somatic embryos.Cell suspensions obtained from friable calluses cultured in M1 modified medium showed a considerable growth capacity. The packed cell volume was doubled in about 15 days of culture at the exponential phase. the results obtained may be used to design further experiments with the aim of improving somatic embryogenesis.Abbreviations NAA -naphthaleneacetic acid - BA benzyladenine - IBA indolebutyric acid - 2,4-d 2,4-dichlorophenoxyacetic acid     

Photocontrol of somatic embryogenesis and polyamine content in Araujia sericifera petals

The effects of photoperiod and end-of-day phytochrome control on somatic embryogenesis and polyamine (PA) content in Araujia sericifera petals have been studied. Petals from immature flowers were cultured under 16- and 8-h photoperiods. Far red (FR), red (R) and FR followed by R light treatments were applied at the end of the photoperiods for three weeks. The number of somatic embryos, callus weight and the levels of free and bound PAs in the cultured petal explants were determined 40 days after the beginning of light treatments. Long day (LD) promoted somatic embryogenesis but did not have any significant effect on PA content. Short day (SD) reduced somatic embryogenesis and enhanced total PAs, mainly in the form of bound spermidine. End-of-day FR treatment increased PA content and inhibited somatic embryogensis under LD but had no significant effect under SD. This effect of FR on PA levels was cancelled by R and was independent of the presence of silver thiosulphate in the medium. End-of-day R treatment reduced the total PA content under SD. However, end-of-day R increased or reduced somatic embryogenesis under SD depending on the presence or absence of silver in the medium. The results suggest a photoperiodic control of somatic embryogenesis and PA content in A. sericifera. The effects of end-of-day R and FR treatments depend on the length of the photoperiod. This finding and the FR/R photoreversibility of end-of-day treatments indicate that phytochrome may be involved in both somatic embryogenesis and accumulation of PA.     

Embryogenesis induction in petals of Araujia sericifera

The embryogenic capacity of Araujia sericifera petals and some of the factors involved in the induction of embryos was investigated. The influence of 6-benzyladenine and α-naphthalene acetic acid, light intensity (90 or 5 μmol m^-2 s^-1) and silver thiosulphate (inhibitor of ethylene action) were studied. It was found that petals are an easy system in which to induce somatic embryogenesis. Plants were recovered from somatic embryos. Although 6-benzyladenine is essential for inducing an efficient response, a high dosage increased callogenesis and reduced embryogenesis. The highest rate of embryogenesis is induced with high light intensity (90–100 μmol m^-2 s^-1), even though the presence of silver thiosulphate in the medium markedly reduced embryo induction. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of light quality on somatic embryogenesis of quince leaves

The effect of light quality on somatic embryogenesis in quince BA 29 was investigated. 2,4-D induced leaves were exposed for 25 days to the following light quality treatments: dark, far-red, far-red+blue, far-red+red, blue, white, red+blue, red. After a further 20 days of white light exposure, somatic embryo production was recorded. Somatic embryogenesis was highest in cultures subjected to red light treatment, and decreased progressively with the transition to red+blue and to white. Overall, embryogenic competence showed a correlation with photoequilibrium. Phytochrome appeared to be inductive although this effect was adversely influenced by the blue absorbing photoreceptor, in particular at low photoequilibrium. Independently of light treatments applied, somatic embryos frequently showed severe morphological abnormalities. Conversion of somatic embryos to plantlets was not observed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of end-of-day irradiations on polyamine accumulation in petal cultures of Araujia sericifera

We have studied photoperiodic control and the effect of phytochrome photoconversion at the end-of-day (EOD) on polyamine (PA) accumulation in petal explants of Araujia sericifera . Petals from immature flowers were cultured under long (LD) and short (SD) days. Light was provided by Gro-lux fluorescent lamps (90–100 µmol m⁻² s⁻¹). Red (R), far red (FR), red followed by far-red (R-FR) and far-red followed by red (FR-R) light treatments were applied daily at the end of the photoperiod. The free and bound putrescine (Put), spermidine (Spd) and spermine (Spm) fractions in petal explants were determined 40 days after the beginning of the culture. We also aimed to clarify the involvement of PA changes by using two inhibitors of PA biosynthesis: D- l -α-difluoromethylarginine (DFMA) and methylglyoxal bis (guanylhydrazone) (MGBG). We found PA accumulation to be under photoperiodic control, and the inhibitory effect of DFMA on this accumulation suggests that arginine decarboxylase (ADC) is the major pathway for Put biosynthesis. Polyamine levels were higher under LD, mainly as a result of the accumulation of free and bound Put. FR-EOD treatment, which dramatically reduced the R : FR ratio after LD, increased the accumulation of PA, mainly as free Put and free and bound Spd. Sequential R-FR and FR-R-EOD treatments strongly increased bound Spd. The concentration of MGBG used increased total PA accumulation, mainly as Put. However, all EOD light treatments dramatically reduced Put accumulation in the presence of MGBG. This may be due to a dual role of FR light in PA accumulation: (1) FR per se stimulates PA production, probably via ADC, and (2) in the presence of MGBG, FR inhibits Put accumulation, probably via ethylene production.     

Recent advances in conifer somatic embryogenesis: improving somatic embryo quality

Somatic embryogenesis of coniferous species was first reported more than 20 years ago. Since then, there has been an explosion of research aimed at developing and optimizing protocols for efficient regeneration of plantlets. Although routinely used both as a means of propagation, as well as a valuable model system for investigating the structural, physiological, and molecular events occurring during embryo development, in vitro embryogenesis is still problematic for some coniferous species. Major problems include: low number of embryos generated; and low frequency of mature embryos able to convert into viable plantlets. Until recent years, despite the fact that embryogenesis is comprised of a sequence of defined steps which include proliferation of embryogenic tissue, embryo maturation, and germination, attempts at improving the whole procedure have been made almost exclusively during the maturation stage. This strategy was based on the assumption that successful regeneration is related to treatments provided during the development of the embryos. Major optimizations of the maturation medium have involved judicious selections of type and concentration of growth regulators, namely abscisic acid, and adjustments of the osmoticum of the culture medium. Extensive work has been conducted in defining the effects of plasmolysing and non-plasmolysing osmoticum agents during maturation, as well as in improving desiccation techniques required for the completion of the maturation program. In the last 2 years, however, work on spruce has clearly demonstrated that the early events in embryogenesis are crucial for the successful completion of the overall embryogenic program. The use of cell tracking techniques, implemented by physiological and molecular studies, has revealed that manipulations of the culture conditions early in the process can increase both number and quality of embryos produced in culture. Additional manipulations of the germination medium can also enhance germination and conversion frequency of somatic embryos matured in a sub-optimal environment. These new findings, together with the unraveling of molecular mechanisms involved in the control/regulation of embryo development hold considerable promise for clonal propagation in conifers.     

Effects of glycerol on somatic embryogenesis in Cichorium leaves

 Direct somatic embryogenesis was induced in leaf cells of a Cichorium hybrid (Cichorium intybus L var. sativum×Cichorium endivia L. var. latifolia) through a two-step procedure. Leaf tissue explants were cultured for 5 days in M17 liquid medium supplemented with 30 mM sucrose and 330 mM glycerol (M17S30Gly330 medium). Synchronised divisions of embryogenic cells occurred after transfer for 7 days onto glycerol free-medium (M17S30). By doubling the sucrose concentration (60 mM) in the presence of glycerol (M17S60Gly330) during the induction step, embryogenesis increased and the length of the induction step was reduced from 5 to 4 days. Compared to sucrose, glycerol as carbon source during the induction and the expression steps had an inhibitory effect on the embryogenic response. During culture, glycerol was not detected in M17S60 medium and was at a low level in leaf fragments incubated in this medium. Initially supplied as an osmoticum, glycerol disappeared from M17S60Gly330 medium during the 4-day induction period and penetrated into the tissues where most of was metabolised. Furthermore, glycerol modified it carbohydrate metabolism, particularly during the induction period of embryogenesis. Sucrose hydrolysis was affected in the medium and sucrose and hexose contents in tissues were higher than in glycerol-free medium. The effects of glycerol as osmoticum and as a molecule itself are discussed. Received: 30 January 1998 / Accepted after revision 25 February 1999     

Effects of boron on somatic embryogenesis of Daucus carota

Although the micronutrient boron (B) apparently exerts no influences on the induction of somatic embryogenesis of cultured carrot petiole explants, strong influences on the development of somatic embryos were observed (0–8 mg l⁻¹ B). At lower B concentrations the development of roots is promoted with simultaneous retardation of shoot development and at higher B concentrations shoot development is favored at the expenses of the development of the root system. Parallel to this, the ratios of concentrations of endogenous indole-3-acetic acid to total cytokinins (with one exception) changes continuously from 4 (zero B) to 0.22 (8 mg l⁻¹ B). Analogies to morphogenetic reactions following the application of various ratios of auxins/cytokinins to the nutrient medium of cultured tissue (Skoog and Miller, 1957 and others) are suggested. An increase in the B concentration in the nutrient medium results generally in a reduced concentration of endogenous abscisic acid.     

Effects of brassinosteroid on cotton regeneration via somatic embryogenesis

Brassinolide (BR), which is the most biologically active brassinosteroid, was used to examine the potential effect of hormone on cotton somatic embryogenesis. Ten-day-old cotton (Gossypium hirsutum L., cv. Cooker) seedlings were used for explant source and hypocotyls were removed and cultured on MS basal medium with B₅ vitamins supplemented with 1 mg/L 6-benzylaminopurine + 0.5 mg/L kinetin for callus induction. After one month proliferating calli pieces were collected and cultured on MS basal medium containing various concentrations of BR (0.1, 0.5, 1.0 μM) with their controls. BR treatments were negatively effective on the fresh weight of calli when compared to control. Differential somatic embryogenesis maturation rates due to BR treatment were observed. Somatic embryogenesis was stimulated especially for transition to cotyledonary phase at 0.5 mg/L BR. Histological preparations from embryogenic calli and somatic embryos at different stages of development revealed the spontaneous polyploidisation during early somatic embryogenesis on BR-treated calli. Present results suggest that BR negatively effected calli growth, however, had a stimulating role in maturation of somatic embryos.     

Effects ofVerticillium albo-atrum culture filtrate on somatic embryogenesis in alfalfa

Cell suspensions derived from young petioles of alfalfa (Medicago sativa L.) were cultured in the presence and absence of aVerticillium albo-atrum culture filtrate (20% v/v) for 6 cycles. The frequency of somatic embryogenesis and the growth rate of the suspension cultures were investigated at each cycle. Somatic embryogenesis in the filtrate-treated cultures declined but was still at a relatively high level after 6 subcultures, compared to controls cultures which virtually lost the capacity for embryo formation in the same period. The decline in the embryogenic capacity of filtrate treated-cultures was accompanied by a six-fold increase in the rate of growth of the cultures.     

Effects of kanamycin on tissue culture and somatic embryogenesis in cotton

The aminoglycoside antibiotic kanamycin was evaluated for its effects on callus initiation from hypocotyl and cotyledon explants, proliferation of non-embryogenic and embryogenic calli, initiation and development of somatic embryos in cotton (Gossypium hirsutum L.). On this basis, the potential use of kanamycin as a selective agent in genetic transformation with the neomycin phosphotransferase II gene as the selective marker gene was evaluated. Cotton cotyledon and hypocotyl explants, and embryogenic calluses were highly sensitive to kanamycin. Kanamycin at 10 mg/L or higher concentrations reduced callus formation, with complete inhibition at 60 mg/L. Kanamycin inhibited embryogenic callus growth and proliferation, as well as the initiation and development of cotton somatic embryos. The sensitivity of embryogenic callus and somatic embryos to kanamycin was different during the initiation and development stages. Kanamycin was considered as a suitable selective agent for transformed callus formation and growth of non-embryogenic callus. Forty to sixty mg/L was the optimal kanamycin concentration for the induction and proliferation of transformed callus. The concentration of kanamycin must be increased (from 50 to 200 mg/L) for the selection of transformation embryogenic callus and somatic embryos. A scheme for selection of transgenic cotton plants when kanamycin is used as the selection agent is discussed.     

Effects of mutagens on somatic embryogenesis and plant regeneration in groundnut

The embryogenic calli (EC) were obtained from hypocotyl explants of groundnut (Arachis hypogaea L.) cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) in combination with 0.5 mg dm⁻³ 6-benzylaminopurine (BAP). The EC were exposed to γ-radiation (10–50 Gy) or treated with 1–5 mM of ethyl methane sulphonate (EMS) or sodium azide (SA). The mutated EC were subcultured on embryo induction medium containing 20 mg dm⁻³ 2,4-D. Somatic embryos (SE) developed from these calli were transferred to MS medium supplemented with BAP (2.0 mg dm⁻³) and 0.5 mg dm⁻³ 2,4-D for maturation. The well-developed embryos were cultured on germination medium consisting of MS salts with 2.0 mg dm⁻³ BAP and 0.25 mg dm⁻³ naphthaleneacetic acid (NAA). Well-developed plantlets were transferred for hardening and hardened plants produced normal flowers and set viable seeds. The fresh mass of the EC, mean number of SE per explant and regeneration percentage were higher at lower concentrations of mutagens (up to 30 Gy/3 mM). Some abnormalities in regenerated plants were observed, especially variations in leaf shape.     

Effects of carbohydrate addition on the induction of somatic embryogenesis in Hevea brasiliensis

Plant Cell, Tissue and Organ Culture (PCTOC) - The effect of different carbohydrates was tested on early somatic embryogenesis of Hevea brasiliensis. Sucrose was replaced with maltose, fructose or...     

Effects of cytokinins and auxins on cassava shoot organogenesis and somatic embryogenesis from somatic embryo explants

Somatic embryos of cassava (Manihot esculenta Crantz) cultivar ‘Nanzhi 188’ were isolated and cut into fragments to be cultured on media with various cytokinins and auxins. Cytokinin induced of cassava shoot organogenesis, while auxin stimulated somatic embryogenesis. The effectiveness on organogenesis was different based on different cytokinins and in combination with auxins. Benzyladenine and thidiazuron stimulated more shoot organogenesis than kinetin and N-isopentenyladenine. This revised version was published online in June 2006 with corrections to the Cover Date.     

植物激素对棉花体细胞胚胎发生的诱导及调节作用

选用11种激素研究了外源激素对棉花胚性愈伤组织增殖、胚胎发生和发育的调控作用。结果表明不同激素对棉花胚性愈伤组织增殖、胚胎发生与发育的影响不同。除2,4-D和BA对棉花胚性愈伤组织的增殖影响不大外,其他激素对棉花胚性愈伤组织的增殖均具有抑制作用,且具有一定的时间效应,同时还受基因型的影响。激素对棉花体细胞胚的形成和发育的影响极大,2,4-D既抑制了体细胞胚的形成,又抑制了体细胞胚的发育;TDZ的作用与2,4-D相似,显抑制了体细胞胚的形成,且诱导获得的体细胞胚均停留在球形胚阶段;GA也抑制了体细胞胚的形成,且不利于体细胞的成熟与萌发;BU-30对棉花体细胞胚形成与发育的影响不大。其他7类生长素类物质和细胞分裂素类物质对棉花体细胞胚的形成均具有促进作用,且依IBA、ABA、IAA、BA、KT、ZT、2iP序增强,其总胚数为对照的1．193—3．852倍;其中2iP的促进作用最大,可使产生的体细胞胚数提高2．852倍。     

Soybean somatic embryogenesis: Effects of hormones and culture manipulations

Somatic embryos were induced in cultures of immature soybean (Glycine max (L.) Merr) embryos, or isolated cotyledons on MS modified medium supplemented with NAA and 2,4-D, BAP and ABA. When NAA and 2,4-D were compared at similar concentrations (25 and 23 M), 2,4-D produced larger number of somatic embryos, however, embryogenesis efficiency was improved in media containing NAA by using higher levels (100–150 M) of the auxin. Somatic embryo morphology varied with auxin type: NAA-induced embryos more closely resembled zygotic embryos than did 2,4-D-induced embryos. Additions of BAP or ABA to auxin-containing media had either no effect or reduced embryo production, although ABA altered the morphology of 2,4-D-induced embryos. In media containing both NAA and 2,4-D, the latter was dominant in terms of embryo morphology. The effects of subculture frequency and of transfers between 2,4-D and NAA media were investigated: Subculture frequency influenced mainly the frequency of normal embryos, while preculture on 2,4-D increased subsequent embryogenesis efficiency on NAA medium but reduced the frequency of normal embryos.Abbreviations Em^-2 s^-1 microEinsteins per square meter per second - NAA -naphthalene acetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid - ABA abscisic acid - BAP benzylamino purine This paper (No. 86-3-96), is published with the approval of the director of the Kentucky Agricultural Experiment Station.     

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.     

Developmental pathways of somatic embryogenesis

Somatic embryogenesis is defined as a process in which a bipolar structure, resembling a zygotic embryo, develops from a non-zygotic cell without vascular connection with the original tissue. Somatic embryos are used for studying regulation of embryo development, but also as a tool for large scale vegetative propagation. Somatic embryogenesis is a multi-step regeneration process starting with formation of proembryogenic masses, followed by somatic embryo formation, maturation, desiccation and plant regeneration. Although great progress has been made in improving the protocols used, it has been revealed that some treatments, coinciding with increased yield of somatic embryos, can cause adverse effects on the embryo quality, thereby impairing germination and ex vitro growth of somatic embryo plants. Accordingly, ex vitro growth of somatic embryo plants is under a cumulative influence of the treatments provided during the in vitro phase. In order to efficiently regulate the formation of plants via somatic embryogenesis it is important to understand how somatic embryos develop and how the development is influenced by different physical and chemical treatments. Such knowledge can be gained through the construction of fate maps representing an adequate number of morphological and molecular markers, specifying critical developmental stages. Based on this fate map, it is possible to make a model of the process. The mechanisms that control cell differentiation during somatic embryogenesis are far from clear. However, secreted, soluble signal molecules play an important role. It has long been observed that conditioned medium from embryogenic cultures can promote embryogenesis. Active components in the conditioned medium include endochitinases, arabinogalactan proteins and lipochitooligosaccharides.     

Noninvasive evaluation of somatic embryogenesis

Callus Suspension Cultures of Ipomea batates Poir. cv. White Star were grown in an airlift bioreactor. A machine vision system was used to monitor nondestructively callus growth during a 10 day culture period. Growth data obtained with this system included the overall reactor population and population estimates for the 200-1200-mum fractions at 200-mum intervals. A model of callus growth was developed to explain the mechanics of callus enlargement. The model was based on the assumptions that (1) the calli could not subdivide or shrink, (2) there was a fixed percentage of the initial population for each fraction that was nonviable, and (3) growth rates did not vary with time during the culture period. It was determined that the growth rates and nonviable ratios decreased as fraction size increased.     

Genomic influence on somatic embryogenesis in the Triticeae

Somatic embryogenesis is generally accepted to be under genetic control. The influence of genome interactions is not well understood given the difficulties of obtaining the appropriate vegetal material. Synthetic alloploids in which genomes from two species are fused together are suitable subject material to analyse this factor. In the Triticeae tribe amphiploids can be easily synthesised, which provides the opportunity to carry on this type of study. Crossing three autotetraploids, Hordeum chilense (HHHH), Triticum tauschii (DDDD) and Secale cereale (RRRR), we obtained three tetraploid amphiploids, ×Tritordeum (HHDD), ×Triticale (DDRR) and ×Hordecale (HHRR). Somatic embryogenesis from immature inflorescence and flag leaves were scored on parents and amphiploids. Immature inflorescences had a higher embryogenic response than the flag leaves. The amphiploids showed higher regeneration ability than their parents. The best genomic combination was the tetraploid triticale DDRR for every inflorescence and flag leaf size tested, followed by HHDD and HHRR. Heterosis was found to be the main genetic factor affecting the in vitro culture response although there are clear differences among the three amphiploids tested. Received: 11 February 1998 / Revision received: 8 April 1998 / Accepted: 22 November 1998