Histology of early somatic embryogenesis inHevea brasiliensis: The importance of the timing of subculturing

Somatic embryos ofHevea brasiliensis can be obtained by culturing thin sections of inner tegument of seed on two successive different media, MH1 and MH3. Histological study showed that in calli cultured on non-renewed medium MH1 for 40 days, the embryogenesis process initiated on the 20th day did not produce results owing to early degeneration of the cells involved in the embryogenic pathway. However, typical embryogenic cells formed when medium MH1 was renewed once during the first phase of culture (between day 20 and day 30). Proembryos developed when the calli were subcultured on medium MH3 10–15 days later. Embryogenic cells did not form when there was frequent renewal of medium MH1 or early subculturing on MH3 after less than 40 days of culture on MH1. Methodical histological monitoring of the development of embryogenic quality of calli thus made it possible to define the optimum culture sequences for the embryogenesis process and which are favourable for regular obtaining of proembryos.     

Water status of callus from Hevea brasiliensis during induction of somatic embryogenesis

To improve somatic embryogenesis in Hevea brasiliensis , the water and plant growth regulator status of the culture medium was studied. Induction of embryogenic tissue from the internal integument of immature seeds was clearly favored by stabilizing the water potential of the culture medium at –0.7 MPa, by using low and decreasing concentrations of 3,4-dichlorophenoxyacetic acid and benzyladenine or by incorporating 10^-7 M abscisic acid in the medium. Each of these changes in the medium favored a specific water status in the callus, namely a high relative water content (93 to 95%) and an elevated water potential (–0.9 MPa). These characteristics were apparently important for initiating somatic embryogenesis, and their decrease corresponded to the loss of embryogenic potential in the callus. Thus, the relative water content and water potential of callus appear to be good markers of its embryogenic state.     

Mitochondrial DNA polymorphism and phylogenetic relationships inHevea brasiliensis

Using fourteen random mitochondrial DNA probes, we have examined restriction fragment length polymorphism (RFLP) in wild and cultivatedHevea brasiliensis. A total of 395 accessions, including 345 from various prospectings collected in Brazil, Colombia and Peru and 50 cultivated clones, were analyzed. Two other species (H. benthamiana andH. pauciflora) were also included in the study for comparison. The high level of mitochondrial polymorphism allowed us to divide all the accessions analyzed into 212 distinct genotypes. The genetic variability of cultivated clones was limited to four genotypes forming two clusters. In contrast, considerable genetic variation was found in the wild collections. In almost all cases, accessions displaying the same RFLP profile were restricted to the same geographical area (same or neighbor administrative districts). In addition, accessions whose genetic closeness was predicted by RFLP profiles were also clustered according to geographical origin. In a few cases, however, similar RFLP profiles were found for accessions originating from geographically distant districts. This discrepancy can be explained either by seed dispersion (by river) or possibly by similar genetic events occurring independently in different geographical locations. Chloroplast DNA RFLP was also analyzed in 217 accessions, representative of 126 distinct mitochondrial genotypes. Very few differences were found, indicating that the chloroplast genome is more highly conserved than the mitochondrial genome.     

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...     

Callus induction and somatic embryogenesis of Phalaenopsis

Callus induction and plant regeneration through somatic embryogenesis in Phalaenopsis Richard Shaffer `Santa Cruz' were examined. Protocorm-like body (PLB) segments formed calli in Vacin and Went medium with sucrose. The optimal concentration of sucrose was 40 g ⋅ l^–1. Medium containing 200 ml ⋅ l^–1 coconut water together with 40 g ⋅ l^–1 sucrose was effective for callus induction. Gellan gum was suitable than agar as a gelling agent for callus induction. The calli easily formed PLBs after being transferred to a medium without sucrose. Histological observation suggested that the PLBs were somatic embryos. No variation was observed in the flowering plants regenerated through somatic embryogenesis. Received: 11 June 1997 / Revision received: 6 October 1997 / Accepted: 18 October 1997     

Improvement of somatic embryogenesis in zonal geranium

A number of media constituents including sucrose, ammonium nitrate and plant growth regulators were evaluated in an attempt to improve somatic embryo production in zonal geranium (Pelargonium ×hortorum) cv. Scarlet Orbit Improved. Somatic embryo production was characterized by the quantity and type of somatic embryo induced by the treatments. Sucrose at 4% supported the highest number of total somatic embryos while improving the proportion of the morphologically normal cotyledon-stage somatic embryos. Addition of ammonium nitrate also improved embryo production. With 1.89 mM ammonium nitrate, normal cotyledon-stage embryo development was increased by 53%; the proportion of normal cotyledon-stage embryos decreased and abnormal embryos with leaves or serrated margins in cotyledons (fringed-shoot type) increased with higher ammonium nitrate concentrations. The effect of plant growth regulators on somatic embryogenesis indicated that exogenous supply of indole-3-acetic acid (IAA) at a range of 0.25 to 4 μM failed to promote somatic embryogenesis. In contrast, benzyladenine (BA) up to 2.0 μM increased the total embryo number and the proportion of desirable cotyledon-stage embryos. There was no interaction between IAA and BA. Our research has demonstrated that improvement in both quantity and quality of somatic embryos can be achieved in zonal geranium.     

Improvement of somatic embryogenesis in Medicago arborea

A number of medium constituents were evaluated in an attempt to improve somatic embryo production in Medicago arborea ssp. arborea, using cotyledons, petioles and leaves as explants. Two culture steps were applied: in the first stage (2 months), Murashige–Skoog (MS) medium was used, containing 2,4 dichlorophenoxyacetic acid (9M 2,4-D) and kinetin (9 M KIN) together with different nitrogen sources (alanine, glutamine, proline or tryptophan (2.5 and 5 mM); casein hydrolysate (100, 500 and 1000 mg l^–1; nitrate (4.69 and 9.39 mM) or casein hydrolysate (100 mg l^–1) and nitrate (4.69 mM)), polyalcohols (mannitol at 164 and 328 mM or sorbitol at 219 and 438 mM), sucrose (43.8 and 175.4 mM) or calcium (1.5 and 6 mM). In the second stage (3 months of cultivation), calli were transferred to a kinetin-free MS medium with 2,4-D (2.25 M) only. The inclusion of proline (2.5 mM) was the most effective treatment for the induction of somatic embryos, with the petiole being the best explant. Treatment with casein hydrolysate (100 mg l^–1) also improved the embryonic efficiency. The rest of the treatments neither affect nor inhibit the embryonic response.A special treatment with sorbitol (219 mM) in the second stage of cultivation produced a slight increase in embryogenesis, but less than that obtained with proline.     

植物凝胶和蔗糖对橡胶树体胚植株再生的影响

为了筛选巴西橡胶体胚植株最为适合的植物凝胶和蔗糖用量,该研究以巴西橡胶树无性系热研7-33-97成熟体细胞双子叶次生胚状体为材料,以添加0.23μmol·L~(-1)KT,0.11μmol·L~(-1)IAA和8.7μmol·L~(-1)GA3的MS培养基为植株再生培养基,研究了添加不同用量的植物凝胶和蔗糖对巴西橡胶树体胚植株再生和生长的影响。结果表明:在橡胶树体细胞胚植株再生培养基中,不同用量的植物凝胶对植株再生频率和植株生长状况有显著影响,较低浓度(0~1 g·L~(-1))时,随着用量增加,植株再生频率提高,但较高浓度(1~4 g·L~(-1))时,随着用量增加,植株生长受到抑制。植物凝胶添加1 g·L~(-1)时植株生长最好,植株再生率为(86.4±5.7)%,株高5 cm以上的占(53±9.4)%,带叶植株为(81.7±3)%;而蔗糖对植株再生频率影响不显著,但对再生植株生长的影响显著,低蔗糖(20~30 g·L~(-1))时促进植株抽叶但抑制茎干伸长,高蔗糖(70~80 g·L~(-1))时显著抑制抽叶但促进茎干伸长。蔗糖添加50 g·L~(-1)时植株生长最好,株高5 cm以上的占(57.6±5.4)%,株高5 cm以上带叶植株占(46.3±12.3)%,均为最高且从外观来看,在50 g·L~(-1)时植株茎干和根都较为粗壮。因此,在橡胶树体细胞胚植株再生培养基中,植物凝胶和蔗糖最佳用量分别为1 g·L~(-1)和50 g·L~(-1)。     

Effect of strictly plant-related factors on the response ofHevea brasiliensis andTheobroma Cacao nodal explants culturedIn vitro

Summary Nodal cuttings (primary nodal explants) were used to initiate microcutting cultures ofHevea brasiliensis andTheobroma cacao. The physiological stage of growth reached by the branches from which the explants were taken had a considerable influence on culture initiation and response. In both species, the buds of the nodal explants taken from dormant branches (stages 11, 12 and 13 inTheobroma; stage D inHevea) revealed greater opening abilityin vitro and shoot elongation than buds taken from branches in a phase of active meristem growth inTheobroma (stages F1 and F2) or during a foliar growth phase inHevea (stage C). InHevea, the branch growth phase effect persisted into the multiplication phase when shoots developedin vitro were used as the source of secondary nodal explants. However, it disappeared during subcultures of primary nodal explants. In both species, the position of the nodes on the branch with respect to the apex also had an effect on culture initiation. The buds of nodes furthest from the apex were more likely to develop into shoots. InHevea, the position of secondary nodal explants obtained from the primary shoot was seen to vary in effect depending on growth regulator applications during primary culture. These results are analyzed in terms of variations in the balance between endogenous growth promoters/inhibitors in the buds, depending on morphogenic stages and the bud position on the branch.     

Effect of calcium on callus friability and somatic embryogenesis in Hevea brasiliensis Mll. Arg.: relations with callus mineral nutrition, nitrogen metabolism and water parameters

Changes in culture medium calcium content modified the texture,embryo development, and water and mineral status of Hevea brasiliensis(Mll. Arg.) calli. With concentrations of 0 to 9 mM CaCl₂ inthe callogenesis medium, high percentages of embryogenic calliwere obtained with only a slight change in their compactness.Higher concentrations led to friable calli whose ability toproduce embryos then depended on low calcium concentrationsin the embryogenesis medium; in effect, only low calcium contentsallowed embryo development. Given that 12 mM CaCl₂ in the firstculture medium resulted in friable calli, it was assumed thatthere was an interaction between the calcium and growth regulatorsin the medium. The change in texture and morphogenetic abilitywas combined with a drop in turgidity potential and water availability,despite an increase in water content. This change was also associatedwith a change in mineral nutrition, notably a drop in nitrateuptake, combined with an increase in osmotic potential and adrop in protein synthesis. Key words: Calcium, somatic embryogenesis, friability, nitrogen metabolism, water status     

Plant regeneration of Chorispora bungeana via somatic embryogenesis

Summary A method was developed for in vitro regeneration of plants via somatic embryogenesis in Chorispora bungeana, an alpine plant with freeze-tolerance, using cell suspensions initiated from leaf-derived callus. Primary calli were induced from leaves of C. bungeana grown on Murashige and Skoog (MS) media supplemented with 4.0 mg l⁻¹ gibberellic acid (GA₃), 0.2 mgl⁻¹ α-naphthaleneacetic acid (NAA) and 0.2 mgl⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Suspension culture was initiated by incubating the callus particulates in liquid MS medium supplemented with 1.0 mgl⁻¹ kinetin (KT) and 0.2 mgl⁻¹ NAA. Individual early cotyledonary-stage somatic embryos isolated from cell suspension developed into whole plants on medium containing high levels of sucrose (60 and 90 gl⁻¹), whereas lower sucrose concentrations (0 and 30 gl⁻¹) were inhibitory to main root development. On the MS medium with 90 gl⁻¹ sucrose, one regenerated plant exhibited hetero-morphologic leaves, while other plants grown on different media showed a transformation from stem to root.     

Somatic embryogenesis and plant regeneration inMedicago suffruticosa

A successful procedure has been designed for the regeneration of plantlets from leaf sections of the self-pollinating species,Medicago suffruticosa. Callus growth was promoted by a 4-week culture period on liquid Kao's medium containing 4.9 M benzyladenine and 4.5 M 2,4-dichlorophenoxyacetic acid (2,4-d), followed by a 4-day treatment in which the benzyladenine was elevated to 44.4 M. Shoots/plantlets were observed after 3–4 weeks culture on growth regulator-free agar-solidified medium. Under these conditions, the regeneration frequency from callus was 18% and a histological study showed that this regeneration was through somatic embryogenesis. The growth regulator treatment, with a relatively high concentration of growth regulators (44.4 M benzyladenine) for a short time period (4 days), is important for inhibiting polyphenol compounds and for stimulating callus growth and plant regeneration.Abbreviations 2,4-d 2,4-dichiorophenoxyacetic acid - BA benzyladenine - NAA -napthaleneacetic acid     

Characterization of maize endosperm-derived suspension cells throughout the culture cycle and enhancement of tissue friability

Batch suspension cultures derived from developing maize (Zea mays L.) endosperm were examined throughout the culture cycle to determine the interaction between the tissue and the medium in relation to sugar transport and the effect of subculturing procedures on growth and friability. The growth rate and friability were improved by increasing the frequency of subculture or by physical screening during transfer. An increase in the conductivity of the medium preceded a decrease in fresh weight associated with tissue senescence. Sucrose in the medium was rapidly hydrolysed, and fructose was depleted more rapidly than glucose. Tissue sucrose concentration expressed on a dry weight basis was higher during the middle of the growth cycle, but hexose, starch, zein, lipid, and soluble protein levels changed very little. The medium pH declined from 5.2 to about 4.5 within one day of subculture. Medium pH changed to 4.5 within one day regardless of initial pH (3.0 to 7.0), indicating regulation of external pH rather than passive acidification. Results are consistent with studies of sugar uptake by these cultures, and indicate that cell clump size can be manipulated without exogenous auxin. The characterization of this tissue line establishes its suitability as a model system for studies of sugar transport and other biochemical events in developing maize endosperm.     

Callus production, somatic embryogenesis and plant regeneration of Lycium barbarum root explants

A new micropropagation system for Lycium barbarum (L.) was developed using root explants as starting material. Callus can be produced from root explants on Murashige and Skoog (MS) medium containing 0.2 mg dm⁻³ 2,4-dichlorophenoxyacetic acid. After three subcultures on the same medium, callus was then transferred onto the MS medium supplemented with 500 mg dm⁻³ lactalbumin hydrolysate to induce somatic embryogenesis (SE). After 20 d, about 60 somatic embryos per 0.25 g(f.m.) of embryogenic callus were obtained but only about 10 % of embryos converted into plantlets. After acclimated in the greenhouse, all of the randomly selected plantlets had survived and were similar phenotypically to zygotic seedlings. In addition, the effects of irradiance, photoperiod, growth regulators, explant age and cold treatment on SE of root-derived callus were evaluated.     

Comparison of somatic embryogenesis and embryo conversion in commercial soybean cultivars

Six commercially important soybean cultivars and one control cultivar were compared for differences in induction-efficiency of somatic embryogenesis, primary embryo yield, and embryo conversion. Cotyledons from immature seeds of similar developmental stage for all soybean cultivars were used for embryo induction. The experiments utilized a Latin square design to exclude the effect of differential lighting and position due to plate location in the growth chamber on the embryogenesis process. Results indicated that the efficiency of embryo induction and yield of primary somatic embryos were genotype-dependent. In contrast, no dependence on genotype was observed for the conversion of embryos to form roots and shoots. The percentage of cotyledons that gave a positive embryogenic response ranged from 26 to 89% for the soybean cultivars tested. The average number of primary globular-stage embryos per responding cotyledon after one month on induction medium ranged from 6 to 13 among the seven cultivars. Conversion frequencies for all genotypes ranged from 27 to 45%.     

Ultrastructure of P-protein inHevea brasiliensis during sieve-tube development and after wounding

Summary P-protein and the changes it undergoes after wounding of sieve tubes of secondary phloem in one- to two-year old shoots ofHevea brasiliensis has been studied using electron microscopy. The P-protein in the form of tubules with a diameter of 8–9 nm and a lumen of 2–2.5 nm occurred in differentiating sieve elements and appeared as compact bodies which consisted of small aggregates of the tubules. As the sieve elements matured, these P-protein bodies dispersed with a disaggregation of the tubules before they turned into striated fibrils, 10–11 nm in diameter. In wounding experiments, as the mature sieve elements collapsed after cutting, their striated P-protein converted into tubules. These tubules were the same in ultrastructure as the tubules in differentiating sieve elements and they often were arranged in crystalline aggregates.     

Effect of primary culture medium composition on high frequency somatic embryogenesis in different Coffea species

High frequency somatic embryogenesis can be obtained over a 7–8 month culture period when using current routing coffee tree micropropagation protocols. To reduce this response time and improve the embryo formation yield, eight different media were tested for primary culture. These media differed from the classically used ones by their mineral nitrogen and plant growth regulator concentrations. An increase from 0.66 to 0.75 in the NO₃/NO₃+NH₄ ratio and a 2-fold lower plant growth regulator concentration in the primary culture medium led to substantial improvements in terms of rapidity and embryo/plantlet regeneration frequencies. Embryo development time was reduced by up to 3 months with a 5-fold increase in the number of formed embryos. These results were obtained for the two cultivated coffee tree species, Coffea canephora and C. arabica, and for a wild one, C. heterocalyx, but not for a second wild species, C. sp. Moloundou showing a species-specific␣response. The new conditions described in this paper led to a substantial enhancement that should be particularly helpful for clonal propagation and genetic engineering of cultivated coffee plants.     

巴西橡胶树的分子生物学研究进展

巴西橡胶树是一种重要的热带经济作物,由于橡胶树体内橡胶含量多,且容易采收,所以橡胶树一直是天然橡胶的商业来源。相比于模式植物和粮食等经济作物来说,分子生物学研究显滞后。,而且橡胶树是多年生乔木,经济性状多集中于胶乳,因此研究难度大,研究也不多,本就巴西橡胶的分子生物学方面的研究进行综述。     

Induction of somatic embryogenesis and embryo development in Rumex acetosella L.

Axillary buds of the dioecious plant Rumex acetosella L. were isolated and cultured in vitro. The callus tissue which developed at the basal parts of the explants displayed a high capacity for shoot formation. This morphogenetic pattern was predominant on Murashige and Skoog (MS) medium supplemented with 2% sucrose, 2.2 mgl^-1 benzylaminopurine and 0.17 mgl^-1 indole-3-acetic acid. Somatic embryogenesis was induced when the osmolality of the medium was increased by adding 6% sucrose instead of 2%, or hexitols in addition to 2% sucrose. Most of the embryogenic calli were formed on the basal parts of leaf laminae and bracts. Development and maturation was strongly promoted by transferring the tissue to a solid or liquid medium lacking benzylaminopurine and indole-3-acetic acid and supplemented with 10 mgl^-1 gibberellic acid. The embryos germinated and developed into normal rosette plants when transferred to vermiculite moistened with hormone-free, half-strength MS salt solution. The histology of successive embryogenic stages is presented.     

Influence of atmospheric gases,particularly ethylene,on somatic embryogenesis of Hevea brasiliensis

The atmosphere of the culture vessel is an important factor for successful somatic embryogenesis in Hevea brasiliensis (Müll. Arg.). Considerable release of carbon dioxide and ethylene occurred during the development of calli. By avoiding the accumulation of gas, unconfined conditions were the most favourable for inducing somatic embryogenesis. Trapping of ethylene was as favourable for calli development and for somatic embryogenesis as unconfined conditions. Inhibition of ethylene synthesis by the adding of aminooxyacetic acid to the medium, also favoured the embryogenic process, and inhibition of ethylene action by the adding of silver nitrate to the medium enhanced significantly embryo production.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AOA amino-oxyacetic acid