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.     

植物组织培养外植体褐变的研究进展

外植体的褐变是组织培养过程中的主要障碍。目前一般认为影响褐变的因素主要有外植体材料、培养基和培养条件。为了防止褐变的发生,主要可以从外植体和培养条件、进行细胞筛选和材料的预处理、抑制剂或吸附剂的加入等方面入手。酚、酚酶的区域性分布假说、自由基伤害假说、保护酶系统假说是褐变机理方面较为成熟的3种假说。     

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.     

Efficient and repetitive production of leaf-derived somatic embryos of Oncidium

Oncidium cultivars gave different embryogenic responses of leaf explants when affected by auxins (2,4-D, IAA, IBA and NAA), cytokinins (2iP, BA, kinetin, TDZ and zeatin), sucrose, NaH₂PO₄, casein hydrolysate, peptone, and glutamine. The best embryogenic responses of cv. Sweet Sugar were at 20 g dm⁻³ sucrose, 85 mg dm⁻³ NaH₂PO₄ and 3 mg dm⁻³ kinetin, respectively. The development of somatic embryos on leaf explants of cv. Sweet Sugar was delayed for about 10 – 20 d in comparison with cv. Gower Ramsey. On growth regulator-free medium, about 40 % of leaf derived embryos of cv. Gower Ramsey were fused together in their basal parts and so called multiple-state embryos. However, under the same condition, the embryos of cv. Sweet Sugar were all in multiple-state form.     

Effect of humidity on somatic embryogenesis in cotyledon explant culture of carrot

In order to clarify the influence of low humidity culture on the structure and function of somatic embryo the cotyledon expiants ofDaucus carrota L. cv. Hongshim were cultured in the petridish whose lids had holes sealed with millipore filters. In the low humidity culture, the production of somatic embryos was enhanced and their maturation promoted but the cotyledon structure of somatic embryos were nearly similar to control. In addition, the low humidity culture improved the germination of somatic embryos. Especially, the germination frequency of jar-shaped embryos was much improved (68%) in comparison with that of jarshaped embryos formed in constant humidity culture (23%). But low humidity culture at its extreme became an obstacle to normal plant regeneration in that precocious embryos were generated and the primary embryos turned into callus and formed secondary embryos. Therefore it is suggested that moderately low humidity culture (80–90% R.H.) is important to the higher production and better-quality of somatic embryos.     

Effects of GA3, ancymidol,cycocel and paclobutrazol on direct somatic embryogenesis of Oncidium in vitro

To study the effects of GA, ancymidol, cycocel and paclobutrazol on direct somatic     

The importance of the explant on regeneration in thin cell layer technology

Summary The basic factor underlying the success of the tissue culture, large-scale micropropagation and genetic transformation of any plant species is regeneration. This has been achieved over the years through the use of various-sized explants ranging from protoplasts (small scale) to entire organs (large scale). Inherent problems underlie the use of either extreme, leading to both nonspecific morphogenic reactions in the latter, or to undesired necrosis in the former. This review investigates the importance of different aspects of a thin cell layer (TCL) explant, from its source to its size. TCLs, as a results of their size and origin, in combination with other controllable factors such as media and environmental conditions, have shown this system to be superior to the use of conventional explants. Numerous species that were previously unsuccessfully tissue-cultured have, with the use of TCL technology, resulted in their successful micropropagation and regeneration. These successes, based on the inherent qualities of the TCL explant—specific for a given species—are also examined.     

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.     

Enhanced somatic embryogenesis and plant regeneration in leaf explant cultures of Ostericum koreanum on medium of varying pH

The leaf explants of Ostericum koreanum were cultured on MS medium supplemented with 5.37 M NAA and 0.44 M BA and did not need transfer to growth regulator–free medium for somatic embryogenesis. The pH level of medium dropped after autoclaving and at the beginning of explant culture, then rose back to the normal pH level of medium. The low pH level of medium, pH 4.0 or 4.3, before autoclaving rose to pH 5.2 or 5.3 and pH 6.1 or 6.2 after the 1 and 8 weeks from culture initiation, respectively, and this level was variable around pH 5–pH 6 during culture period. The explants were exposed to low pH for only several days at the early period of culture. On medium of pH 4.3, the production of somatic embryos was enhanced to six times in comparison with that on medium of pH 5.8. The average regeneration rate of total somatic embryos produced on medium of low pH was over 10% higher than that at pH 5.8. The regeneration of cup-shaped embryos was improved from 33% on medium of pH 5.8 to 67% on medium of pH 4.3. Therefore, the production and regeneration of somatic embryos were enhanced by the temporary exposure of leaf explant to medium of low pH, even though somatic embryogenesis substantially occurred on medium of nearly routine pH.     

The effect of genotype and explant age on somatic embryogenesis of coffee

We demonstrate that embryogenic capacity of coffee (Coffea arabica) depends on the genotype, and that it is a character fixed at the early generations F3 or F4. Therefore, the embryogenic capacity F5 lines can be predicted in a breeding program if it is possible to evaluate the F3 of F4 ancestors, or lines with the same parents on those generations.Regeneration via somatic embryogenesis of genotypes from a C. arabica cv. Caturra by Timor Hybrid cross were evaluated, to determine the effect of the pedigree, month of leaf collection, as well as its interaction with the genotype. Large variations in embryogenic capacity among genotypes were detected with rates ranging from 4.8 to 72.7. There was association between embryogenic response of the progenies and the progenitor from which they proceeded. Significant differences were also found in embryogenic responses depending on the month of leaf explant collection, as well as a significant interaction of genotype by planting month.The results are relevant for breeding and tissue culture purposes of coffee because they will allow speed up of the breeding program and save effort by being able to predict embryogenic capacity of a line, based on the response of either its progenitors or the F3 or F4 lines of the same origin. In addition, high embryogenic capacity lines can be considered directly for genetic transformation or for massive multiplication (bioreactors).     

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.     

Characterization of competence during induction of somatic embryogenesis in alfalfa tissue culture

Systematic manipulations of the culture protocol leading to somatic embryogenesis in alfalfa petiole-derived callus were performed to study the competence phase during somatic embryogenesis in alfalfa. These demonstrated a requirement for the acquisition of competence prior to the induction of the embryogenesis pathway. Induction was triggered by a number of synthetic auxins including 2,4-dichlorophenoxyacetic acid (2,4-d). Competence could be acquired in the presence of these auxins as well as phenylacetic acid (PAA), an auxin that did not induce embryogenesis. Different degrees of competence were apparently acquired by exposure to 2,4-d and PAA. Some degree of competence was acquired in the absence of auxin treatment. The current understanding of the concept of competence is discussed.PRC contribution no. 1431     

The effect of explant material on somatic embryogenesis of Cyclamen persicum Mill

Somatic embryos of Cyclamen persicum Mill. could be produced through a callus phase from juvenile explant material including anthers, ovaries and zygotic embryos. The auxin 2,4-D (1.0–1.5 mg l^-1) and coconut milk (10% v/v) in MS medium were important factors for the induction of somatic embryogenesis. Somatic embryos germinated into plantlets in MS medium without growth regulators. The plants grew well in the greenhouse and flowered normally. The plants were phenotypically identical to the mother plants with a few exceptions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthylacetic acid - IAA 3-indoleacetic acid - BA 6-benzyladenine - ABA abscisic acid - CM coconut milk     

Direct somatic embryogenesis on leaf explants of Oncidium Gower Ramsey and subsequent plant regeneration

 Segments taken from young leaves of an orchid (Oncidium Gower Ramsey) produced clusters of somatic embryos directly from epidermal and mesophyll cells of leaf tips and wound surfaces without an intervening callus within 1 month when cultured on a Gelrite^TM-gelled 1/2-MS basal medium supplemented with a low dosage (0.3–1 mg/l) of thidiazuron. Subculturing of these embryo clusters produced more embryos and subsequent plantlet formation on the same medium. The high-frequency embryogenesis of these leaf cells in this orchid is strong evidence of their totipotency, and further modification of the protocol for plant formation could be useful for the mass propagation and transformation of selected elite lines. Received: 16 September 1998 / Revision received: 16 February 1999 / Accepted: 26 February 1999     

A continuous culture system of direct somatic embryogenesis in microspore-derived embryos of Brassica juncea

Summary An efficient culture system has been developed for repeated cycles of somatic embryogenesis in microspore-derived embryos of Brassica juncea without a callus phase. Haploid embryos produced through anther culture showed a high propensity for direct production of somatic embryos in response to 2 mgL^–1 BA and 0.1 mgL^–1 NAA. The embryogenic cultures which comprised the elongated embryonal axis of microspore-derived embryos when explanted and grown on the medium of same composition produced a large number of secondary embryos. These somatic embryos in turn underwent axis elongation and produced more somatic embryos when explanted and cultured. This cycle of repetitive somatic embryogenesis continued with undiminished vigour passage after passage and was monitored for more than a year. Somatic embryos from any passage when isolated at cotyledonary stage and grown on auxin-free medium for 5 days and then on a medium containing NAA (0.1 mgL^–1), developed into complete plants with a profuse root system and were easily established in the soil. The cytology of the root tips of these plants confirmed their haploid nature. The total absence of callus phase makes the system ideal for continuous cloning of androgenic lines, Agrobacterium-mediated transformation and mutation induction studies.     

High frequency somatic embryogenesis and plant regeneration in root explant cultures of carnation

Root explants excised from carnation plants maintained in vitro formed off-white, friable calluses after three weeks of culture on Murashige and Skoog (MS) medium supplemented with 1 mg l⁻¹ thidiazuron (TDZ) and 1 mg l⁻¹ α-naphthalaneacetic acid (NAA). These calluses were subsequently transferred to MS basal medium where, after an additional four weeks of culture, approximately 50% of the calluses formed somatic embryos. However, calluses formed on root explants that had been cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid did not produce somatic embryos upon transfer to MS basal medium. Somatic embryos developed into plantlets and subsequently were grown to maturity. These results indicate that root explants have a high competence for somatic embryogenesis in carnation. J. Seo and S.W. Kim contributed equally to this work.     

Somatic embryogenesis in pea (Pisum sativum L.): effect of explant, genotype and culture conditions

In this study 16 cultivars of pea (Pisum sativum L.) were screened in vitro for the formation of somatic embryos which were dependent on the genotype, culture conditions and explant source used. The cultivars Stehgolt, Maro and Progreta showed the highest tendency to form somatic embryos (c. 25%) while Alaska, Rondo and Ascona did not show any embryo production. Using the cultivar Belman, the highest embryo production was achieved by using nodal explants of shoots (10.6%) and a cotyledon-free embryo as explant source (14.1%) in the light (15.8%) compared to using apices as explants (1.8%) and a seedling as the explant source (9.4%) in the dark (3.3%). Media containing picloram (0.75 mg/litre) followed by BA (1 mg/litre) or kinetin (1 mg/litre), each for four weeks gave the highest somatic embryo production. The development of embryos to whole plants was unreliable and some 90% of the embryos induced did not develop any further, died, recallused or formed secondary embryos. The size of the embryo at separation and the timing of the separation from the original callus were important factors determining success for complete development to whole plant. Regeneration of 184 plants was achieved with ensuing flowering, pod formation and viable seed production from the techniques described.     

Influence of explant,temperature and different culture vessels on <Emphasis Type="Italic">in vitro </Emphasis>culture for germplasm maintenance of four mint accessions

Two experiments were conducted with four accessions of mint (Mentha spp.) on MS medium for their in vitro performance. In the first experiment apical and nodal explants were cultured at both 20 °C and 25 °C. Data was recorded at second and at fifth week. Both apical and nodal explants of mint showed better leaf production at 25 °C than 20 °C. Nodal explants of mint cultured at 25 °C in both cultivation periods exhibited the highest number of leaves. In the second experiment apical explants were cultured in four different culture vessels viz., industrial glass jar (IG), magenta vessel (MV), Erlenmeyer flask (EF) and culture tube with 1(CT1) and 2(CT2) explants at 25 °C for 6 weeks. The highest weight loss from the media, evapo-transpiration and fresh weight gain were recorded in IG and next in MV. The lowest weight loss from the media and fresh weight gain both were found in CT2. However the lowest evapo-transpiration was noted in EF. The highest numbers of leaves were recorded from MV. Without explants, depletion of medium and increase of headspace were both higher in IG than in the other vessels. Overall, Magenta vessel GA 7 showed the best in vitro performance.     

Picomolar concentrations of salicylates induce cellular growth and enhance somatic embryogenesis in Coffea arabica tissue culture

Embryogenic cell suspension cultures of Coffea arabica cv. Caturra Rojo were treated with salicylic acid (SA). Two concentrations, 10^-12 and 10^-10 M, had a significant effect on the growth rate of the cell cultures when compared to the control, and this effect was concentration-dependent. These two SA concentrations also had a dramatic effect on both the number of somatic embryos and quality, in terms of embryo size and development. In general, the use of SA had a positive effect on cellular growth and somatic embryogenesis, causing a twofold increase in both processes. The increase in the number of somatic embryos could be a reflection of an increase in the number of embryogenic cells induced with SA treatment.     

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