青扦胚性细胞悬浮培养中影响体细胞胚发生因素的研究

试验以青扦（Ｐｉｃｅａｗｉｌｓｏｎｉ）的胚性愈伤组织为材料,以改良５９基本成分附加２４－Ｄ１ｍｇ／Ｌ及ＫＴ１ｍｇ／Ｌ为培养介质,比较了液体悬浮与半固体二种培养方式对胚性愈伤组织增殖和体细胞发生的影响,研究了液体悬浮培养过程中影响体细胞胚发生的因素。结果表明：液体悬浮培养好于半固体培养,它的胚性愈伤组织的生长率为２６８％,是半固体培养的１２４倍;体细胞胚的分化率为９３％,是半固体培养的２２倍;悬浮培养较佳的培养条件为：初始细胞密度为２％（鲜重）,蔗糖浓度为２０ｇ／Ｌ,摇床转速为１００ｒ／ｍｉｎ,ｐＨ为５８。经过两个月悬浮培养,将培养物转至１／２改良５９附加ＡＢＡ１ｍｇ／Ｌ的分化培养基上,３个月后每ｇ培养物上可获得２８５个正常的子叶期体细胞胚。     

Improvement of Citrus somatic embryo development by temporary immersion

Liquid medium improves and facilitates somatic embryo development from Citrus deliciosa Ten. suspension cultures. Three different culture conditions were compared to determine a means of overcoming poor somatic embryo development. Somatic embryos derived from suspension cultures were plated on solid medium, maintained in suspension culture or temporarily immersed. About 60% of somatic embryos plated on solid medium developed to the cotyledonary stage, but were hyperhydric. Continuous growth in suspension culture at 100 rpm hindered cotyledon and protoderm formation, and somatic embryos were unable to develop beyond the globular stage. Temporary immersion promoted somatic embryo development, i.e. 66% of the somatic embryos produced were cotyledonary, and were morphologically similar to nucellar embryos. This latter culture system also improved regeneration synchronization by hampering secondary embryogenesis at the onset of germination. Irrespective of the culture system used, most cotyledonary somatic embryos studied had no caulinary meristem or starch and protein reserves, thus explaining the low germination rates obtained. This revised version was published online in June 2006 with corrections to the Cover Date.     

Differences in protodermal cell wall structure in zygotic and somatic embryos of <Emphasis Type="Italic">Daucus carota</Emphasis> (L.) cultured on solid and in liquid media

The ultrastructure, cuticle, and distribution of pectic epitopes in outer periclinal walls of protodermal cells of Daucus carota zygotic and somatic embryos from solid and suspension culture were investigated. Lipid substances were present as a continuous layer in zygotic and somatic embryos cultured on solid medium. Somatic embryos from suspension cultures were devoid of cuticle. The ultrastructure of the outer walls of protodermis of embryos was similar in zygotic and somatic embryos from solid culture. Fibrillar material was observed on the surface of somatic embryos. In zygotic embryos, in cotyledons and root pectic epitopes recognised by the antibody JIM5 were observed in all cell walls. In hypocotyls of these embryos, these pectic epitopes were not present in the outer periclinal and anticlinal walls of the protodermis. In somatic embryos from solid media, distribution of pectic epitopes recognised by JIM5 was similar to that described for their zygotic counterparts. In somatic embryos from suspension culture, pectic epitopes recognised by JIM5 were detected in all cell walls. In the cotyledons and hypocotyls, a punctate signal was observed on the outside of the protodermis. Pectic epitopes recognised by JIM7 were present in all cell walls independent of embryo organs. In zygotic embryos, this signal was punctate; in somatic embryos from both cultures, this signal was uniformly distributed. In embryos from suspension cultures, a punctate signal was detected outside the surface of cotyledon and hypocotyl. These data are discussed in light of current models for embryogenesis and the influence of culture conditions on cell wall structure.     

Changes in the Brown Seaweed Ascophyllum Nodosum (L.) Le Jol. Plant Morphology and Biomass Produced by Cutter Rake Harvests in Southern New Brunswick, Canada

Shoots and clumps of shoots of the commercial brown seaweed Ascophyllum nodosum (“rockweed”) add to the benthic complexity of the intertidal environment, providing an important habitat for invertebrates and vertebrates. To protect the structure of this habitat, management plans for the rockweed harvest of southern New Brunswick include restrictions on gear type and exploitation rates limited to 17% of the harvestable biomass. However, owing to physical and environmental factors, the harvest is not homogeneous, creating patches of exploitation ranging from 15 to 50%.A direct relationship existed between clump vulnerability, weight and length in a controlled harvest at 50% exploitation within 8 m by 8 m plots. At this exploitation rate, the gear rarely impacted clumps below 50 g or 60 cm in length. Clumps larger than 300 g and 130 cm were reduced by up to 55% of their length and 78% of their biomass. The overall impacts of the harvest on intertidal habitat is however of short duration as biomass recovers after a year of the experimental harvest. The rapid recovery is mostly due to a stimulation of growth and branching of the suppressed shoots of the clumps. Some harvested plots showed biomass even higher than initial levels, suggesting an increase in productivity at least during the first year after the harvest.     

白Pian体细胞胚悬浮培养的动力学研究

白(ＰｉｃｅａｍｅｙｅｒｉＲｅｈｄ.ｅｔＷｉｌｓ.)是我国特有的云杉属树种,在林业生产和环境绿化中均具有重要地位。其体细胞胚胎发生的研究,一方面可用于优良种质的大规模快速繁殖,为植树造林和园林绿化提供优质苗木;另一方面可作为遗传转化的再生系统,进行树种遗传...     

A key developmental switch during Norway spruce somatic embryogenesis is induced by withdrawal of growth regulators and is associated with cell death and extracellular acidification.

The biotechnology of somatic embryogenesis holds considerable promise for clonal propagation and breeding programs in forestry. To efficiently regulate the whole process of plant regeneration through somatic embryogenesis, it is of outmost importance to understand early developmental events when somatic embryos are just formed. In Norway spruce, somatic embryos transdifferentiate from proembryogenic masses (PEMs). This work describes the developmental dynamics (frequency distribution of PEMs and early somatic embryos) of the whole embryogenic suspension culture growing in the presence and absence of plant growth regulators (PGRs), auxin and cytokinin. The experiments have shown that PEM-to-somatic embryo transition is a key developmental switch that determines the yield and quality of mature somatic embryos and ultimately plant production. This switch was induced by the withdrawal of PGRs in cell suspension leading to a rapid accumulation of early somatic embryos (to a maximum of 75% of the entire population of suspension culture) and concomitant degradation of PEMs. The latter was evident from increased level of cell death measured through spectrophotometric Evans blue staining assay. Proembryogenic mass-to-embryo transition and concomitant activation of cell death were mediated by strong extracellular acidification. Therefore, buffering PGR-free culture medium at high (pH 5.8) or low (pH 4.5) levels of pH inhibited both PEM-to-embryo transition and cell death. The yield of mature somatic embryos on abscisic acid (ABA)-containing medium was increased up to 10-fold if the suspension culture had been pretreated for 1 to 9 days in unbuffered PGR-free medium. In this case a large proportion (75%) of the total number of mature embryos was formed within a short, 5-week, contact with ABA. The latter is practically important because prolonged contact with ABA suppresses the growth of somatic embryo plants. Based on these results, an improved method for regulating somatic embryogenesis was set up and tested for nine genotypes of Norway spruce. Over 800 plants regenerated from all tested genotypes demonstrated a good performance in the greenhouse and they were transferred to the field.     

Scale-up of somatic embryogenesis in alfalfa (Medicago sativa L.) I subculture and indirect secondary somatic embryogenisis

Three methods of increasing the productivity of somatic embryogenesis in Medicago sativa L. were investigated. In the basic procedure, somatic embryos were initiated from young petioles and carried through several phases: callus formation, suspension culture, selection of the embryogenic fraction by sieving, development, maturation, desiccation and storage. The suspensions were normally separated into three fractions by sieving. Fraction I (<200 m) containing nonembryogenic cells or cell clusters was discarded. Fraction II (200–500 m) consisting of embryogenic cell clusters was collected for embryo development and maturation. Fraction III (over 500 M) containing the mixture of petiole residues with large pieces of calli and globular somatic embryos was usually discarded. Several methods to scale-up the suspension phase were unsuccessful. Direct subculture of the entire suspension by the addition of fresh liquid medium resulted in the loss of embryogenic capacity by the third subculture. Subculture of fraction II decreased embryogenic cell mass, and hence reduced total productivity. The recycling of fraction III back to fresh B₅g liquid medium resulted in high productivity in the first culture but further subculture of this fraction resulted in a rapid decline in the embryogenic capacity.As an alternative, somatic embryos from the first tissue culture cycle were also used as explants for the initiation of secondary embryogenic callus. The embryogenic capacity of these somatic embryo explants declined rapidly as they matured. More than 100 secondary somatic embryos could be induced from embryo explants removed from development medium at 10 days after sieving the suspension, but only 40 somatic embryos were produced from each mature somatic embryo explant, and 13 from desiccated embryos. The secondary somatic embryos were comparable to the primary embryos in quality according to germination tests. The implications of the results to the efficiency of somatic embryo production of Medicago are discussed.Abbreviations ABA abscisic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - DAS days after sieving - PPF photosynthetic photon flux density - SE somatic embryo     

Production of vigorous,desiccation tolerant white spruce (Picea glauca [Moench.] Voss.) synthetic seeds in a bioreactor

Summary This report describes a low-cost method for generating large numbers of high quality mature white spruce (Picea glauca [Moench.] Voss) somatic embryos which survived desiccation and grew to plantlets more vigorously than excised zygotic embryos cultured in vitro. Somatic embryos from suspension culture were supported within a culture chamber on a flat absorbent pad above the surface of a liquid culture medium containing 20–50 M abscisic acid and 7.5 % polyethylene glycol. Throughout a 7 week culture period 3 L of fresh medium was pumped into one end of the chamber, while the spent medium exited by gravity from the opposite end. Over 6,300 cotyledonary stage white spruce somatic embryos were recovered after this time from a single culture chamber without manual manipulation. The somatic embryos were of excellent appearance with well developed cotyledons, and possessed high levels of storage lipids. They survived drying to about 8 % moisture content following treatment for 4 weeks at 63 % relative humidity, and following imbibition converted to normal plantlets at a frequency of 92 %, compared to 80 % for embryos grown in Petri dishes. Somatic embryos cultured within the bioreactor developed to plantlets that were 20 % longer than zygotic embryos excised from mature seed and grown in vitro, and were 38 % longer than somatic embryos cultured upon agar medium in Petri dishes.Plant Research Centre contribution No. 1523     

Callus formation and plantlet regeneration from protoplasts derived from suspension cultures of wheat (Triticum aestivum L.)

Protoplasts were isolated from anther-derived suspension cultures of commercial wheat (Triticum aestivum L. cv. Chris). The protoplasts were released enzymatically and isolated by centrifugation on a sucrose cushion. The isolated protoplasts were initially cultured in a liquid medium in the dark. Numerous microcalli were produced under these conditions, some of which differentiated into globular embryos. Upon transfer to a solid medium and exposure to 16h/8h light/dark cycle, the protocalli proliferated and many of the somatic embryos matured. Complete plantlets were obtained and maintained in sterile culture.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - MES 2-[N-morpholino] ethanesulfonic acid     

Somatic embryo formation and germination from immature embryo-derived suspension-cultured cells of Angelica sinensis (Oliv.) Diels

Embryogenic callus was induced from immature embryos of Angelica sinensis cultured on Murashige and Skoog (MS) basal medium. Embryogenic callus growth was more rapid on MS basal medium than on B5 or White medium. Embryogenic callus was used to establish a suspension culture and somatic embryos and germinating embryos developed during the culture. A shaking speed of 80 rpm was found to be optimal for establishing suspension cultures, while 100 rpm produced more somatic embryos and germinating embryos with an initiation cell density of 0.2 ml packed cell volume/25 ml medium. Adding 0.3% agar to the liquid medium also stimulated the formation of somatic and germinating embryos. While no plant growth regulators were needed for culture initiation and plant regeneration, the addition of 0.5–1 mg/l 2,4-dichlorophenoxyacetic acid was needed to maintain the embryogenic suspension culture by preventing embryo germination. Forty percent of the germinating embryos survived after culturing on filter paper moistened with liquid half-strength MS medium containing 3% sucrose. The plants were successfully transferred into soil. Received: 19 March 1997 / Revision received: 21 November 1997 / Accepted: 19 January 1998     

Application of bioreactor system for large-scale production of Eleutherococcus sessiliflorus somatic embryos in an air-lift bioreactor and production of eleutherosides

Embryogenic callus was induced from leaf explants of Eleutherococcus sessiliflorus cultured on Murashige and Skoog (MS) basal medium supplemented with 1 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D), while no plant growth regulators were needed for embryo maturation. The addition of 1 mg l(-1) 2,4-D was needed to maintain the embryogenic culture by preventing embryo maturation. Optimal embryo germination and plantlet development was achieved on MS medium with 4 mg l(-1) gibberellic acid (GA(3)). Low-strength MS medium (1/2 and 1/3 strength) was more effective than full-strength MS for the production of normal plantlets with well-developed shoots and roots. The plants were successfully transferred to soil. Embryogenic callus was used to establish a suspension culture for subsequent production of somatic embryos in bioreactor. By inoculating 10 g of embryogenic cells (fresh weight) into a 3l balloon type bubble bioreactor (BTBB) containing 2l MS medium without plant growth regulators, 121.8 g mature somatic embryos at different developmental stages were harvested and could be separated by filtration. Cotyledonary somatic embryos were germinated, and these converted into plantlets following transfer to a 3l BTBB containing 2l MS medium with 4 mg l(-1) GA3. HPLC analysis revealed that the total eleutherosides were significantly higher in leaves of field grown plants as compared to different stages of somatic embryo. However, the content of eleutheroside B was highest in germinated embryos. Germinated embryos also had higher contents of eleutheroside E and eleutheroside E1 as compared to other developmental stages. This result indicates that an efficient protocol for the mass production of E. sessiliflorus biomass can be achieved by bioreactor culture of somatic embryos and can be used as a source of medicinal raw materials.     

Effect of serum concentration, method of trypsinization and fusion/activation utilizing transfected fetal cells to generate transgenic dairy goats by somatic cell nuclear transfer

This work was performed within a commercial nuclear transfer program to investigate different methods for synchronizing donor cell cycle stage, for harvesting donor cells, and for fusion and activation of reconstructed caprine embryos. Primary fetal cells isolated from day 35 to day 40 fetuses were co-transfected with DNA fragments encoding both the heavy and light immunoglobulin chains of three different monoclonal antibodies and neomycin resistance. Four neomycin resistant cell lines for each antibody were selected, expanded, and aliquots were both cryopreserved for later use as karyoplast donors or used for further genetic characterization. Transfected fetal cells were cultured in 0.5% FBS to synchronize G0/G1 cell cycle stage cells, then re-fed with 10% FBS prior to use to allow donor cells to re-enter the cell cycle. Alternatively, transfected fetal cells were grown to confluence in 10% FBS to induce contact inhibition to synchronize G0/G1 cell cycle stage cells. Adherent monolayers of transfected fetal donor cells were harvested by either partial or complete trypsinization. Donor cells were simultaneously fused and activated with enulceated in vivo produced ovulated oocytes from superovulated does. Half of the fused couplets received an additional electrical activation pulse and non-fused couplets were re-fused. Four live offspring were produced from 587 embryos generated from cell lines cultured in 0.5% FBS, while one live offspring was produced from 315 embryos generated from cell lines cultured in 10% FBS (0.7% versus 0.3% embryos transferred, respectively, P > 0.05). Five offspring were produced from 633 embryos generated from cell lines harvested by partial trypsinization (0.8% embryos transferred), and no offspring were produced from 269 embryos generated from cell lines harvested by complete trypsinization. Four live offspring were produced from 447 embryos generated from re-fused couplets, and one live offspring was produced from 230 embryos generated from fused couplets that received an additional electrical activation pulse (0.9% versus 0.4% embryos transferred, respectively, P > 0.05). These results suggest that low-serum culture of transfected goat fetal cells and harvest by partial trypsinization may be more efficient methods for generating transgenic goats by somatic cell nuclear transfer. In addition, re-fusion of non-fused couplet or an additional activation step was successful for producing live offspring.     

Somatic embryogenesis and plant regeneration in avocado (Persea americana Mill.): influence of embryogenic culture type

Embryogenic cell lines of Persea americana were classified as SE- or PEM-type based on their morphology in maintenance medium. PEM-type cultures consisted of proembryonic masses (PEMs) with occasional development of proembryos and early-stage somatic embryos, while SE-type cultures consisted of somatic embryos from globular to cotyledonary stages with only a low frequency of proembryos and PEMs. Moreover, histological analysis revealed signs of cellular organisation, with a higher proportion of small cells in peripheral regions of the callus mass in the SE-type, but not in PEM-type cell lines. The effects of time in suspension, inoculum density and maintenance medium on the ability of avocado cell lines to produce mature somatic embryos were evaluated. Morphological differences observed during proliferation were correlated to the subsequent capacity of cultures to develop mature somatic embryos. The period of time in suspension and inoculum density were critical factors that influenced the ability of cultures to undergo maturation. Optimal conditions differed between SE-type cultures and PEM-type cultures. In SE-type cultures, the highest number of somatic embryos was observed in cultures grown in suspension for 9?days and 0.4?g inoculum; in PEM-type cultures a slight increase in mature somatic embryos production could be observed with 4?g inoculum size and 14?days in suspension. The presence of agar in the maintenance medium of SE-type cultures was essential for the maturation of somatic embryos.     

Soluble Signals from Cells Identified at the Cell Wall Establish a Developmental Pathway in Carrot

Cells in a plant differentiate according to their positions and use cell-cell communication to assess these positions. Similarly, single cells in suspension cultures can develop into somatic embryos, and cell-cell communication is thought to control this process. The monoclonal antibody JIM8 labels an epitope on cells in specific positions in plants. JIM8 also labels certain cells in carrot embryogenic suspension cultures. We have used JIM8 and secondary antibodies coupled to paramagnetic beads to label and immunomagnetically sort single cells in a carrot embryogenic suspension culture into pure populations. Cells in the JIM8(+) population develop into somatic embryos, whereas cells in the JIM8(-) population do not form somatic embryos. However, certain cells in JIM8(+) cultures (state B cells) undergo asymmetric divisions, resulting in daughter cells (state C cells) that do not label with JIM8 and that sort to JIM8(-) cultures. State C cells are competent to form somatic embryos, and we show here that a conditioned growth medium from a culture of JIM8(+) cells allows state C cells in a JIM8(-) culture to go on and develop into somatic embryos. JIM8 labels cells in suspension cultures at the cell wall. Therefore, a cell with a role in cell-cell communication and early cell fate selection can be identified by an epitope in its cell wall.     

Conservation Management of the Mixed Species Nontimber Forest Product of “moss”—Are they Harvesting What we <Emphasis Type="Italic">think</Emphasis> they’re Harvesting?

Management to preserve biodiversity while allowing resource extraction must be tailored to the taxa impacted by harvest. Yet millions of kilograms of mosses, liverworts, some lichens and an occasional vascular plant are harvested from the forests of the Pacific Northwest of North America each year without an adequate understanding of which species are impacted by harvest. Research has focused on epiphytic bryophytes as the primary target of harvest, but the habitats and substrates actually impacted by harvest and the species sold as “moss” have never been determined. To determine if the species being researched are the same as those that are actually sold, we compared the relative abundance and species composition of 17 commercially sold bags of moss with those from 33 field sites in the Oregon Coast and Cascade Ranges on which moss harvest research had previously been conducted. Most species found in the purchased bags had been previously reported from the field studies of what were thought to be commercially harvestable species, including all of the most frequently harvested taxa. However, several new species were found to be commercially harvested and more log and valley hardwood species were found than were previously believed to be harvested. Proper management will require harvest impact studies for the species found in these additional habitats. All nontimber forest products involving mixtures of various species should be periodically surveyed at the point of sale to determine which species are actually being harvested.     

Stable transformation via particle bombardment in two different soybean regeneration systems

The Biolistics^® particle delivery system for the transformation of soybean (Glycine max L. Merr.) was evaluated in two different regeneration systems. The first system was multiple shoot proliferation from shoot tips obtained from immature zygotic embryos of the cultivar Williams 82, and the second was somatic embryogenesis from a long term proliferative suspension culture of the cultivar Fayette. Bombardment of shoot tips with tungsten particles, coated with precipitated DNA containing the gene for -glucuronidase (GUS), produced GUS-positive sectors in 30% of the regenerated shoots. However, none of the regenerants which developed into plants continued to produce GUS positive tissue. Bombardment of embryogenic suspension cultures produced GUS positive globular somatic embryos which proliferated into GUS positive somatic embryos and plants. An average of 4 independent transgenic lines were generated per bombarded flask of an embryogenic suspension. Particle bombardment delivered particles into the first two cell layers of either shoot tips or somatic embryos. Histological analysis indicated that shoot organogenesis appeared to involve more than the first two superficial cell layers of a shoot tip, while somatic embryo proliferation occurred from the first cell layer of existing somatic embryos. The different transformation results obtained with these two systems appeared to be directly related to differences in the cell types which were responsible for regeneration and their accessibility to particle penetration.     

A temporary immersion system improves in vitro regeneration of peach palm through secondary somatic embryogenesis

Background and Aims

Secondary somatic embryogenesis has been postulated to occur during induction of peach palm somatic embryogenesis. In the present study this morphogenetic pathway is described and a protocol for the establishment of cycling cultures using a temporary immersion system (TIS) is presented.

Methods

Zygotic embryos were used as explants, and induction of somatic embryogenesis and plantlet growth were compared in TIS and solid culture medium. Light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to describe in vitro morphogenesis and accompany morpho-histological alterations during culture.

Key Results

The development of secondary somatic embryos occurs early during the induction of primary somatic embryos. Secondary somatic embryos were observed to develop continually in culture, resulting in non-synchronized development of these somatic embryos. Using these somatic embryos as explants allowed development of cycling cultures. Somatic embryos had high embryogenic potential (65·8 ± 3·0 to 86·2 ± 5·0 %) over the period tested. The use of a TIS greatly improved the number of somatic embryos obtained, as well as subsequent plantlet growth. Histological analyses showed that starch accumulation precedes the development of somatic embryos, and that these cells presented high nucleus/cytoplasm ratios and high mitotic indices, as evidenced by DAPI staining. Morphological and SEM observations revealed clusters of somatic embryos on one part of the explants, while other parts grew further, resulting in callus tissue. A multicellular origin of the secondary somatic embryos is hypothesized. Cells in the vicinity of callus accumulated large amounts of phenolic substances in their vacuoles. TEM revealed that these cells are metabolically very active, with the presence of numerous mitochondria and Golgi apparatuses. Light microscopy and TEM of the embryogenic sector revealed cells with numerous amyloplasts, large nuclei and nucleoli, and numerous plasmodesmata. Plantlets were obtained and after 3 months in culture their growth was significantly better in TIS than on solid culture medium. However, during acclimatization the survival rate of TIS-grown plantlets was lower.

Conclusions

The present study confirms the occurrence of secondary somatic embryos in peach palm and describes a feasible protocol for regeneration of peach palm in vitro. Further optimizations include the use of explants obtained from adult palms and improvement of somatic embryo conversion rates.     

Enhancement of American chestnut somatic seedling production

Somatic embryogenesis holds promise for mass propagation of American chestnut trees bred or genetically engineered for resistance to chestnut blight. However, low germination frequency of chestnut somatic embryos has limited somatic seedling production for this forest tree. We tested the effects of culture regime (semi-solid versus liquid), cold treatment, AC and somatic embryo morphology (i.e., cotyledon number) on germination and conversion of the somatic embryos. Cold treatment for 12 weeks was critical for conversion of chestnut somatic embryos to somatic seedlings, raising conversion frequencies for one line to 47%, compared to 7% with no cold treatment. AC improved germination and conversion frequency for one line to 77% and 59%, respectively, and kept roots from darkening. For two lines that produced embryos with one, two or three-plus cotyledons, cotyledon number did not affect germination or conversion frequency. We also established embryogenic American chestnut suspension cultures and adapted a fractionation/plating system that allowed us to produce populations of relatively synchronous somatic embryos for multiple lines. Embryos derived from suspension cultures of two lines tested had higher conversion frequencies (46% and 48%) than those from cultures maintained on semi-solid medium (7% and 30%). The improvements in manipulation of American chestnut embryogenic cultures described in this study have allowed over a 100-fold increase in somatic seedling production efficiency over what we reported previously and thus constitute a substantial advance toward the application of somatic embryogenesis for mass clonal propagation of the tree.     

Application of bioreactors for large-scale micropropagation systems of plants

Summary The application of bioreactor culture techniques for plant micropropagation is regarded as one of the ways to reduce production cost by scaling-up and automation. Recent experiments are restricted to a small number of species that, however, demonstrate the feasibility of this technology. Periodic immersion liquid culture using ebb and flood system and column-type bubble bioreactors equipped with a raft support system to maintain plant tissues at the air and liquid interface were found to be suitable for micropropagation of plants via the organogenic pathway. Balloon-type bubble bioreactors proved to be fit for micropropagation via somatic embryogenesis with less shear stress on cultured cells. Several cultivars of Lilium were successfully propagated using a two-stage culture method in one bioreactor. A large number of small-scale segments were cultured for 4 wk with periodic immersion liquid culture to induce multiple bulblets from each segment, then the bulblet induction medium was changed into bulblet growth medium by employing a submerged liquid bioreactor system. This culture method resulted in a nearly 10-fold increase in bulblet growth compared to conventional culture with solid medium. About 20 000 cuttings of virus-free potato could be obtained from 120 singlenode explants in a 20-liter balloon-type bubble bioreactor after 8 wk of culture. The percentage of ex vitro survival and root induction of the cuttings was more than 95%. Other successful results were obtained from the micropropagation and transplant production of chrysanthemum, sweetpotato, Chinese foxglove. Propagation systems via somatic embryogenesis in Acanthopanax koreanum and thornless Aralia elata were established using a liquid suspension of embryogenic determined cells. More than 500 000 somatic embryos in different stages were harvested from a 10-liter balloon-type bubble bioreactor after a 6-wk culture. Further development of these embryos in solid medium and eventually in the field was successful. The bioreactor system could reduce initial and operational cost for micropropagation, but further development of sophisticated technology might be needed to apply this system to plant micropropagation industries.