The dynamics of nutrient utilization and growth of apple root stock ‘M9 EMLA’ in temporary versus continuous immersion bioreactors

The present study investigated the dynamics of nutrient utilization and various growth and physiological parameters during in vitro proliferation of apple root stock ‘M9 EMLA’ in two different bioreactor systems, i.e. temporary and continuous immersions. Individual shoots obtained from temporary immersion system had higher dry mass and were of better quality than those obtained from continuous immersion. In continuous immersion bioreactor, apple shoots appeared to utilize more nutrients from liquid culture medium than that from temporary immersion. The shoot growth was limited by the availability of phosphate and nitrogen in continuous immersion system. The shoots produced in temporary immersion bioreactor showed higher photosynthetic rate, maximum quantum yield of photosystem-II and slow but steady rate of nutrient absorption, indicating the occurrence of higher photomixotrophic metabolism. The study also showed that high level of antioxidant scavenging enzymes in shoots grown in continuous immersion system induced physiological changes to foster adaptation to stresses.     

Production of biomass and flavonoid of Gynura procumbens (Lour.) Merr shoots culture in temporary immersion system

Gynura procumbens (Lour.) Merris one of medicinal plant which was carried out used as antioxidant, anticancer, anti-inflammatory, hepatoprotective, and antimicrobial. Many strategies were used to increase the production of biomass and valuable compounds. This study was to investigate the variation effect of growth regulators and immersion frequency on production of biomass and flavonoid contained of G. procumbens shoots culture in temporary immersion bioreactor. Stem nodes were used as an explants and induction of shoots were done in solid MS medium supplemented with many kinds of growth regulator. The best treatments were used to produce biomass and flavonoid compounds in temporary immersion bioreactor; there are combination of IAA 2?mg/L and BA 4, 6, 8?mg/L and immersion frequency (5 min each 3?h; 15?min each 12?h). Results showed that the growths of G. procumbens shoots in solid MS medium were influenced by supplementation of growth regulators. MS medium supplemented with single cytokinine (6 mg/L kinetin) and combination of auxin (IAA) and cytokinine (BA) caused increasing of shoots growth. Production of biomass of G. procumbens in temporary immersion bioreactor was achieved in long immersion interval (12?h) and highest flavonoid production was obtained in combination treatment of immersion frequency 15?min each 12?h and MS medium supplemented with IAA 2?mg/L, BA 8?mg/L.     

Morphogenetic evolution of hydroid colony pattern

A scheme of evolution of hydrozoan colony pattern is proposed based upon the consideration of macro-morphogenesis. Four main processes play decisive roles(1) hard skeleton formation by soft tissues, (2) changes in duration of the growth phase relative to the transition to differentiation in interdependent zones of growth, (3) ratio in growth rates between adjacent zones of growth within the rudiment, the shoot, or the whole colony, and (4) spatial relationships among growth zones. The main tendency in morphological evolution of the hydroids is an increasing integration of the colony as revealed by increasing complexity of its structure. That is from a temporary colony towards the permanent one with highly organised shoots, as hydranths and branches are localised in a strictly arranged manner. An analysis of diverse data allows one to state that the main morphogenetic mechanism of increasing complexity in the hydroid colony is convergence, then fusion, of adjacent growth zones, a variant of heterochrony.     

Kanamycin selection in temporary immersion bioreactors allows visual selection of transgenic citrus shoots

In mature citrus transformation, the nptII gene is most commonly used for selection and it is confounded by the high number of non-transformed, escaped shoots that develop on semi-solid kanamycin selection medium, even at high concentrations. Selection in liquid medium with kanamycin in temporary immersion bioreactors might provide a better means of distinguishing between transformed and non-transformed shoots. A dose-response curve was constructed for wild-type Carrizo rootstock in liquid medium to evaluate the effects of kanamycin concentration on the number and the length of microshoots. Kanamycin at 200 mg/l was chosen as the optimal concentration for selection of transgenic mature citrus shoots in bioreactors. At this dose, most non-transgenic microshoots turned yellow and their lengths and numbers were significantly reduced in comparison to the no kanamycin controls. Selection of transgenic shoots in bioreactors was tested after Agrobacterium transformations of mature Carrizo and Valencia using three different binary vectors containing nptII as the selectable marker. Shoots developed on semi-solid medium and were transferred to temporary immersion bioreactors containing liquid MS medium with 200 mg/l kanamycin. After two weeks of culture in bioreactors, 21 dark green shoots were visually selected on the basis of color from a total of 6882 microshoots, and 17 of them (81%) were confirmed as transgenic with either the GUS histochemical assay, GFP fluorescence or PCR. Yellow shoots (5675) to be discarded from pTLAB21 and pCAMBIA2301 transformations were also tested for GUS or GFP expression and only one (0.01%) was positive. Kanamycin selection of mature transgenic shoots in temporary immersion bioreactors permitted transgenics to be visually distinguished on the basis of color, and reduced labor and consumable costs for PCR screening, particularly when reporter genes were not used.     

Pineapple (Ananas comosus L. Merr) micropropagation in temporary immersion systems

A procedure for the mass propagation of pineapple plants (Ananas comosus L. Merr) using a temporary immersion technique is described. This procedure involved three distinct phases in the automated temporary immersion system: shooting, bud differentiation and elongation. To establish this protocol, we used in vitro shoots obtained from established liquid culture as starting materials. Three culture methods (solid, liquid and temporary immersion) were compared. Temporary immersion increased the multiplication rate and fresh and dry weight after 42 days. Conventional micropropagation (liquid medium) and temporary immersion were compared in combination with paclobutrazol. Paclobutrazol promoted the formation of compact bud clusters with limited leaf development. The highest multiplication rate (106) was found when ex-plants were cultured in shooting medium (MS+2.1 mg/l BA+0.3 mg/l NAA) supplemented with 1 mg/l PB for 7 weeks. A 10-l temporary immersion bioreactor was used to test two approaches during elongation stage: reduction of the shoot-formation period or decrease of the initial number of explants. The highest number of competent and uniform plants (191.8 plant/l) was achieved when shoots were cultured for 4 weeks in shooting medium supplemented with PB. Received: 4 February 1998 / Revision received: 22 June 1998 / Accepted: 14 August 1998     

Improving knowledge of plant tissue culture and media formulation by neurofuzzy logic: a practical case of data mining using apricot databases

Plant tissue growth can be regulated and controlled via culture media composition. A number of different laborious and time-consuming approaches have been used to attempt development of optimized media for a wide range of species and applications. However, plant tissue culture is a very complex task, and the identification of the influences of process factors such as mineral nutrients or plant growth regulators on a wide spectrum of growth responses cannot always well comprehended.This study employs a new approach, data mining, to uncover and integrate knowledge hidden in multiple data from plant tissue culture media formulations using apricot micropropagation databases as an example. Neurofuzzy logic technology made it possible to identify relationships among several factors (cultivars, mineral nutrients and plant growth regulators) and growth parameters (shoots number, shoots length and productivity), extracting biologically useful information from each database and combining them to create a model. The IF-THEN rule sets generated by neurofuzzy logic were completely in agreement with previous findings based on statistical analysis, but advantageously generated understandable and reusable knowledge that can be applied in future plant tissue culture media optimization.     

Effect of immersion cycles on growth,phenolics content,and antioxidant properties of Castilleja tenuiflora shoots

Castilleja tenuiflora, a species highly valued for its medicinal properties, is threatened in the wild. We evaluated the effects of six different immersion cycles in a temporary immersion bioreactor on C. tenuiflora shoot growth, proliferation rate, phenolics content, flavonoid content, and antioxidant activity. We also evaluated the regeneration capacity of the shoots. The highest proliferation rate (nine shoots per explant) was obtained using an immersion cycle of 5 min every 12 h, and the longest shoots (38.8?±?1.9 mm) were obtained using an immersion cycle of 5 min every 24 h. Shoots obtained from immersion cycles of 30 min every 24 h or 5 min every 24 h showed 100% rooting efficiency. Shoots obtained from immersion cycles of 30 min every 3 h or 30 min every 12 h accumulated H₂O₂, developed abnormal stomata, and showed symptoms of hyperhydricity. These characteristics were associated with a low survival rate (16–80%) when the plants were transferred to potting mix. The shoots from an immersion cycle of 30 min every 24 h showed the highest total phenolics content, which coincided with the highest antioxidant activity in the 2,2′-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) free-radical scavenging assay (161.74?±?10.06 μmol Trolox/g dry weight (DW)). The shoots from an immersion cycle of 5 min every 24 h showed the highest activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging assay, and those from an immersion cycle of 5 min every 3 h showed the strongest reducing power. These results show that temporary immersion culture represents a reliable and efficient method for in vitro micropropagation of C. tenuiflora.     

Reconstruction of a Lower Cretaceous conifer

Remains of Pseudofrenelopsis parceramosa (Fontaine) Watson (Cheirolepidiaceae) from the Wealden of the Isle of Wight have been examined from the point of view of the branching and mode of growth of the plant. Evidence is presented that the tree exhibited seasonal growth and that the young extension shoots bore numerous temporary ultimate branch? is of limited growth. The tree was probably adapted to warm seasonally arid conditions.     

A simple method for mass propagation of Spathiphyllum cannifolium using an airlift bioreactor

Summary A method for the micropropagation of Spathiphyllum cannifolium is presented using shoot tip proliferation onto Murashige and Skoog (MS) medium supplemented with different plant growth regulator concentrations and combinations. The proliferation responses were significantly influenced by the cytokinin type and concentrations. Supplementation of the medium with benzyladenine (BA; 4.44–13.32 μM) increased the shoot proliferation rate significantly as compared to other treatments. When cytokinins were used with auxin (indole-3-butyric acid, IBA and naphthalene acetic acid. NAA), the number of shoots per explant increased in comparison with treatments with BA alone. The largest number of shoots, 9.3 per explant, was obtained with 13.32 μM BA and 4.9 μM IBA. Different MS medium strengths and sucrose concentrations were used with the aim to stimulate in vitro shoot proliferation. Full MS medium with 30 gl⁻¹ sucrose was found to be suitable for shoot tip culture of Spathiphyllum. Comparative studies between gelled medium and bioreactor culture [continuous immersion (with or without net) and temporary immersion in liquid media using ebb and flood] revealed that shoot multiplication and growth were more efficient in continuous immersion (with net) bioreactor with low cytokinin-supplemented media. Plantlets from the bioreactor were cultured hydroponically for 30 d and 100% of plants were rooted and acelimatized successfully. Rapid and efficient multiplication rate in bioreactor and successful transfer to greenhouse makes this protocol suitable for large-scale multiplication of this important foliage plant.     

High frequency axillary bud multiplication and ex vitro rooting of Wedelia chinensis (Osbeck) Merr.--a medicinal plant

An efficient protocol was achieved for rapid propagation of Wedelia chinensis (Osbeck) Merr. through axillary bud proliferation and ex vitro rooting. Murashige and Skoog (MS) medium supplemented with benzyladenine (BA; 8.87 microM) and indole-3-butyric acid (IBA; 2.46 microM) was optimal for axillary bud proliferation, which developed a mean of 8.3 shoots/node. Excision and culture of node segments from in vitro shoots on medium supplemented with the same concentration of growth regulators developed more than 30 shoots within 40 days. Excision and culture of nodes in succession enhanced the number of shoots. Shoot multiplication did not exhibit decrease in the number of shoots even at 10th subculture. Nevertheless, the shoots exhibited a tendency towards stunted nature. But reduction of BA to 4.44 or 2.22 microM resumed normal growth of shoots. Half strength MS medium fortified with IBA (2.46 microM) induced the highest number of roots. All in vitro rooted shoots survived in field. Dipping of the basal end of shoots collected from multiplication medium in IBA (2.46 microM) solution for 7 days induced roots and its transfer to small pots facilitated the survival of all rooted shoots (100%). Rooting ex vitro by direct transfer of shoots from multiplication medium exhibited 89.2 per cent survival. Use of commercial sugar and tap water and also the omission of in vitro rooting reduce the propagation cost 50-70 per cent. The protocol enables to harvest more than 50,000 plantlets within 150 days starting from a single node explant.     

Changes in Enzyme Regulation during Growth of Maize: I. Progressive Desensitization of Homoserine Dehydrogenase during Seedling Growth

The sensitivity of homoserine dehydrogenase (EC 1.1.1.3) to inhibition by the feed-back modifier, l-threonine, was examined in preparations derived from etiolated shoots, roots, and lightgrown tissues of Zea mays L. var. earliking. A progressive decrease in enzyme sensitivity was observed during seedling growth. Enzyme derived from internode tissue retained a greater sensitivity to the effector than enzyme derived from apical portions of etiolated shoots, whereas enzyme from root tips was characteristically more sensitive than that prepared from mature cells of the root. Enzyme desensitization occurred rapidly during culture of excised shoots and the activities of both homoserine dehydrogenase and aspartokinase (EC 2.7.2.4) declined during shoot culture under a variety of conditions. The initial enzyme levels and the characteristic sensitivity of homoserine dehydrogenase were preserved during culture at 5 to 7 C, but desensitization was not prevented by inclusion of cycloheximide in the culture medium.Results of control experiments provide evidence that desensitization occurs in vivo. No alteration of the enzyme properties was detected during extraction or concentration of sensitive or insensitive enzyme or during coextraction of enzyme from mixed populations of different age shoots; nor was a differential distribution of inhibitors or activators indicated during assay of mixed preparations. The change in enzyme sensitivity was apparent under a variety of assay conditions and was not accompanied by changes in the apparent affinity of the enzyme for the substrate, homoserine. It is suggested that systematic changes in the regulatory characteristics of certain enzymes could be an important level of metabolic regulation during cellular differentiation.Three forms of maize homoserine dehydrogenaase were detected after acrylamide gel electrophoresis of samples derived from 72-hr shoots. Similar analysis of samples from older shoots revealed a broad asymmetric band of enzyme activity, suggesting that changes in the relative distribution of specific forms of the enzyme could be related to the growth-dependent changes in the sensitivity of maize homoserine dehydrogenase.     

Flow cytometry of the differentiation of Physarum polycephalum myxamoebae to cysts

Myxamoebae of Physarum polycephalum, strain Cld, were grown on agar lawns on live bacteria. Myxamoebae were harvested, fixed and stained with propidium iodide. Flow cytometry showed that, as in the case of Physarum plasmodia, there is no G1 phase during rapid exponential growth. However, an apparent G1 phase was observed at the end of exponential growth when the culture arrested with the G1 DNA content for about a day between growth and differentiation. Most myxamoebae differentiated into cysts, but some formed microplasmodia and others appeared to lose DNA. The cysts possessed the G2 phase DNA content and there was an S phase connecting the G1-arrested state with the encysted state. Encystment was blocked by hydroxyurea (HU) suggesting that DNA synthesis is essential for encystment. The natural temporary synchronization in G1 phase may provide the basis of a method for selecting mutants with a conditional block in G2 or M phases.     

Axillary shoot proliferation and growth of Populus deltoides shoot cultures

Shoot cultures of four genotypes of Populus deltoides Bartr. ex Marsh. were established from adventitious shoots regenerated from internodal stem explants. Stable shoot cultures for all four genotypes were maintained in a continuous culture regime for over one year. The stable shoot cultures were used as explants to investigate the effects of zeatin concentration and genotype on axillary shoot production and growth. The concentration of zeatin significantly affected the production of axillary shoots, with 1.0 mgL^–1 zeatin producing the greatest number of shoots (31.0 shoots per culture vessel) while 0.25 mgL^–1 zeatin produced the greatest growth (5.9 mg per axillary shoot) when measured by dry weight accumulation per shoot. Genotypic differences were significant in the production and growth of axillary shoots.Abbreviations DKW Driver and Kuniyuki Walnut medium - PAR Photosynthetically Active Radiation Journal Series No. 9111, Agricultural Research Division, University of Nebraska     

Temporary immersion systems for Amaryllidaceae alkaloids biosynthesis by Pancratium maritimum L. shoot culture

The alkaloid patterns of sea daffodil (Pancratium maritimum L.) shoot culture, cultivated in a temporary immersion cultivation system were investigated. The shoots accumulated maximal amounts of biomass (0.8 g dry biomass/L and Growth Index?=?1.6) at immersion frequency with 15 min flooding and 12 h stand-by periods. At this regime P. maritimum shoots achieved the highest degree of utilization of carbon source. Twenty-two alkaloids, belonging to narciclasine, galanthamine, haemanthamine, lycorine, montanine, tazettine, homolycorine and tyramine types were identified in intracellular and extracellular alkaloid extracts. The immersion frequency affected strongly the capacity of alkaloid biosynthesis in P. maritimum shoots and at the optimum conditions of cultivation, the total intracellular alkaloid content reached up to 3,469 μg/g dry biomass. The main biosynthesized alkaloids were haemanthamine (900.1 μg/g) and lycorine (799.9 μg/g). The obtained results proved that temporary immersion technology, as a cultivation approach, and P. maritimum shoots, as a biological system, are prospective for producing wide range bioactive alkaloids.     

Characterization of the antioxidant system during the vegetative development of pea plants

The antioxidative system was studied during the development of pea plants. The reduced glutathione (GSH) content was higher in shoots than in roots, but a greater redox state of glutathione existed in roots compared with shoots, at least after 7 d of growth. The 3-d-old seedlings showed the highest content of oxidised ascorbate (DHA), which correlated with the ascorbate oxidase (AAO) activity. Also, the roots exhibited higher DHA content than shoots, correlated with their higher AAO activity. The activities of antioxidant enzymes were much higher in shoots than in roots. Ascorbate peroxidase (APX) activity decreased during the progression of growth in both shoots and roots, whereas peroxidase (POX) activity strongly increased in roots, reflecting a correlation between POX activity and the enhancement of growth. Catalase activity from shoots reached values nearly 3 or 4-fold higher than in roots. The monodehydroascorbate reductase (MDHAR) activity was higher in young seedlings than in more mature tissues, and in roots a decrease in MDHAR was noticed at the 11^th day. No dehydroascorbate reductase (DHAR) was detected in roots from the pea plants and DHAR values detected in seedlings and in shoots were much lower than those of MDHAR. In shoots, GR decreased with the progression of growth, whereas in roots an increase was seen on the 9^th and 11^th days. Finally, superoxide dismutase (SOD) activity increased in shoots during the progression of growth, but specific SOD activity was higher in roots than in shoots.     

蓝莓离体叶片胚状体高效发生及其组织学观察

以高灌蓝莓试管苗叶片为外植体、以改良WPM为基本培养基,研究了外源激素TDZ、ZT及其组合对离体叶片胚状体发生的影响,同时也探讨了蔗糖浓度、水解酪蛋白、椰汁等对胚状体发生、丛芽形成的影响.结果表明:不同浓度的外源激素TDZ、ZT及其组合对胚状体的发生频率、丛芽的形成和生长起重要作用;蔗糖浓度对胚状体的发生及丛芽的生长影响较大,而添加有机质则对胚状体的发生及丛芽的生长没有明显影响.适合高灌蓝莓叶片胚状体发生及成苗的培养基为WPM TDZ 0.04 mg/L ZT 0.25～2.0 mg/L 蔗糖20～40 g/L,而培养基WPM ZT 0.5～1.0 mg/L 蔗糖20 g/L适合于丛芽继代生长.组织学观察表明,蓝莓叶片胚状体发生主要起源于叶上表皮细胞和部分叶肉细胞,可能为多细胞起源,历经多细胞原胚、原球胚、梨形胚、心形胚、子叶胚等发育阶段,并能直接发育成苗.     

Suppression of the Tumorous State in Crown Gall Teratomas of Tobacco A Clonal Analysis

Two types of experimental approaches designed to test the possible involvement of chimeras in the persistent but readily reversible suppression of the tumorous state and in a recovery from that state in crown gall teratomas of tobacco were investigated. These studies showed that the tumorous state was suppressed in specialized cells present in the different tissues derived from each of the three layers of the apical meristem of teratoma-derived shoots of clonal origin. Although differences existed in the exogenous requirements needed for the reestablishment of the tumorous state in the different specialized cell types present in the different tissues, all again acquired a capacity for autonomous growth when appropriately stimulated. No evidence was found for the existence of periclinal or sectoral chimeras in teratoma-derived shoots of a single cell origin. Chimerism was, however, found to exist in one noncloned teratoma-derived shoot. Analysis of more than 500 clones derived from leaf mesophyll cells of cloned teratoma shoots showed that the vast majority of them (> 99%) were tumorous, while all of the 144 seed-grown plants from these shoots had completely recovered from the tumorous state. The results of these studies suggest that (a) a teratoma shoot does not depend for its development on the presence of significant numbers, if any, of cells that had recovered from the tumorous state; (b) the tumorous state may be suppressed in even the most highly specialized cells; (c) some special mechanism exists in the meiotic process for the elimination of all or most of the foreign DNA from transformed cells.     

Effects of temperature,photoperiod and culture vessel size on adventitious shoot production of in vitro propagated <Emphasis Type="Italic">Huernia hystrix</Emphasis>

High production costs due to low growth rate in vitro and high labour costs are among factors limiting commercial application of micropropagation techniques. The low growth rate could be due to unfavourable or sub-optimal environmental and chemical conditions of the cultures. The effects of temperature, photoperiod and culture vessel size were investigated on adventitious shoot production of Huernia hystrix. There were significant increases in shoot proliferation with increased temperature in cultures maintained under a 16 h photoperiod. Slow growth observed at low temperatures (15 and 20°C) offers a potential strategy for cost-effective in vitro storage of H. hystrix germplasm. The maximum adventitious shoots produced per explant and percentage of explants producing shoots (4.2 ± 0.74 and 94% respectively) were observed in cultures maintained at 35°C, the optimum temperature for photosynthesis in plants possessing crassulacean acid metabolism (CAM). The nocturnal accumulation of organic acids in cultures incubated under a 16 h photoperiod further suggest the presence of CAM in this species. On the other hand, cultures kept under continuous light appear to shift to a C-3 photosynthetic pathway. There was a significant decrease in fresh weight of adventitious shoots regenerated per explant as temperature increased. The use of larger culture vessels further increased the shoot proliferation to 5.6 shoots per explant with a potential production of 3,429 shoots per m² in the growth room compared to 2,750 shoots per m² using culture tubes.     

In vitro propagation of Dendrobium hybrids using flower stalk node explants

Large-scale in vitro propagation protocol for Dendrobium hybrids Sonia 17 and 28, two highly prized commercial cut flower cultivars through shoot multiplication using flower stalk node explants and protocorm-like bodies (PLBs) formation was accomplished. Both hybrids did not exhibit significant differences in initiation, multiplication, rooting, and field establishment. Flower stalk nodes cultured on half strength Murashige and Skoog (MS) medium supplemented with 6.97 microM kinetin (Kn), or 15% coconut water (CW) or 13.3 microM of N6-benzyladenine (BA) evoked bud break. Kn showed better growth of the initiated bud. Excision and culture of the initiated shoots on medium having same amount of Kn developed more than 5 shoots per shoot directly from the base. Subsequent culture enhanced the rate of shoot induction. Transfer of isolated shoots onto 44.4 microM of BA enriched medium displayed induction of more than 6 PLBs from the base within 60 days. PLBs underwent rapid multiplication upon transferral to medium having the same concentration of BA (44.4 microM). Subsequent culture increased the proliferation of PLBs. No decline was observed in the proliferation of shoots as well as PLBs up to 15th subculture. PLBs transferred onto half strength MS medium with 6.97 microM of Kn underwent conversion of more than 90% PLBs to shoots. The shoots were rooted at the best on half strength MS medium with 2 g l(-1) activated charcoal. Survival rate of the plantlets of the two hybrid cultivars after acclimatization was more than 80%.     

文心兰原球茎液体增殖培养研究

以茎尖诱导形成的原球茎(protocorm-like bodies,PLBs)为外殖体,采用液体培养方式比较了不同浓度的激素配比、蔗糖浓度和添加不同量的新鲜椰汁对文心兰PLBs增殖的影响,并比较了相同培养基成分时液体培养PLBs增殖、分化成苗和固体培养PLBs增殖和分化成苗的差异。试验结果表明:不同浓度的外源激素及其配比对文心兰PLBs增殖生长影响较大,6-BA0.5 mg/L Ad 0.05 mg/L NAA0.05 mg/L的激素组合比较适合文心兰PLBs增殖;蔗糖浓度对文心兰PLBs增殖的影响也较大,适合文心兰PLB在液体培养条件下增殖的蔗糖浓度为7.5 g/L;添加5%新鲜椰汁不仅对文心兰PLBs增殖有促进作用,而且能改善PLBs的质量;适合文心兰PLBs增殖的培养基为MS 6-BA0.5 mg/L Ad 0.05 mg/L NAA0.05 mg/L 5%椰汁 蔗糖7.5 g/L。文心兰PLBs在5周内的增殖生长曲线呈倒"V"字形,第3周的增殖速度达最高峰,而固体培养基PLBs增殖速度较慢,生长曲线几乎成直线。液体增殖的PLBs分化成苗较固体培养增殖的PLBs差。