Transgenic tea [Camellia sinensis (L.) O. Kuntze cv. Kangra Jat] plants obtained by Agrobacterium-mediated transformation of somatic embryos

A protocol for the production of transgenic tea [Camellia sinensis (L.) O. Kuntze cv. Kangra Jat] was developed via Agrobacterium-mediated genetic transformation of somatic embryos. Two disarmed Agrobacterium tumefaciens strains, EHA 105 and LBA 4404, both carrying the binary plasmid p35SGUSINT with the nptII gene and gus-intron were evaluated as vector systems. A number of parameters were tested with respect to maximizing transformation efficiency. While pre-culture, wounding and acetosyringone treatment were inhibitory, the bacterial growth phase (optical density; OD₆₀₀ = 0.6), cell density (10⁹/ml), co-cultivation period (5 days) and pH of the co-cultivation medium (5.6) had positive effects on transformation. Following co-cultivation, globular somatic embryos were placed on multiplication medium and stressed with kanamycin (50 µg/ml). Further selection occurred in the maturation and germination medium at an elevated kanamycin level (75 µg/ml). An average of 40% transient expression was evident based on the GUS histochemical assay. Kanamycin-resistant, GUS-positive embryos were germinated, and the resulting microshoots were multiplied in vitro. Integration of the transgenes into the tea nuclear genome was confirmed by PCR analysis using nptII- and gus-specific primers and by Southern hybridization using an nptII-specific probe. The transgenic shoots were micrografted onto seed-grown rootstocks of cv. Kangra Jat and eventually hardened in a walk-in polyhouse. This is the first report on the production of transgenic tea.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated production of transgenic plants of<Emphasis Type="Italic"> Muscari armeniacum</Emphasis> Leichtl. ex Bak.

A system for the production of transgenic plants has been developed for the Liliaceous ornamental plant Muscari armeniacum Leichtl. ex Bak via Agrobacterium-mediated transformation of embryogenic cultures. Leaf-derived embryogenic cultures were co-cultivated with each of three A. tumefaciens strains, all of which harbored the binary vector carrying the neomycin phosphotransferase II (nptII), hygromycin phosphotransferase (hpt) and intron-containing #-glucuronidase (gus-intron) genes in the T-DNA region. Following co-cultivation, the embryogenic cultures were cultured on a medium containing 500 mg l^-1 cefotaxime for 1 week followed by a medium containing 75 mg l^-1 hygromycin in addition to cefotaxime. After 4-5 weeks, several hygromycin-resistant (Hyg^r) cell clusters were produced from the co-cultivated embryogenic cultures. The highest efficiency of production of Hyg^r cell clusters was obtained when embryogenic cultures were inoculated with A. tumefaciens EHA101/pIG121Hm in the presence of 100 µM acetosyringone (AS) and 0.1% (v/v) of a surfactant (Tween20) followed by co-cultivation in the presence of 100 µM AS. Hyg^r embryogenic cultures developed into complete plants via somatic embryogenesis, and most of them were verified to be transgenic by GUS histochemical assay and polymerase chain reaction analysis. Southern blot analysis revealed the integration of one to five copies of the transgene into the genome of transgenic plants, but most of them had one or two copies.     

Effect of NaCl Salinity on Somatic Embryo Development in Sapindus trifoliatus L.

The effect of NaCl salinity on growth and development of somaticembryos of Sapindus trifoliatus L. was examined. Incorporationof 25 and 50 mol m^–3 NaCl into the medium greatly increasedthe growth and development of somatic embryos and both theseconcentrations favoured the production of secondary embryoids.However, supplementation of 100 mol m^–3 NaCl to the mediumdid not have any significant effect on the growth and developmentof somatic embryos. On the other hand, the culturing of proembryostructures in medium containing 200 mol m^–3 NaCl resultedin complete death within 7 d of salt exposure. Analysis of somatic embryos revealed that, upon salinization,they accumulated Na⁺ and Cl^– in significant amounts butthe content of Na⁺ was much less compared to that of Cl^–.Addition of NaCl (up to 50 mol m^–3) in the medium resultedin a considerable increase in the K⁺ content of somatic embryos.The content of proline in somatic embryos, however, increasedsubstantially in response to salinization. The amount of freesterols, steryl glycosides, steryl esters, and phospholipidsalso rose to higher values in salt-affected somatic embryos.The results suggest that somatic embryos of S. trifoliatus cantolerate concentrations of NaCl up to 100 mol m^–3 withoutaffecting growth and that they have sufficient cellular mechanismsto tolerate salinity at relatively high levels. Key words: Salinity, somatic embryo, sterols, phospholipids     

Induction of high-frequency somatic embryos in cassava for cryopreservation

Methods for inducing high-frequency somatic embryos in cassava on cotyledons and 33 clonal accessions by the addition of supplementary copper sulphate to the induction medium were investigated. The addition of copper sulphate enhanced primary embryo induction and significantly increased secondary embryo production. All accessions from Latin America (CIAT) were embryogenically competent on medium supplemented with 8 mg l^-1 2,4-dichlorophenoxyacetic acid (2,4-D) plus 1 µM copper sulphate as were 15 of the 18 accessions from Africa. The percentage of calli producing somatic embryos ranged from 7.5% in M. Bra 12 to 100% in M. Col. 1505, while the number of embryos produced per callus ranged from 0.3 in M. Bra 383 to 13.5 in TEK. The frequency of embryo production was dependent on the concentration of copper sulphate. The number of primary embryos produced per callus was also comparatively higher in the medium supplemented with copper sulphate than in the controls. The optimal concentration of copper sulphate for number of embryos produced in most accessions was 5 µM, and at this concentration the number of embryos produced was double that of the controls. Copper sulphate also reduced the maturation time of somatic embryos to 25 days from embryo initiation. High levels of 2,4-D were detrimental to embryo production. Similarly, fragmented embryos incubated in the dark produced more embryos tan those incubated under light conditions. On the basis of these results, the use of cassava somatic embryo micropropagules for germplasm conservation and synthetic seed development seems to be a strong possibility.     

An efficient leaf-disc culture method for the regeneration via somatic embryogenesis and transformation of grape (Vitis vinifera L.)

By manipulating hormone levels, light intensities and temperature, we have developed an efficient leaf-disc method for the regeneration of plants via embryogenesis and for transformation in four genotypes of Vitis vinifera L. In MS basal medium supplemented with 1 mg l^-1 6-benzylaminopurine (BAP) and 0.1 mg l^-1 2,4-dichlorophenoxyacetic acid, leaf discs cultured for 2 weeks under dark conditions produced calli in over 80% of the cultures. These subsequently differentiated into pro-embryos and embryos only if kept under conditions of low light intensity (15 µE m^-2 s^-1) for 2 weeks before being transferred to conditions of high light intensity (60 µE m^-2 s^-1). If the calli were directly transferred to high light intensity, the differentiation into embryos was blocked and the calli turned pink. The somatic embryos germinated at a frequency of about 10% on NN basal medium and about 32% on NN medium supplemented with 1 mg l^-1BAP and 0.1 mg l^-1 indole-3-butyric acid. The embryos, however, germinated when pre-exposed to a low temperature of 4°C for 2 weeks. If they were transferred directly to room temperature under conditions of high light intensity (60 µE m^-2 s^-1), shoot buds were produced, whereas under conditions of low light intensity (15 µE m^-2 s^-1) secondary embryogenesis was induced. About 90-95% of the in vitro grown plantlets could be successfully transferred to soil. The above method was also applicable for developing transgenic embryos whose transgenic nature was monitored using #-glucuronidase as a reporter gene.     

Optimized fed-batch fermentation of L-β-hydroxy isobutyric acid by Yarrowia lipolytica

Yarrowia lipolytica KCCM50506, which transforms isobutyric acid to L-#-hydroxy isobutyric acid (L-#-HIBA), was screened. Chemostat cultures were carried out in jar fermentors at dilution rates of 0.02 h^у to 0.12 h^у. L-#-HIBA fermentation-regulating factors were determined to be specific growth rate, and concentrations of glucose and isobutyric acid in fermentor from analysis of steady-state data. The specific productivity of L-#-HIBA increased as the specific growth rate increased, apparently as a growth-associated type of product formation. A fed-batch culture was carried out under optimum conditions where the concentrations of glucose and isobutyric acid in the fermentor were maintained at 23 g l^у and 9 g l^у, respectively. The concentrations of cells and L-#-HIBA obtained at the end of fermentation were 20 g l^у and 49 g l^у, respectively, corresponding to 2.0 and 2.7 times more than concentrations in batch culture.     

Inhibition of ethylene production and stimulation of carrot somatic embryogenesis by salicylic acid

The effects of salicylic acid (SA) and other phenolic compounds, acetylsalicylic acid (ASA), benzoic add (BA) and sulfosalicylic acid (SSA), on ethylene production and somatic embryogenesis by carrot (Daucus carota L.) cell cultures were studied. SA and ASA, at concentrations of 10 μM and 100 μM, significantly stimulated somatic embryogenesis and effectively inhibited ethylene production by carrot cell suspension cultures. The observed increase of embryo number was proportional to the inhibition rate of ethylene production. However, BA and SSA affected neither ethylene production nor somatic embryogenesis. The role of SA in somatic embryogenesis is discussed.     

Effect of Corn steep liquor supplementation and scale up on Lactococcus starter production

Summary Three Lactococcus strains (Lactococcus ssp. lactis var. diacetylactis, Lactococcus ssp. lactis cremoris and Lactococcus ssp. lactis var. lactis) isolated from the Tunisian lben were grown at constant pH on CSL medium in stirred fermentors for lactic starters production. The agitation required to homogenate alkali used to pH control should be low because it affects the Lactococcus growth. Scale up from 20-liter fermentor to 400-liter fermentor was carried out at constant impeller tip speed below 150 cm s^у. The CSL supplementation and fed-batch with glucose increased the yield in the upper 10¹⁰ cfu/ml. The consumed glucose during fermentation was converted into lactic acid and cell. Before fed-batch, the maximum specific growth rate of Lactococcus ssp. lactis var. diacetylactis was around 1 h^у and the number of cells increased 20 to 40 times according to inoculum size. After fed-batch, the glucose consumption rate remains constant but specific growth rate decreased and number of cell trebled only.     

Induction of multiple shoots by thidiazuron from caryopsis cultures of minor millet (Paspalum scrobiculatum L.) and its effect on the regeneration of embryogenic callus cultures

Caryopsis culture of a minor millet (Paspalum scrobiculatum L. cv. PSC 1) on N₆ medium supplemented with high concentrations of thidiazuron (TDZ, 11.25 µM and 22.5 µM), a phenylurea derivative known to simulate cytokinin action, resulted in the formation of multiple shoots from the base of the seedling. This is the first time that multiple-shoot formation by a seedling cultured on TDZ without a callus interphase has been reported in graminaceous crop plants. The presence of a cytokinin, 6-benzylaminopurine (BAP), at low or high concentrations failed to evoke any morphogenic response. The presence of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D, 4.5 µM) either alone or with BAP (4.5 µM) resulted in the formation of embryogenic callus from the base of the seedlings, which subsequently differentiated into somatic embryos. The combination of TDZ and the auxin (4.5 µM, 2,4-D) in the medium stimulated the differentiation of shoot buds in embryogenic callus cultures. This effect of TDZ, noted for the first time in a monocotyledonous plant, was evident in terms of a significant increase in the frequency of shoot-bud formation in embryogenic callus cultures and occurred only at a high concentration of TDZ (11.25 µM). This requirement for a high concentration of TDZ for the induction of multiple shoots from cultured seedlings or shoot buds in an embryogenic callus culture of a monocot is contrary to its effect at low concentrations in dicotyledonous plants. Complete plantlets, derived either from somatic embryos or shoot buds, could be regenerated on hormone-free basal medium or on basal medium fortified with activated charcoal (0.5%). Following a gradual acclimatization in a culture room, these regenerants survived on transfer to soil and ultimately set seed.     

Enhanced production and germination of somatic embryos by temporary starvation in tissue cultures of Daucus carota

Embryogenic callus was induced from cotyledonary explants of Daucus carota L. cultured on solidified MS medium supplemented with 1 mg l^-1 2,4-D. Following callus initiation somatic embryos were developed from the callus on MS medium without 2,4-dichlorophenoxyacetic acid. To stimulate the production and germination of somatic embryos we cultured the callus under physically and chemically modified conditions during subculture. When the embryogenic callus was cultured on half-strength MS medium or MS medium without sucrose or cultured under conditions of reduced humidity (69.3%), the production of embryos increased 3.4- to 4.5-fold compared to culture on MS medium containing 3% sucrose (control). Embryogenic callus cultured on MS medium after 5 days of starvation (by being placed in empty 12-well tissue culture plates) showed a 20-fold increase in somatic embryo production and enhanced maturation and germination of embryos. An important point is that the germination of somatic embryos with cup-shaped cotyledons, after a period in culture without medium, was remarkably improved (92%) compared to that of the controls (23%).Thus, we were able to show that stress by starvation without medium led to the enhanced production and increased germination of somatic embryos.     

Plant regeneration from protoplasts of dessert banana cv. Grande Naine (Musa spp., Cavendish sub-group AAA) via somatic embryogenesis

Protoplast culture and plant regeneration of the dessert banana cultivar Grande Naine (Musa spp., Cavendish sub-group AAA) were achieved through somatic embryogenesis. Protoplasts were isolated from cell suspensions at a yield of 3᎒⁷ protoplasts/ml packed cell volume (0.5 g). For the induction of cell divisions, two banana cell suspensions, SF265 (AA) and IRFA903 (AA), were used as feeder layers. SF265 (AA) was found to be more efficient for inducing cell divisions than IRFA903 (AA). The first embryogenic cell suspensions were established from protoplast-derived microcalli. The transfer of microcalli and protoplast-derived cell suspensions onto regeneration medium containing plant growth regulators slightly increased the number of embryos relative to those maintained on a feeder layer with growth regulators. Plant regeneration was achieved in the same regeneration medium.     

Influence of xylem development on axial hydraulic conductance within Prunus root systems

The effect of secondary growth on the distribution of the axial hydraulic conductance within the Prunus root system was investigated. Secondary growth resulted in a large increase in both the number (from about 10 to several thousand) and diameter of xylem vessels (from a few micrometres to nearly 150 µm). For fine roots (<3 mm), an increase in root diameter was correlated with a slight increase in the number of xylem vessels and a large increase in their diameter. Conversely, for woody roots, an increase in root diameter was associated with a dramatic increase in the number of xylem vessels, but little or no change in vessel diameter. The theoretical axial conductivity (Kh, m⁴.s^-1.MPa^-1) of root segments was calculated with the Poiseuille-Hagen equation from measurements of vessel diameter. Kh measured using the tension-induced technique varies over several orders of magnitude (7.4᎒^-11 to 5.7᎒^-7 m⁴.s^-1.MPa^-1) and shows large discrepancies with theoretical calculated Kh. We concluded that root diameter is a pertinent and useful parameter to predict the axial conductance of a given root, provided the root type is known. Indeed, the relationship between measured Kh and root diameter varies according to the root type (fine or woody), due to differences in the xylem produced by secondary growth. Finally, we show how the combination of branching pattern and axial conductance may limit water flow through root systems. For Prunus, the main roots do not appear to limit water transfer; the axial conductance of the main axes is at least 10% higher than the sum of the axial conductance of the branches.     

Modeling of growth and energy metabolism of Pichia pastoris producing a fusion protein

A fusion protein composed of a cellulose binding domain from Neocallimastix patriciarum cellulase A and Candida antarctica lipase B (CBD-lipase) was produced by Pichia pastoris methanol utilization plus phenotype in high cell-density cultures. The genes expressing CBD-lipase were fused to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. To control the repression and induction of AOX1 and oxygen demand at high cell density, a four-stage process was used. Batch growth on glycerol was used in the first step to provide biomass (28 g L^-1) while product formation was prevented due to repression of the AOX1. The second stage was exponential fed-batch growth on glycerol, which caused a slight increase of the enzyme alcohol oxidase activity due to derepression of the AOX1. This procedure resulted in smooth transition to exponential fed-batch growth on methanol, the third stage, in which the AOX1 was strongly induced. The fourth stage was constant fed-batch growth on methanol used to control the oxygen demand at the high cell density. A kinetic model was developed that could predict biomass growth and oxygen consumption in processes with and without oxygen-enriched air. With oxygen enrichment to 34% O₂ in the inlet air the methanol feed rate could be increased by 50% and this resulted in 14% higher final cell density (from 140 to 160 g L^-1 cell dry weight). The increased methanol feed rate resulted in a proportionally increased specific rate of product secretion to the medium. After an initial decrease, the synthesis capacity of the cell was kept constant throughout the cultivation, which made the product concentration increase almost constantly during the process. The kinetic model also describes how the low maintenance demand of P. pastoris compared with E. coli enables this organism to grow to such high cell densities.     

Growth regulator pretreatment improves somatic embryogenesis from leaves of squash (Cucurbita pepo l.) and melon (Cucumis melo l.)

Leaf explants of squash (Cucurbita pepo L.) and melon (Cucumis melo L.) were pretreated initially with 113.1, 226.2 or 452.4 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 46.5, 93 or 186 µM kinetin or a combination of both at the above concentrations, for 6, 24 or 48 h. After pretreatment, explants were transferred to an agar-solidified medium that was not supplemented with growth regulators or to a species-specific standard induction medium. Control explants from each species were incubated directly on the species-specific standard induction medium. Initial pretreatment of squash explants with 186 µM kinetin and of melon explants with 226.2 µM 2,4-D for 48 h significantly promoted the formation of somatic embryos which developed further to the torpedo-shape stage and germinated. Under these conditions at least four plants can be regenerated per square centimeter of explant surface, thus achieving an increase over non-pretreated cultures of 143% and 130% for squash and melon, respectively.     

Methyl laurate and 6-benzyladenine promote the germination of somatic embryos of a hybrid rose

Globular stage somatic embryos were induced in callus cultures of Rosa Heritage 2 Alister Stella Gray on medium containing 13.5 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and developed to the cotyledonary stage on medium containing 9 µM 2,4-D. Cotyledonary-stage embryos were transferred to germination media with or without 1.5 µM 6-benzyladenine (BA) and with or without 44 µM methyl laurate (Mela). BA and Mela both promoted the development of shoots and roots and increased the frequency of bipolar germinations. An average of 56.5% (SEdž.1%) embryos on medium containing both BA and Mela underwent bipolar germinations compared with less than 20% in treatments where either or both were excluded. The effectiveness of BA and Mela was reduced if Mela was included in the development medium or if the concentration of salts and vitamins in the germination media was sub-optimal. There was evidence that growth at one pole of the somatic embryo promoted development at the other.     

花楸合子胚诱导体细胞胚胎发生研究

分别以完整成熟胚、切去一个子叶的成熟胚和切下的子叶为外植体,以MS为基本诱导培养基、1/2MS为基本分化培养基,进行了花楸体细胞胚胎发生研究。结果表明:以完整合子胚作为外植体的体胚诱导率最高,为100%,最佳植物生长调节剂组合为5 mg.L-1NAA+2 mg.L-16-BA;NAA和6-BA浓度及二者的交互作用对愈伤组织和体胚诱导率的影响极显著;光照配合延长继代间隔时间有利于体胚发生。实体观察结果表明,花楸体胚发生方式有直接发生和间接发生两种;体胚发育经历了球形期、心形期、鱼雷形期和子叶期。组织学观察结果表明,体胚具有两极性,子叶期体胚结构完整。     

Stimulation ofDaucus carota somatic embryogenesis by inhibitors of ethylene synthesis: cobalt and nickel

The effects of Co²⁺ and Ni²⁺ on ethylene production and somatic embryogenesis by carrot (Daucus carota L.) cell cultures were studied. At concentrations of 10 M to 50 M, CoCl₂ effectively inhibited ethylene production by embryogenic cultures and significantly stimulated somatic embryogenesis. The observed increase of embryo number was proportional to the inhibition level of ethylene production. However, CoCl₂ had no effect when Ethephon was supplied. Nickel also reduced ethylene production, but to a slightly lesser extent than CoCl₂, bringing about a lower increase in the number of somatic embryos. The role of ethylene on somatic embryogenesis is discussed.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - SAM S-adenosyl-methionine     

Shear stress effects on cell growth and <Emphasis Type="SmallCaps">L</Emphasis>-DOPA production by suspension culture of <Emphasis Type="Italic">Stizolobium hassjoo</Emphasis> cells in an agitated bioreactor

Suspension cultures of Stizolobium hassjoo cells were cultivated in a 7l bioreactor. The growth rate and intracellular L-DOPA content of the cells using two different turbine impellers were compared. There were distinct differences in growth behavior and L-DOPA productivity in the range of 100 to 500 rpm for flat-blade turbine impeller. Disk turbine retarded significantly the cell growth but not so significantly for L-DOPA production in the range of 200 to 300 rpm. The shear force intensity of the two impellers at various rotational rates was compared with shear force index (SFI), and power input per unit mass and eddy length scale. There was good consistency among the three indexes for shear force intensity. Thus with SFI the shear force intensity of bioreactor can be indirectly estimated. A critical shear stress that may cause sublytic effect in cells was identified for flat-blade turbine operated at 400 rpm. The common effect between the shear stress and the proton elicitation in the bioreactor was elucidated with a hypothesis of signal transduction by second messenger, H⁺. Our results suggested that H⁺ transduced the signal to protoplast when S. hassjoo cells were stimulated by shear stress. This resulted in an increase of H⁺ which triggered a similar reaction to the pH control of culture broth and enhanced the L-DOPA production.     

The effect of oxygen partial pressure in bioreactors on cell proliferation and subsequent differentiation of somatic embryos of Cyclamen persicum

The effect of the relative oxygen partial pressure (pO₂) in bioreactors on cell proliferation and subsequent differentiation of somatic embryos from suspension cultures of Cyclamen persicum Mill. was investigated. The growth rate of cell line 3738-VIII in growth-regulator containing medium in bioreactors at 5% pO₂ was slightly reduced in comparison to 10% and 20% pO₂. Cultures growing at 40% pO₂ had a lower growth rate, a markedly reduced cell viability and showed a decrease of the medium pH to 3.5. Because a pH-control with a setpoint of 3.3 caused cell death within 4 days, it was assumed, that the reason for the poor cell proliferation and viability in the cultures at 40% pO₂ was an effect of medium acidification rather than of the high O₂ partial pressure. A significantly higher number of germinating embryos was obtained from the cultures grown at 40% pO₂ than from those grown in flasks or in bioreactors at 5%, 10% and 20% pO₂. These results were specific for cell line 3738-VIII. Another cell line, 3736-12, did not show marked differences in cell proliferation, viability, pH or subsequent regeneration of somatic embryos when grown at different O₂ partial pressures. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effects of cambial age and position within the stem on specific conductivity in Douglas-fir (Pseudotsuga menziesii) sapwood

Specific conductivity (k_s, m²s^-1MPa^-1) describes the permeability of xylem and is determined by all aspects of xylem anatomy that create resistance to the flow of water. Here we test the hypothesis that k_s is a function of radial and vertical position within the stem, rather than solely a function of cambial age (ring number from the pith), by measuring k_s on samples excised from 35-year-old Douglas-fir [Pseudotsuga menziesii var. menziesii (Mirb.) Franco] trees at six heights and two or three radial positions. Sapwood k_s decreased from the cambium to the heartwood boundary, and the difference between outer and inner sapwood increased with height in the tree. Beneath the live crown, inner sapwood had 80-90% the k_s of outer sapwood, but only 55% just 10 m higher in the stem (about 10 nodes down from the tree top). Outer sapwood k_s peaked near the base of the crown and declined toward both the base and top of the stem. These patterns can be explained by two superimposed effects: the effect of cambial age on the dimensions of tracheids as they are produced, and the effect of xylem aging, which may include accumulation of emboli and aspiration of bordered pits. Tracheid lumen diameter and earlywood and latewood density and width, all factors known to vary with cambial age, were measured on different trees of the same age and from the same stand. Lumen diameter increased with cambial age, whereas the proportion of latewood and growth ring density increased after an initial decrease in the first 5 years. Our results suggest that the effect of cambial age on xylem anatomy is not sufficient to explain variation in k_s. Instead, physical position (both vertical and radial) in the stem and cambial age must be considered as determinants of conductivity.