Somatic Embryogenesis and Plant Regeneration from Suspension Cultures of Pearl Millet (Pennisetum americanum)

Immature embryos of Pennisetum americanum (pearl millet), culturedin the presence of 2,4-dichlorophenoxy acetic acid (2,4-D) produceda pale-yellow and compact callus tissue by proliferation ofthe scutellum. Teased pieces of the compact callus were placedin a liquid medium on a gyrotory shaker to establish suspensioncultures. The cultures were composed of large, elongated andhigly vacuolated cells, and a population of richly cytoplasmiccells. The latter, here termed embryogenic cells, containednumerous plastids with starch, and occurred in tight groupsof four or more cells, and occasionally as single cells. Structuresresembling various stages of embryogenic development were foundin the suspension cultures. When the cultures were plated ina 2,4-D-free agar medium containing abscisic acid, embryoidswith the typical organization of cereal embryos were produced.The embryoids ‘germinated’ in vitro to give riseto plantlets, which were successfully transferred to soil. Theregenerated plants showed the normal diploid chromosome numberof 14. Embryoids apparently arose from single embryogenic cells,either directly or after the formation of a proembryonal massof cells. embryogenesis, pearl millet, Pennisetum americanum, regeneration, suspension culture     

Dried Cryopreserved Somatic Embryos of Two Picea Species Provide Suitable Material for Direct Plantlet Regeneration and Germplasm Storage

The present study aimed to develop a cryopreservation methodfor long-term storage of mature somatic embryos of Picea spp.The effects of drying rate, embryo water content prior to cryopreservation,thawing rate, and rehydration on survival of cryopreserved somaticembryos were investigated. Emphasis was placed on the capacityof cryopreserved somatic embryos to germinate and regenerateplantlets directly, or to reinduce embryogenic tissue for newembryo production. Firstly, a slow drying rate at 97 or 88%relative humidity (RH) was needed to achieve high germination(96.7–100%) and high plantlet conversion rates (26.7–46.7%)(not different from controls). Secondly, somatic embryos hadto reach a water content of 0.23 g H₂O g^-1d.wt (48 h of desiccationat 97% RH) before immersion in liquid nitrogen to germinateat high frequency (93.8%). Thawing techniques had no effecton embryo survival. Dried and cryopreserved somatic embryosof Picea can also be used to reinduce embryogenic tissue andstart new embryo production. Best reinduction frequency (66.7%)was obtained from cryopreserved embryos dried at 97% RH andrehydrated at 100% RH for 12 h prior to reinduction. No differencein embryo production was noticed between the parent line (1stembryogenic cycle) and the sub-lines (2nd embryogenic cycle).Second generation embryos germinated and regenerated into plantletsat rates similar to controls. The optimal cryopreservation methodwas successfully applied to severalP. mariana and P. glaucagenotypes. Copyright 2000 Annals of Botany Company Picea mariana(Mill) B.S.P., Picea glauca(Moench) Voss, desiccation, embryo water content, thawing, rehydration, embryogenic tissue, genotypes, conifers, clonal propagation     

Morphogenic and genetic stability in longterm embryogenic cultures and somatic embryos of Norway spruce (Picea abies {L.} Karst)

Embryogenic cultures were initiated from mature zygotic embryos of Picea abies. The somatic embryos in the embryogenic cultures were first stimulated to mature and then either to develop further into plantlets or to differentiate new embryogenic cultures. The procedure was repeated three times during two years. The ability to give rise to new embryogenic cultures or to develop into plantlets was similar for all somatic embryos irrespective of how long they had been cultured in vitro. The nuclear DNA content, measured in a flow cytometer, was estimated at 32 pg/G1 nuclei in seedings developed from zygotic embryos. Nuclei isolated from embryogenic cultures and from plantlets regenerated from somatic embryos had the same DNA content as those isolated from seedlings.Abbreviations N⁶-benzyladenine BA - 2,4-dichlorophenoxyacetic acid 2,4-D - abscisic acid ABA     

Expression of the gus A gene in embryogenic cell lines of Sitka spruce following Agrobacterium-mediated transformation

Transformation of Picea sitchensis (Bong) Carr. was investigatedby incubating embryogenic cell lines, initiated from immatureand mature zygotic embryos, with a supervirulent strain of Agrobacteriumtumefaciens. The latter carried a gus A-intron gene. Transientgene expression was determined histochemically by recordingthe number of distinct areas of ß-glucuronidase (GUS)activity. Maximum expression of the gus gene was achieved witha bacterial suspension with an OD₆₀₀ of 0.8–1.1 dilutedwith an equal volume of MPM medium, Inoculation of cells withbacteria for 30 mm, 72 h co-cultivation period and exposureof Agrobacterium and plant cells to 50 µM acetosyrmngone.These results are discussed in relation to Agrobacterium-mediatedgene delivery for the stable transformation of Sitka spruceand other conifers. Key words: Sitka spruce, Agrobacterium, transformation, embryonal suspensor masses, GUS activity     

Regeneration of transgenic cassava plants (Manihot esculenta Crantz) through Agrobacterium-mediated transformation of embryogenic suspension cultures

A protocol was developed for Agrobacterium-mediated transformation of embryogenic suspension cultures of cassava. The bacterial strain ABI containing the binary vector pMON977 with the nptII gene as selectable marker and an intron-interrupted uidA gene (encoding β-glucuronidase) as visible marker was used for the experiments. Selection of transformed tissue with paromomycin resulted in the establishment of antibiotic-resistant, β-glucuronidase-expressing lines of friable embryogenic callus, from which embryos and subsequently plants were regenerated. Southern blot analysis demonstrated stable integration of the uidA gene into the cassava genome in five lines of transformed embryogenic suspension cultures and in two plant lines.     

The Influence of Composition of the Basal Medium on the Growth and Morphogenesis of Cultured Sitka Spruce (Picea sitchensis) Tissues

In vitro culture of Picea sitchensis (Bong.) Carr. needle explantson a number of basal culture media indicated that a complexmixture of organic additives was neither essential nor stimulatory.Adventitious bud production occurred at inorganic nitrogen levelsof 15–60 x 10^–3M and 7.5–30 x 10^–3 Min the adventitious bud induction and elongation media, respectively,when a well-balanced ratio of NH₄⁺:NO₃^– was maintained.However, necrosis was increased at the highest level of inorganicnitrogen. Organogenesis was more sensitive to changes in theratio of NH₄⁺:NO₃^–. Complete replacement of NH₄⁺ withNO₃^– during the adventitious bud induction passage severelyinhibited organogenesis, indicating that a reduced form of nitrogenmay be essential for adventitious bud differentiation. Conversely,a high proportion of NH₄⁺ in either the adventitious bud inductionor elongation medium increased tissue necrosis and inhibitedbud induction, reflecting the potential toxicity of this ion.Explants from different individual trees were found to varyconsiderably in their morphogenetic responses, but NH₄⁺:NO₃^–ratios of 1:5 or 1:2 were o ptimal for all individual treeswhich developed adventitious buds from needle cultures. Picea sitchensis, Sitka spruce, tissue culture, nitrogen     

Somatic Embryogenesis and Plant Regeneration from Freely-suspended Cells and Cell Groups of Panicum maximum Jacq

Embryogenic calluses derived from cultured immature embryosand young inflorescences of Panicum maximum Jacq. were placedin Murashige and Skoog's liquid medium supplemented with 1 mg1^–1 2, 4- dichlorophenoxyacetic acid (2, 4-D) and 2.5per cent coconut water, to initiate suspension cultures. Suspensionsconsisted of two types of cells: small, richly-cytoplasmic andoften starch-containing embryogenic cells, and large, vacuolatednon-embryogenic cells. A presumed sequence of developmentalstages from single embryogenic cells to globular and heart-shapedstages of embyrogenesis was observed in the suspension cultures.Plantlets were produced from the embryoids when the suspensionswere plated in an agar medium without any hormone or with only0.2 mg 1^–12, 4-D or naphthalene acetic acid. Embryogenicsuspension cultures derived from immature embryos as well asfrom inflorescence segments gave rise to plants which showedthe normal somatic chromosome number of 2n = 4x = 32. Panicum maximum Jacq., Guinea grass, embryogenesis, regeneration, suspension culture     

Flow Cytometric Characterization of Embryogenic and Gametophytic Development in Brassica napus Microspore Cultures

Microspore cultures are ideal systems for studying plant embryogenesisbecause the resulting embryos are very similar to zygotic embryos,all the stages of development are readily accessible and theprocess can be induced by a simple heat treatment. However,not all microspores are embryogenic and the mixture of cellsthat develops in the cultures complicates the use of this system.Brassica napus microspore cultures cultured at 30°C (induced)and at 25°C (non-induced) were compared by flow cytometryto obtain structure and function information for several typesof cells in the culture. Clear differences in light scatterand fluorescence were found between induced and noninduced culturesthat are related to early stages of embryo development. Viable,round cells that were unique to induced cultures were sortedinto culture media and developed into embryos confirming thatthey were embryogenic. The present study provided flow cytometricidentifiers for embryogenic and gametophytic cells, demonstratedhow flow sorting can be used to isolate specific cell typesand defined benchmarks for assessing the embryogenic potentialof microspore cultures. (Received July 9, 1997; Accepted December 10, 1997)     

Somatic embryogenesis and plant regeneration in different organs ofEuterpe edulis mart. (Palmae): Control and structural features

Somatic embryogenesis and further plant regeneration were observed using zygotic embryos, young inflorescences and young leaves ofEuterpe edulis (Palmae) as explants. Both for the cultures of zygotic embryos and inflorescences, activated charcoal in the medium was essential for the establishment of viable cultures. Embryogenesis was induced by using a gelled basal medium with MS or Euwens salts supplemented by high 2, 4-D levels (50–100 mg L⁻¹). The embryogenic process was direct without a callus stage. For further development, cultures with globular or post-globular embryos were transferred to the basal medium with 2-iP (2.5 mg L⁻¹) and NAA (0.1 mg L⁻¹). To convert embryos to plantlets, cultures were transferred to a third medium in which sucrose and salts were reduced to the half-strenght of the basal medium, without growth regulators. In the case of liquid medium, with either 2, 4-D or NAA (10–20 mg L⁻¹). The developmental stage of each explant was critical for the induction of embryogenesis. The histological study of embryogenic cultures revealed that in the case of zygotic embryos, somatic embryos arise directly from the surface of the cotyledonar node, or from subepidermal tissues. In the inflorescences, a pro-embryogenic tissue is formed at the floral primordium region; in the leaves, the first morphogenic event is cell proliferation in the vascular parenchyma.     

Somatic Embryogenesis and Plant Regeneration from Cultured Leaf Explants of Zea mays

Young leaf segments of Zea mays L. seedlings were cultured onMurashige and Skoog's basal nutrient medium supplemented with2 mg l^–1 2, 4-D and sub-cultured on medium containing8 mg l^–1 2,4-D. Two types of callus tissues appeared—embryogenicand non-embryogenic. The embryogenic callus tissue producednumerous somatic embryos which on transfer to media containinglow amounts of 2,4-D or ABA produced plantlets. Callus tissuesexhibited embryogenic potential for more than 1 year. Zea mays L. cv. Ageti-76, Zea mays L. cv. N-L-D-Comp., maize, leaf, callus, somatic embryogenesis, regeneration     

Genotype dependent blue and red light inhibition of the proliferation of the embryogenic tissue of Norway spruce

Summary The influence of light quality on the proliferation of embryogenic tissue of three genotypes of Norway spruce (Picea abies [L.] Karst), with different capacities for mature somatic embryo production, was studied. The proliferating tissues were subjected to light from commercially available light sources: Philips TLD Warm White 36W/29, Philips TLD Blue 18W/18, Philips TLD Red 36W/15, Osram L Fluora 36W/77 and Sylvania Far Red 7080, for 18 h a day with the photon flux (PAR) at 30 μmol m⁻²s⁻¹. The effect of light quality on the growth of embryogenic tissue was strongly genotype dependent. In genotype 164-4 tissue proliferation was strongly inhibited by blue and red light. Genotype 86∶52 reacted in a similar way, but not as strongly as 164-4, whereas the tissue of genotype 186-3 was almost insensitive to light quality and grew fast in all light conditions.     

Somatic Embryogenesis in Sugarcane (Saccharum officinarum L.): Growth and Plant Regeneration from Embryogenic Cell Suspension Cultures

Embryogenic cell suspension cultures were established from calliderived from young leaves of sugarcane (Saccharum officinarumL.) by placing them in liquid medium containing 5 per cent coconutwater (CW), 2–3 mg 1^–1 2, 4-D and 500 mg 1^–1casein hydrolysate (CH). The cultures were maintained by transferring2.5–5.0 ml of the suspension to 35 ml of fresh mediumevery 4–5 days. Organized structures resembling the earlystages of embryogeny were formed when 2, 4-D in the medium waslowered (0.1–1.0 mg 1^–1) but these did not developbeyond the globular or early scutellar stages. High levels ofsucrose (6–10 per cent) promoted the formation of proembryoids.Plating of the suspension on MS agar medium supplemented with0.25–2.0 mg 1^–1 2, 4-D, 5 per cent CW, 500 mg 1^–1CH, with or without activated charcoal, resulted in the formationof embryogenic calli. A large number of embryoids were formedin media containing lower 2, 4-D concentrations. Transfer ofembryoids to half-strength MS medium with 6 per cent sucroseestablished plantlets which were successfully transferred tosoil. Saccharum officinarumL, sugarcane, suspension culture, embryogenesis, regeneration     

Regeneration of plants from cryopreserved embryogenic cell suspension and callus cultures of cotton (Gossypium hirsutum L.)

Successful regeneration of cotton (Gossypium hirsutum L.) plants from cryopreserved embryogenic callus and cell suspension cultures is described. The cryoprotectant mixture consisting of a modified Murashige and Skoog (1962) medium with sucrose (5% w/v), DMSO (5% v/v) and glycerol (5% v/v) gave the highest survival rate (70%) from cell suspension cultures cryopreserved in liquid nitrogen after slow cooling (0.5 to 1.0°C/min). A cooling rate of 0.5°C/min provided a satisfactory recovery rate (30%) from cryopreserved embryogenic callus cultures and was superior to a cooling rate of 1°C/min. Regenerated plants from cell suspension and embryogenic callus cultures cryopreserved for more than four years exhibited normal morphology, growth and boll set upon transfer to soil.Abbreviations DMSO dimethylsulfoxide - MS Murashige and Skoog (1962) - MMS modified MS - NAA -naphthaleneacetic acid     

Myo-inositol Biosynthesis and Galactose Utilization by Wild Carrot (Daucus carota L. var. carota) Suspension Cultures

Wild carrot (Daucus carota var. carota) cell suspensions (63–120µm in diameter) were grown on a mineral salt medium containingdifferent carbon sources in the presence (10 mM) and absenceof myo-inositol. The data obtained after 14 and 21 days of growthshow that an external supply of myo-inositol is not essentialfor growth and development of wild carrot embryos. A linearrelationship was found between growth (d. wt) and embryo numberin the presence and absence of myo-inositol. Standard stock cell suspensions never exposed to exogenous myo-inositoland grown in the absence of 2, 4-D with glucose or galactoseas the carbon source synthesized radioactive myo-inositol whenexposed to D-[1–¹⁴C]glucose or D-[1–¹⁴C]galactose.Gas chromatographic analyses revealed the presence of myo-inositolin the bulk tissue grown in the presence of 2.25 µM 2,4-D with glucose, galactose, fructose or mannose as the solecarbohydrate. We could not detect any component indicating anisomer or a methylated derivative of an inositol in the tissueextracts. Stock cultures were maintained (with 2, 4-D) successfully forat least three successive sub-cultures on D-galactose as thesole carbohydrate. The growth achieved over this culture periodshowed that wild carrot cells used by us could quickly adaptto grow on D-galactose as rapidly as they grow on sucrose. Daucus carota L., wild carrot, suspension cultures, myo-inositol, galactose     

Susceptibility of embryogenic and organogenic tissues of maritime pine (Pinus pinaster) to antibiotics used in Agrobacterium-mediated genetic transformation

The effects of antibiotics commonly used in Agrobacterium-mediated transformation were studied on Pinus pinaster tissues. Embryogenic tissue growth from three embryogenic lines and adventitious bud induction from cotyledons from three open-pollinated seed families were analysed. Cefotaxizme, carbenicillin and timentin commonly used for Agrobacterium elimination, at concentrations of 200–400 mg l ⁻¹ did not inhibit the embryogenic tissue growth on filter paper nor as clumps. Adventitious bud induction and bud number were significantly reduced for one of the tested families when using 400 mg l⁻¹ cefotaxime or timentin. The selection agent kanamycin significantly inhibited growth of embryogenic tissue on filter paper in all the embryogenic lines␣and concentrations tested (20–50 mg l⁻¹). Kanamycin also inhibited growth of embryogenic clumps after two subcultures at 5–50 mg l⁻¹. In␣cotyledons, kanamycin inhibited adventitious bud␣formation in the three seed families used, regardless of the concentrations tested (5–25 mg l⁻¹). There was a significant effect of the seed family on the bud induction and the number of adventitious buds produced. From the results obtained, we propose the use of timentin to eliminate Agrobacterium in transformation experiments, at concentrations of 400 mg l⁻¹ for embryogenic tissues and of 300 mg l⁻¹ for cotyledons. For selection of transformed tissues carrying the kanamycin resistance gene, kanamycin should be used at 20 mg l⁻¹ for embryogenic tissues on filter paper, at 5 mg l⁻¹ when clumps are in direct contact with the selection medium, and bellow 5 mg l⁻¹ for adventitious bud induction.     

Evaluation of somaclonal variation during somatic embryogenesis of interior spruce (Picea glauca engelmannii complex) using culture morphology and isozyme analysis

Somaclonal variation during interior spruce (Picea glauca engelmannii complex) somatic embryogenesis was evaluated using culture morphology and isozyme analysis. Genotype-specific abscisic acid-dependent developmental profiles and isozyme patterns were similar for subclone and parent line embryogenic cultures and cotyledonary somatic embryos. Extensive analysis of fifteen hundred subclone embryos of one genotype revealed no isozyme pattern variation. Initiation of embryogenic cultures was dependent on the developmental stage of the explant although cultures derived from different stages were morphologically similar. The embryogenic cultures initiated from interior spruce embryos show a high degree of genetic stability in that the morphological behavior and isozyme phenotype were always consistent with that of the explant genotype. These results support the conclusion that this culture system is appropriate for clonal propagation of interior spruce.     

Analysis of Shoot Apical Growth of Picea sitchensis Seedlings

During the first 100 days after sowing (March-June) the followingchanges took place at the terminal shoot apices of Picea sitchensisseedlings: plastochrones (T) decreased from over 24 h to 4 h;apical domes enlarged from less than 0·20 mm to 0·45mm diameter (D); the ‘projected’ area of tissuesproduced by the apical domes (i.e. viewed from above) increasedin amount from less than 0·012 to 0·024 mm² day^-1;about 15 per cent of this tissue was re-invested in the apicaldomes, the rest was used to produce primordia; and the volume-doublingtimes of the apical dome tissues decreased from over 150 h to50 h. After 100 days there was no further re-investment in theapical domes, but the domes did not decrease abruptly in size.Less tissue was produced per day, but the primordia were smallerso that the rate of primordia formation did not fall precipitously.Plastochrone ratios were inversely related to D, but the relationshipbetween T and D depended on whether T was decreasing or increasing.Progenies which were known to be fast growing tended to build-uptheir apical domes rapidly (i.e. have large ‘re-investmentratios’) and to be capable of producing small primordia.These attributes can evidently be evaluated on seedlings andcould help to lessen the cost of tree breeding progeny-testprogrammes. meristem, Picea sitchensis, Sitka spruce, growth, shoot apex     

Biolistic Transformation of Rose (Rosa hybridaL.)

A reproducible method has been developed for the Biolistic transformationand regeneration of transgenic plants from embryogenic callusof rose (Rosa hybridaL.) cv. Glad Tidings. DNA delivery wasoptimized using the ß-glucuronidase (gus) gene. Thedistance between the stopping screen and target explants andsupplementation of pre-and post-bombardment culture media with0.25Mmyo-inositol influenced the transformation efficiency.Prior to culture on selection medium containing 250 mg l^-1kanamycinsulphate, embryogenic calli were bombarded, using optimizedgene delivery parameters, with a plasmid carrying the neomycinphosphotransferase (nptII) gene. Somatic embryo-derived kanamycin-resistantplants were regenerated and subsequently transferred to glasshouseconditions. Transformation was confirmed by kanamycin resistanceof calli and plants, NPT II ELISA assay and Southern analysis.All transgenic plants were morphologically normal (true-to-type).Copyright1998 Annals of Botany Company Biolistic; genetic engineering; rose;Rosa hybridaL.; transformation.     

Cryopreservation and plant regeneration via somatic embryogenesis using shoot apical domes of mature Pinus roxburghii sarg, trees

Summary The present investigation reports optimized parameters for somatic embryogenesis and cryopreservation of embryogenic cultures using shoot apical domes from mature trees of Pinus roxburghii Sarg. Embryogenic tissue of P. roxburghii Sarg. was cryopreserved for 24 h, 10 d, and 8 wk using sorbitol and dimethylsulfoxide (DMSO) as cryoprotectants. Results indicate that 0.2M sorbitol and 5% DMSO had the best cryoprotecting effect. The recovered tissue showed luxuriant growth on maintenance medium (II). Partial desiccation of thawed embryogenic tissue for 24 h prior to transfer to maturation medium enhanced the maturation of somatic embryos. Maturation frequency increased from 1.3 to 18.3% after 12 h desiccation treatment, and from 18.3 to 61.8% after 24 h of desiccation. However, non-desiccated embryogenic tissue produced the least number of somatic embryos (1.3%) on the maturation medium with the same abscisic acid and Gellan gum concentration. All the three embryogenic lines produced plantlets and had the same appearance and normal growth as compared to unfrozen controls.     

Tree Competition and Species Coexistence in a Sub-boreal Forest, Northern Japan

The growth and mortality patterns and the mode of competitionof six tree species forming a sub-boreal climax forest in Hokkaido,northern Japan, were investigated based on the diffusion modelat the level of the individual tree 2 m height in a 2·3-hastudy site. Picea jezoensis, Picea glehnii, Betula ermanii andAbies sachalinensis were dominant species, occupying approx.94% of the total basal area. Sorbus commixta and Acer ukurunduensewere subordinate species occupying approx. 6% of the total basalarea. A model for individual growth was developed, consideringboth intra- and inter-specific competition and the degree ofcompetitive asymmetry. Asymmetry was found in intraspecificcompetition of Sorbus commixta and Acer ukurunduense. Piceajezoensis, Betula ermanii and Abies sachalinensis showed symmetricintraspecific competition. There was little interspecific competitionamongst Picea jezoensis, Picea glehnii and Betula ermanii. Abiessachalinensis competed symmetrically with Picea jezoensis (onlyvery weakly, P < 0·1) and Betula ermanii (P < 0·01).Picea glehnii gave no indication of inter- or intra-specificcompetition. The growth of the four dominant species was neveraffected by the two subordinate species; the growth of the twosubordinate species was governed by the abundances of the fourdominant species, the sum of which almost amounted to standcrowdedness (i.e. symmetric competitive effect and one-sidedcompetitive direction). On the scale of 2·3 ha of thesub-boreal forest, symmetric competition prevailed over one-sidedor asymmetric competition although statistical evidence forany competitive effects was rather weak. This was probably dueto the relatively low tree density and stand crowdedness ofthis climax forest. Little competition between the dominantspecies suggested by relatively low proportions of r²-valuesattributable to competitive effects indicates weak organizationamongst the component species (i.e. species were more or lessindependent of each other) at the level of the individual tree 2 m height on the 2·3-ha scale.Copyright 1995, 1999Academic Press Climax forest, diffusion model, individual growth, one-sided competition, size structure, symmetric competition