Plant regeneration from protoplasts of sugar beet (Beta vulgaris)

Mesophyll protoplasts from two of five sugar beet lines tested were regenerated into plants. Mesophyll protoplasts of all lines showed high plating efficiencies up to 4.0% developed hard compact callus, and two of the lines also developed white, soft and friable callus consisting of starch grain-containing cells. Whereas the compact callus never regenerated into plants, the white friable ones frequently developed globular structures, which became green in the light and formed adventitious shoots after cytokinin (BAP or thidiazuron) treatment. Genetic analysis by PCR-fingerprinting and flow cytometry showed uniformity and unchanged ploidy levels in 15 independently regenerated plantlets in line NF. but altered ploidy level (from diploid to triploid) in a regenerated plantlet of clone VRB.     

IgG stability in fresh and conditioned medium of tobacco (Nicotiana?tabacum) and larch (Larix?decidua) embryogenic suspension cultures

To explore the feasability of larch (Larix decidua Mill.) embryogenic cell culture as alternative plant expression system, protein stability in fresh and conditioned medium was characterized in this study and compared to tobacco BY2 suspension culture. Fresh and conditioned media were spiked with 1 μg human IgG and IgG content was determined by ELISA after 24 h incubation. In fresh media, IgG recovery rate decreased to 12–23%. Adsorption on vessel walls probably is the best explanation for this IgG loss and EDTA in the medium strongly influenced wall adsorption. A high IgG recovery rate occurred in all conditioned cell culture media (7 or 14 days after inoculation). Changes in the low molecular weight-constitution of conditioned medium, rather than co-secreted polymers, are responsible for IgG stability in the cell suspension cultures.     

Stable Agrobacterium-mediated transformation of embryogenic tissues from Pinus pinaster Portuguese genotypes

Protocols for genetic transformation of maritime pine (Pinus pinaster Sol. ex Aiton) embryogenic tissues were developed using the Agrobacterium C58pMP90/pPCV6NFGUS. This is the first report of Agrobacterium-mediated T-DNA integration in P. pinaster confirmed by Southern blot analysis. The omission of casein hydrolysate from culture medium during cocultivation and subsequent subculture was crucial to control Agrobacterium growth. Two different transformation protocols were compared: (1) bacterial drops were spread over embryogenic clumps; (2) a mixture of bacterial and embryogenic cell suspensions was plated on filter paper. The highest frequency of transformation (22 independent transformed lines per g fresh weight, for embryogenic clone 31/668/00) was obtained with Protocol 2. The same basic procedure allowed transformation of embryogenic cell suspensions, which was dependent on subculture age. From 52 hygromycin-resistant independent lines obtained, 47 showed stable uidA gene expression and were PCR-positive for uidA gene and 42 for hpt gene. No residual Agrobacterium was detected in the transformed lines. Transgene integration was achieved using both protocols, as confirmed by Southern hybridization. From 38 (90%) transformed lines successfully cryopreserved and recovered, 71% regrown replicates have maintained the frequency of cell aggregates and early-formed embryos with uidA expression. Maturation of 44 transformed lines gave rise to 3 mature somatic embryos, each one coming from a different transformed line. Our results show the high potential of Protocol 2 for application to different culture systems.     

Developmental patterns during direct somatic embryogenesis in protoplast cultures of european larch (Larix decidua Mill.)

Summary Protoplasts isolated from embryogenic suspension cultures of European larch (Larix decidua Mill.) were cultured in thin alginate layers using a nylon mesh to enable a monitoring of the development of single cells. The patterns of cell division and differentiation are characterized and compared with zygotic embryogenesis to which homologies can only be drawn to some extent when the protoplasts grow in an auxinfree environment. Already at 2.5 M both 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid cause vacuolation and elongation of individual cells, thus disturbing the process of somatic embryogenesis which generally lacks the precise quantitative patterns occurring in vivo. Prior to the formation of an embryo, a proembryonal mass develops. Oligonucleated products of spontaneous protoplast fusions are able to cellularize even without preceding karyokinesis and perform a normal embryogenic program.Abbreviations BAP N⁶-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MES 2-(N-morpholino)ethanesulfonic acid - PEM proembryonal mass     

A parenchymatic medium solidifier for plant in vitro culture

A new material for the solidification of liquid culture media was prepared from plant parenchyma tissues by mechanical subdivision, solute extration and dessication from ethanol. It is suitable for in vitro culture and propagation of callus as well as shoot tip cultures. The following plant materials have been grown by means of the new medium solidifier: shoot cultures of Betula pendula Roth, Gerbera jamesonii H. Bolus ex Hook and Floribunda rose "Triumph", callus tissues of Daucus carota L. and Chenopodium album L. The new solidifying material has special advantages over agar for application in the rooting phase of in vitro propagation.Abbrevations PMS parenchymatic medium solidifier - MS Murashige and Scoog's medium - IAA Indole-3-acetic acid - B biotin - K kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid - ch caseine hydrolysate     

Somatic embryogenesis in Araucaria angustifolia

Immature and mature zygotic embryos were used as source of explants for induction of somatic embryogenesis in Araucaria angustifolia. Embryogenic cultures (EC) were only obtained from immature zygotic embryos. Basic medium, carbon source, and genotype showed a significant influence on the formation of stage I somatic embryos (SE). When EC were submitted to maturation conditions, SE continued their individual development until stage II, but mature embryos were not obtained. Proteins secreted by embryogenic cultures were, to a certain degree, genotype specific and included an extracellular class IV chitinase and β-1-3-glucanase.     

Effect of Fusicoccin on Adventitious Root Formation of Birch Shoot Tips (Betula pendula ROTH) Cultured in vitro

The effect of fusicoccin (FC) on adventitious root formationwas investigated using in vitro shoot tip cultures of birch(Betula pendula ROTH) as test system. Treatment with 10^–7–10^–5M FC hastened root appearance as well as 5 ? 10^–6 M IAAdid. Optimal FC concentrations also promoted rooting by increasingthe root number per cutting. FC application during the first48 hours of culture was enough to obtain these effects. Usinginternode segments without any bud it was shown that FC couldnot replace the root inducing activity of endogenous auxin asapplied IAA did, but FC lowered the threshold concentrationof IAA for rooting response and stimulated adventitious rootformation if it was applied with IAA simultanously. Root growthwas enhanced in the early phase but inhibited later by continuoustreatment with FC. Some aspects of possible FC IAA interactionsare discussed. (Received September 4, 1986; Accepted November 24, 1987)     

Boron controls suspensor development in embryogenic cultures of Larix decidua

Suspensor formation in somatic embryos of Larix decidua Mill. was. studied in relation to its dependence on nutritional factors of the culture medium. Protoplasts exhibiting embryogenic competence were prepared from an embryogenic suspension culture, immobilized in Ca-alginate and cultured in liquid media. Developmental patterns showed qualitative differences in the two basic media used. In one medium, containing 100 μ M boric and, compact colonies developed, most of which displayed formation of polar suspensors after 3 to 4 weeks. In contrast, suspensor development never occurred in the other medium, containing 10 μ M borate, yet colonies of loosely connected cytoplasmically dense cells were regenerated without loss of embryogenic competence. Detailed examination of the constituents of the two media led to the conclusion that suspensor development is dependent on boron concentration: below 35 μ M boron, suspensor formation is blocked completely, whereas with an increase up to 1 μ M boron there is a progressively faster development of suspensor-bearing embryos.     

Influence of light on adventitious root formation in birch shoot culturesin vitro

The influence of light of different spectral composition and levels of irradiance (2-40 Wm-²) on adventitious root formation (ARF) in birch shoot segments was investigated. Spontaneous rooting of shoot segments occurred in segments with intact apical or axillary meristems. Concerning ARF shoot meristems could be substituted by application of auxin. The very low rooting percentage of shoot segments in darkness was improved considerably by auxin application. Irradiation of cuttings was a requirement for a high percentage of spontaneous rooting. The promoting effect of light was dependent on its spectral composition and was the highest under red followed by white and blue light. The low rooting response under blue light was enhanced almost to the red light level by shielding the root-forming cutting base from light.