Franz Hoffmann

Plant regeneration from protoplasts of common buckwheat (fagopyrum esculentum).

Protoplasts were isolated from hypocotyls of etiolated seedlings from a diploid and the corresponding autotetraploid variety of common buckwheat (Fagopyrum esculentum). The isolated protoplasts started to divide after 4 days in culture in a modified MS medium. Maximum plating efficiency was approximately 1%. Regenerated calli derived from the tetraploid genotype developed roots easily but were recalcitrant to form shoots. Eighteen months following the initiation of cultures, tetraploid embryoids and shoots emerged after 3 weeks on an MS medium containing 0.1 mg/l gibberellic acid.

Laser microbeams for the manipulation of plant cells and subcellular structures

Laser microsurgery has been used in plants to study physiological, cell biological and genetical questions for over 10 years. More recently, the optical trap became available as an additional tool. Specific areas of research include membrane physiology, motility, transformation and protoplast fusion. Compared to the data reported in animal systems, the contributions of laser microbeam manipulations in plant biology are rather limited. However, with increased awareness of the enormous potential of the technology and better accessibility to less expensive and more user-friendly equipment, the next decade should be more productive.

Selection of a super-growing legume root culture that permits controlled switching between root cloning and direct embryogenesis

Abstract Root cultures, displaying vigorous growth and high embryogenic capacity, were established in the legume forage species Lotus corniculatus (bird’s-foot trefoil). Root cloning as well as plant regeneration was achieved on hormone-free medium, in agitated culture in the dark or under stationary conditions in the light, respectively. These qualities of vigorous growth and regeneration faded with time in hormone-free culture, with slow-growing roots turning brown in color. Addition of the synthetic cytokinin-like hormone benzylaminopurine to the culture medium, however, re-established the aging tissue’s capacity for somatic embryogenesis and plant formation. During continuous initiation of new cultures, it was possible to obtain one root culture (selected from 11 960 seeds at a 65% germination rate) which did not show the typical decline of qualities after prolonged proliferation but distinguished itself by displaying even faster growth and more vigorous embryogenic plant production on hormone-free medium. There was no decline since its initiation 9 months earlier. This super-growing root culture produces plants that show no morphological differences as compared to wild-type regenerants or seedlings. Roots, dissected from plantlets derived from super-root embryogenesis, expressed all the super-root qualities again when cultured in vitro. This is the first report on somatic embryogenesis from sustained root cultures without exogenous hormone application. Such a hormone-free, continuous root culture should provide a superior experimental system for genetic or developmental studies that might be sensitive to exogenous hormones, such as somaclonal variation in transgenesis or, since introduced in a legume species, nodulation in vitro.

Transgenic superroots of Lotus corniculatus can be regenerated from superroot-derived leaves following Agrobacterium-mediated transformation

Super-growing roots (superroots; SR), which have been established in the legume species Lotus corniculatus, are a fast-growing root culture that allows continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely growth regulator-free culture conditions. These features are unique for non-hairy root cultures, and they are now stably expressed since the culture was isolated more than 10 years ago (1997). Attempts to achieve direct and stable transformation of SR turned out to be unsuccessful. Making use of the supple regeneration plasticity of SR, we are reporting here an indirect transformation protocol. Leaf explants, derived from plants regenerated from SR, were inoculated with Agrobacterium tumefaciens strain LBA4404 harboring the binary vector pBI121, which contains the neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes as selectable and visual markers, respectively. After co-cultivation, the explants were selected on solidified MS medium with 0.5 mg/L benzylamino purine (BAP), 100 mg/L kanamycin and 250 mg/L cefotaxime. Kanamycin-resistant calli were transferred to liquid rooting medium. The newly regenerated, kanamycin-resistant roots were harvested and SR cultures re-established, which exhibited all the characteristics of the original SR. Furthermore, kanamycin-resistant roots cultured onto solidified MS medium supplemented with 0.5 mg/L BAP produced plants at the same rate as control SR. Six months after gene transfer, PCR analysis and histochemical locating indicated that the NPTII gene was integrated into the genome and that the GUS gene was regularly expressed in leaves, roots and nodules, respectively. The protocol makes it now possible to produce transformed SR and nodules as well as transgenic plants from transformed SR.

Plants from protoplasts isolated from a long-term root culture (Super Root) of Lotus corniculatus

Summary In the legume birds-foot trefoil ( Lotus corniculatus L.), a super-growing root (super root) culture has been recently introduced. This system is unique because it allows continuous root cloning, somatic embryogenesis and mass regeneration of plants under entirely hormone-free culture conditions. Here we report the isolation and culture of protoplasts from this long-term root culture (more than three years old) and the regeneration of plants from super root-derived protoplasts. Treatment of pre-cultured super roots with an enzyme mixture containing 4.0 % cellulase and 0.1 % pectolyase in 0.25 mol/L mannitol/0.25 mol/L sorbitol plus 0.1 % CaCl 2 led to the separation of the root tips from the rest of the roots and, within four hours, to the isolation of approximately 3.0 × 10 6 protoplasts per gram root tissue. Protoplasts were released almost entirely from the separated root-tips and cultured, embedded in small agarose disks, in a modified KM8P medium supplemented with 0.05 mg/L BAP, 0.1 mg/L 2,4-D and 0.5 mg/L NAA. After five to seven days, the first divisions were observed, and after four weeks micro calli of 0.5 to 1 mm could be recovered. Calli were placed on MS medium containing relatively high concentrations of BAP and NAA (both 1.5 mg/L) for four weeks, resulting in prolific shoot formation. Elongated shoots rooted easily on hormone-free medium, and plants could be established. Roots from regenerated plants and roots formed directly on protoplast-derived calli were used to establish new root cultures. These newly initiated cultures expressed all the super root qualities again, including prolific shoot regeneration upon transfer to light, indicating that the super-growing character is not lost through protoplast isolation and regeneration. The super root pathway of regeneration, from protoplasts to callus, roots, root culture and plants, allows the virtually unlimited mass regeneration of plants from root protoplasts. Regenerating super root protoplasts add an important component to tissue culture systems for legume/ Rhizobium research. Furthermore, this is the first report on plant regeneration from protoplasts isolated from a true root culture rather than excised roots.

Design, synthesis and application of surface-active chemicals for the promotion of electrofusion of plant protoplasts

Abstract To provide milder, and thus less destructive, electrical conditions without reduced fusion rates a combination of chemofusion and electrofusion techniques was successfully applied to isolated plant protoplasts. Chemical adjuvants were employed for pretreatment of the protoplasts, added to the fusion medium during electroapplication and left in the culture medium in order to maintain the most obvious advantage of electrofusion, i.e. direct culture in the fusion medium without the need for washing procedures to remove fusion-promoting substances. Eight chemicals were investigated in the chemostimulated electrofusion and five proved to be positively effective and non-toxic to the cells in culture. Calluses were successfully induced. These chemicals are l -α-lyso(stearoyl/palmitoyl)phosphatidyl cholinium chloride (LLCC), quaternary starch (QS), di-[poly(oxyethylene)ethanol]-stearylammonium chloride (DSC), di-[poly(oxyethylene)ethanol]-stearylamine (DSA) and polyvinylalcohol (PVA). Among these five chemicals, the latter three gave the most promising results. DSC can cause a relative fusion rate of up to 2.15 with a normal (non-reduced) electrical field and 1.94 with a milder less damaging field. DSA gave the highest relative fusion rate under reduced electrofusion conditions (1.72). PVA triggered relative fusion rates as high as 2.21 under normal electrical conditions and 1.26 under milder conditions.

FOX-superroots of Lotus corniculatus, overexpressing Arabidopsis full-length cDNA, show stable variations in morphological traits.

Using the full-length cDNA overexpressor (FOX) gene-hunting system, we have generated 130 Arabidopsis FOX-superroot lines in birds-foot trefoil (Lotus corniculatus) for the systematic functional analysis of genes expressed in roots and for the selection of induced mutants with interesting root growth characteristics. We used the Arabidopsis-FOX Agrobacterium library (constructed by ligating pBIG2113SF) for the Agrobacterium-mediated transformation of superroots (SR) and the subsequent selection of gain-of-function mutants with ectopically expressed Arabidopsis genes. The original superroot culture of L. corniculatus is a unique host system displaying fast root growth in vitro, allowing continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely hormone-free culture conditions. Several of the Arabidopsis FOX-superroot lines show interesting deviations from normal growth and morphology of roots from SR-plants, such as differences in pigmentation, growth rate, length or diameter. Some of these mutations are of potential agricultural interest. Genomic PCR analysis revealed that 100 (76.9%) out of the 130 transgenic lines showed the amplification of single fragments. Sequence analysis of the PCR fragments from these 100 lines identified full-length cDNA in 74 of them. Forty-three out of 74 full-length cDNA carried known genes. The Arabidopsis FOX-superroot lines of L. corniculatus, produced in this study, expand the FOX hunting system and provide a new tool for the genetic analysis and control of root growth in a leguminous forage plant.

Cortical microtubules and protoplast fusion: Effect and fate of microtubular lattices

Abstract Cortical microtubular lattices of isolated plant protoplasts retain their pattern during PEG-induced protoplast fusion and integrate into each other as shown by indirect immunofluorescence for protoplasts of Nicotiana glutinosa fused with those of Daucus carota . In fused and unfused cultures, microtubules assume a parallel orientation and a pre-prophase band-like pattern develops. There is a negative correlation between the presence of a cortical microtubular net in protoplasts and the fusion rate.

High-frequency embryogenesis from cotyledons of bird's-foot trefoil (Lotus corniculatus) and its effective utilization in Agrobacterium tumefaciens-mediated transformation

Summary We have developed a rapid and simple procedure to regenerate birds-foot trefoil ( Lotus corniculatus L.) plants from hypocotyl and cotyledon expiants of 10 to 14-days-old seedlings. Both expiant types regenerated well on the same medium with only minimal interfacing callus formation but with different modes of morphogenesis. While hypocotyls responded primarily by forming shoot apices, cotyledons mostly underwent embryogenes is. Remaining on the original explantation medium, both structures developed into rootless shoots. The highest shoot regeneration frequency was obtained on B5-based medium containing 0.5 mg/L 6-benzylaminopurine with approximately 80% of all expiants forming on average 19 shoots in four weeks. Only two media were used in the entire regeneration process, the second being the rooting medium of half-strength B5 supplemented with 0.01 mg/L α-naphthaleneacetic acid. Rooting frequency and survival rate after potting were about 100%. This regeneration protocol has been successfully applied to Agrobacterium tumefaciens -mediated transformation. Cotyledon segments were used as acceptor tissue. The expiants were co-cultivated with A. tumefaciens strain LB4404 carrying the plasmid vector pBI121. This vector contains the neomycin phosphotransferase II gene (NPTII) and β-glucuronidase reporter gene (GUS), both under the control of the CaMV 35S promoter. Kanamycin-resistant plants regenerated within 45 days after transfer to selective media. On a selection medium containing 100 mg/L kanamycin, shoots were formed by 19.0% of the expiants. The histological GUS assay showed that 7.0% of the resistant shoots also expressed the GUS gene in a variety of tissues. The stable integration of this gene was confirmed by polymerase chain reaction (PCR) analysis. Using the embryogenic cotyledon regeneration system transgenic birds-foot trefoil plants were obtained within 2–3 months.

Transgenic antibiotic resistance may be differentially silenced in germinating pollen grains.

Transgenic tobacco (Nicotiana tabacum L.) plants, carrying the neomycin phosphotransferase (NPT II) gene from E. coli, are resistant to kanamycin when grown from seeds on kanamycin containing medium. Tissue and cell cultures derived from those transformants also express resistance and regenerate complete plantlets in the presence of the antibiotic. This unspecific response to the selective condition has led to the belief that the foreign gene is continuously active or uniformly inducible in all cells of the transgenic plant. However, our experiments show that this view is not true for pollen grains during in vitro germination. Pollen grains isolated from kanamycin resistant tobacco plants carry and transmit the foreign gene but do not express resistance when germinating in vitro. This data presents evidence for differential silencing of a foreign gene in a mature gamete. On the other hand, immature pollen grains (microspores) appear to express resistance. The point of the downregulation of the neomycin transferase gene during pollen maturation is discussed.