J. B. Power

Electro-enhancement of division of plant protoplast-derived cells

SummaryElectric field pulses, ranging from 250 to 2000 V and of 10 to 50 μsec duration, were assessed for their effect on the growth in culture of isolated protoplasts ofGlycine canescens, Prunus avium × pseudocerasus, Pyrus communis, Solanum dulcamara andSolanum viarum. Three successive voltage pulses between 250 and 1000 V caused a small decrease in protoplast viability, but promoted cell division and enhanced significantly the plating efficiency. A higher percentage of electro-pulsed protoplasts showed sustained growth in culture to the microcallus stage compared to untreated protoplasts. The rate of cell division was also stimulated in electro-treated protoplasts. These observations are discussed in relation to present knowledge of the effects of electrical treatments on plant and animal cells.

Isolation, culture and plant regeneration of colt cherry (Prunus avium × pseudocerasus) protoplasts

Abstract Large numbers of viable protoplasts were isolated from leaf mesophyll tissues and cell suspension cultures of Colt cherry using 1% (w/v) Onozuka R-10, 0.2% (w/v) Macerozyme R-10, 0.1% (w/v) Driselase, 1% (w/v) polyvinylpirrolidone (av. MW 10 000) (PVP-10) and 2% (w/v) Meicelase, 2% (w/v) Rhozyme HP-150 and 0.03% (w/v) Macerozyme R-10. Culture media, based on Murashige and Skoogs (MS) salts, supplemented with 9% (w/v) mannitol and various combinations of α-naphthalene acetic acid (NAA), 6-benzylaminopurine (BAP) and zeatin (Z) promoted cell wall regeneration followed by cell colony and callus formation. Protoplasts of both sources were compared in relation to their cultural requirements. Protoplast-derived callus underwent organogenesis.

Electroporation-mediated improvement of plant regeneration from colt cherry (Prunus avium × pseudocerasus) protoplasts

Abstract Results are presented that show a promotory carry-over effect, of an electroporation treatment of isolated cell suspension protoplasts of Colt cherry ( Prunus avium × pseudocerasus ), on the growth of protoplast-derived calli and on plant regeneration capacity. Callus from protoplasts subjected to three successive exponential pulses at 250 V or 500 V showed the largest fresh weight increases between subcultures, and also exhibited the highest frequency of plant regeneration based on the number of shoots per callus. These shoots, in turn, produced a more prolific root system when compared to those derived from non-electropulsed protoplasts.

Transformation of sugarcane protoplasts by direct uptake of a selectable chimaeric gene.

Sugarcane protoplasts were transformed to kanamycin resistance at a frequency of approximately 8 in 107 following PEG-induced uptake of Sma1 linearised pABD1 plasmid. DNA-treated protoplasts were cultured in agarose droplets, and protoplast-derived transformants selected on 80 μg ml−1 kanamycin. Transformed tissues maintained on this level of antibiotic expressed APH(3′)II activity, and contained DNA that hybridised to a probe with the APH(3′)II gene.

Somatic Hybrid Plants of Lycopersicon esculentum Mill.and Lycopersicon peruvianum Mill.

Summary Fertile somatic hybrid plants between tomato and one of its wild relatives ( L.peruvianum ) have been produced and were characterised by comparative morphology and fraction 1 protein analysis.All the hybrids were hexaploids and since backcrossing is possible, these hybrids are likely to be of use in tomato improvement.This demonstration of somatic hybridisation opens up the possibility of incorporating novel traits from wild sources to cultivated tomato.

Plant Regeneration from Protoplasts of Apple Rootstocks and Scion Varieties (Malus X domestica Borkh.)

Summary Large numbers of viable protoplasts were isolated from leaves of apple rootstocks M.9, MM. 106 and the scion variety Spartan and also from leaf tissues, callus and cell suspensions of Bramleys Seedling apple. Upon culture, using modified MS-based media (for rootstocks M.9 and MM.106) or modified KM media (for the scions Spartan and Bramleys Seedling), protoplasts entered division to give callus. Shoot buds were regenerated, from the protoplast-derived calli of M.9, MM.106 and Spartan with the production of rooted, autotrophic plants, for M.9 and Spartan apple.

An alternative approach to plant regeneration from protoplasts of sour cherry (Prunus cerasus L.)

Abstract Mesophyll protoplasts, of sour cherry ( Prunus cerasus ), clones CAB 4D, CAB 5H and CAB 11E, gave differential cultural responses. Protoplasts in media based on Murashige and Skoog (MS) salts, supplemented with 9% (w/v) mannitol plus growth regulators underwent cell wall regeneration, cell colony and callus formation. Zeatin (Z) was needed in order to induce cell division for all three sour cherry clones. The protoplast-derived calli, of clones CAB 4D and CAB 5H, underwent rhizogenesis as an intermediate step towards shoot bud differentiation. Clone CAB 5H also gave direct shoot bud regeneration from the protoplast callus.

An improved procedure for plant regeneration from indica and japonica rice protoplasts

Plant regeneration from protoplasts of two commercially cultivated Indian indica rice varieties, Pusa Basmati 1 and Java, has been accomplished by plating embryogenic cell suspension-derived protoplasts on the surface of filter membranes overlying agarose-embedded feeder cells of Lolium multltiflorum and Oryza ridleyi, combined with the use of a maltose-containing shoot regeneration medium. Embryogenic cell suspension cultures of Pusa Basmati 1 and Jaya were initiated from mature seed scutellum-derived calli in liquid R2 medium modified by the addition of 560 mg l−1 of proline and 1.0 % (w/v) maltose. In both varieties, protoplast plating efficiencies up to 0.4 % were obtained, depending on the nature of the feeder cells. L. multiflorum feeder cells induced a 6-fold higher plating efficiency than feeder cells of O. ridleyi. In combination, O. ridleyi and L. multiflorum feedercells further enhanced protoplast plating efficiency. Protoplast-derived cell colonies were not obtained from protoplasts of either indica varieties in the absence of feeder cells. MS-based medium containing kinetin (2.0 mg l−1) and α-naphthaleneacetic acid (0.5 mg 1−1), together with sucrose and maltose both at 1.5 % (w/v), induced green shoot regeneration in 44 % of protoplast-derived tissues, depending on the feeder cells used for protoplast culture. In both varieties, tissues obtained using O. ridleyi feeder cells were more morphogenic than tissues obtained using L. multiflorum feeder cells, either alone or in combination with cells of O. ridleyi. In the japonica rice variety Taipei 309, this new procedure resulted in a 30-fold increase in plant regeneration from protoplasts compared to previous published procedures.

Over-expression of a gibberellin 2-oxidase gene from Phaseolus coccineus L. enhances gibberellin inactivation and induces dwarfism in Solanum species

Gibberellins (GAs) are endogenous hormones that play a predominant role in regulating plant stature by increasing cell division and elongation in stem internodes. The product of the GA 2-oxidase gene from Phaseolus coccineus (PcGA2ox1) inactivates C19-GAs, including the bioactive GAs GA1 and GA4, by 2β-hydroxylation, reducing the availability of these GAs in plants. The PcGA2ox1 gene was introduced into Solanum melanocerasum and S. nigrum (Solanaceae) by Agrobacterium-mediated transformation with the aim of decreasing the amounts of bioactive GA in these plants and thereby reducing their stature. The transgenic plants exhibited a range of dwarf phenotypes associated with a severe reduction in the concentrations of the biologically active GA1 and GA4. Flowering and fruit development were unaffected. The transgenic plants contained greater concentrations of chlorophyll b (by 88%) and total chlorophyll (11%), although chlorophyll a and carotenoid contents were reduced by 8 and 50%, respectively. This approach may provide an alternative to the application of chemical growth retardants for reducing the stature of plants, particularly ornamentals, in view of concerns over the potential environmental and health hazards of such compounds.

Isolation, culture and callus regeneration of protoplasts of wild and cultivated Helianthus species.

Protoplasts were isolated from seedling roots, hypocotyls, and cotyledons of four cultivars of Helianthus annuus and from leaves of axenic shoot cultures of the wild species H. praecox, H. scaberimus and H. rigidus. Optimal culture conditions were established for the respective protoplast systems, using the agarose bead method of culture. Protoplast division was induced for all the species examined. In the case of the cultivars of H. annuus, hypocotyl and cotyledon protoplast division was sustained leading to callus formation, which in turn, could be induced to produce roots and organised meristematic regions in the presence of NAA and 6-BAP.