Effects of electric cell fusion treatment among leaf protoplasts of Populus alba and alnus firma on growth, leaf morphology, and rapd pattern of eleven acclimatized plants

Summary This study reports the characterization of 11 plants regenerated from electrically fused protoplasts between Populus alba and Alnus firma. Growth characteristics of five regenerated plants (AP-1-AP-5) in terms of shoot height and leaf color showed small differences compared with those of P. alba grown, in pots, and showed no difference in shoot height and diameter compared with those grown in nursery field. There was also no difference in the RAPD pattern between the plants regenerated from interfamilial protoplast fusion and P. alba. In contrast, the lately regenerated plants (AP-6-AP-11) grown in pots showed a marked difference in leaf morphology and RAPD pattern. There was a variation in the ratio of longitudinal to transverse length of leaves among the 11 plants from interfamilial fusions compared with that of protoclones and intraspecific fused protoplasts of P. alba.     

Development of novel elongated fiber-structure in protoplast cultures of Betula platyphylia and Larix leptolepis

Summary Novel elongated fiber-structures were repeatedly found both in leaf protoplast culture of two clones of Betula platyphylla and in protoplast culture of embryogenic cells of Larix leptolepis. Suboptimum culture conditions for cell division appeared to lead to fiber formation when using multi-well plate culture with varying medium compositions The suboptimum conditions for cell divisions were brought about by (1) plant growth regulators: auxins and cytokinins; (2) pH: 3.5, 4.5, 5.8; (3) divalent cations: CaCl₂ and MgCl₂; and (4) sugars: sucrose and mannitol. Divalent cations had the most profound effect on fiber formation. Calcium ions were preferred by Betula and magnesium ions were preferred by Larix. Single fiberpurification and micro-staining methods using a micromanipulator were developed. The fibers fluoresced when stained with Calcofluor White and Aniline Blue, which suggested that they were composed of cell wall component(s), including callose (β-1,3-glucan). Electron microscopy showed that fiber bundles of Larix fibers had helical substructures.     

In vitro plantlet regeneration of “dwarf” Indian olive (Elaeocarpus robustus Roxb.): a fruit plant of Bangladesh

A plantlet regeneration protocol was developed on pot-grown mature plants of Elaeocarpus robustus Roxb. cv. Dwarf from nodal and leaf explants. The best yield of adventitious shoots was achieved from the leaf-derived calli in a modified MS (MMS₁, half strength of major salts, full strength of minor salts, and vitamins) medium containing 4.0 μM BA + 4.0 μM Kn + 0.5 μM NAA + 15% coconut water (CW). The shoot multiplication rate was amplified about twofold per culture after the addition of 15% CW to the medium. The rate of shoot multiplication reached maximum at the 5th subculture, and it maintained this rate throughout the 3 subsequent subcultures. The best rooting in vitro was investigated by subculturing the microcuttings in an MMS₂ (half strength of both major salts and minor salts and full strength of vitamins) medium containing 1.0 μM IBA in the dark for one initial week at 30°C, followed by subculturing them in a plant-growth regulator (PGR)-free medium in the light. The plantlets raised in vitro were successfully established under ex vitro conditions.     

Interfamilial cell fusion among leaf protoplasts of Populus alba, Betula platyphylla and Alnus firma: assessment of electric treatment and invitro culture conditions

The optimal conditions for fusion of leaf protoplasts of Populus alba, Betula platyphylla, and Alnus firma by electric treatment were alternate current (AC) 200 V cm⁻¹ in 2.5 mM CaCl₂ for a pearl chain formation and direct current (DC) pulse of 100 μs at 2 kV cm⁻¹ After interfamilial cell fusion treatment, colonies were obtained using liquid media containing 2,4-D or NAA as an auxin and BA or CPPU as a cytokinin at 0.1, 1, or 10 µM in MS (Murashige and Skoog (1962) Physiol. Plant. 15: 473--497), 1/2salt MS, or NH₄NO₃-free MS containing 0.6 M mannitol and 3% sucrose (totaling 147 combinations). Two shoots after electric cell fusion treatment between P. alba and B. platyphylla, and 12 regenerated plants after electric cell fusion between P. alba and A. firma, were obtained from colonies induced on agar medium containing NAA, IBA, CPPU, and BA. Seven lines of the latter 12 plants which were regenerated later cultured in vitro had serrated leaves different from those of P. alba.     

Callus proliferation from leaf protoplasts using phenylurea type cytokinin, 4-PU,inBetula platyphylla VAR.Japonica

Summary Protoplasts were isolated from leaves ofBetula platyphylla var.japonica using a 0.6M mannitol solution containing 1% Cellulase Onozuka R-10 and 1% Driselase. The cell division and colony formation were largely enhanced using Murashige and Skoog (1962) liquid medium at half strength (1/2 MS), containing 0.6M mannitol, 0.09M sucrose, and factorial combinations of 0.1–30 μM N-(2-chloro-4-pyridyl)-N′-phenylurea (4-pu) and 0.1–10 μM 1-naphthaleneacetic acid (NAA) or 0.1–30 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The optimal protoplast density was 5–7 × 10⁴/ml. Continuous callus proliferation from protoplasts was achieved by transferring colonies to fresh 1/2 MS agar medium containing 1 μM NAA and 1 μM 4-pu with no mannitol. It appeared that supplementation of the medium with phenylurea type cytokinin, 4-pu gave the successful callus proliferation from the protoplasts ofB. platyphylla.     

Plantlet regeneration from mesophyll protoplasts ofBetula platyphylla var.japonica

Summary InBetula platyphylla var.japonica, colonies were induced efficiently from mesophyll protoplasts cultured in half strength MS (1/2MS) liquid medium containing 0.6 M mannitol, 0.09M sucrose and 1 M 4-PU and 1 M NAA at a cell density of 5 × 10⁴/ml. The colonies grew actively and developed into callus after 3 months of culture.Roots differentiated from the protoplast-derived white calluses cultured on the 1 /2MS solid media supplemented with 0.1–1 M 4-PU and 1 M NAA, and 10 M zeatin with no supplementation of NAA. Furthermore, the protoplast-derived green callus differentiated shoots with 1/2MS solid medium containing 1 M 4-PU or 10 M zeatin with no supplementation of NAA. When shoots obtained were cultured on the cytokinin-free MS solid medium with 2.5 M IBA and 0.1 M NAA, they rooted and developed into plantlets after one month of culture.The phenylurea-type cytokinin, 4-PU, was effective for plantlet regeneration from the mesophyll protoplasts ofB. platyphylla var.japonica. This suggests that there is potential for the use of 4-PU in the culture of protoplasts in many forest tree species.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - IBA indole-3-butyric acid - 2ip N ⁶-(2-isopentenyl)-adenine - NAA 1-naphthaleneacetic acid - 4-PU N-(2-chloro-4-pyridyl)-N–phenylurea - TDZ thidiazuron