Plant regeneration from cultures of fused protoplasts in brood-leaved trees

在桤木、白桦和白杨3种速生乔木之间进行原生质体融合。原生质体融合的最佳条件为：细胞密度为5 × 10^4/ml；融合缓冲液，0.5M甘露醇+ 2.5mM CaCl_2；交流，200v/cm；交流频率，1MHz；直流，2kV/cm；在此条件下，杉木、白桦和白杨的原生质体融合率分别为31%、34%和36%。由桤木-桦木融合细胞培养而来的细胞悬浮培养物发育成白桦样的白色培养物，表明桤木和桦木之间存在杂交培养。从白桦x杨原生质体培养中发现，在含有1 μ M NAA的MS培养基和含有0.1 μ M 4-PU的MS培养基中，有部分幼芽再生，说明这些幼芽是由两种原生质体融合产生的，因为白桦在含有NAA的MS培养基中不能分化任何幼芽。桤木与杨木的融合细胞培养形成愈伤组织，并分化出一些芽。这些苗在含有0.1亩NAA和2.5亩IBA的MS培养基上生根，发育成5株不同性状的苗。

Protoplast fusion was done between 3 fast-grown trees, Alnus firma, Betula platyphylla, and Populus alba. The optimal condition of protoplast fusion was as follows : cell density, 5x10^4/ml ; fusion buffer, 0.5M mannitol + 2.5mM CaCl_2 ; Alternate current, 200v/cm ; AC frequency, 1MHz ; Direct current, 2kV/cm ; pluse width, 100 mu sec.This condition of protoplast fusion gave fusion ratios of 31% for Alnus firma, 34% for Betula platyphylla, and 36% for Populus alba.Cell suspension cultures derived from fused cells of Alnus x Betula developed into white cultures of Betula-like form, suggesting the hydrid cultures between Alnus and Betula.From protoplast cultures of Betula x Populus, some shoots regenerated in MS medium containing 1 mu M NAA and 0.1 mu M 4-PU,suggesting that these shoots obtained were derived from fused protoplasts between both species, because Betula species can not differentiate any shoots in Ms medium containing NAA.Fused cell cultures of Alnus x Populus developed into callus from which some shoots differentiated. These shoots rooted in MS medium containing 0.1 mu M NAA and 2.5 mu M IBA and developed into 5 plantlets with different characters.