Control of morphogenesis in tobacco protoplast cultures: organogenesis vs embryogenesis

The morphogenetic pathway leading to plant differentiation in tobacco mesophyll protoplasts could be regulated. The course of development via organogenesis or embryogenesis was controlled by manipulating nutrient media, culture conditions and hormone requirements. A lowering of molarity of medium after 5 weeks of protoplast culture, inclusion of GA3 (0.5 mg/l) in the medium for first 8 weeks of culture and exclusion of reduced nitrogen in the medium resulted in shoot organogenesis, while maintenance of higher molarity of the medium till 8 weeks, reduced nitrogen in the medium and removal of 2, 4-D after 5 weeks of culture induced embryogenesis. Regenerability of viable plants was obtained by both developmental pathways. The implications of tobacco embryogenesis system in plant molecular genetics were highlighted.

Characterization of a new lectin involved in the protoplast regeneration of Bryopsis hypnoides

A group of coenocytic marine algae differs from higher plants, whose totipotency depends on an intact cell (or protoplast). Instead, this alga is able to aggregate its extruded protoplasm in sea water and generate new mature individuals. It is thought that lectins play a key role in the aggregation process. We purified a lectin associated with the aggregation of cell organelles in Bryopsis hypnoides. The lectin was ca. 27 kDa with a pI between pH 5 and pH 6. The absence of carbohydrate suggested that the lectin was not a glycoprotein. The hemagglutinating activity (HA) of the lectin was not dependent on the. presence of divalent cations and was inhibited by N-Acetylgalactosamine, N-Acetylglucosamine, and the glycoprotein bovine submaxillary mucin. The lectin preferentially agglutinated Gram-negative bacterium. The HA of this lectin was stable between pH 4 to pH 10. Cell organelles outside the cytoplasm were agglutinated by the addition of lectin solution (0.5 mg ml(-1)). Our results suggest that the regeneration of B. hypnoides is mediated by this lectin. We also demonstrated that the formation of cell organelle aggregates was inhibited by nigericin in natural seawater (pH 8.0). Given that nigericin dissipates proton gradients across the membrane, we hypothesize that the aggregation of cell organelles was proton-gradient dependent.

Optimisation of protoplast production in white lupin

The influence, was investigated, of abiotic parameters on the isolation of protoplasts from in vitro seedling cotyledons of white lupin. The protoplasts were found to be competent in withstanding a wide range of osmotic potentials of the enzyme medium, however, -2.25 MPa (0.5 M mannitol), resulted in the highest yield of protoplasts. The pH of the isolation medium also had a profound effect on protoplast production. Vacuum infiltration of the enzyme solution into the cotyledon tissue resulted in a progressive drop in the yield of protoplasts. The speed and duration of orbital agitation of the cotyledon tissue played a significant role in the release of protoplasts and a two step (stationary-gyratory) regime was found to be better than the gyratory-only system.

Optimization of protoplast production in white lupin

The influence, was investigated, of abiotic parameters on the isolation of protoplasts from in vitro seedling cotyledons of white lupin. The protoplasts were found to be competent in withstanding a wide range of osmotic potentials of the enzyme medium, however, −2.25 MPa (0.5 M mannitol), resulted in the highest yield of protoplasts. The pH of the isolation medium also had a profound effect on protoplast production. Vacuum infiltration of the enzyme solution into the cotyledon tissue resulted in a progressive drop in the yield of protoplasts. The speed and duration of orbital agitation of the cotyledon tissue played a significant role in the release of protoplasts and a two step (stationary-gyratory) regime was found to be better than the gyratory-only system.

Constitutive expression of the cold-regulated Arabidopsis thaliana COR15a gene affects both chloroplast and protoplast freezing tolerance

Cold acclimation in plants is associated with the expression of COR (cold-regulated) genes that encode polypeptides of unknown function. It has been widely speculated that products of these genes might have roles in freezing tolerance. Here we provide direct evidence in support of this hypothesis. We show that constitutive expression of COR15a, a cold-regulated gene of Arabidopsis thaliana that encodes a chloroplast-targeted polypeptide, enhances the in vivo freezing tolerance of chloroplasts in nonacclimated plants by almost 2°C, nearly one-third of the increase that occurs upon cold acclimation of wild-type plants. Significantly, constitutive expression of COR15a also affects the in vitro freezing tolerance of protoplasts. At temperatures between −5 and −8°C, the survival of protoplasts isolated from leaves of nonacclimated transgenic plants expressing COR15a was greater than that of protoplasts isolated from leaves of nonacclimated wild-type plants. At temperatures between −2 and −4°C, constitutive expression of COR15a had a slight negative effect on survival. The implications of these data regarding possible modes of COR15a action are discussed.

Parasite-host interaction of the protoplast isolates of Entomophthora egressa with the eastern hemlock looper and the eastern spruce budworm

Hemocytes of the insects Lambdina fiscellaria fiscellaria and Choristoneura fumiferana did not adhere to the protoplasts of ~he fungus EntomoEhthora egressa. Hemocyte reaction for both insect species to test-particles was not suppressed by the protoplasts. The spherule cells of _-L. fiscellaria fiscellaria adhered to the spherical hyphal bodies and hyphae of ~· ~gressa. The granular cells of -c. fumiferana adhered to the hyphae of ~· egress~. Protoplasts exposed to papain were attacked by the granular ·cells of -c. fumiferana. Spent growth medium of both protoplast isolates produced paralysis when injected into -c. fumiferana larvae. Evidence suggests that heat-stable proteins may be involved. Protoplast isolates showed differences in the growth rates and regeneration sequences using coagulated egg yolk medium, a highly modified version of Grace's insect tissue . culture medium (MGM) and modifications of MGM and in the presence of C0₂. The isolates also differed in the changes that they induced in MGM composition during protoplast growth and in the rates of glucose utilization and protein secretion. The serum of c. fumiferana larvae contained protein(s) which we believe adhere to the cell membranes of the protoplasts of E. egressa. Evidence is presented for hemocyteplasn~ interaction in the presence of protoplasts. Components in the larval serum were found to influence protoplast growth patterns. The possibility of antiprotoplast serum activity is presented. Melanin, toxic levels of ninhydrinpositive compounds and antiprotoplast proteins may have been involved in this activity. The granular cells of -L. fiscellaria fiscellaria and Q• fumiferana adhered to the hyphae of ,Rhizopus ~i$rican~. Spores of Absidia repens and the bacteria Escherichia coli and Bacillus cereus adhered to the granular cells of both species of· insects. The granular cells and plasmatocytes of -c. fumiferana were capable of phagocytosing -B. cereus. Adhesion of .A... . repens spores to c. fumiferana granular cells ~ . - was stimulated by N-acetylglucosamine and glucosamine, moderately reduced by D-fucose, D-arabinose, D-mannose, D-galatose and sucrose and mildly reduced by D-glucose, D-fructose and trehalose. There was no evidence of humoral opsonins in larval hemolymph favoring test-particle-hemocyte interaction. Granular cells of c. fumiferana exposed to papain had reduced affinities for A. repens spores.

Characterisation of the subgenomic RNAs of an Australian isolate of barley yellow dwarf luteovirus

We have characterised the subgenomic RNAs of an Australian isolate of BYDV-PAV. Northern blot analyses of infected plants and protoplasts have shown that this isolate synthesises three subgenomic RNAs. Precise mapping of the transcription start sites of all three subgenomic RNAs and translational analyses of subgenomic RNA 2 and 3 have revealed a number of features. First, the transcription start site of subgenomic RNA 1 in this isolate differs markedly from the start site determined for an Illinois isolate of BYDV-PAV. Second, the start sites of subgenomic RNA 1 and 2 occur at a sequence that closely resembles the 5' end sequence of the genomic RNA (5'AGUGAAGA). Third, subgenomic RNA 2 appears to express ORF 6 of BYDV-PAV but the gene product is truncated due to the appearance of a new stop codon in the sequence. Last, subgenomic RNA 3, which is abundantly transcribed and encapsidated by the virus particle, appears to have no coding ability. We postulate that this novel subgenomic RNA has a regulatory function.

A simple method for the isolation of plant protoplasts

A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been described. The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplast-releasing enzymes that are stable and efficient at higher temperatures. Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum (mannitol or MgSO4), and a protectant (CaCl2 2H2O), all dissolved in distilled water. The protocol has ensured reproducibility, higher yields and is gentle on protoplasts as the protoplasts obtained were amenable to cell wall regeneration and cell division. Plant regeneration was demonstrated forNicotiana tabacum cv. Thompson from protoplasts isolated by this method. Wall regeneration and cell division were obtained in other species. The merits of the protocol are, simple and easy-to-handle procedure, non-requirement of preconditioning of donor plant and explants, incubation without agitation, satisfactory yields, culturability of the protoplasts isolated and applicability of the protocol to a large number of species including mucilage-containing plants.

玉米原生质体培养、超低温保存和组织培养的研究

本文叙述了两个玉米基因型（小八趟×水白和白17）原生质体培养的植株再生;用基因型（小八趟×水白）为材料研究影响玉米原生质体培养的各种因素，并此基因型的原生质体经超低温保存后获得植株再生;以及用多种基因型玉米幼胚为材料诱导愈伤组织与植株再生。 影响玉米原生质体游离、分裂与植株再生的因素是多方面的。酶液组合0.2% Onozuka RS + 1% Hemicellulase + 0.1% Pectolyase，利用继代8-16天的愈伤组织，所获原生质体的数量与质量最佳。在原生质体植板率方面，结果表明：N6作为基本培养基是理想的;氮源中，NO3-具有明显的促进作用，而NH4+具有明显的抑制作用;有机氮源是不能缺少的，所使用的四种有机氮源中L-脯氨酸效果最明显。2,4-D浓度以1.0 mg/l最佳。原生质体培养后的渗透压浓度降低的时间以培养四星期后为宜。利用三步诱导，成功地获得胚胎发生的植株再生，并且还指出原生质体起始材料的保存年限大大影响原生质体所再生愈伤组织的分化。 采用上述筛选出的最佳游离、培养以及植株再生的方法，成功地培养了基因型（白17）的原生质体，并获得植株再生。原生质体再生细胞培养4-5天后开始一次分裂;培养15天后，植板率为3-4%。一个月后，原生质体所再生的肉眼可见的愈伤组织，分步转至分化培养基。最后，愈伤组织通过胚胎发生获得植株再生，频率约10%。 玉米原生质体，利用5%DMSO与0.55 M葡萄糖作为混合保护剂，经慢速（1 ℃/分钟）降至－40 ℃，停留二小时后直接投入液氮保存。保存3天后，原生质体在40 ℃的温水浴中快速化冻，成活率高达30-40%。成活原生质体培养后生长正常，植板率高达8-10%。培养5-6星期后，再生愈伤组织转至分化培养基;最后获得植株再生，频率为5-10%。 本文最后叙述了玉米七种基因型的幼胚诱导获得愈伤组织，再生植株频率可达70-80%。 上述各方面的研究结果，对玉米的遗传操作和细胞抗寒性研究、生理代谢的研究等都是十分有价值的。

青花菜原生质体培养和根癌农杆菌介导的遗传转化

从4-5天龄青花菜(Brassica oleracea var．italica)下胚轴游离原生质体，经纯化后培养在简化的KMsP培养基上，原生质体分裂形成了细胞团;同时，对影响外源DNA导入子叶和下胚轴原生质体后瞬间表达强度的若干因素作了较详细的研究，这些因素包括转化介质中二价阳离子的种类和浓度、PEG溶液的浓度以及PEG溶液的pH值， 为进一步进行原生质体水平上的细胞遗传转化创造了条件。 以青花菜(Brassica oleracea var．italica)子叶和下胚轴为外植体材料，进行了根癌农杆菌介导的遗传转化研究。在建立了子叶和下胚轴外植体组织培养的高频率植株再生系统的基础上，用携带有双元载体质粒的根癌农杆菌(Asrobacterium,tumefaciens)A208sE感染青花菜子叶和下胚轴，对根癌农杆菌的感染过程以及影响抗性芽分化频率的诸多因素作了详细研究，再生了具有卡那霉素抗性的完整转化植株。Dot Blot分析表明NPTⅡ酶活性的存在;以pROA93经EcoRI /HindⅢ酶切产生的gus基因片段(约2.6Kb)为探针进行Southern Blot分子杂交，结果表明gus基因已整合到植物细胞基因组中，并且得到了表达。

普通烟草与毛曼陀罗族间体细胞杂交的研究

采用PEG／DMSO融合法，从普通烟草(Nicotiana tabacum L.cv.Xanthi nc)叶肉和毛曼陀罗(Datura innoxia Mill.)茎或叶愈伤组织原生质体融合获得I5株花盆中健康生长的族间体细胞杂种植株;其中11株是在没有选择压力的条件下获得，4株则是在有IOA和R6G选择系统存在下获得。同时获得一株试管开花且形态异常的植株和一株花盆中生长的嵌合植株。lOmM IOA和I5μ g／ml R6G均能分别有效地抑制烟草叶肉和毛曼陀罗愈伤组织原生质体的分裂;10% DMSO能显著提高原生质体融合率;PEG种类并不重要，但浓度则很重要;BAP较ZT，KT对植株分化有更好的诱导效果。杂种的形态、细胞、同工酶、Southern杂交，花粉育性分析结果如下：1、l5株杂种较双亲普遍株型矮小，生长缓慢，形态接近烟草但不很正常，根据形态特征可分为两种类型：(1)共有8株，其叶片大小、形状、颜色、开花习惯、花类型（单花）等均与毛曼陀罗接近，但子房败育;(2)共有7株，其株型、叶片形状、颜色、光滑度、花形状、类型（圆锥状花序）、颜色更接近烟草，但少数杂种开单花或先单花后圆锥状花序或先单花后两种花并存，且开花时间不一，部分子房败育。2、杂种染色体数目大都在60～90之间，个别者较少(48条)或较多(125条)，没有一株为双二倍体(2n=96)，并全部为混倍体。3、15株杂种植株均有双亲的细胞色素氧化酶同工酶特征谱带;大部分都有双亲的过氧化物酶同工酶特征谱带，少都仅具烟草的谱带。4、Hae Ⅲ／水稻rDNA的Southern杂交分析表明杂种1较双亲多一条谱带，杂种2较双亲也多一条弱带，其它杂种尚待定。5、花粉活力测定表明毛曼陀罗（种子再生而来）的为99%，烟草（原生质体再生而来）为80～90%，而杂种的为24～61%，育性普遍低于双亲。