Synthesis of male sterile,triazine-resistant Brassica napus by somatic hybridization between cytoplasmic male sterile B. oleracea and atrazine-resistant B. campestris

Summary Fusion of leaf protoplasts from an inbred line of Brassica oleracea ssp. botrytis (cauliflower, n=9) carrying the Ogura (R1) male sterile cytoplasm with hypocotyl protoplasts of B. campestris ssp. oleifera (cv Candle, n=10) carrying an atrazine-resistant (ATR) cytoplasm resulted in the production of synthetic B. napus (n=19). Thirty-four somatic hybrids were produced; they were characterized for morphology, phosphoglucose isomerase isoenzymes, ribosomal DNA hybridization patterns, chromosome numbers, and organelle composition. All somatic hybrids carried atrazine-resistant chloroplasts derived from B. campestris. The mitochondrial genomes in 19 hybrids were examined by restriction endonuclease and Southern blot analyses. Twelve of the 19 hybrids contained mitochondria showing novel DNA restriction patterns; of these 12 hybrids, 5 were male sterile and 7 were male fertile. The remaining hybrids contained mitochondrial DNA that was identical to that of the ATR parent and all were male fertile.     

Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion

Summary With the idea to develop a selection system for asymmetric somatic hybrids between oilseed rape (Brassica napus) and black mustard (B. nigra), the marker gene hygromycin resistance was introduced in this last species by protoplast transformation with the disarmed Agrobacterium tumefaciens strain C58 pGV 3850 HPT. The B. nigra lines used for transformation had been previously selected for resistance to two important rape pathogens (Phoma lingam, Plasmodiophora brassicae). Asymmetric somatic hybrids were obtained through fusion of X-ray irradiated (mitotically inactivated) B. nigra protoplasts from transformed lines as donor with intact protoplasts of B. napus, using the hygromycin resistance as selection marker for fusion products. The somatic hybrids hitherto obtained expressed both hygromycin phosphotransferase and nopaline synthase genes. Previous experience with other plant species had demonstrated that besides the T-DNA, other genes of the donor genome can be co-transferred. In this way, the produced hybrids constitute a valuable material for studying the possibility to transfer agronomically relevant characters — in our case, diseases resistances — through asymmetric protoplast fusion.     

A comparison of anther and microspore culture as a breeding tool in Brassica napus

Summary A direct comparison of microspore culture and anther culture was made in Brassica napus using F₁ crosses of Regent (canola) by Golden (rapeseed), and their reciprocals, as well as a hybrid between Reston and a highly embryogenic, canola-quality breeding line (G231) as donor plants. The study confirmed that microspore culture can be ten times more efficient than anther culture for embryo production. Embryo yields from cultures initiated from the Reston x G231 were four-fold greater than those initiated from the Regent x Golden crosses, and significant differences were also detected among cultures initiated from the different Regent x Golden crosses. These results illustrate the influence that donor plant genotype has on embryo production. However, superior embryogenic potential among donor material was not always coincident with superior plant production. The average haploid-todiploid ratio in microspore-derived regenerates was 21 for the population obtained from the Regent x Golden crosses but 11 for the Reston x G231 cross. For both types of material, the frequency of diploids increased upon repeated cycles of explanting. A field study showed that there were no differences between the populations of anther-derived and microspore-derived spontaneous diploid and doubled haploid lines, with respect to the days required for them to flower or to mature. The information is valuable for canola breeding programs considering the use of haploidy.     

Regeneration of plants from hypocotyl protoplasts of rapessed (Brassica napus L. var. oleifera) cultivars

Protoplast cultures were prepared from hypocotyls of ten spring rapeseed cultivars. Protoplasts from all genotypes tested formed calli, and shoots were regenerated from calli of nine of the genotypes at frequencies varying from 15 to 76%. The regenerating cultivars fell into a high regenerating group (>60% and a low regenerating group <25%).     

Growth and shoot formation in protoplast-derived calli of Brassica oleracea ssp. acephala and ssp. capitata

The effect of media components and environmental factors on growth and organogenesis of protoplast-derived calli of curly kale and cabbage were tested. Optimal growth (fresh weight increase of calli, shoots and roots) was found at 60 mM sucrose. Lower sucrose concentrations (3–30 mM) were favourable for shoot formation. Nitrate concentrations from 23 to 100 mM in combination with 8 or 21 mM ammonium were optimal for shoot formation. However, growth was reduced by high (100 mM) nitrate concentration. The effects of various organic nitrogen compounds at 0.5 and 2 mM were tested. Glutamine did not influence shoot formation and barely growth. Proline at 0.5 mM stimulated growth of cabbage calli but decreased growth of curly kale calli, and at 2 mM, proline also inhibited shoot production. Adenine sulphate decreased growth of cabbage calli at 0.5 mM, and at 2 mM shoot production was also reduced. Spermidine and spermine inhibited both growth and differentiation. Putrescine resulted in about 50% higher fresh weights, and also increased the number of calli producing shoots by about 35%. More calli produced shoots in white light than in blue or red light or in darkness. The length of the photoperiod or intensity of light was not critical for shoot production.     

原生质体来源的大白菜悬浮细胞系的超低温保存

原生质体来源的大白菜 Brasstca campessris var.pekinsis 悬浮细胞系在二甲亚砜的保护下,能在液氮中(-196℃)长期冻存。加入山梨醇能增强保护作用;而加入甘露糖则降低保护作用。培养基对冻存也有明显的影响。在液氮中存放的时间长短对细胞存活率没有多大影响。冻后相对活性最高可达75.4％,恢复生长快,化冻后重新悬浮培养6天,生长量可达300-500％。遮光比不遮光对恢复更有利。冻存后恢复生长的悬浮细胞,能与未经冰冻的对照一样进行原生质体分离和培养。     

Plant regeneration from callus and protoplasts of Brassica nigra (IC 257) through somatic embryogenesis

A method to obtain plants from embryogenic callus of Brassica nigra and protoplasts of hypocotyl expiants is described. Callus was initiated on Murashige and Skoog medium containing kinetin (kn) and 2,4-dichlorophenoxy acetic acid (2,4-D). Lowering of auxin induced embryo formation. Supplementation with gibberellic acid (GA₃) enhanced embryogenic response tenfold. Passage through liquid medium devoid of growth regulators was essential for the growth of embryos. Secondary embryos were produced on transfer to solid basal medium. Embryogenic callus retained its morphogenic ability even after 12 subcultures. Both primary and secondary embryos produced fertile plants. Hypocotyl-derived protoplasts were also regenerated to plants following the same protocol. The survival of plants on transfer to soil was about 80%. The seeds from plants derived from callus and protoplasts were viable.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA naphthalene acetic acid - IAA indole acetic acid - kn kinetin - GA₃ gibberellic acid     

Microsporogenesis in a normal line and in the ogu cytoplasmic male-sterile line of Brassica napus

Summary In the ogu cytoplasmic male-sterile (CMS) line of Brassica napus, stamen morphology was influenced by temperature conditions. Under a high temperature regime (27° C/23° C; day/ night) CMS stamens had a near-normal morphology, but microsporogenesis proceeded to a maximum of the microspore stage. However, compared to the normal stamens, the occurrence of sporopollenin-like deposits in the tapetum and deposition of exine on the microspores was sparse. Also, the tapetal cells of the CMS line were often highly vacuolate and failed to degenerate at the same stage as the normal. Ultrastructural changes in the mitochondrial matrix and cristae plus dilation of the endoplasmic reticulum, which occurred during development in sporogenous tissues of the normal line, were often lacking or mistimed in the mutant. Due to extensive variation, even between adjacent locules, the cytological differences between the normal and CMS anthers cannot be ascribed as the cause of male sterility in the ogu CMS line of B. napus, rather they may be the consequence of it.     

甘蓝型油菜芥酸和二十碳烯酸含量的基因效应

以甘蓝型油菜的4种纯合芥酸基因型之间所有可能的6个杂交组合的P_1、P_2、F_1、P_2、B_1和B_2世代为材料,用生统遗传学方法研究了芥酸和二十碳烯酸的基因作用形式及效应。发现无论亲本是单基因差异还是二基因差异,F_1和F_2代的芥酸含量都接近中亲值,F_1略大于中亲值和F_(20)世代均值分析表明,芥酸含量的遗传符合加性显性模型,加性效应占绝对优势,显性效应不显著。用数量遗传学方法估计的芥酸基因数与已知的结果相近。     

Organogenesis and plant formation from cotyledon explant cultures of wild turnip rape (Brassica tournefortii L.)

Cotyledon explants of Brassica tournefortii L. were excised from germinated seedlings and cultured on Murashige & Skoog's [6] basal medium supplemented with various combinations of cytokinins and auxins, Both cytokinin and auxin were required for induction of shoot organogenesis. Of the three cytokinins tested (in combination with a low concentration of IAA), kinetin was found to be the best for shoot regeneration. On this medium, cotyledonary explants invariably underwent callusing followed by multiple shoot formation. NAA in combination with any of the three cytokinins yielded a reduced number of shoots or none, but favoured good callus growth. Callus so produced also regenerated shoots when subcultured on media containing high concentration of KIN or ZEA and low concentration of IAA. Shoots were rooted during prolonged incubation on the same medium or on MS medium free of growth regulators. Mature plants were grown in the greenhouse.