In Vitro Selection for Salt Tolerance in Brassica juncea L. Using Cotyledon Explants, Callus and Cell Suspension Cultures

Salt-tolerant Brassica juncea L. cell lines or plants have beenselected by screening callus pieces, cell suspension culturesand cotyledon explants in vitro on high concentrations of NaCl.Callus-based selection was unsatisfactory, as only two out ofseven isolated clones retained tolerance after 3 months of subcultureon NaCl-free medium. Selections made via plated cell suspensionswere found to be more stable for salt-tolerance. AH selectedtolerant cell lines, however, failed to regenerate plantlets.A third selection method, employing cotyledon explants was basedon their high potential for regenerating multiple shoots. Outof a total of 2620 explants cultured on high salt media, threesurvived, showed sustained callus proliferation and each regeneratedone shoot. The salt-selected shoots withstood the stabilitytest after 3 months of growth and axillary bud multiplicationon NaCl-free medium. While one of these somaclones was morphologicallyabnormal and sterile, the other two could be reared to maturitywith normal seed set. Brassica juncea, tissue culture, in vitro selection, salt-tolerance, plant regeneration     

Agrobacterium tumefaciens-mediated transformation of cauliflower,Brassica oleracea var. botrytis

We have developed an efficient and simpler method for genetic transformation and regeneration of cauliflower, Brassica oleracea var. botrytis plants. Explants from 4-day old seedlings were inoculated and cocultivated with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector with the neomycin phosphotransferase-II gene under the regulatory control of nopaline synthase promoter and terminator sequences, permitting transformed shoots to be selected on kanamycin containing medium. After three months rooted transformed plantlets were successfully transferred and grown under glasshouse conditions. Higher numbers of transformed plants were obtained from cotyledon than hypocotyl explants, presumably indicating cotyledons of cauliflower are more amenable to genetic transformation. Integration and expression of the introduced transgene were analysed by DNA gel blot and PCR analysis and NPT-II expression assay. Factors influencing transformation efficiency include explant age, concentration of bacterium used for infection, duration of infection and cocultivation with Agrobacterium. Transgenic plants of three commercial genotypes of cauliflower were produced using this method. We also show that introduction of antisense Bcp1 (pollen-specific gene) linked to a pollen-specific promoter (Lat52) resulted in the expected sterility of 50% pollen carrying this transgenic construct.     

Temporal and spatial root development of cauliflower (Brassica oleracea L. var. botrytis L.)

Row crops are often inefficient in utilizing soil resources. One reason for this appears to be inefficient rooting of the available soil volume. Five experiments were performed to study the temporal and spatial root development of cauliflower (cv. Plana). The crop was grown with 60 cm between rows, and root development was followed in minirhizotrons placed under the crop rows, 15 cm, and 30 cm from the crop rows. Soil was sampled and analyzed for nitrate content at the final harvest and once during growth. In two of the experiments N fertilizer rate was varied and in two of the other experiments two cultivars were compared (cv. Plana and Siria).The rooting depth of cauliflower was found to be linearly related to temperature sum, with a growth rate of 1.02 mm day^-1 °C^-1. Depending on duration of growth this leads to rooting depths at harvest of 85–115 cm. Soil analysis showed that the cauliflower was able to utilize soil nitrogen down to at least 100 cm.With Plana differences in root growth between row and interrow soil were only observed during early growth, but with Siria this difference was maintained until harvest. However, at harvest both cultivars had depleted row and interrow soil nitrate equally efficient. Nitrogen fertilizer did not affect overall root development significantly.The branching frequency of actively branching roots was increased in all soil layers from about 6 to 10 branches cm^-1 by increasing N fertilizer additions from 130 to 290 kg N ha^-1. Increasing N supply increased the number of actively branching roots in the topsoil and reduced it in the subsoil.The average growth rate of the roots was always highest in the newly rooted soil layers, but fell during time. At 74 days after planting very few roots were growing in the upper 60 cm of the soil whereas 70% of the root tips observed in the 80–100 cm soil layer were actively growing. Within each soil layer there was a large variation in growth rate of individual root tips.     

Root, Callus, and Cell Suspension Cultures, from Atropa belladonna, L. and Atropa belladonna, Cultivar lutea Doll

Root, callus, and cell suspension cultures have been establishedfrom seedlings of Atropa belladonna, L. and Atropa belladonna,cultivar lutea Döll. The growth of these cultures is described.Callus cultures transferred to auxin (-naphthaleneacetic acid)-freemedium initiated roots and shoots. Excised root cultures havebeen established from such roots and plants from such shoots.Extracts of the cultures have been submitted to the Vitali—Morinreaction and following chromatography, to the Dragendorff reaction.Cultured excised roots and plants raised from shoots initiatedon cultured callus were shown to contain atropine (hyoscyamine)and reactive substances corresponding in Rf to hyoscine andcuscohygrine. These alkaloids were absent from cultured callusand cultured cell suspensions and from leaves when initiatedwithout roots on callus. The cultured calluses and cell suspensionscontained choline (0.022–0.027 g per 100 g dry weightof root callus). The growth of cell suspension cultures wasnot inhibited by incorporating atropine sulphate, L-hyoscyamine,L-hyoscine hydrobromide, or DL-scopoline nitrate in the culturemedium at 250 mg/I. These alkaloids were absorbed by the cells,a high proportion of the added alkaloid could be recovered fromthe cultures even after 4 weeks' growth and no evidence wasobtained of the presence of degradation products of the alkaloids.The suppression of alkaloid formation in actively growing callusand cell suspension cultures is discussed.     

Changes in Lipid Composition during Callus Differentiation in Cultures of Oilseed Rape (Brassica napus L.)

The lipid composition of different callus cultures of Brassicanapus varied according to their state of differentiation. Photomixotrophiccallus was characterized by the ability to synthesize relativelyhigh levels of triacylglycerol (TAG) which was rich in oleate.Glycosyldiacylglycerols were also detected. In contrast, heterotrophiccallus was found to possess high proportions of membraneousphospholipids which were rich in palmitate, linoleate, and linolenate.Moreover, the lipid content was considerably less than thatof photomixotrophic callus. Caulogenesis was achieved in bothtypes of callus strains and the lipid composition of the regeneratedleaves contained a much higher proportion of chloroplast glycosyldiacylglycerolsand thus resembled more those of the parent plant. Some callientered a senescent phase whereby there was considerable degradationof the constituent membrane lipids. Senescent callus also exhibiteda high proportion of polyploid nuclei. In this study we havebeen able to cause large changes in the morphology of calluscultures. These morphological changes were accompanied by significantalterations in the quality and quantity of acyl lipids. In photomixotrophiccells the lipid changes resembled those seen for developingseed tissues where high rates of TAG deposition are accompaniedby an altered fatty acid pattern. Thus, the selection of differentcallus types should be of use for investigations of the regulationof lipid biosynthesis under controlled culture conditions.     

甘蓝自交不亲和反应的细胞信号转导

S受体激酶(S—receptor kinase,SRK)和S位点富含半胱氨酸(S-locus cysteine-rich,SCR)分别是甘蓝柱头和花粉中导致自交不亲和反应的决定性蛋白质因子。本文就SRK、SCR的结构和功能加以综述,阐明两者在细胞信号转导中的作用。     

生姜离体叶片愈伤组织悬浮细胞系建立与植株再生

以‘莱芜大姜’为试材,研究了生姜离体叶片愈伤组织的诱导以及细胞悬浮系建立与植株再生。结果表明,以生姜试管苗叶片为外植体,接种到MS+1.0 mg/L 2,4-D+0.5 mg/L 6-BA+30 g/L蔗糖的培养基上,可有效诱导出生长迅速、质地疏松的愈伤组织。将获得的愈伤组织接种到MS+0.15 mg/L 2,4-D+6.0 mg/L 6-BA+30 g/L蔗糖的液体培养基上,25℃黑暗条件下震荡培养25-30 d,可建立分散性好、生长迅速的悬浮细胞系,细胞悬浮系培养的适宜参数为:初始接种量为1.0-1.5 g,继代培养的适宜间隔期为15 d,继代培养液体培养基更新比例为3/4。将悬浮细胞接种到固体培养基MS+0.2 mg/L NAA+10.0 mg/L 6-BA+30 g/L蔗糖上可获得再生植株。     

Effect of Different Sugars on Photosynthesis and Chlorophyll Fluorescence in Photoautotrophic Tomato Suspension Cell Cultures

The effects of metabolisable sugars sucrose and glucose along with non-metabolisable isomers of sucrose palatinose and turanose were tested. Rate of oxygen evolution (P), electron transport rate (ETR), and photochemical quenching (q_p) showed substantial decrease after 24 and 48 h by glucose and sucrose treatments, whereas there was no effect on all these parameters by the treatment with palatinose and turanose. Also the F_v/F_m ratio remained constant through the time of studies revealing that the maximal photochemical capacity of the cells was unchanged. Non-photochemical quenching (q_N) showed a decrease compared to the control values by all the treatments. Hence P and Chl fluorescence parameter were affected only by those sugars which are used in the metabolic pathways and not by sugar analogues.     

The Growth, Anatomy and Morphogenetic Potential of Callus and Cell Suspension Cultures of Hevea brasiliensis

Callus cultures have been initiated from stem explants of youngplants of Hevea brasiliensis and maintained over long periodsat 30 ?C by serial subculture in Murashige and Skoog mediumcontaining 2 mg 1^–1 2,4-D and 0.5 mg 1^–1 kinetin.Newly-initiated cultures spontaneously initiated roots but,on serial subculture, this property was lost and the culturesbecame heterogeneous (consisting of proliferating light segmentsand darker compact non-growing segments). Serially propagatedcultures continued to differentiate a few scattered latex vesselscontaining particulate material similar to that in the rootlaticifers. This callus (O callus) did not yield a growing cellsuspension when transferred to agitated liquid medium. However,the large cell aggregates which could be recovered after twopassages in liquid medium, when again grown on solid mediumyielded a highly friable light-coloured fast-growing homogeneouscallus (R callus) which retained its distinctive character onsubculture. This callus when transferred back to agitated liquidmedium yielded a fine rapidly growing cell suspension culturewhich could be serially propagated at 30 ?C in the same mediumas that used for callus culture. Both the O and R cultures were2,4-D dependent, but differed in their responses to 2,4-D. Bothretained their diploid character when serially propagated. Serially-propagatedsuspensions came to contain a proportion of polyploid cells.When the suspensions were maintained for several months withoutsubculture the larger cell aggregates which developed gave riseto embryo-like structures. Attempts to promote the further developmentof these embryo-like structures into plantlets were unsuccessful.     

Activity of Wall-bound Enzymes in Callus Cultures of Gossypium hirsutum L. During Growth

Activities of - and ß-glucosidase, - and ß-galactosidase,-mannosidase, ß-1,3-glucanase, acid and neutral invertaseswere detected in the cytoplasmic fraction as well as in cellwalls isolated from callus cultures of cotton. Activity of ß-mannosidase,however, could not be detected in the cell walls. Transfer ofcallus to a fresh medium did not immediately influence the activitiesof -glucosidase and ß-galactosidase but increasedsignificantly ß-glucosidase, -mannosidase, acid andneutral invertases. Addition of cycloheximide (1 and 100 mgl^–1) further stimulated acid and neutral invertases butnot other enzymes tested. Sodium chloride (NaCl) was effectivein extracting a-glucosidase, ß-glucosidase, ß-galactosidase,acid and neutral invertases. EDTA extracted most of the -galactosidase,-mannosidase, ß-1,3-glucanase and some -glucosidase.But, NaCl and EDTA could not extract some of the - and ß-glucosidasesand also acid and neutral invertases as evidenced from the residualand extra cellular activity. Studies with whole cells as a sourceof enzyme revealed that some of these enzymes were associatedwith the cell surface. Callus, glycosidases, glucanase, growth, Gossypium hirsutum     

Changes in Peroxidase Activity in Bean Suspension Cultures after B. cinerea and Elicitor Treatment

Treatment of cell suspension cultures of bean ( Phaseolus vulgaris ) c.v. 'Gold Saxa') with Botrytis cinerea resulted in the increase in extra- and intracellular peroxidase activity. A similar effect was obtained by treatment of cell suspension cultures with an elicitor active extract from fungal mycelium.
It was shown by means of electrofocusing that the infection- or elicitor-related increase in total intracellular peroxidase activity was associated with the increase in the activity of a specific isozyme with an isoelectric point of 4.8. This anionic peroxidase did not differ in substrate specificity to guaiacol, syringaldazine and chlorogenic acid.     

Plant Regeneration of Protoplasts of Trifolium lupinaster L. from Cell Suspension Cultures

paper deals with regeneration of protoplasts in cell suspension cultures of hypocothl from Trifolium lupinaster L. on the SL2 basal medium with BA 0.1 mg/L and picloram 0.06 mg/L for 3--4 month,s. The protopiasts were isolated from suspensions cells subcultured for 3 days and were recuhured in modified liguid medium 8p. The first division of the regenerated cell occurred 3 days after being cultured in medium Bp. Small calli could be seen with naked eyes by one month. The calli when grew up to 2 mm long, were transferred in succession differentiation medium A and B for organ differentiation. The differentiated shoots formed their roots on 1/2 MS supplamented with NAA 1.0mg/L and then grew into plantlets.     

怀牛膝细胞悬浮培养及多糖含量变化的研究

采用正交实验设计研究基本培养基、碳源、2,4-D、6-BA、CH及接种量对怀牛膝悬浮培养细胞生长和细胞中多糖含量的影响。结果表明,(1)6-BA是影响怀牛膝细胞生长的关键因子,其影响效应依次为6-BA>CH>接种量>基本培养基>2,4-D>碳源,细胞生长的适宜培养基为MS 30 g/L葡萄糖 2 mg/L 2,4-D 1 mg/L 6-BA 0.5 g/L CH,适宜接种量为30 g/L。(2)碳源是影响细胞中多糖含量的关键因子,其影响效应依次为:碳源>2,4-D>6-BA>CH>接种量>基本培养基,利于细胞中多糖含量提高的适宜培养基为:LS 30 g/L蔗糖 0.5 mg/L 6-BA 0.5 g/L CH,适宜接种量为30 g/L。     

青花菜花粉母细胞减数分裂及雄配子体发育

采用染色体制片及爱氏苏木精染色-冬青油透明技术对青花菜花粉母细胞减数分裂及雄配子体发育过程进行了细胞学研究.结果表明:花粉母细胞减数分裂的细胞质分裂为同时型,四分体为正四面体型或十字交叉型;中期Ⅰ和中期Ⅱ,少数细胞可见赤道板外染色体;后期Ⅰ和后期Ⅱ部分细胞出现染色体桥及落后染色体;四分体时期可观察到二分体、三分体及含微核的异常四分体.雄配子体发育过程包括2次有丝分裂,成熟花粉为3细胞型,具3个萌发孔.减数分裂过程中染色体行为异常的花粉母细胞约占10.28%;雄配子体发育过程中异常频率约为3.2%,败育主要发生在单核期.     

Characterization of Growth, Water Relations, and Proline Accumulation in Sodium Sulfate Tolerant Callus of Brassica napus L. cv Westar (Canola)

Unselected and sodium sulfate tolerant callus cultures of Brassica napus L. cv Westar were grown on media supplemented with mannitol, NaCl, or Na₂SO₄. In all cases, growth of tolerant callus, measured on a fresh weight or dry weight basis, was greater than that of unselected callus, which was also subject to necrosis on high levels of salt. Tissue water potential became more negative in both unselected and tolerant callus grown in the presence of mannitol or Na₂SO₄. Water potentials in unselected callus were more negative than those of the tolerant tissues; but over a range of Na₂SO₄ concentrations both cultures displayed osmotic adjustment, maintaining relatively constant turgor. Proline accumulation in both unselected and tolerant callus was low (15 to 20 micromoles per gram dry weight) in the absence of stress, but increased on media supplemented with mannitol, NaCl, or Na₂SO₄. Increases in proline concentration were approximately linear in tolerant callus, reaching a maximum of 130 to 175 micromoles per gram dry weight. In unselected callus, concentrations were higher, reaching 390 to 520 micromoles per gram dry weight. Proline accumulation was correlated with inhibition of growth, and there was a negative correlation between proline concentration and culture age for tolerant callus.     

Organogenesis in Cell Suspension Cultures of Atropa belladonna L. and Atropa belladonna Cultivar lutea Doll

Callus cultures have been initiated from excised cultured rootsof Atropa belladonna and A. belladonna cultivar lutea and usedto establish suspension cultures in a synthetic culture medium(referred to as SSM) containing 2.0 mg/1 -naphthaleneaceticacid (NAA). Visible cellular aggregates develop in these suspensioncultures and when such aggregates are cultured in an NAA-omittedmedium, large numbers of roots arise superficially on the aggregates.Root development is enhanced by incorporating into the auxin-freemedium either tropic acid or I-naphthoxyacetic acid and boththese substances can promote root initiation in the presenceof levels of NAA which alone suppress organogenesis. The aggregatesalso give rise under appropriate conditions of culture to shootsand to embryo-like structures. The nature and frequency of suchmorphogenesis in the aggregates is dependent not only upon thecomposition of the culture medium but upon the number and thelength of previous culture passages through which the callusand suspension cultures have been propagated in the SSM. Theultimate loss of morphogenetic potential in serially propagatedcallus is discussed. The structure of the aggregates in SSM and in the media promotingroot initiation is described. Examination of the aggregatesfor their content of alkaloids indicates that the major belladonnaalkaloids can only be detected in cultures forming roots.     

GA_3对老化花椰菜种子活力和几种相关生理生化性状的影响

用10、50、100、150和200mg·L-15种浓度赤霉素（GA3）溶液处理于10℃冰箱中贮藏5年的花椰菜老化种子。结果表明,100mg·L-1GA3浸种22h的效果最佳,老化种子的发芽率、发芽势、活力指数、根长均有提高,畸形苗率下降;过氧化物酶（POD）、过氧化氢酶（CAT）和脱氢酶活性提高,可溶性蛋白和叶绿素含量也提高,种子浸出液的电导率和丙二醛（MDA）含量则下降。另外,100mg·L-1GA3处理后的种子在温度为25℃条件下贮藏时间不宜超过25d。