紫薯愈伤组织诱导和培养条件优化及绿原酸类化合物的积累

以紫薯叶片为外植体,通过筛选培养基和培养条件建立了紫薯愈伤组织培养体系。研究6-苄基氨基嘌呤（6-BA）及萘乙酸（NAA）不同浓度组合对紫薯愈伤组织诱导的影响,之后分别考察基础培养基MS、1/2MS、B5、WPM、White及NAA、6-BA、2,4-二氯苯氧乙酸（2,4-D）三种激素对紫薯愈伤组织继代生长的影响。确定最适基础培养基后,对激素进行单因素实验后再做正交实验。结果表明1/2MS基本培养基中添加1.5 mg/L 6-BA和0.4 mg/L NAA适合紫薯愈伤组织诱导,诱导率为81.82%。MS为优选基础培养基,通过正交实验优化的激素配比组合为1.0 mg/L NAA,0.4 mg/L 6-BA,1.0 mg/L 2,4-D。最佳继代培养基为MS+1.0 mg/L NAA+0.4 mg/L 6-BA+1.0 mg/L 2,4-D+6 g/L琼脂+30 g/L蔗糖,此优化激素条件下愈伤组织的生物量增长倍数为23.46倍,3-咖啡酰奎宁酸、3,5-二咖啡酰宁酸、4,5-二咖啡酰宁酸三种绿原酸类化合物含量分别为0.30%、1.91%、1.10%,三者总含量3.31%,因此,通过紫薯愈伤组织培养生产绿原酸类化合物是可行的。      

EMS诱变甘蔗愈伤组织的初步研究

为了探索EMS对甘蔗愈伤组织进行化学诱变的合适浓度和时间组合,以ROC22和粤糖93-159甘蔗愈伤组织为材料,研究了不同浓度(8、24、40μmol/L)、不同时间(2、4、6 h)诱变处理对愈伤组织的每克鲜重相对增重量和相对分化率的影响。并通过分化试验选择适合诱变后愈伤组织分化的培养基。结果表明,适合诱变处理后愈伤组织分化的培养基为:MS基本培养基+2 mg/L KT+2 mg/L NAA+3.0%蔗糖(W/V)+0.8%琼脂(W/V)。适合EMS诱变甘蔗的处理为:24μmol/L诱变处理2 h或8μmol/L诱变处理6 h。     

Effects of explant source,culture medium: Strength and growth regulators on the in-vitro propagation of three Jamaican yams: (Dioscorea cayenensis,D trifida and d rotundata)

Explants of tuber, meristem and vines from three widely cultivated yam species in Jamaica—D cayenensis, D rotundata and D trifida—were examined for their responses to mineral media strength, inorganic ammonium and growth regulator supplements. Tuber pieces (5mm³) showed some positive growth responses but did not produce in-vitro plantlets on all the media tested. Meristem tips of D trifida grew rapidly on basal media (BM) supplemented with either 0.1 mglitre^?1 6-benzylamino purine (BAP) and 0.01 mg litre^?1indole butyric acid or 0.2 mg litre^?1 BAP and 1.0 mg litre^?1 naphthalene acetic acid (NAA) producing plantlets by 28 weeks. The nodal explants grew rapidly with plantlets obtained from all the cultivars within 4 weeks. Use of young, vigorously growing vines of 8 weeks or less, as explant source, gave low contamination levels (16–25%) in culture when sterilised for 30 min in 200 g litre^?1 NaOCl in the case of D trifida and 300 g litre^?1 NaOCl in the case of D cayenesis prior to culturing. Initiation of growth was optimal when explants were taken from monopodial vines grown in October or January and placed on BM supplemented with 0.5 mg litre^?1 BAP (BM0.5BAP). Addition of 0.5 mg litre^?1 kinetin to the BM or 0.05 mg litre^?1 NAA to BM0.5BAP depressed shoot production, while 5.0 mg litre^?1 kinetin increased swelling of the nodal region in explants from sympodial shoots and from vines grown in March. The results suggest that nodal segments excised from young, fast growing vines of these species are the best explant source for the purpose of commercial micropropagation.     

紫苏细胞悬浮培养生产迷迭香酸条件研究

通过诱导紫苏下胚轴和子叶外植体产生愈伤组织,建立细胞悬浮培养体系,以提高细胞产量及细胞中迷迭香酸含量。结果表明,在MS液体培养上添加3.0mg/L 6-芐氨基嘌呤(6-BA)＋0.3mg/L萘乙酸(NAA)培养基上,下胚轴愈伤组织呈嫩黄色松散状态,出愈时间短,出愈率可达100%。紫苏细胞悬浮培养产生迷迭香酸的最佳条件为培养时间7d、接种量鲜质量浓度20g/L、摇床转速110r/min、蔗糖30g/L、L-苯丙氨酸0.15g/L,此条件下可获得高达2.283mg/g的迷迭香酸。     

Study on Flax Genetic Transformation Mediated by Agrobacterium tumefaciens

Abstract

Flax is an important natural material for the linen spinning industry and as an oil crop in China. Flax products worth US $100 million are exported every year. Recently, with the development of a market economy and China's entry into the WTO, farmers and flax factories have shown a need for new varieties that have a short vegetation period, are resistant to herbicides and lodging, and have a high fiber content. In order to resolve these problems quickly, we have studied flax genetic transformation since 1998.

The paper is about flax genetic transformation by Agrobacterium tumefaciens strain EHA105. The explants were hypocotyl segments from 5-7 day old seedlings. The segments cannot form a callus when the concentration of kanamycin is 30 mg/L and 50 mg/L. Therefore, 50 mg/L of kanamycin was selected as an efficient concentration to select the transgenic plants. The segments of hypocotyl were treated with EHA105 and then were inoculated in different media (Ms, B5, N6). The experiment showed that Ms is a preference medium for flax genetic transformation. Agrobacteriumtieated segments of hypocotyl were cultured on the surface of modified Ms medium (Ms agar medium supplemented with 3.5 mg/L IAA, 2 mg/L KT, 150 mg/L LH). Callus was formed by 71.4%-81.6% of the hypocotyls. Callus inoculated in a regenerative medium of Ms with little BA and NAA could regenerate at the rate of 31.8%-40.2%, and 87.4% of regenerative plants could root in the medium of 1/2 Ms supplemented with 0.001 mg/L NAA. This transformation system has been tested by the GUS-INT gene. After co-cultivation of the segments of hypocotyl and GUS-INT, the segments of hypocotyl were moved to callus medium. After 3 days, the transformation rate of new callus was 72.0%; after 4 weeks the transformation rate of callus was 24.0%; the transformation rate of regenerative plants was 7.5%. The experiment allowed for the establishing of a flax genetic transformation system mediated by Agrobacterium tumefaciens.     

花生组织培养及高频率植株再生

以14个花生品种为材料,对花生组织培养及植株再生进行了研究。将花生成熟胚幼叶、子叶和下胚轴外植体接种到添加0—2mg／LNAA和0～10mg／LBAP的MSB,（MS无机盐＋B5有机）培养基上诱导愈伤,再转到添加0～10mg／LBAP的分化培养基上诱导不定芽。结果表明,诱导培养基中添加的激素及其浓度对以后在分化培养基上不定芽分化有重要作用,以添加1mg／LNAA和6mg／LBAP最利于不定芽分化;分化培养基中添加4mg／LBAP利于不定芽伸长及植株再生;幼叶外植体分化不定芽频率明显高于子叶和下胚轴;不同基因型不定芽分化率存在明显差异,白沙1016和花育23幼叶外植体获得了较高的不定芽分化率,分别达到91．8％和88．5％。再生苗经生根培养和驯化后,移栽花盆可正常开花结果。     

Vodka production from potato (Solanum tuberosum L.) using three Saccharomyces cerevisiae isolates

The efficacy of three strains of Saccharomyces cerevisiae in the production of vodka from potato hydrolysate was evaluated. For each isolate, two treatments were performed: one containing potato hydrolysate (11% w/w of soluble solids) and the other containing potato hydrolysate supplemented with sucrose (17% w/w) in the fermentation medium. The best results were obtained using a baker's yeast in the second treatment, which showed higher substrate‐to‐product conversion (0.47 g ethanol g^?1 total reducing sugars), higher fermentation yield (91.4%) and a higher ethanol content (6.05% v/v). Following fermentation, the medium was clarified using centrifugation, and two successive distillations and filtrations were conducted. The results were examined by referring to the standards for vodka, as established by Brazilian legislation. The ethanol concentration (39.7% v/v) fell within the legally stipulated range, and the levels of copper and furfural remained undetectable. However, the methanol (35.04 mg 100 mL^?1 of anhydrous alcohol) content and the levels of some secondary compounds (148.33 mg 100 mL^?1) were both higher than the guidelines (20 and ≤50 mg 100 mL^?1, respectively). Copyright © 2016 The Institute of Brewing & Distilling     

AN IN SITU METHOD TO STUDY THE REACTION CATALYZED BY ALKALINE PHOSPHATASE ON DNPP

The reaction catalyzed by alkaline phosphatase on disodium‐p‐nitrophenyl phosphate was studied in a reaction media composed of sucrose (43.5 and 53.5%, w/w) and sucrose (43.5%, w/w) + polysaccharide (10%, w/w) added with agar‐agar (0.5%, w/w) at low temperatures (20, 0 and ?5C). The results show that temperature and concentration of sucrose and sucrose + dextran in the media affect the enzymatic reaction. The presence of 10% dextran (molecular weight of 10⁴ g/mol, w/w) in the reaction medium decreased the reaction rate. This could be explained by the increase in the viscosity of the medium, due to the presence of this polysaccharide. The experimental data were compared with predicted values, calculated from Atkin's theory. The results show that in our experimental conditions described previously, the reaction catalyzed by alkaline phosphatase cannot be predicted by Atkin's theory, probably because the viscosity was not the main parameter governing the reaction.     

In vitro culture of Pandanus amaryllifolius and enhancement of 2‐acetyl‐1‐pyrroline,the major flavouring compound of aromatic rice,by precursor feeding of L‐proline

Shoots, plantlets and semi‐differentiated callus (SDC) cultures of Pandanus amaryllifolius capable of producing high levels of basmati rice flavour were established in vitro using Murashige and Skoog nutrient medium. A total of 10% of the initial explants responded to produce shoot cultures in the presence of benzylamino purine (BAP) (0.5 mg L^?1) and glutamine (100 mg L^?1). Leaf explants and basal portions of shoots produced SDC whereas elongated in vitro shoots could be continuously multiplied, using BAP (1.5 mg L^?1) and kinetin (Kn) (1.0 mg L^?1), and rooted in half‐strength medium for ex vitro cultivation leading to a process of micropropagation. Steam‐distillation extraction (SDE) followed by gas chromatography‐mass spectrometry (GC‐MS) analysis of various cultured organs and spent liquid medium used for SDC revealed the presence of 2‐acetyl‐1‐pyrroline (2‐AP) to various extents. This 2‐AP compound has been identified as the major flavouring compound of scented basmati and other scented rice varieties. 2‐AP was found to be highest, on a fresh weight basis, in SDC (19.7 mg kg^?1) on the 40th day, whereas in vitro roots, shoots and field leaves (of one‐year‐old plant) had lower levels of 15, 6.8 and 14 mg kg^?1, respectively. Further enhancement of 2‐AP in SDC using precursor was possible by feeding into medium 1 mmol L^?1 of L ‐proline where a highest level of 21.67 ppm of 2‐AP accumulated on the seventh day whereas a higher level of 2 mmol L^?1 of L ‐proline suppressed 2‐AP levels. The present report is the first on the tissue culture studies of P. amaryllifolius where continuous production of plantlets as well as synthesis of high levels of 2‐AP has been documented. Copyright © 2005 Society of Chemical Industry     

基因工程方法抗甜菜丛根病病毒育种的研究：（Ⅰ）甜菜坏死黄脉病毒外 …

采用含有甜菜坏互黄脉病毒外壳蛋白（ＢＮＹＶＶ ＣＰ）基因的农杆菌，分别以不同甜菜品系的叶柄、下胚轴及子叶为外植体材料进行基因转化研究。含有ＢＮＹＶＶ ＣＰ基因和卡那抗性筛选基因的农杆菌与甜菜组织不同外植体共培养后，经过诱导分化培养，在含有卡那霉素的培养基上，从叶柄及下胚轴分别直接诱导出抗卡那霉素再生芽，而从子叶上只诱导出紧密型绿色愈伤组织，未分化出不定芽。从叶柄及下胚轴诱导两生芽的诱导培养基分别为     

预防组培中蓖麻子叶节玻璃化的研究

为了预防蓖麻子叶节在组织培养过程中的玻璃化现象,以建立其高效的遗传转化体系,本实验对影响子叶节生长状态的几个因素进行了初步研究。结果表明,在胚轴、子叶均为淡黄色的时期切取蓖麻子叶节,接种在1/8MS、内含10g/L琼脂粉、20g/L蔗糖、8.0mg/LZT、1.0mg/LNAA的培养基中进行培养,能有效地预防蓖麻子叶节玻璃化。     

祈州漏芦的组织培养与快繁技术研究

目的：对祁州漏芦愈伤组织诱导培养基和生长的培养条件进行研究,构建祁州漏芦快速繁殖体系。方法：通过筛选外植体,添加不同种类及浓度的生长调节剂对祁州漏芦进行愈伤组织诱导、芽增殖培养及生根培养。结果：诱导愈伤组织适宜培养基：MS＋0.3mg/L NAA＋3.0mg/L 6-BA;芽增殖培养以MS＋0.3mg/L NAA＋5.0mg/L 6-BA为宜;生根培养以1/2MS＋0.1mg/L NAA为佳。结论：利用组织培养技术能够实现祁州漏芦快速繁殖。     

Optimization of Medium Composition for Nisin Fermentation with Response Surface Methodology

ABSTRACT:  Nisin is an effective food biopreservative widely used in food industry. However, 1 problem of concern is limited production rate and final nisin concentration. A nisin-producing strain, L. lactis Lac2, a mutant strain with high yield of nisin, was obtained in our laboratory recently. In the present study, a fractional factorial design was applied to investigate the main factors that affect the yield of L. lactis Lac2. Central composite experimental design and response surface methodology were adopted to derive a statistical model for optimizing the composition of the medium. The results showed that the optimum medium for nisin production of L. lactis Lac2 was composed of 2.68% sucrose (w/v), 0.5% tryptone (w/v), 1% yeast extract (w/v), 0.3% Tween-80 (w/v), 0.02% MgSO₄·7H₂O (w/v), 0.81% NaCl (w/v), 1.91% K₂HPO₄ (w/v), 0.05% ascorbic acid (w/v), and 2% agar (w/v) (if necessary) at pH 6.5. When cultured in the optimum medium, the nisin yield is an average of 3381.81 IU/mL, which nearly doubled the yield when incubated in the initial medium. Also, the concentration of tryptone was decreased while that of the sucrose was increased when compared with CM broth, which means a reduction of the fermentation cost.     

Factors affecting the in-vitro establishment of Jamaican yams (Dioscorea spp) from nodal pieces

Factors such as inorganic ammonium, growth regulator supplements, propagule type and subculture period affecting the in-vitro establishment of Dioscorea cayenensis and D trifida have been examined. The results indicate that for D cayenensis, transfer of whole plantlets to fresh initiation media prior to cutting into nodal segments and transfer to establishment media was beneficial. Addition of 800 mg litre^?1 NH₄NO₃ to basal medium (BM) containing 0.5 mg litre^?1benzylamino purine (BAP) caused a significant improvement in nodal numbers and resulted ir. 11.8 propagules per original explant after 8 weeks or 184.1 propagules after 16 weeks in-vitro. Rapid establishment of D trifida cv Short Neck Yampie depended on the original explant source. Explants obtained from small tuber vines grew faster on establishment than those obtained from leafy cuttings. Initiation on BM supplemented with 0.5 mg litre^?1 kinetin (BM0.5K) and establishment on BM supplemented with 0.5 mg litre^?1 BAP (BM0.5BAP) produced the optimum result for the vines obtained from small tubers (24.0 propagules after 8 weeks of culture). For explants obtained from leafy cuttings, initiation on BM0.5BAP and establishment on BM0.5K gave optimum results (15.8). Quick establishment of initiated plantlets shown by increased shoot response and high multiplication rates were achieved in this study.     

Antimicrobial Effects of Lactoferrin, Lysozyme, and the Lactoperoxidase System and Edible Whey Protein Films Incorporating the Lactoperoxidase System Against Salmonella enterica and Escherichia coli O157:H7

Lactoferrin (LF), lysozyme (LZ), the lactoperoxidase system (LPOS), and edible whey protein isolate (WPI) films incorporating LPOS were studied for inhibition of Salmonella enterica and Escherichia coli O157:H7. Antimicrobial effects of LF (5 to 40 mg/mL), LZ (1 to 20 mg/mL), and LPOS (0.5% to 5.0% [w/v] [0.03–.25 g/g, dry basis]) were examined by measuring turbidity of antimicrobial‐containing media after inoculation and by examining cell inhibition by WPI films incorporating LPOS (LPOS‐WPI films) on an agar recovery medium. Elastic modulus (EM), tensile strength (TS), percent elongation (%E), oxygen permeability (OP), and Hunter L, a and b of WPI films incorporating 0.03 to 0.25 g/g of LPOS were compared with those of plain WPI films without LPOS. The growth of S. enterica and E. coli O157:H7 (4 log colony‐forming units [CFU]/mL) in tryptic soy broth (TSB) was not prevented by LF at ≥20 and ≥40 mg/mL, respectively. S. enterica and E. coli O157:H7 in TSB were not inhibited by LZ at ≥ 6 and ≥ 20 mg/mL, respectively. LPOS at concentrations of 2.75% (w/v) and 1.0% (w/v) reduced S. enterica and E. coli O157:H7 to below the limit of detection (1 CFU/mL) in TSB, respectively. LPOS‐WPI films (0.15 g/g) completely inhibited S. enterica and E. coli O157:H7 (4 log CFU/cm²), inoculated either onto agar before placing the film disc or onto top of the film disc. Incorporation of 0.25 g/g of LPOS decreased EM, TS, and %E. The oxygen barrier property of WPI films was improved with the incorporation of LPOS at 0.15 to 0.25 g/g.     

Dihaploid Production in Flax by Anther and Ovary Cultures

Abstract

The response of flax anther cultures is still very genotype dependent. We screened different flax genotypes (Szegedi 30, Flanders, Carolin, PR FGL 77, Viking and Red Wing) for androgenic response. The highest androgenic response for each genotype was achieved on N&N medium supplemented with 6% sucrose and combination of growth hormones (1 mg l^?1 NAA and 1 mg l^?1 BAP) after the anthers were cold pretreated at 8°C for 7 days. The highest number of calli and shoots were derived with genotype Red Wing. In case of Red Wing even embryo-like structures were regenerated from the calli.

For gynogenesis this is the first report on ovary cultures in flax. The obtained response (number of ovaries producing calli) ranged from 4% in genotype PR FGL 62 to 64% in genotype AC Emerson on N6 medium supplemented with the same sucrose and hormone combination and concentration as for anther cultures. Yellow calli were formed within two to four months and in course of cultivation turned green. Regeneration was realized via shoot formation. Cells in the yellow calli were preferentially diploid (2n) and in green ones tetraploid (4n) and also higher ploidy level was observed (up to 16n) in course of cultivation.     

二步培养和AgNO3对甘蓝型油菜子叶和下胚轴芽再生的影响

以甘蓝型油菜（Brassica napus L．）品种浙双758为材料,研究二步培养及添加AgNO3对甘蓝型油菜子叶和下胚轴外植体离体再生的影响。外植体先在含0．5—1．5mg／L2,4-D的MS培养基上预培养3d或7d诱导愈伤组织的产生,再转到含有3mg／LBA和0．15mg／LNAA及添加或不添加2．5mg／LAgNO3的分化培养基上诱导芽的分化。结果表明,外植体愈伤组织诱导率和芽再生率与2,4-D浓度、预培养时间和AgNO3密切相关;分化培养基中添加银离子可显著增加不定芽的再生频率;二步培养及添加AgNO3可使半子叶、完整子叶和下胚轴外植体芽再生频率分别达到了96．1％,96．7％和96．7％。     

Studies on “sugar-reactivity” of agars extracted from some Indian agarophytes

Sugar reactivity was observed within the sugar–agar complexes in presence of sucrose and glucose with agars of Indian agarophytes viz. Gelidiella acerosa, Gracilaria edulis, Gracilaria crassa and Gelidium pusillum. The sugar reactivity was more pronounced in presence of sucrose than glucose. Oxoid agar was used as the reference material. Control agar gel contained 1.12% agar (w/w) in water. Sucrose–agar and glucose–agar gels in water consisted of 50% (w/w) sucrose and 50% (w/w) glucose, respectively along with 1.12% (w/w) agars of the four seaweeds mentioned above. Addition of sucrose resulted in increase (ca. 25–45%) in gel strength; increase (2–3 °C) in gelling and melting temperatures was observed in the gels prepared with agars from all the agarophytes and Oxoid agar. On the other hand, addition of glucose resulted in increase (19–34%) in the gel strength and gelling and melting temperatures of the agar gels of Oxoid as well as of all other agars decrease (2–3 °C). Maximum sugar reactivity was observed with the 50% level of sucrose and glucose in agar gels. Rheological and thermogravimetric characteristics of these gel samples were studied. The latter showed two patterns e.g. control agar gel of Oxoid agar was thermally less stable than the four control agar samples studied; in sugar–agar gel samples it followed a reverse pattern. To our knowledge, this is the first report of “sugar reactivity” of agar of Indian agarophytes. Sugar reactivity of agar in presence of glucose is also reported for the first time. The results of this study will be useful in bioprospecting as well as in exploring new applications.     

云烟87烤烟品种突变株的离体快繁技术研究

以云烟87烤烟品种突变巨型株的休眠芽为试材,对其进行了打破休眠促使萌芽、愈伤组织诱导、丛生芽增殖和生根培养的研究。结果表明:打破休眠促使其萌芽的最佳培养基配方为MS+2.0mg/L 6-BA+1.0mg/L NAA;诱导产生愈伤组织效果最佳的培养基配方为MS+3.0mg/L 6-BA+0.3mg/L NAA;丛生芽增殖效果最佳的培养基为MS+1.0mg/L 6-BA+0.3mg/L NAA;MS+0.6mg/L NAA对根系的生长最为有利。      

杜仲疏松愈伤组织诱导条件的优化筛选

杜仲愈伤组织生长过于致密从而导致难以实现悬浮培养,因此获得疏松、生长快速愈伤组织是实现杜仲细胞悬浮培养的前提.本文研究了不同外植体、激素和培养基对杜仲愈伤组织诱导率、疏松程度以及褐化率的影响,结果表明：上胚轴和子叶更容易被诱导为疏松愈伤组织,MS培养基是最适诱导培养基;适当增加6-BA浓度有助于褐化的减轻且提高愈伤组织疏松程度,杜仲疏松愈伤组织诱导最适激素配比为1.0mg/L 2,4-D＋1.0mg/L NAA＋1.0mg/L 6-BA.