Enhanced regeneration of rice (Oryza sativa L.) embryogenic callus by light irradiation in growth phase

The effect of light irradiation on the growth and regeneration of embryogenic rice calli was investigated. Rice calli grew slightly better in the light than in the dark during the growth stage. When calli subcultured under light irradiation were transferred into a liquid regeneration culture system, the number of plantlets with shoots regenerated from calli subcultured in the light was higher than in the case of those subcultured in the dark, whereas there was no obvious difference between the light and dark subcultures when a solid medium was used for the regeneration. The number of regenerated plantlets increased with increasing light intensity up to 2000 lx, but remained the same when the light intensity was raised further. The maximum number of regenerated plantlets was obtained under a photoperiod of 16 h/d. About 8800 plantlets per liter-medium were regenerated from light-cultured calli, which is 1.5 times more than the number regenerated from calli grown in the conventional dark culture.     

Development of a bioreactor suitable for embryogenic rice callus culture

Embryogenic rice calli induced from mature rice (Oryza sativa L., Sasanishiki) were cultured in a jarfermentor with disk turbine impellers, an air-lift reactor, and a turbine-blade reactor (TBR(R)), and the effects of agitation were investigated. In all cases, the growth inhibition was observed, though a slightly improved regeneration frequency was obtained in the TBR. To overcome the growth inhibition, small cubes of polyurethane foam were used as immobilization supports in the TBR culture. Supports accumulated around the cylindrical stainless-steel mesh in the reactor and rice calli were observed growing in them. Polyurethane foam with an average pore size of 1.3 mm gave the maximum ratio of immobilized cells during 1-week culture. When 5- or 10-mm cubes were used, supports were observed floating on the medium surface, but 3-mm cube supports accumulated uniformly around the stainless-steel mesh and were found to be suitable for the immobilized culture of rice calli. Three-millimeter cube supports corresponding to 5% by volume were added to 600 ml medium in the TBR and the effects of the agitation speed on the cell growth and regeneration frequency were investigated. At all the agitation speeds examined, no significant decrease in either the cell growth or the regeneration frequency occurred. From these results, it was concluded that the TBR is a suitable reactor for the propagation of embryogenic rice calli and that immobilization supports with 1.3 mm of average pore size are effective in preventing hydrodynamic stress as well as in supplying nutrients to the immobilized calli.     

Release of embryogenic carrot cells with high regeneration potency from immobilized alginate beads

For the mass-production of regenerated carrot plantlets, embryogenic carrot callus immobilized in calcium alginate gel beads was cultivated in a growth medium and the regeneration frequency of cells released from alginate gel beads was compared with that in a suspension culture. Cells released in the immobilized culture were regenerated at a frequency which was about 1.5 times higher than that obtained in the suspension culture. When CaCl2 was added to the growth medium at 5 mM, repeated batch culture for plantlet production continued for 245 d with no significant decrease in the productivity (1.6 x 10(5) plantlets/l-medium/d).     

Enhanced and prolonged production of plantlets regenerated from carrot callus in a viscous additive-supplemented medium

To reduce the hydrodynamic stress in plant cell culture and enhance the production of regenerated plantlets, a liquid medium containing a viscous additive was newly designed and plantlet production from embryogenic carrot callus cultivated in the medium was examined. Na-alginate or carboxymethyl cellulose (CMC) was used as the viscous additive. The viscosity of the medium increased with increasing additive content and the number of regenerated plantlets also increased. When carrot calli were cultivated in the medium containing 0.4% CMC, designated as N medium (viscosity, 3 mPa.s), the maximum enhancement of plantlet regeneration, approximately 2.5 times higher than that in the control medium, was obtained. Enlargement of callus size observed in N medium is considered to be the main reason for the enhanced plantlet regeneration. Regeneration enhancement was sufficiently induced after calli were cultivated once in N medium, but this regeneration ability rapidly disappeared after once cultivation in the conventional medium. In repeated batch culture using N medium, plantlet production continued at a high level for 18 batches (250 d) with no significant decrease, while in the control culture without CMC the number of plantlets produced dropped to almost zero by the sixth batch (84 d).     

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。     

甘蔗离体培养抗除草剂突变体的筛选研究简报

本试验采用桂糖11号细胞团和幼苗作离体培养，用Co^60-γ射线35Gy剂量参照后，分别转入含除草剂的培养基中，用除草剂作为选择压力，研究离体培养条件下抗除草剂突变体的筛选技术和方法。实验结果表明，经诱变培养法处理的细胞团及幼苗的存活率比经共培养法处理的高。     

Mass propagation and essential oil analysis of Artemisia vulgaris

Artemisia vulgaris L. (Mugwort) is a threatened and valuable medicinal plant. Attempts have been made in this research to mass propagate its plantlets through in vitro liquid culture technology using Murashige and Skoog (MS) basal medium supplemented with 6-benzyl adenine (BA) (0.44-8.88 microM). Initially, 22.6 shoots (99.9% shooting frequency) developed from shoot tip explants cultured in MS with 4.44 microM BA at 100 ml flask capacity. This was further subcultured at increasing flask capacity (150, 250, and 500 ml) for shoot proliferation. Of the different concentrations of BA and flask capacities tested, 4.44 microM BA and 500 ml flask capacity were found to produce a maximum of 85.5 shoots after 30 d of culture. Shoot proliferation was found to increase with increasing flask capacity whereas shoot number decreased with increasing BA concentration (>4.44 microM). Individual shoots were isolated and rooted on MS medium containing 8.56 microM indole-3-acetic acid (IAA). Then the plantlets were acclimatized under standard laboratory conditions and later under greenhouse conditions. Fresh leaves were collected from greenhouse-grown plants and subjected to essential oil analysis by the simultaneous distillation and extraction method. GC-MS results revealed the presence of 88 components and the extracted oil was rich in camphor (16.8%), alpha-thujone (11.3%), germacrene D (7.2%), camphene (6.5%), 1,8-cineole (5.8%) and beta-caryophyllene (5.4%). This in vitro strategy can be a reliable method for the steady production of a large number of plants for essential oil production, which is reported for the first time for A. vulgaris.     

苯乙酸对烟草单倍体植株叶脉组织培养一步成苗的效应研究

本研究探明了苯乙酸(PAA)对烟草单倍体植株叶脉切段培养一步成苗的效应,发现PAA与NAA、6-BA配合使用能有效促进一步成苗的发生。试验证明PAA对一步成苗的促进效应与各种激素浓度配比和基本培养基的种类有关,与基因型的关系不明显。烟草单倍体植株叶脉切段培养一步成苗的最佳培养基配方为MS+PAA 20 mg/L+NAA 0.5 mg/L+6-BA 1.0 mg/L+谷氨酰胺50 mg/L+麦芽糖30 g/L。试验还证明,与2,4-D诱导产生愈伤组织途径的多级成苗培养相比,本研究建立的PAA一步成苗培养具有以下优点:1)显著提高愈伤组织的诱导率;2)显著提高愈伤组织的质量,有效提高愈伤组织分化率;3)成苗率显著高于多级成苗;4)再生植株具有更好的幼苗素质;5)再生植株的染色体数目变异率也显著低于多级成苗;6)获得双单倍体植株的频率显著高于多级成苗;7)能有效缩短培养周期、节约成本。      

Production of recombinant protein by baculovirus-infected insect cells in immobilized culture using porous biomass support particles

Immobilization of insect cells using porous biomass support particles (BSPs) and production of a recombinant protein by the immobilized cells after infection with a baculovirus were investigated in a shake-flask culture. Sf9 cells were passively immobilized in reticulated polyvinyl formal (PVF) resin BSPs (2 x 2 x 2 mm cubes) with matrices of 60 mum mean pore diameter in situ in shake-flasks. The cell density in the BSPs was over 5 x 10(7) cells/cm3-BSP in cultures with regular replacement of the culture medium, as estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After infection with a recombinant baculovirus carrying the beta-galactosidase gene, immobilized cells within the BSPs showed a high specific productivity, comparable to the maximum productivity in shake-flask cultures of non-immobilized cells, as long as nutrients in the medium were not depleted. Even when immobilized cells at a high density of 5 x 10(7) cells/cm3-BSP were infected with the baculovirus, efficient beta-galactosidase production with a high specific productivity was possible by replacing the medium at appropriate intervals to avoid nutrient depletion.     

花生胚小叶的离体再生及筛选压力选择

以花生胚小叶为外植体优化离体再生条件,通过对再生苗进行筛选,为外源基因的遗传转化提供实验依据。研究表明,不同基因型胚小叶的再生能力差异显著,在供试基因型中以中花8号不定芽诱导率和成苗率最高,且外植体状态最好。中花8号胚小叶再生的最佳诱导培养基为MSB（MS无机盐＋B5有机成分）＋6-BA（4.5mg/L）＋NAA（1mg/L）＋AgNO3（2mg/L）,最佳诱导培养时间为21d。对中花8号再生苗进行筛选浓度实验,草丁膦（PPT）为20mg/L时,可筛选出抗性苗;300mg/L的卡那霉素（Km）可用于较小幼苗筛选,而对较大幼苗则需将Km的浓度提高至400mg/L以上。     

Selection of embryogenic sugarcane callus by image analysis

In the cultivation of plant calli on solid media, two kinds of calli such as compact and friable calli, which are a bright yellow and a whitish clump, respectively, are often obtained. Distinction of these calli is of much importance in the regeneration step. The image analysis system associated with a Charge Coupled Device (CCD) camera and microscopy were used to distinguish sugarcane calli. The original images from compact and friable calli were input to a computer via an image analysis board. At first, the brightnesses of trichromatic colors, red (R), green (G) and blue (B), of each pixels were extracted and the average brightness value for each color was calculated. From these values of the trichromatic colors, compact and friable calli could not be clearly distinguished. Next, the brightness of yellow, Br(Y), and white, Br(W), were defined using Br(R), Br(G) and Br(B), and the difference between Br(Y) and Br(W), Br(Y-W), which can be used to express the yellowish grade, was calculated. When Br(Y-W) was determined from all pixels of the original images of both calli, the compact calli were found to be clearly distinguished from the friable calli by the frequency distributions of Br(Y-W). Average brightness center value, Av(C(Y-W)), was calculated from the frequency distributions. It was found that the calli with less than 10 units of Av(C(Y-W)) was never regenerated and a proportional relationship between Av(C(Y-W)) and the regeneration frequency of the callus line was obtained.     

固定化Amycolatopsis sp.ST2710分段发酵无锡他汀

用海藻酸钠对Amycolatopsis sp.ST2710细胞进行固定化处理。以固定化细胞的机械强度以及分段发酵无锡他汀的转化率为指标,考察了海藻酸钠浓度、CaCl2浓度、固定化时间和包埋菌体量对固定化细胞分段发酵无锡他汀的影响,得到最佳的固定化条件为:海藻酸钠浓度20 g/L、CaCl2浓度10 g/L、固定化时间1 h、包埋菌体量2 mL菌悬液/10 mL凝胶。对固定化细胞分段发酵无锡他汀的特点进行了研究,结果显示:Amycolatopsissp.ST2710经过固定化处理后,大幅度提升了其对底物洛伐他汀的耐受性,显著降低了对发酵培养基中的淀粉需求量,可重复利用。当发酵两阶段pH分别为7.5和5.5、发酵时间分别为48 h和10 h、洛伐他汀添加量3 g/L、转化培养基中淀粉浓度15 g/L时,中间产物Ⅰ对洛伐他汀转化率为64%,无锡他汀对中间产物Ⅰ的转化率为75%。连续发酵5批后,产物转化率仍能维持较高的水平。     

Xylitol production from rice straw hemicellulose hydrolyzate by polyacrylic hydrogel thin films with immobilized Candida subtropicalis WF79

Xylose from rice straw hemicellulose hydrolysate was fermented for xylitol production using Candida subtropicalis WF79 cells immobilized in polyacrylic hydrogel thin films of 200 mum thickness. Cell immobilization was conducted by first suspending the yeast cells in a mixture of 2-hydroxyethyl methacrylate (HEMA, hydrophilic monomer), polyethylene glycol diacrylate (PEG-DA, crosslinking agent), and benzoin isopropyl ether (photoinitiator). The mixture was then allowed to form polyacrylic hydrogel thin films, between two pieces of glass sheets, by UV-initiated photopolymerization. The hemicellulose of rice straw was hydrolyzed using dilute sulfuric acid at 126 degrees C. The hydrolysate was neutralized with calcium hydroxide. After separating the solid residues and calcium sulfate precipitates by filtration, the hydrolysate was treated with charcoal to partially remove potential inhibitory substances, followed by vacuum concentration to obtain solutions of desired xylose concentrations for yeast fermentation. The thin films with immobilized yeast cells were submerged in the xylose solution from rice straw hydrolysate for fermentation in an Erlenmeyer flask. The maximum yield was 0.73 g of xylitol per gram of xylose consumed. In the 52.5-day long durability test, after 40 d of repeated batchwise operation, the fermentation activities of the cell immobilized in thin films began to decline to a yield of 0.57 g/g at the end.     

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     

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％。     

ZIF-8原位合成法固定化脂肪酶CRL

以ZIF-8为固定化载体,通过原位合成法固定褶皱假丝酵母脂肪酶(CRL),研究了不同固定化条件对固定化酶活力的影响以及固定化酶的稳定性,并对固定化酶进行了结构表征。结果表明:在10 m L 40 mmol/L硝酸锌溶液、10 m L 160 mmol/L 2-甲基咪唑溶液、2-甲基咪唑溶液初始p H8、酶添加量2.75 mg、-48℃冷冻干燥条件下,获得的固定化酶CRL@ZIF-8蛋白回收率为93%,比活力为1.08 U/mg;底物的分子大小对固定化酶的催化性能有明显影响,在以相对小分子的乙酸对硝基苯酯为底物时,重复使用9次后固定化酶仍保持72.8%的相对活力;通过对固定化酶进行场发射扫描电镜和氮气吸附脱附表征,表明脂肪酶CRL在载体表面和内部均有分布,固定化酶颗粒粒径约400 nm。     

多聚半乳糖醛酸酶(PG)反义基因转化加工番茄

通过农杆菌(Agrobacterium tumefaciens)LBA4404介导将多聚半乳糖醛酸酶(PG)反义基因导入新疆加工番茄(代号:99-162混)。卡那霉素抗性筛选,获得移栽成活的10株再生植株,PCR和Southern blot检测表明,其中4株再生植株的染色体中有外源基因的插入。所得4株转基因加工番茄在基因转化处理当代表现不同;果实饱满有光泽;Northern杂交结果表明,转反义PG基因加工番茄果实外果皮PG基因表达水平比对照低。转基因植株的表型观察和分子检测结果相吻合。     

Characterization of glucoamylase immobilized on magnetic nanoparticles

Magnetic support was prepared by precipitation from an alkaline solution of divalent and trivalent iron ions and subsequently was modified with 3‐aminopropyltriethoxysilane. FTIR analysis showed existence of a new Si–O–Fe bond in obtained particles. Scanning electronic microscopy images shows that the nanoparticles of all samples have particle size below 30 nm. Glucoamylase AMG 300L was immobilized onto the modified magnetic support using glutaraldehyde as a coupling agent. Obtained preparations had specific activity of 148 U/g of the support when measured at 55°C using maltose as substrate. The immobilized enzyme exhibited mass transfer limitation as reflected by a higher apparent K_m value and a lower energy of activation. The immobilization was almost completely terminated after 30 min of the reaction at 30°C. The highest immobilization yield of the enzyme was achieved at glutaraldehyde concentration of 10 mM. The immobilization did not influence considerably on optimum pH and temperature of substrate hydrolysis catalyzed by investigated enzyme (55°C, pH 4.5). Moreover, immobilized glucoamylase was easily separated from the reaction medium by an external magnetic field and retained about 60% of initial activity after nine repeated cycles of enzyme reaction followed by magnetic separation.     

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.     

脂肪酶固定化及其在食品领域中应用的研究进展

为构建新型固定化酶催化体系,以聚甲基丙烯酸缩水甘油酯为载体,利用贻贝仿生技术——多巴胺/聚乙烯亚胺共沉积进行修饰,采用扫描电镜（SEM）、能谱（EDS）、Zeta电位及红外光谱（FT-IR）表征所得材料,并研究其固定化近平滑假丝酵母CICC 33470所产脂肪酶的表征及酶学性质。最佳酶固定化条件为：固定化温度为30 ℃,固定化pH为7.0,固定化时间为5 h,初始酶活为337.76 U/mL,载体添加量为0.2 g。固定化酶最佳反应温度为50 ℃,最佳反应pH为8.0,最佳反应时间为10 min,最优条件下固定化酶酶活为484.42±5.97 U/g-载体。固定化酶的稳定性明显提高,重复使用8次后,固定化酶仍有39.22%的初始酶活。进一步将固定化酶用于催化乙酰丙酸与十二醇的酯化反应,转化率可达75.94%,充分证明聚甲基丙烯酸缩水甘油酯经修饰后是固定化脂肪酶的优良载体,为未来扩大脂肪酶的应用范围提供了基础数据。