红豆杉愈伤组织超代温保存有关因素的研究

对红豆杉愈伤组织超低温保存中几个主要因素进行了比较,试验证明：预培养时间、预培养其中蔗糖浓度、保护剂的组合以及冰冻降温方法与超低温保存后的相以细胞活力密切相关。试验结果表明,在含8％蔗糖的62号液体培养基中振荡预培养6d,红豆杉愈伤组织在超低温保存后细胞活力保持最高。有效的冷冻保护剂为10％山梨醇＋10％DMSO,冷冻方法以分步冷冻和慢冻较为适宜,而经快冻的愈伤组织复苏后活力低下。     

仙客来愈伤组织的超低温保存

为了避免连续继代造成仙客来愈伤组织的变异, 对仙客来愈伤组织进行了超低温冷冻保存研究。以继代后处于对数生长期的愈伤组织为实验材料, 首先在含有不同蔗糖浓度的培养基上预培养不同时间, 转至不同的冰冻保护剂中直接液氮冷冻或-20oC预冷冻2 h, 然后液氮超冷冻保存, 37oC水浴迅速解冻, 并用相应蔗糖浓度的液体培养基洗涤, 以中性红染色测定细胞的存活率, SPSS13.0软件进行统计学分析。结果表明: 预培养基中蔗糖浓度、预培养时间、降温方式、冷冻保护剂等对解冻后材料相对存活率存在不同程度的影响, 筛选出4%蔗糖浓度预培养3 d、9号保护剂、0oC停留30 min后直接冷冻为超低温保存的最佳方案, 通过简单的方法获得了较好的愈伤组织保存效果。     

仙客来愈伤组织的超低温保存

为了避免连续继代造成仙客来愈伤组织的变异, 对仙客来愈伤组织进行了超低温冷冻保存研究。以继代后处于对数生长期的愈伤组织为实验材料, 首先在含有不同蔗糖浓度的培养基上预培养不同时间, 转至不同的冰冻保护剂中直接液氮冷冻或-20oC预冷冻2 h, 然后液氮超冷冻保存, 37oC水浴迅速解冻, 并用相应蔗糖浓度的液体培养基洗涤, 以中性红染色测定细胞的存活率, SPSS13.0软件进行统计学分析。结果表明: 预培养基中蔗糖浓度、预培养时间、降温方式、冷冻保护剂等对解冻后材料相对存活率存在不同程度的影响, 筛选出4%蔗糖浓度预培养3 d、9号保护剂、0oC停留30 min后直接冷冻为超低温保存的最佳方案, 通过简单的方法获得了较好的愈伤组织保存效果。     

水稻愈伤组织超低温保存体系的建立

水稻种质资源的长期有效保存对于世界粮食安全至关重要,而超低温保存是实现这一目标的最佳方式.本研究以水稻的胚性愈伤组织为材料,利用冻存后恢复培养阶段的新生愈伤率和新生愈伤分化率来评价不同品种不同粒级大小的胚性愈伤组织、不同浓度蔗糖预培养,以及高糖预培养和高糖预培-玻璃化处理2种预处理方式对水稻愈伤组织超低温保存效率的影响...     

防风愈伤组织的玻璃化法超低温保存及再生

为避免连续继代造成的愈伤组织变异,探索新的种质资源保存方法,对防风愈伤组织进行了超低温冷冻保存及植株再生研究。以关防风3周龄的愈伤组织为材料,单一变量法研究适宜的玻璃化法超低温保存程序。结果显示:(1)防风愈伤组织超低温保存的最佳方案为:4℃条件下于MS+1.0mg/L 6-BA+1.0mg/L NAA+5%DMSO的继代培养基中预培养3d,60%PVS2常温装载20min,100%PVS2于2℃脱水45min后直接投入液氮。(2)防风愈伤组织经超低温保存后的相对存活率最高为79.24%,其中预培养和脱水是实现超低温冻存的关键环节,且1.0mol/L蔗糖的MS溶液洗涤、暗培养14d以上有助于冻后愈伤组织恢复生长。研究表明,玻璃化超低温冻存可以作为防风愈伤组织的保存方法,冻后愈伤可以恢复生长并再生成完整植株。     

江西铅山红芽芋胚性愈伤组织的玻璃化法超低温保存及植株再生

以江西铅山红芽芋胚性愈伤组织为材料,研究各种因素对其玻璃化法超低温保存的影响。结果表明:江西铅山红芽芋胚性愈伤组织玻璃化法超低温保存较佳的预培养条件为0.3mol·L-1蔗糖预培养3d,较佳的60%PVS2装载时间为20min,较佳的100%PVS2脱水条件为25℃脱水30min,较佳的化冻温度为40℃,较佳的洗涤液蔗糖浓度为1.2mol·L-1,较佳的冻后培养条件为暗培养7d再转到光周期中培养。红芽芋胚性愈伤组织包埋玻璃化超低温保存后的平均成活率约为70%。红芽芋胚性愈伤组织冻后再生苗没有发生形态学、生理学和细胞学的变异。     

唐菖蒲愈伤组织超低温保存（简报）

唐菖蒲愈伤组织在培养了 20～25天后为最佳冷冻材料,通过含5％ DMSO的培养基预培养5天和10％ DMSO 10％甘油的冷冻保护剂处理。都能显著提高冷冻后愈伤组织的存活率,而且分步冷冻较快速冷冻效果更好。经过冷冻后的愈伤组织成功地得到增殖和植株再生。     

江西铅山红芽芋胚性愈伤组织的包埋干燥法超低温保存

对江西铅山红芽芋（Colocasia esculenta var．cormosus cv．Hongyayu）胚性愈伤组织包埋干燥法超低温保存进行了初步的研究。结果表明：江西铅山红芽芋胚性愈伤组织包埋干燥法超低温保存较佳的条件为：0．75mol·L-1蔗糖预培养3d;脱水方式为空气干燥7h或硅胶干燥11h;化冻温度为37℃（2min）;冻后培养条件为暗培养7d再转到光周期中培养。此方法超低温保存后的平均成活率约为45％。超低温保存时间以及是否去除包裹的褐藻酸钙对其成活率无显著性影响。形态学和细胞学检测表明红芽芋胚性愈伤组织冻后再生苗与母本材料相比没有发生变异。     

江西铅山红芽芋胚性愈伤组织的包埋玻璃化超低温保存

为长期安全保存江西铅山红芽芋种质资源,本文以江西铅山红芽芋的胚性愈伤组织为对象,研究了包埋玻璃化冻存过程中各因素对细胞活力和愈伤组织成活率的影响,优化建立了江西铅山红芽芋胚性愈伤组织包埋玻璃化超低温保存体系。将约0.2 g胚性愈伤组织块包埋成海藻酸钙凝胶珠后,在25℃下转入MS+2 mg/L TDZ+1 mg/L NAA+0.75 mol/L蔗糖的培养基中于14 h/d光周期下预培养1 d;预培养后的胚性愈伤组织块用2 mol/L甘油和0.4 mol/L蔗糖的混合物在25℃下装载40 min;采用PVS2在25℃下脱水30 min,更换PVS2后直接投入液氮保存1 d;再将胚性愈伤组织块置于37℃恒温水浴中化冻3 min,然后用MS+2 mg/L TDZ+1 mg/L NAA+1.2 mol/L蔗糖的液体培养基洗涤3次,每次10 min;洗涤后的胚性愈伤组块转入MS+2 mg/L TDZ+1mg/L NAA固体培养基上先暗培养7 d再转到14 h/d光周期中培养。7 d后胚性愈伤组织块开始恢复生长,并且在30 d内分化出胚状体;将胚状体再次转入MS+2 mg/L TDZ+1 mg/L NAA固体培养基上,60 d后形成完整的植株。红芽芋胚性愈伤组织包埋玻璃化超低温保存后的平均成活率约为60%,并且红芽芋胚性愈伤组织冻后再生苗没有发生形态性状和染色体数目的变异,此结果为长期安全保存江西铅山红芽芋种质资源奠定了良好的基础。     

江西铅山红芽芋胚性愈伤组织小滴玻璃化法超低温保存技术的研究

探索江西铅山红芽芋胚性愈伤组织的小滴玻璃化法超低温保存,为其种质资源的超低温保存提供技术基础和理论依据。植物组织培养和单因子试验的方法。江西铅山红芽芋胚性愈伤组织小滴玻璃化法超低温保存的较佳程序为:约0.2 g胚性愈伤组织在25℃下转入MS+2 mg·L-1TDZ+1 mg·L-1NAA+0.5 mmol·L-1蔗糖的培养基中,于14 h·d-1光周期下预培养3 d后用MS+2 mmol·L-1甘油+0.4 mmol·L-1蔗糖在25℃下装载20min,然后用PVS2在0℃下脱水40 min,吸取5滴PVS2到铝箔条上,将脱水后的红芽芋胚性愈伤组织块转到铝箔条上的PVS2液滴里。附有胚性愈伤组织块的铝箔条在液氮里蘸一下,然后迅速将其转入2 mL装满液氮的冷冻管中,再投入液氮保存1 d。从液氮中取出冷冻管中的铝箔条,浸入用40℃温水预热过的洗涤液(MS+2 mg·L-1TDZ+1 mg·L-1NAA+1.2 mmol·L-1蔗糖)中,使胚性愈伤组织块从铝箔条上脱落下来,然后再将胚性愈伤组织块转入新鲜的洗涤液中洗涤3次,每次10 min。洗涤后转入MS+2 mg·L-1TDZ+1 mg·L-1NAA固体培养基上,先暗培养7 d再转到14 h·d-1光周期中培养。30 d后将分化出的胚状体再次转入MS+2 mg·L-1TDZ+1 mg·L-1NAA固体培养基上可再生完整植株。红芽芋胚性愈伤组织包埋玻璃化超低温保存后的平均成活率约为80%。红芽芋胚性愈伤组织冻后再生苗没有发生形态学、生理学和细胞学的变异。红芽芋胚性愈伤组织小滴玻璃化法超低温保存可以保证其遗传资源的稳定性,为江西铅山红芽芋种质资源的离体保存提供了一条新的途径。     

Phenol content, acidic peroxidase and IAA-oxidase during somatic embryogenesis in Theobroma cacao L.

Calli were induced in cacao cotyledon explants on a half-strength Murashige and Skoog medium containing 6 × 10-2 g m-3 saccharose and various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) with kinetin (kin), benzylaminopurine (BAP) or 2-isopentenylphosphate (2-iP). Experiments were carried out on two clones of cacao differing in their susceptibility to black pod disease. The highest percentage of explants forming callus and the most rapid callus development were obtained with 10-6 g m-3 2,4-D and 0.5× 10-6 g m-3 kin. Somatic embryogenesis and rhizogenesis were induced by transferring 3-week-old callus in a half strength Murashige and Skoog medium containing 3 × 10-2 g m-3 saccharose and NAA or IBA in the 0 to 5 × 10-6 g m-3 concentration range. No differentiation could be observed when the medium was supplemented with kin or BAP. The conversion of callus into somatic embryos and roots was accompanied by a drop in phenol content and an increase in peroxidase and IAA-oxidase activities. Moreover, cell differentiation was characterized by the persistence in the callus of one acidic soluble isoperoxidase which was not detected in nondifferentiating callus. Although some differences were noticed between the clones, alterations responsible for cell differentiation were the same in both genotypes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Grain growth in wheat ears cultured in saccharose solution

Ears of uniculm wheat (Triticum aestivum L. cv. Gigas) grown in liquid medium for 11d absorbed more solution when the saccharose concentration was 2 % than when it was 10 %. When the ears were grown in 6 % saccharose solution, the rate of uptake from the solution was between that from the 2 and 10 % saccharose medium. Dry mass per grain increased with the saccharose concentration in the medium and the reduced uptake of solution did not decrease the moisture percentage of the grain. The culture of ears decreased pH of the solution with 2 % saccharose more than with 10 %. Addition of 0.5% chloramphenicol to the culture solution had no adverse effect on grain mass; it prevented contamination of the solution and maintained a higher pH     

东北红豆杉组织培养的研究 Ⅰ．叶片愈伤组织的诱导

东北红豆杉（ＴａｘｕｓｃｕｓｐｉｄａｔａＳＩＥＢＥＴＺＵＣＣ）是天然抗癌药物资源,其树皮中含有一种抗癌药物（ＴＡＸＡＬ紫杉酚）引起各国的注意。由于红豆杉生长缓馒,往往遭到砍伐,远远不能满足抗癌药物研究和医疗药用的需要;加之资源又贫乏,因此确定此项研究。开展此项研究结果表明：黑龙江省、辽宁省及哈尔滨地区采样诱导率哈尔滨居首位,采样时间以五月份愈伤组织诱导率高达５６．５％;叶片不同部位以叶前部和后部为好;叶脉方向对诱导率无明显差导。诱导愈伤组织中注意及时转移、继代、防止在培养中的褐变。克服褐变需对愈伤组织选择出组织紧密、褐变速度慢、同时在培养基中加入一些抗氧化剂,其效果明显。目前愈伤组织三颗粒状,已见绿芽点,现转入分化试验。     

Organogenesis andin vitro flowering ofEchinochloa colona. Effect of growth regulators and explant types

Echinochloa colona regeneration via organogenesis in callus cultures derived from leaf base and mesocotyl expiants andin vitro flowering were achived. Shoot bud regeneration was achieved on Murashige and Skoog’s (MS) basal medium supplemented with 6.66 μM 6-benzylaminopurine (BAP), 2.68 μM 1-naphthalene acetic acid (NAA) and 3 % (m/v) saccharose. Regenerated shoots were rooted on half strength basal MS medium with 2 % (m/v) saccharose devoid of growth regulators. About 90 -95 % of rooted plantlets survived in the greenhouse.In vitro flowering was induced in the regenerated shoots derived from callus on half strength MS medium supplemented with 4.4 μM BAP, 74.07 μM adeninesulphate, 0.72 μM gibberellic acid, and 3 % (m/v) saccharose. The frequency ofin vitro flowering was 80 – 90 % in three repeated experiments. Fertile seeds were recovered fromin vitro grown plantlets which were subsequently germinated into plants. Acknowledgement: The authors wish to thank to the Department of Environment and Forests, Government of India for financial assistance to undertake this investigation.     

Effect of picloram and methyl jasmonate on growth and taxane accumulation in callus culture of Taxus × media var. Hatfieldii

We have analysed the effect of some culture conditions and media components on callus growth rate and production of taxanes in callus of Taxus × media var. Hatfieldii. For callus induction and maintenance a Gamborg B5 medium and a White - Rangaswamy medium (WR) with different modifications were used. On an improved WR medium (containing 10 μM picloram) the callus growth factor increased up to 5.8 fold (fresh weight). Picloram only enhanced the growth of callus, but not taxane production. On WR medium with (100 μM) methyl jasmonate the paclitaxel content increased from 2.37 μg g-1 to 90 μg g-1 and cephalomannine from 5.14 μg g-1 to 29.14 μg g-1 (dry weight), whereas growth of the cultures ceased. The presence of paclitaxel and cephalomannine was established by high performance liquid chromatography. This revised version was published online in June 2006 with corrections to the Cover Date.     

Production and cryoconservation of embryogenic cultures of mandarin and mandarin hybrids

Assays were performed to obtain embryogenic callus lines from nine mandarin and mandarin hybrid cultivars by in vitro culture of ovules collected from immature fruits six weeks after anthesis. All cultivars produced embryos and loose friable callus. The small proliferations of nucellar callus from cultured ovules were suitable to recover embryogenic callus lines by periodical subculturing to fresh medium. The embryogenic callus cultures were subjected to cryoprotection with 10% (v/v) (DMSO), freezing by slow cooling, storage in liquid nitrogen and thawing by fast warming. Whole plants from these cryopreserved cultures were recovered through embryogenesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Organogenesis and plant regeneration in Taxus wallichiana (Zucc.)

We describe an efficient process for regeneration of Taxus wallichiana plants via shoot organogenesis from callus cultures derived from zygotic embryos. Zygotic embryos cultured on half strength Lloyd and McCown's basal medium supplemented with SH vitamin ((1/2) WPMSH), 0.5 mg l(-1) 6-benzyladenine (BA) in combination with 1.0-2.0 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D) or alpha-Napthaleneacetic acid (NAA) produced two morphologically distinct types of calli-compact, green callus (CG) and compact, yellow (CY) callus after 4 weeks of culture. Optimum frequency (63%) of adventitious shoot bud induction was achieved in CG callus (3.0+/-0.67 shoot buds per gram of CG callus) when cultured on (1/2) WPMSH basal medium supplemented with 2.5 mg l(-1) BA after 4 weeks. The inclusion of 1% activated charcoal (AC) to (1/2) WPMSH basal medium (shoot elongation medium) led to maximum shoot elongation (2.15 cms). Microshoots rooted in high frequency (40%) in MS basal medium in which the concentration of nitrates was reduced to one-fifth the normal concentration after 4 months of culture.     

Plant Regeneration Through Somatic Embryogenesis in Taxus wallichiana

We describe an efficient process for regeneration of Taxus wallichiana (Zucc) plants from callus cultures derived from zygotic embryos. Zygotic embryos cultured on half strength Lloyd and McCown’s basal medium supplemented with SH vitamin (1/2 WPMSH), 0.5 mg I^?1 6-benzyladenine (BA) and 1.0–2.0 mg I^?1 á-Napthaleneacetic acid (NAA) produced compact yellow (CY) callus after 4 weeks of culture. The 8-week-old CY call! (lines CY-A and CY-B) were initially slow growing but proliferated on transfer to WPM basal medium supplemented with 8.0 mg I^?1 2,4-D, 0.1–0.9 mg^?1 NAA and 0.3–1.0 mg^?1 BA after 4 weeks. Four morphologically distinct calli lines were obtained, of which only two call! lines, CY-B-FW and CY-B-FY were embryogenic. The 12-week-old callus line CY-B-FW developed globular somatic embryos on transfer to secondary medium after 8 weeks and matured in maturation medium after 4 weeks. Only 10% of the mature somatic embryos regenerated into complete plantlets after 4 weeks on conversion medium. Although the frequency of conversion was low, complete regenerated plantlets via somatic embryogenesis were obtained after 7–8 months of initiation of culture. Taxane analysis showed that the paclitaxel accumulation was higher in embryogenic callus than in non-embryogenic callus.     

Cryopreservation of cell suspension cultures of <Emphasis Type="Italic">Taxus</Emphasis> × <Emphasis Type="Italic">media</Emphasis> and <Emphasis Type="Italic">Taxus floridana</Emphasis>

Different lines of cell suspension cultures of Taxus × media Rehd. and Taxus floridana Nutt. were cryopreserved with a two-step freezing method using a simple and inexpensive freezing container instead of a programmable freezer. Four to seven days old suspension cell cultures were precultured in growth medium supplemented with 0.5 M mannitol for 2 d. The medium was then replaced with cryoprotectant solution (1 M sucrose, 0.5 M glycerol and 0.5 M dimethylsulfoxide) and the cells incubated on ice for 1 h. Before being plunged into liquid nitrogen, cells were frozen with a cooling rate of approximately −1 °C per min to −80 °C. The highest post-thaw cell viability was 90 %. The recovery was line dependent. The cryopreservation procedure did not alter the nuclear DNA content of the cell lines. The results indicate that cryopreservation of Taxus cell suspension cultures using inexpensive freezing container is possible.     

Improved Taxol production by combination of inducing factors in suspension cell culture of Taxus baccata

To date enormous attempts have been devoted to improve Taxol production exploiting various methodologies from bioprocess engineering to biotechnological and synthetic approaches. We have developed a 2-stage suspension cell culture of Taxus baccata L. using modified B5 medium in order to improve cell growth as well as productivity. After callus induction and cell line selection, B5 medium was supplemented with vanadyl sulfate (0.1 mg/l), silver nitrate (0.3 mg/l) and cobalt chloride (0.25 mg/l) at the first day of stage I culture to maximize cell growth. This medium was further supplemented with sucrose (1%) and ammonium citrate (50 mg/l) on day 10 and sucrose (1%) and phenylalanine (0.1 mM) on day 20 (i.e., biomass growth medium). At stage II (day 25), two different concentrations of several elicitors such as methyl jasmonate (10 or 20 mg/l), salicylic acid (50 or 100 mg/l) and fungal elicitor (25 or 50 mg/l) were added to the biomass growth medium with the aim of improving cellular productivity. For morphological analysis, microscopic inspection was carried out during cultivation. Cell-associated and extracellular amount of Taxol were detected and measured using HPLC methodology. At stage I, overall Taxol amount of biomass growth medium was 13.75 mg/l (i.e., 5.6-fold higher than that of untreated B5 control). At stage II, treated cells with methyl jasmonate (10 mg/l), salicylic acid (100 mg/l) and fungal elicitor (25 mg/l) produced the highest amount of Taxol (39.5 mg/l), which is 16-fold higher than that of untreated B5 control (2.45 mg/l). Microscopic analyses of Taxus cells in suspension cultures showed various positional auto-fluorescence showing direct correlation with Taxol production. Our studies revealed that intervallic supplementation of B5 medium with combination of biomass growth factors at stage I and mixture of elicitors at stage II could significantly increase Taxol production. Thus, we suggest that the exploitation of this methodology may improve the production of Taxol since demands for Taxol pharmaceuticals are increasingly growing and resource paucities have limited its direct harvesting from Taxus trees.