大花卷丹的组织培养及限制生长保存

离体保存是植物种质保存的重要手段之一,为实现对大花卷丹的保护性利用,本文对其组织培养体系及限制生长离体保存技术进行了研究。结果表明,在常温（23±2）℃、光照强度约为40μmol.m-2.s-1、光照时间14h.d-1的条件下,大花卷丹鳞片在MS＋6-BA1.0mg.L-1＋NAA0.2mg.L-1培养基中生长情况较好,能直接诱导芽,且小鳞茎的生长速度较快。将诱导出的小鳞茎切割后,接种到1/2MS＋NAA0.5mg.L-1＋活性炭2g.L-1生根培养基2～3周即能生根,生长状况良好。提高MS培养基中蔗糖达90和110g.L-1时可以抑制其生长,能够保存大花卷丹试管苗10个月,保存过程中生长正常,株高生长缓慢,但根长势较快。在蔗糖浓度90g.L-1基础上再添加30g.L-1甘露醇的培养基能进一步抑制试管苗根的生长。6个月后,转移到正常培养基上培养均能恢复生长,其鳞片在诱导培养基上能正常分化。因此,采用大花卷丹鳞片组织培养可以形成种苗,在培养基中添加高蔗糖浓度和甘露醇可以使其试管苗保存1年以上。     

巴戟天种质离体保存研究

以巴戟天试管苗为材料,研究不同浓度的无机盐、蔗糖和四种植物生长调节剂(CCC、PP333、ABA、MH)对巴戟天试管苗保存的影响。结果表明:巴戟天试管苗在无生长调节剂的MS、1/2MS、1/4MS三种无机盐浓度培养基上均能保存培养360d,存活率达90%;蔗糖浓度为20~40g/L时,植株生长健壮,能延长保存时间;四种生长调节剂均能诱导试管苗侧芽的生成,抑制顶芽和叶片的生长。其中以PP333促进壮苗效果最好,能提高试管苗素质,在1/2MS+PP3330.5~1.0mg/L+蔗糖30g/L培养基上,试管苗能保存480d,存活率达100%。     

药用植物红根草种质资源的离体保存

以红根草试管苗为材料,研究了不同培养基(MS、1/2MS、1/4MS)、蔗糖浓度和植物生长抑制剂(CCC、PP333、ABA、MH)在红根草试管苗保存中的作用。结果表明:培养基1/2MS对红根草保存最好,保存270d后存活率最高。培养基中不添加蔗糖较添加一定浓度蔗糖时植株的形态和色泽差,但保存时间更长,转继代后能正常恢复生长。添加生长抑制剂能减缓生长速度,延长保存时间,最佳浓度分别为:CCC1.2～1.6mg/L;PP3331.6mg/L;ABA0.5～4.0mg/L;MH0.5mg/L。其中,ABA0.5～4.0mg/L对植株的生长最好,CCC浓度为1.2mg/L和1.6mg/L时,保存时间长,360d时,存活率达90%。     

铁皮石斛快速繁殖和离体种质保存的研究

对铁皮石斛种子发芽、原球茎增殖、丛芽分化和壮苗培育进行了试验、观察和分析,研究了培养基、植物激素、光强和添加剂等对其分化和生长的影响。结果显示种子在1/2MS+蔗糖2%的培养基上,30d萌发95%以上。原球茎在1/2MS+椰子汁25%+蔗糖3%的培养基上,45~60d原球茎增殖速度可达1∶10。丛芽分化较适宜的培养基为1/2MS+BA2mg·L-1+NAA0.2mg·L-1+IBA0.1mg·L-1+蔗糖3%,45~60d芽丛增殖速度为1∶4~5。试管苗在MS+香蕉泥20%+蔗糖2%培养基上,大约60d苗快速长高,茎粗壮且根系发达。离体保存材料可采用试管丛芽和原球茎两种方式,以保持其遗传多样性。保存方法是在15℃左右条件下,保存离体材料,继代间隔期为12~18个月;也可以采用室温保存,在1/2MS+蔗糖1%培养基上,继代间隔期可延长至10~12个月。     

铁皮石斛原球茎常温保存研究

以铁皮石斛原球茎为材料,通过不同培养基、蔗糖浓度、继代周期、保存时间等多种因素对原球茎在保存过程中增殖生长和分化成苗的影响,进行铁皮石斛原球茎常温保存的研究。结果表明:铁皮石斛原球茎常温((25±2)℃)保存的适宜培养基为1/2MS、蔗糖浓度为1%,继代周期可达10个月;原球茎在5年内能保持分化和增值能力,随着保存年限的增加,分化率越来越低,可通过复壮和成苗培养提高分化成苗率。     

鸢尾的组织培养

植物名称:鸢尾(Iris tectorum)。材料类别:种子。培养条件:培养基:(1)MS+IBA0.5mg/l(单位,下同)+BA1+KT1;(2)MS 0(无生长素的MS培养基)。培养物置于25℃恒温,光照度1000—2000lx,日照10—12小时的条件下培养。生长与分化情况:将一粒种子播于培养基(1)上,进行静置培养。约经2个月后种子萌发,伸出子叶,成为无根子叶苗。子叶苗下胚轴接触培养基处的组织形成膨大的小块突起,形似球状,以后小块数目逐渐增多,形成结节状组织。在播种后的第四个月,可见到结节组织的球状突起处分化出小苗,     

紫花苞舌兰类原球茎诱导及植株再生

为建立紫花苞舌兰类原球茎离体再生体系,本研究以其腋芽为外植体,研究了不同激素浓度配比、添加剂对其类原球茎诱导、分化及生根诱导的影响。结果表明,外植体在1/2 MS+6-BA2.0 mg/L+NAA0.2 mg/L+AC(活性炭)0.2 g/L+CW(椰汁)10%培养基上培养35 d后,获得无菌苗,其成活率达80%以上;无菌苗的顶端分生组织在1/2 MS+10 mg/L Picloram培养基上的类原球茎的诱导率最高,达75%;类原球茎在1/2 MS+6-BA 2.0 mg/L+IBA 0.2 mg/L培养基上培养45 d后分化出小苗,分化率为54%;小苗在1/2 MS+6-BA 0.5 mg/L+IBA 1.0 mg/L+AC 0.2 g/L培养基上生根培养35 d后,生根率达85%以上;小苗驯化移栽成活率达95%,长势良好。     

纪伊潮菊离体保存及其遗传稳定性分析

通过在基本培养基中添加不同浓度配比的蔗糖和矮壮素(CCC)对纪伊潮菊(Chrysanthemum shiwogiku var.kinokuniense)离体保存的影响进行研究,并对保存材料再生后代的遗传稳定性进行分子标记鉴定与分析.结果表明:在(23±2)℃、2 000～3 000 lx光照强度、12 h/d的光照培养条件下,MS+0.5 mg·L~(-1) BA+0.1 mg·L~(-1) NAA+琼脂6.5 g·L~(-1)培养基中添加30 g·L~(-1)蔗糖和1 500～2 000 mg·L~(-1)的CCC能够保存试管苗12个月,存活率为92.86%~96.43%,且恢复生长后试管苗长势良好,其再生后代的形态特征、过氧化物酶(POD)活性和ISSR分子标记扩增图谱与对照株无差异.     

黄花蒿组培快繁与种质离体保存的研究

以带侧芽的黄花蒿(Artemisia annua L.)茎段为外植体,以MS为基本培养基,进行组织培养和种质保存研究.结果表明,培养基MS 6-BA 1.0 mg L-1 IBA 0.1 mg L-1、MS 6-BA 0.5 mg L-1 IBA 0.1 mg L-1和MS NAA0.1 nag L-1 IBA 0.5 rng L-1可分别用于黄花蒿的芽诱导、增殖和生根培养,培养20 d的增殖倍数为5.5倍,生根率98.3%.培养基MS CCC 1.0 mg L-1、MS CCC 2.0 nag L-1、MS PP3334.0 mg L-1可用作离体保存,连续保存200 d的存活率分别达72.3%、77.0%、69.2%.活力检测表明,黄花蒿种质经保存后的增殖、生根能力没有下降.因此,可通过诱导腋芽增殖建立黄花蒿快繁体系,及在培养基中添加CCC或PP333拼能使材料长期保存.     

蝴蝶兰原球茎增殖分化影响因子探讨

采用3个月胚龄的种胚播种形成的原球茎,初代培养时间在2.5~3个月之间换瓶转接,原球茎生长状况较好,分化形成的芽苗生长健壮;以1/2MS为基本培养基培养30d,原球茎增殖率达332%,MS培养基更有利于分化,培养2个月分化发芽率达90.6%;附加不同细胞分裂素原球茎的增殖率为6-BA >KT >Ad+KT >Ad;NAA浓度在0~1.0mg/L,对原球茎增殖分化影响不明显。     

Droplet-vitrification and morphohistological studies of cryopreserved shoot tips of cultivated and wild pineapple genotypes

Germplasm conservation of pineapple [Ananas comosus (L.) Mer.] is crucial to preserve the genus’ genetic diversity, to secure material for genetic improvement and to support innovative and new research. Long-term conservation is accomplished through cryopreservation, that is done by storing cells or tissues at ultra-low temperature in liquid nitrogen (−196 °C). Droplet-vitrification, a combination of droplet freezing and solution-based vitrification, was used to establish a protocol for cryopreservation of pineapple genetic resources. This protocol was tested on cultivated and wild pineapple genotypes to establish a long-term germplasm security duplicate as well as to investigate cryo-injuries in the tissues by means of histological techniques. Excised shoot tips (0.5–1 mm with one primordial leaf) of different pineapple genotypes were precultured for 48 h on solid MS medium containing 0.3 M of sucrose. Three PVS2 exposure times (30, 45 and 60 min) were tested. The results showed high post cryopreservation survival for all genotypes evaluated. The best PVS2 exposure time varied according to genotype, although 45 min gave the best survival for the majority of genotypes. The technique was highly efficient in cryopreserving meristem shoot tips of different pineapple genotypes, and was also less laborious than techniques previously reported. This is a first report on application of the droplet-vitrification technique to diverse genotypes of cultivated and wild pineapples and the first report on histological changes occurring in cryopreserved Ananas tissue.

     

Alginate-encapsulation of nodal segments for propagation, short-term conservation and germplasm exchange and distribution of Eclipta alba (L.)

Nodal segments obtained from in vitro proliferated shoots of Eclipta alba (L.) Hassk, were encapsulated in calcium alginate beads for large-scale clonal propagation, short-term conservation and germplasm exchange and distribution. The best gel complexation was achieved using 3% sodium alginate and 100 mM CaCl₂·2H₂O. Maximum percent response (100%) for conversion of encapsulated nodal segments into plantlets was obtained on 0.7% agar-solidified full-strength MS medium containing 0.88 μM BAP. Encapsulated nodal segments could be stored at low temperature (4°C) up to 60 days with a survival frequency of 51.2%. The well-developed plantlets regenerated from encapsulated nodal segments were hardened-off successfully with 90% survival frequency.     

Encapsulation of nodal segments of Cassia angustifolia Vahl. for short-term storage and germplasm exchange

The present study described the encapsulation of nodal segments of Cassia angustifolia Vahl. excised from 1-month-old in vitro raised cultures for short-term conservation and propagation. Various concentrations and combinations of gelling matrix (sodium alginate) and complexing agents (calcium chloride) were tested to prepare uniform beads. The ideal beads were obtained through a combination of 3 % sodium alginate and 100 mM calcium chloride. The maximum conversion response (94 %) of encapsulated beads was obtained in Murashige and Skoog’s medium (MS medium) supplemented with 2.5 μM benzyladenine (BA) and 0.4 μM α-naphthalene acetic acid (NAA) after 6 weeks of culture. The encapsulated and non-encapsulated nodal segments were also stored at 4 °C for different time periods (0, 1, 2, 4, 6 and 8 weeks). The regenerated microshoots were best rooted in optimized rooting medium that comprised half-strength MS + 1.0 μM indole-3-butyric acid (IBA) + 5.0 μM phloroglucinol (PG) for the production of complete plantlets. The regenerated plantlets were successfully hardened and acclimatized in natural conditions with 70 % survival rate.     

High-frequency shoot multiplication in Artemisia vulgaris L. using thidiazuron

An in vitro propagation system for Artemisia vulgaris L., a traditional medicinal plant, has been developed. The best organogenic response, including adventitious shoot number and elongation, was obtained when hypocotyl segments were cultured onto MS medium supplemented with 4.54 μM TDZ (N-phenyl-N′-(1,2,3-thidiazol-yl) urea). Up to 28 shoots formed per explant for an optimal duration of exposure of 48 days. Regenerated shoots formed roots when subcultured onto a medium containing 8.56 μM IAA (indole-3-acetic acid). Healthy plantlets were transferred to a garden soil:farmyard soil:sand (2:1:1) mixture for acclimatization, which was successful, and subsequent maturity was achieved under greenhouse conditions over a six-month period. The survival rate of the plantlets varied under acclimatization. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of medicinally active constituents of A. vulgaris. This optimized protocol has been successfully employed for genetic transformation studies in A. vulgaris, which are currently underway in our laboratory.     

珍稀药用和观赏植物地涌金莲的组织培养和快速繁殖

以地涌金莲(Musella lasiocarpa)吸芽为外植体建立了有效的快繁体系。外植体经灭菌处理后在MS 6-BA4.0 mg L-1 NAA 0.2 mg L-1 维生素C 150 mg L-1 10%椰子乳 3%蔗糖培养基上能进行不定芽的诱导和增殖,培养60 d后每个芽平均能产生4.10个不定芽。第6代增殖后,丛生芽的增殖系数可达4.23。生根培养基以1/2MS NAA1.0 mg L-1 AC 50 mg L-1的效果较好。以沙:泥炭土:珍珠岩=1:1:1为基质移栽试管苗,成活率达到93.5%以上。经过12个月的组织培养已生产10 000多株试管苗。     

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。     

Plant regeneration via somatic embryogenesis in Drimia robusta

A simple efficient in vitro plant regeneration system was developed by direct and indirect somatic embryogenesis of Drimia robusta, a medicinal plant extensively used in South African traditional medicine. Different developmental stages of somatic embryos (SEs: globular embryos, partial pear-shaped embryos and club-shaped embryos), club-shaped cotyledon initiation, plumule initiation and plantlets were directly obtained from leaf explants on Murashige and Skoog (MS) medium containing 3.5 % (w/v) sucrose and different plant growth regulators (PGRs). In MS medium containing 3.5 % (w/v) sucrose and supplemented with 10 μM picloram, 1 μM thidiazuron (TDZ) and 20 μM glutamine, a higher number of SEs and plantlets were achieved. These were established onto half-strength MS medium followed by successful acclimatization (100 %) in the greenhouse. Liquid somatic embryo medium (SEM_L) containing 500 mg of friable embryogenic callus on MS medium supplemented with different concentrations and combinations of PGRs and organic elicitors produced different stages of SEs. Somatic embryo production was enhanced by 0.5 μM picloram, 1 μM TDZ and mebendazole treatment. The highest number of plantlets (9.0 ± 0.70) was obtained in SEM_L containing 0.5 μM picloram, 1 μM TDZ and 25 mg l^?1 haemoglobin. All the cotyledon and plumule embryos germinated on half-strength MS medium, however 90 % of SEs germinated on half-strength MS medium containing 0.5 μM naphthaleneacetic acid. All plantlets were successfully acclimatized in the greenhouse. This first report of D. robusta somatic embryogenesis provides an opportunity to control extinction threats, ensure germplasm conservation and provides a system for analysis of bioactive compounds and bioactivity.     

半夏缓慢生长法保存及体细胞变异的ISSR检测

以添加了不同浓度的甘露醇、PP333和ABA的培养基对半夏试管苗进行缓慢生长法保存,并对保存材料再生后代的体细胞变异进行检测。结果显示,甘露醇、PP333和ABA均能有效抑制试管苗生长,且存活率高;最佳浓度分别为甘露醇2%～4%,PP3332.0mg·L-1,ABA 2.0～4.0mg·L-1。保存在添加了2%～4%甘露醇或2.0mg·L-1 PP333培养基上的植株未检测到变异,而保存在添加了2.0～4.0mg·L-1 ABA培养基上的植株检测到1条新增标记和1条缺失标记,位点变异率为1.7%,个体变异率为30%。研究表明,ABA不宜用于半夏试管苗的缓慢生长法保存,但有助于新突变体的产生,在种质创新上具有特殊意义。     

Double-phase culture system for large scale production of pineapple

We have developed an efficient and simplified method for shoot proliferation of the pineapple (Ananas comosus L.), using a Double-Phase Culture System (DPS) (semi-solid medium with a layer of liquid medium on the top). Under these conditions, up to 430 microshoots were produced from 5 initial shoots within 5 months, and without shoot manipulation. Thus, >50% shoots proliferated as compared to that on agar-solidified medium. Shoots taken from DPS were rooted within 4 weeks at a frequency of up to 100%, in full-strength or half-strength Murashige and Skoog medium containing 0.5–2.5 μM indole-3-butyric acid. The plantlets were then transferred into soil, and they survived acclimatization with 100% success. Thus, DPS could be useful for large-scale micropropagation of a range of pineapple cultivars, with high production of plantlets at the end of process, and reduced labor costs during the subcultures.