天然有机物对铁皮石斛原球茎生长及有效成分的影响

为铁皮石斛原料药的生产提供一种新方法。以MS为基本培养基,探讨天然有机附加物与培养时间对铁皮石斛原球茎(PLBs)生长及主要药用成分石斛多糖和总生物碱的影响。研究结果表明:在添加不同浓度土豆汁的培养基中,以250 g/L效果最好,其干物率和多糖含量均在30天时达最大值,分别为8. 944%和17. 921%,增殖率在培养50天时最高,为1 028. 94%,而总生物碱含量在40天时积累最多,达0. 031 61%。增殖率、多糖和总生物碱含量均显著高于添加激素的对照组。添加番茄汁的培养基中各指标变化与土豆汁的类似,但原球茎长势差,颜色泛黄,增殖能力较添加土豆汁的差。综上,MS+250 g/L土豆汁+30 g/L蔗糖+7 g/L琼脂条有利于铁皮石斛PLBs生长和有效成分的积累,可考虑作为石斛多糖和石斛碱的生产来源,对铁皮石斛主要药用成分的生产具有重要的实践意义。     

氮源和真菌诱导子对铁皮石斛原球茎悬浮培养的影响

利用正交实验设计研究不同种类和浓度的氮源对铁皮石斛原球茎生长的影响,利用完全随机实验设计研究不同真菌诱导子对铁皮石斛原球茎生长和多糖积累的影响.结果表明硝态氮促进铁皮石斛原球茎鲜重和干重的增加( P ＜0.05);铵态氮促进铁皮石斛原球茎鲜重增加( P ＜0.05).无论鲜重还是干重,硝态氮和铵态氮的影响均不存在互作,最佳组合为:67-V培养液 100 mg/L (NH4)2SO4 800 mg/L KNO3 30 g/L蔗糖 200 g/L马铃薯提取汁.F检验的结果表明,14种真菌诱导子对铁皮石斛原球茎生物量的增加(鲜重与干重)均无显著性影响( P ＞0.05).但与对照铁皮石斛原球茎的多糖含量相比,真菌诱导子g6、g14、g5、g4处理可使其多糖含量分别提高14%、9%、6%、4%.本研究获得了有利于铁皮石斛原球茎生长的硝态氮和铵态氮的最佳搭配方案以及有利于铁皮石斛原球茎积累多糖的四种真菌诱导子,表明通过液体悬浮培养生产铁皮石斛原球茎及其多糖成分具有较好的开发应用前景.     

6-BA和2,4-D对铁皮石斛原球茎增殖、分化和离体保存的影响

本文探讨6-BA和2,4-D对铁皮石斛原球茎增殖和分化的影响。结果表明：铁皮石斛原球茎芽再生的较好培养基为MS,芽增长的较好培养基为MS + 6-BA 1 mg/L + 2,4-D 0.1 mg/L,原球茎再生芽生根和原球茎增殖的较好培养基也为MS。添加0.5 mg/L 2,4-D不利于铁皮石斛原球茎的保存,而不添加2,4–D或添加0.1 mg/L 2,4-D均有利于铁皮石斛原球茎保存,且有持续增长趋势。     

中药材铁皮石斛组培苗不同培养基的筛选与优化

铁皮石斛组培苗在生长各个阶段对营养物质的需求不一样,培养条件不同组培苗生长结果也不一样。本研究以铁皮石斛种子为材料,通过无菌播种进行原球茎诱导、增殖、分化、生根试验,对各阶段培养基进行比较,以期筛选出最佳培养基条件,为将来铁皮石斛走向工厂化、标准化生产提供思考和借鉴。结果表明,种子萌发以及原球茎诱导的最佳培养基配方是1/2 MS培养基,35 d后萌发率达93.1%。原球茎增殖最佳培养基配方组合是MS+6-BA(1.5 mg/L)+NAA(0.5 mg/L),增殖系数达14.8。原球茎分化成苗的最佳培养基配方组合是MS+6-BA(0.5 mg/L)+NAA(0.2 mg/L),分化率达92.1%,且长势良好。组培苗生根的最佳培养基配方组合是1/2 MS+NAA(1.5 mg/L),生根率达97.7%,根系健壮发达。     

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。     

铁皮石斛同源四倍体工厂化快繁工艺研究

以实验室前期诱导的铁皮石斛四倍体为研究材料,利用气孔、叶绿体、染色体计数及流式细胞仪等方法,对铁皮石斛同源四倍体株系的纯合性进行鉴定,在此基础上利用茎段扩繁和原球茎诱导、增殖、分化两种方案比较增殖效率,并对原球茎的倍性稳定性进行再鉴定。结果表明：（1）实验室的‘花自 2’株系为纯合同源四倍体。（2）茎段扩繁最佳增殖培养基为MS＋2.0 mg/L 6 BA+0.5 mg/L NAA+15%香蕉汁,茎段诱导原球茎最佳培养基为MS+1.0 mg/L 6 BA+0.5 mg/L NAA+0.2 mg/L KT,诱导率达到76.66%;原球茎增殖最佳培养基为MS+1.0 mg/L 6 BA+0.5 mg/L NAA+15%马铃薯提取液,原球茎分化最佳培养基为1/2 MS+0.5 mg/L 6 BA+20%马铃薯提取液,原球茎分化率达到85.70%;生根最佳培养基为1/2 MS＋0.5 mg/L NAA＋15%香蕉汁,生根率达到92.77%。（3）原球茎根尖鉴定结果表明,诱导的倍性可以稳定遗传。（4）瓶苗移栽的最佳炼苗时间为5 d,移栽成活率达92.30%。该试验建立了铁皮石斛同源四倍体株系新的快繁技术体系,为四倍体试管苗的工厂化生产和推广奠定了技术基础。     

Study on technology of aseptic sowing and rapid propagation of Demdrobium officinale

以铁皮石斛的蒴果为外植体,采用种子→原球茎→完整植株→移栽的途径快速成苗进行工厂化生产,对各阶段培养基进行筛选,以及其他一些影响因子进行比较研究.结果表明:人工授粉后生长60～180 d的铁皮石斛种子在离体条件下均能萌发,其中授粉150～180 d种子的萌发效果最好,萌发率为87.2%～94.4%,适宜的萌发培养基为MS+6-BA 1.0 mg/L+NAA 0.1 mg/L+马铃薯汁200 g/L+AC 1.0 g/L;原球茎增殖的最佳培养基为MS+6-BA 1.5 mg/L+NAA 0.1 mg/L+香蕉汁100 g/L+AC 1.0 g/L,繁殖系数约为20倍/50 d;原球茎在MS+6-BA 1.0 mg/L+NAA 0.1 mg/L+马铃薯汁200 g/L+AC 1.0 g/L培养基上进行分化培养,分化的同时还能进行一定的增殖;将已分化的芽苗转接到壮苗培养MS+6-BA 0.5 mg/L+NAA 0.2 mg/L+香蕉汁100 g/L+AC 1.0 g/L上培养1代后,转接到生根培养基1/2MS+NAA 0.8 mg/L+无机盐A 0.2～0.5 mg/L +香蕉汁100 g/L+AC 1.0 g/L上,培养50～70 d后,生根率100%,无机盐A可以有效地控制愈伤或原球茎的形成,明显提高生根苗的数量和质量.在桂林地区,生根苗以3～5月和9～10为最佳移栽期,以通过高温处理并堆沤腐熟的松树皮为基质,移栽成活率可达90%.     

铁皮石斛无菌播种产业化繁育技术研究

以铁皮石斛的蒴果为外植体,采用种子→原球茎→完整植株→移栽的途径快速成苗进行工厂化生产,对各阶段培养基进行筛选,以及其他一些影响因子进行比较研究。结果表明:人工授粉后生长60～180d的铁皮石斛种子在离体条件下均能萌发,其中授粉150～180d种子的萌发效果最好,萌发率为87.2%～94.4%,适宜的萌发培养基为MS+6-BA 1.0mg/L+NAA 0.1mg/L+马铃薯汁200g/L+AC 1.0g/L;原球茎增殖的最佳培养基为MS+6-BA 1.5mg/L+NAA 0.1mg/L+香蕉汁100g/L+AC 1.0g/L,繁殖系数约为20倍/50d;原球茎在MS+6-BA 1.0mg/L+NAA 0.1mg/L+马铃薯汁200g/L+AC 1.0g/L培养基上进行分化培养,分化的同时还能进行一定的增殖;将已分化的芽苗转接到壮苗培养MS+6-BA 0.5mg/L+NAA 0.2mg/L+香蕉汁100g/L+AC 1.0g/L上培养1代后,转接到生根培养基1/2MS+NAA 0.8mg/L+无机盐A 0.2～0.5mg/L+香蕉汁100g/L+AC 1.0g/L上,培养50～70d后,生根率100%,无机盐A可以有效地控制愈伤或原球茎的形成,明显提高生根苗的数量和质量。在桂林地区,生根苗以3～5月和9～10为最佳移栽期,以通过高温处理并堆沤腐熟的松树皮为基质,移栽成活率可达90%。     

诱导子CuSO4对金钗石斛原球茎产石斛碱的影响

为了探究在金钗石斛原球茎培养过程中,添加诱导子CuSO₄对其石斛碱产量的影响,并获得原球茎产石斛碱的优化条件,为后续多因素胁迫金钗石斛原球茎高产石斛碱提供数据支撑。采用液体悬浮培养的方式,研究了诱导子CuSO₄在不同添加时间、不同浓度、添加后不同培养时间3种处理因素对金钗石斛原球茎生物量和产石斛碱的影响。结果表明:添加0.25 mmol/L CuSO₄培养10～20 d,对原球茎的抑制作用强于培养30～40 d,其中培养20 d为最佳添加时间,石斛碱含量最高达29.65μg/g。CuSO₄浓度为0.25 mmol/L时,石斛碱含量最高达35.10μg/g,原球茎生物量也最多,达2.72 g,增殖系数达到了0.36;随着CuSO₄浓度的增加,经不同处理的原球茎抑制程度逐渐增加,原球茎也出现褐化死亡。添加诱导子后培养时间处理因素对原球茎的影响不明显,当培养至20 d时,石斛碱含量最高达29.37μg/g。最优的CuSO₄胁迫条件:在原球茎培养20 d时,加入浓...     

低温和外源NO对铁皮石斛原球茎多糖合成的影响

以铁皮石斛(Dendrobium officinale)原球茎为材料,研究了低温(4℃)、外源NO(NO供体SNP)以及NO清除剂(cPTIO)和一氧化氮合酶抑制剂(PBITU)对铁皮石斛原球茎中NO含量、蔗糖合成酶(SS)活性、多糖含量以及蔗糖、果糖、葡萄糖等糖含量的影响,以明确低温和內源NO在多糖合成中的关系。结果显示:(1)4℃低温处理下,铁皮石斛原球茎中NO含量显著上升,SS活性升高,蔗糖、果糖和葡萄糖含量增加,多糖含量也得到提高;SNP(0.5mmol·L-1)处理与4℃低温处理具有相似的效果;且低温诱导的铁皮石斛原球茎中SS活性提高和多糖含量的增加时期均在NO大量产生之后、蔗糖的积累早于果糖和葡萄糖。(2)4℃低温+SNP组合处理能够显著提高铁皮石斛原球茎中SS活性、NO含量以及蔗糖、果糖、葡萄糖和多糖含量,它们分别比对照组显著高出了68.04%,96.20%,60.69%、45.64%、66.90%和67.03%,且比低温和SNP单独处理效果都好。(3)PBITU能够部分抑制低温诱发铁皮石斛原球茎中产生NO,抑制率达到77.15%;同时还抑制了低温对铁皮石斛原球茎中SS活性、多糖合成和蔗糖、果糖、葡萄糖积累的促进作用。(4)SNP+cPTIO和4℃+cPTIO处理组中铁皮石斛原球茎SS活性和蔗糖、果糖、葡萄糖、多糖含量及NO水平,且与对照组差异不显著。研究表明,低温和外源NO对铁皮石斛原球茎多糖的合成均具有促进作用,并且低温可诱导铁皮石斛原球茎产生NO,SS活性提高和多糖含量增加与NO产生相关,说明NO是诱导铁皮石斛原球茎多糖合成所必需的信号分子。     

Tissue culture independent transformation for Corchorus olitorius

An efficient microprogation protocol has been developed for Dendrobium densiflorum Lindl. ex Wall., a traditional medicinal plant, through protocorm-like bodies (PLBs) from nodal stem segments using 6-benzylamino-purine (BAP) and the lanthanoid neodymium. The highest percentage of explants producing PLBs (72%), with an average of 15 PLBs per explant, was induced by culturing stem segments on Murashige and Skoog (MS) medium supplemented with 5.0 mg l⁻¹ BAP. The newly formed PLBs proliferated well on the basal MS medium and completely converted into shoots on MS medium containing 2.0 mg l⁻¹ BAP. Shoots produced an average of 22 roots per plantlet when cultured on MS medium supplemented with 2.0 mg l⁻¹ neodymium nitrate. Healthy plantlets with well-developed roots were successfully acclimatized. The obtained result suggests that the lanthanoids can be used to effectively initiate rooting in the micropropagation and conservation of D. densiflorum.     

硝酸镧和兰科菌根真菌对铁皮石斛生理特性的影响

利用盆栽的方式研究了不同硝酸镧水平(0、1.0、3.0、5.0和7.0mg·L-1)下接种兰科菌根真菌对铁皮石斛生物量、多糖和蛋白质合成的影响,并分析了叶绿素含量、丙二醛(MDA)含量以及超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)活性的变化,以探讨硝酸镧和兰科菌根真菌对铁皮石斛生理特性的影响。结果表明:(1)添加适量的硝酸镧有利于菌根真菌侵染和菌根发育,提高铁皮石斛幼苗生物量。(2)在接种菌根真菌的同时添加5.0mg·L-1的硝酸镧,铁皮石斛的根重、茎叶重和总生物量均达到最大,分别是未添加硝酸镧以及未接种对照组的4.26倍、4.98倍和4.87倍,其菌根侵染率也高达92.8%;而且可显著提高叶片中叶绿素含量,并显著降低细胞内的丙二醛含量。(3)在适量(5.0mg·L-1)的硝酸镧水平下接种菌根真菌能促进铁皮石斛幼苗多糖和蛋白质的合成,并显著提高细胞内SOD、CAT和POD活性。研究认为,菌根真菌与适宜浓度硝酸镧(5.0 mg·L-1)联合使用能显著促进铁皮石斛菌根的形成,增强植株的生理活性和适应能力,提高其生物量和多糖等活性成分的积累,有效改善铁皮石斛的药用品质。     

Micropropagation of Aerides maculosum lindl. (Orchidaceae)

Summary Young leaf segments from plants growing both in vivo and in vitro were cultured on Murashige and Skoog (MS) medium supplemented with auxins [naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D)], cytokinins [kinetin (KN) and N⁶-benzyladenine (BA)] and coconut liquid endosperm (CW). The explants from mature leaves did not show any growth and turned necrotic, while those obtained from juvenile leaves growing in vitro developed protocorm-like bodies (PLBs) at their cut surfaces within 4–8 wk depending on the growth medium. An optimum of 18 PLBs developed from leaf explants on medium supplemented with 2.0 mg l⁻¹ (8.87 μM) BA. Upon subculture in basal MS medium, the PLBs differentiated into plantlets within 6–8 wk. The resulting plantlets were successfully transferred to vermiculite initially and subsequently to potting mixture; 84% of the plantlets survived after 3 mo. of transplantation.     

Effect of plant growth regulators on regeneration of plantlets from bud cultures ofCymbopogon nardus L. and the detection of essential oils from thein vitro plantlets

Cymbopogon nardus L. could be propagated via tissue culture using axillary buds as explants. The aseptic bud explants obtained using double sterilization methods produced stunted abnormal multiple shoots when they were cultured on Murashige and Skoog medium (MS) supplemented with 1.0 mg L^-1 or 2.0 mg L^-1 benzyladenine (BA). Stunted shoots that cultured on MS + 1.0 mg L^-1 BA + 1.0 mg L^-1 N⁶-isopentenyl-adenine (2iP) could induce elongation of shoots from about 60% of the stunted shoots. Normal multiple shoots could be induced at the highest (19.7 shoots per bud) from the bud explants within six weeks when cultured on proliferation medium consisted of MS supplemented with 0.3 mg L^-1 BA and 0.1 mg L^-1 indole-3-butyric acid (IBA). The separated individual shoot produced roots when transferred to basic MS solid medium. The essential oils that were contained in the mature plants namely citronellal, geraniol and citronellol were also found in thein vitro C. nardus plantlets. Citronellal was the main essential oil component in the matured plants while geraniol was the main component in thein vitro plantlets.     

石斛兰转ACS反义基因抗性原球茎及转化植株的筛选与鉴定

该试验就石斛兰转化ACS(1-氨基环丙烷-1-羧酸合成酶)反义基因的不同筛选方法和筛选处理对抗性原球茎筛选的影响,以及石斛兰转基因植株的再生与鉴定进行研究.结果表明:(1)石斛兰原球茎经带有gus报告基因和ACS反义基因的农杆菌LBA4404侵染共培养5d后除菌,采用逐渐提高选择压浓度的延迟筛选,并在低选择压浓度下切割而高选择压浓度下不切割的处理方式为抗性原球茎的最佳筛选途径,抗性原球茎获得率可达14.97％.(2)抗性原球茎繁殖时应逐渐降低选择压浓度,且在低选择压浓度下进行切割处理,繁殖倍数达到1.15倍,且原球茎生长势好.(3)抗性原球茎在1/2 MS+0.5 mg/L 6-BA培养基中的分化率达到73.85％;107株无根小苗培养于1/2 MS+1.0 mg/L NAA+50.0 mg/L Km(卡那霉素)+100.0 mg/L Cef(头孢霉素)培养基中进行生根培养,共获得了13株具有卡那霉素抗性的转化植株,转化效率达到12.15％.(4)转化植株经报告基因产物GUS组织化学检测和gus的PCR检测,证实带ACS反义基因的T-DNA已整合进石斛兰基因组中,且转基因植株在形态上与未转基因植株无明显差别,3株转基因植株移栽2个月后均已成活.     

Rapid propagation of Phalaenopsis from floral stalk-derived leaves

Summary An efficient and rapid in vitro method was developed for regeneration of Phalaenopsis using leaf segments derived in vitro from flower stalk nodes. Leaf segments of four cultivars Tinny Sunshine ‘Annie’, ‘Taisuco Hatarot’, Teipei Gold ‘Golden Star’, Tinny Galaxy ‘Annie’ cultured on Murashige and Skoog medium supplemented with N ⁶-benzyladenine (BA; 88,8 μM) and α-naphthaleneacetic acid (NAA; 5,4 μM) produced an average of 10–13 protocorm-like bodies (PLBs) after 12 wk. PLB proliferation was achieved on a modified Hyponex medium (1 gl⁻¹ 6.5N−4.5P−19K+20N−20P−20K+2gl⁻¹ peplone +3% (w/v) potato homogenate +0.05% activated 1 gl⁻¹ charcoal) and an optimal number of 13–18 PLBs developed from single PLB sections of different cultivars. Plantlet development was also achieved on a modified Hyponex medium. By repeated subculture of PLBs on a proliferation medium, and culturing them in the plantlet regeneration medium, plantlets could be produced continuously. Approximately 6 mo, were required from the initiation of culture to the production of plantlets for transplant to community pots.     

广西五种绞股蓝属植物离体快繁与种质保存研究

以绞股蓝属植物的带芽茎段为材料,研究不同6-BA浓度与NAA 0.02mg·L-1组合对其诱导、分化和增殖的影响,并建立离体快繁体系。结果表明:MS+6-BA 2.0mg·L-1+NAA 0.02mg·L-1最适宜初代诱导,MS+6-BA 2.0mg·L-1+NAA 0.02mg·L-1最适合扁果绞股蓝的增殖培养,而MS+6-BA 1.5mg·L-1+NAA0.02mg·L-1是其它四种植物增殖的最佳培养基,在1/2MS+NAA 1.0mg·L-1上的生根率均达100%。1/2MS与蔗糖40g·L-1对五种植物的保存效果均最好;添加生长抑制剂能有效减缓生长速度,最佳生长抑制剂为ABA和CCC,浓度均为1.0mg·L-1,其中CCC能适合多个物种,连续保存360d的存活率均在94.5%以上;PP333不适合五种植物的保存。活力检测表明,各种质经保存后增殖、生根能力均未下降。     

In Vitro Propagation and Conservation of Dendrobium lituiflorum Lindl Through Protocorm-Like Bodies

Axillary buds obtained from 5-month-old in vitro growing plants of Dendrobium lituiflorum Lindl were cultured in Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-D. Fastest initiation (13.3 days) of protocormlike bodies (PLBs) was observed in cultures containing MS medium supplemented with 0.5 mg l^?1 2,4-D. Maximum explant response of 83% was also observed in the same medium. PLBs obtained in MS medium containing 0.5 mg l^?1 2,4-D showed maximum regeneration potential of seedlings (19 explant^?1) when subcultured in MS medium. Well developed shoots and roots of the seedlings were obtained in the medium containing 0.5 mg l^?1 each of NAA and BAP, in combination. Encapsulated PLBs of D. lituiflorum could be stored at 8°C for 90 days with 80% regeneration. However, it was observed that regeneration potential of encapsulated PLBs reduced with further storage. Seventy seven per cent hardened plants survived and bloomed after 2.5 years of hardening.     

In vitro propagation of <Emphasis Type="Italic">Paphiopedilum</Emphasis> orchid through formation of protocorm-like bodies

Paphiopedilum orchids are among the world’s most popular orchid due to their impressively beautiful flowers. Propagation of these orchid genera has been hampered by the naturally slow growth rate of the plant, which renders it very difficult to be propagated through conventional methods. In vitro culture techniques have provided a useful alternative technology for propagating this recalcitrant species. In this study, the propagation of P. rothschildianum was achieved through the in vitro formation of secondary protocorm-like bodies (PLBs) from the primary PLB that developed from stem-derived callus. The PLBs were cultured on half-strength MS medium supplemented with different concentrations (1.0, 2.0, 3.0, and 4.0 μM) of 6-benzyladenine (BA) and kinetin for the induction of secondary PLBs. The highest number of secondary PLBs formed was obtained on half-strength MS medium supplemented with 4.0 μM kinetin, with an average of 4.1 PLBs per explant after 8 weeks of culture. The secondary PLBs continued to proliferate further and formed 9.5–12.1 new PLBs per secondary PLB after being subcultured onto half-strength plant growth regulator-free MS medium supplemented with 60 g/L banana homogenate (BH). These tertiary PLBs were subcultured onto media containing different organic additives, such as BH, coconut water, potato homogenate, and tomato homogenate, for plantlet regeneration. Among the organic additives tested, the addition of 20% CW to half-strength MS medium resulted in the best average plantlet regeneration percentage from the PLBs, 67.9%, after 8 weeks of culture.     

Efficient production of protocorm-like bodies and plant regeneration from flower stalk explants of the sympodial orchid <Emphasis Type="Italic">Epidendrum radicans</Emphasis>

Summary A simple and efficient micropropagation method was established for direct protocorm-like body (PLB) formation and plant regeneration from flower stalk internodes of a sympodial orchid, Epidendrum radicans. Small transparent tissues formed on surfaces and cut ends of flower stalk internodes on a modified half-strength Murashige and Skoog basal medium with or without thidiazuron (TDZ) after 1–2 wk of culture. In the light, the transparent tissues enlarged and turned into organized calluses on most of the explants. However, PLBs formed only on a medium supplemened with 0.45 μM TDZ within 2 mo. of culture. Sucrose, NH₄NO₃, and KNO₃ were used in media to test their effects on PLB proliferation and shooting. The best response on number of PLBs per tube was 23.6 at 40 gl⁻¹ sucrose, 825 mgl⁻¹ NH₄NO₃, and 950 mgl⁻¹ KNO₃, and the highest number of PLBs with shoots was found at 10 gl⁻¹ sucrose, 825 mgl⁻¹ NH₄NO₃, and 950 mgl⁻¹ KNO₃. Homogenized PLB tissues produced by blending were used to test the effects of four cytokinins [TDZ, N⁶-benzyladenine (BA), zeatin-riboside, and kinetin] on PLB proliferation and shoot formation. The best responses on number of PLBs per tube, proliferation rate, and number of PLBs with shoots per tube were obtained at 4.44 μM BA, 0.28 μM zeatin-riboside, and 1.39 μM kinetin, respectively. Normal plantlets converted from PLBs on the same TDZ-containing medium after 1 mo. of culture. The optimized procedure required about 12–13 wk from the initiation of PLBs to plantlet formation. The regenerated plants grew well with an almost 100% survival rate when acclimatized in a greenhouse.