Tissue Culture and Plant Regeneration of Chicory

[ Objecllve] To establish a high-frequency regeneration system of chicory （ Cichodum intybus L. ） using leaf segments of aseptic seed- lings. E Method] Calluses and adventitious buds of chicory were induced by inoculating explants on MS medium supplemented with 6-BA （6-benayl aminopurine） and NAP, （naphthylacetic acid） at different final concentrations. [ Result] When lower part of leaves derived from 20-day-old seedlings was used as explant and inoculated on MS medium containing 2.0 rng/L 6-BA, 0.5 mg/L NAA and 40 g/L sucrose, the frequency of adventitious bud formation was 90.0%. When the regenerated shoots were cultured in 1/2 MS medium containing 0.1 mg/L NAA, the frequency of root forma- tion was 88.3%. All rooted plants transplanted in pots could survive and grew well without abnormal shape. [ Conclusion] Better differentiation of adventitious buds can be achieved by inoculating the lower part of leaves derived from 20-day-old seedlings on MS medium containing 2.0 mg/L 6- BA, O. 5 mg/L NAP, and 40 g/L sucrose. The 1/2 MS medium containing O. 1 mg/L NAP, is most suitable for rooting.

Study on Plant Regeneration of Wheat Mature Embryos Under Endosperm-Supported Culture

To reveal the suitability of using mature embryos as an explant source in wheat tissue culture, mature embryos from eight common wheat cultivars （Triticum aestivum L. cv.） were cultured with or without endosperm to test their efficiency of callus induction and plant regeneration. When embryos were cultured together with endosperm （endosperm-supported culture, ES）, the percentage of callus induction was significantly lower than that when embryos were cultured in the absence of endosperm （non-endosperm-supported culture, NES）. This pattern was evident in most genotypes, regardless of whether 2 or 8 mg L^-1 2,4-D was added in the NES culture. However, in ES culture, more induced calli were differentiated into distinct green spots and they further developed into plantlets. Thus, more plants were regenerated in ES culture than in the NES treatment. Most of the eight tested genotypes showed a significant difference in callus induction rate and plantlet regeneration in both ES and NES cultures. In addition, the enzymatic activity of oxalate oxidase in the callus of ES culture condition was obviously higher than that in the callus of NES culture condition, suggesting that the activity of oxalate oxidase may be a parameter for selection of calli with potential for plantlet regeneration. These results indicate that wheat mature embryos are valuable explants for highly efficient callus induction and plant regeneration, if proper treatment and medium are used.

Dicamba and Sugar Effects on Callus Induction and Plant Regeneration from Mature Embryo Culture of Wheat

To establish a highly efficient plant regeneration system for wheat genetic transformation, the effects of three different concentrations of dicamba and two different sugar types on callus induction and plant regeneration from mature embryo cultures were evaluated. Callus induction and plant regeneration were obtained from mature embryos of two commercial cultivars Zhoumai 18 and Yumai 34 （Triticum aestivum L.） cultured on L3 basal medium. The results showed that the efficiency of mature embryo culture was significantly influenced by the genotypes, sugar types and dicamba concentrations. 4 mg L^-1 dicamba proved the best effective for inducing embryogenic callus and also gave the highest proportion of plants regenerated across the two cultivars. Substitution of maltose by sucrose significantly improved the plant regeneration efficiency in both cultivars. There was a significant interaction between genotype-by-sugar types, and sugar types-bydicamba concentrations. Overall, Zhoumai 18 gave the highest frequency of plant regeneration （82.65%） when dicamba concentration was 4.0 mg L^-1 and with sucrose in initial callus induction. These results will facilitate genetic transformation work with elite wheat.

Callus Induction and Plant Regeneration from Mature Seeds of Perennial Ryegrass

Objective The aim was to explore callus induction and plant regeneration of perennial ryegrass, as well as provide the foundation for transgenic research on perennial ryegrass.[ Methed] Mature seeds of perennial ryegrass were used as explants to study the effects of different hormone compositions on callus induction, proliferation and plant differentiation. Result The result showed that the induction rate achieved its highest on 2,4-D of 8 mg/L combining with 6-BA of 0.025 mg/L, which was up to 56.42%. Callus were differentiated after two to three generations, the highest differentiation rate 34.14% was achieved in the medium contained MS medium with 6-BA of 2 mg/L, and the differentiation rate was obviously affected by the callus condition after proliferation. The root inducing medium, containing 0.5 mg/L NAA and MS medium with half of macroelement, gained 98% root inducing rate. Conclusien A high frequency genetic regeneration system was established.

Establishment of Plant Regeneration System from Immature Embryos of Maize(Zea mays L.) Inbred Lines

[Objective] The aim was to explore the conditions of high frequency induction of embryonic callus and plant regeneration of maize. [Method] Immature embryos of maize inbred lines were used as explants to study the effect of different 2,4-D concentrations on the induction of callus,the effect of different 6-BA concentrations on the differentiation of test-tube plantlet,as well as the effect of different IBA concentrations on the rooting of test-tube plantlet. [Result] 2,4-D showed obvious effect on the induction of inducement rate of maize,and the optimum induction medium was：N6 ＋ 2 mg/L of 2,4-D ＋ 500 mg/L of CH ＋ 500 mg/L of Pro ＋10 mg/L of AgNO3; the optimum differentiation medium was：N6 ＋ 0.5 mg/L of BA ＋ 500 mg/L of Pro; the optimum for the rooting of test-tube plantlet was 1/2 MS ＋ 0.5 mg/L of IBA. [Conclusion] The study had provided basis for the genetic transformation of maize.

Experiment on Tissue Culture Technique of Saposhnikovia divaricata(Turcz.) Schischk

The study aimed to optimize the induction and differentiation medium by exploreing different tissue culture of Saposhnikovia divaricata （Turcz.） Schischk. In tissue culture with the root, stem segments, young leaf, cotyledonary node and axillary bud of Saposhnikovia divaricata （Turcz.） Schischk as explants, a lot of plantleles were obtained and the corresponding plant regeneration-system was established. The results showed that when use MS＋1.0 mg·L^-1 6-BA＋0.2 mg·L^-1 NAA as callus induction medium, the cotyledonary node had the highest bourgeon rate, and its callus was better than any others; MS＋2 mg·L^-1 6-BA＋0.4 mg·L^-1 NAA was the best adventitious buds induction medium, and the best adventitious buds induced condition was 3% sucrose as carbon source, illumination for 12-14 h·d^-1 and pH 5.8, The best rootage medium was 1/2 MS＋0.5 mg·L^-1 NAA.

Efficient Plant Regeneration with Arabinogalactan-Proteins on Various Ploidy Levels of Cereals

To determine the most effective dose of arabinogalactan-protein （AGP） in regeneration medium, mature embryos of genotypes in three different ploidy levels （Triticum aestivum L. cv. Ikizce-96, Triticum durum Desf. cv. Mirzabey and Hordeum vulgare L. cv. Tokak） were used to establish an efficient plant regeneration system for cereals. Percentage of callus production, capacity of regeneration were calculated, and also culture effect, root, stem, and total plant length of regenerant plants were observed in six different regeneration media （MS control, MS＋2, 5, 7, 10, 12 mg L-1 AGP） in these three different genotypes. According to the results, the highest amount of callus production was found in Ikizce-96 as 93.75% using 5 mg L-1 dicamba and 1 mg L-1 kinetin in induction medium. However, the most improved callus was observed in diploid barley Tokak as 179.95 mg in weight and 6.18 mm in diameter, respectively. The highest regeneration capacity was observed in the dose of 5 mg L-1 AGP in MS of all the genotypes and hexaploid wheat Ikizce-96 gave the best results with the highest regeneration capacity and culture effects （94.86 and 92.5%） in the same dose of AGE These results indicated that effective dose of AGP in regeneration medium increase plant regeneration in calli derived from cereal mature embryos.

In vitro clonal propagation of Achyranthes aspera L. and Achyranthes bidentata Blume using nodal explants

Objective:To develop the reproducible in vitro propagation protocols for the medicinally important plants viz.,Achyranthes aspera(A.aspera)L.and Achyranthes bidentata(A.bidentata)Blume using nodal segments as explants.Methods:Young shoots of A.aspera and A.bidentata were harvested and washed with running tap water and treated with 0.1%bavistin and rinsed twice with distilled water.Then the explants were surface sterilized with 0.1%(w/v)HgCl_2 solutions for I min.After rinsing with sterile distilled water for 3-4 times,nodal segments were cut into smaller segments(1 cm)and used as the explants.The explants were placed horizontally as well as vertically on solid basal Murashige and Skoog(MS)medium supplemented with 3%sucrose,0.6%(w/v)agar(HiMedia,Mumbai)and different concentration and combination of 6-benzyl amino purine(BAP),kinetin(Kin),naphthalene acetic acid(NAA)and indole acetic acid(IAA)for direct regeneration.Results:Adventitious proliferation was obtained from A.aspera and A.bidentata nodal segments inoculated on MS basal medium with 3%sucrose and augmented with BAP and Kin with varied frequency.MS medium augmented with 3.0 mg/L of BAP showed the highest percentage(93.60±0.71)of shootlets formation for A.aspera and(94.70±0.53)percentages for A.bidentata.Maximum number of shoots/explants(10.60±0.36)for A.aspera and(9.50±0.56)for A.bidentata was observed in MS medium fortified with 5.0 mg/L of BAP.For A.aspera,maximum mean length(5.50±0.34)of shootlets was obtained in MS medium augmented with 3.0 mg/L of Kin and for A.bidentata(5.40±0.61)was observed in the very same concentration.The highest percentage,maximum number of rootlets/shootlet and mean length of rootlets were observed in 1/2 MS medium supplemented with 1.0 mg/L of 1BA.Seventy percentages of plants were successfully established in polycups.Sixty eight percentages of plants were well established in the green house condition.Sixty five percentages of plants were established in the field.Conclusions:The results have shown that use of nodal buds is an alternative reproducible and dependable method for clonal propagation of A.aspera and A.bidentata.The high rate of direct shoot-root multiplication and their high rate of post-hardening survival indicate that this protocol can he easily adopted for commercial large scale cultivation.

单瓣长寿花‘萨姆巴’高效快速繁殖体系的构建

选取单瓣长寿花‘萨姆巴’的叶片为外植体,设计正交试验,研究不同种类植物生长激素及其浓度配比的培养基对长寿花叶片愈伤组织的诱导、不定芽的分化和不定芽生根的影响,并研究试管苗炼苗及移栽的成活情况。试验结果表明,诱导长寿花叶片产生愈伤组织的最适培养基是MS+6-BA 1.0 mg/L+NAA 0.2 mg/L+2,4-D 0.2 mg/L,诱导率是83.33%。诱导愈伤组织不定芽分化的最适培养基是MS+6-BA 2.0 mg/L+NAA 0.3 mg/L,增殖系数是21.4。最适的不定芽生根培养基为1/2MS+IBA 0.2 mg/L+NAA 0.2 mg/L,生根系数为7.03。试管苗移栽到蛭石∶营养土=1∶1的基质中培养,移栽的成活率可达100%。

膜荚黄芪组织培养及植株再生体系的建立

以膜荚黄芪幼叶、幼茎和下胚轴为试材,采用在MS培养基中添加不同浓度和不同组合的6-BA、NAA、IAA、IBA和TDZ的方法,研究膜荚黄芪愈伤组织培养和不定芽诱导分化及再生体系的建立,构建膜荚黄芪组织培养及植株再生的完整体系,以期为膜荚黄芪种质资源保护、种苗快速繁育和遗传改良提供参考依据。结果表明:幼茎是诱导愈伤组织的最佳外植体,诱导愈伤组织的最佳培养基为MS+6-BA 1.0 mg·L^(-1)+NAA 0.5 mg·L^(-1)+TDZ 0.2 mg·L^(-1),诱导率为90.00%;愈伤组织诱导不定芽的最佳培养基为MS+6-BA 1.0 mg·L^(-1)+NAA 0.3 mg·L^(-1),诱导率为76.60%;幼茎诱导不定芽的最佳培养基为MS+6-BA 1.5 mg·L^(-1)+IAA 0.2 mg·L^(-1),诱导率为90.30%。生根培养的最佳培养基为1/2MS+IBA 0.5 mg·L^(-1)+IAA 1.0 mg·L^(-1),生根率为39.30%。

扭果花旗杆组织培养与再生体系的建立

【目的】以扭果花旗杆(Dontostemon elegans)根和下胚轴为外植体,建立扭果花旗杆组织培养体系,为后续遗传转化体系的建立提供技术基础。【方法】以MS培养基为基础培养基,利用萘乙酸(NAA)探究根和下胚轴直接诱导不定芽情况,利用2,4-二氯苯氧乙酸(2,4-D)、6-苄氨基嘌呤(6-BA)、NAA、苯基噻二唑基脲(TDZ)、吲哚丁酸(IBA)进行愈伤组织诱导及分化和不定芽生根试验,统计愈伤组织诱导及分化和不定芽生根情况,确定扭果花旗杆组培体系最佳培养基类型。【结果】(1)由根和下胚轴直接诱导不定芽的最适培养基分别为MS+0.3 mg/L NAA和MS+0.6 mg/L NAA,直接诱导不定芽的最佳外植体为根。(2)扭果花旗杆根和下胚轴都能诱导愈伤组织,其中根系诱导愈伤组织速度最快且状态最佳,其次为下胚轴。根系愈伤组织诱导的最佳培养基为MS+1.0 mg/L 2,4-D+0.2 mg/L 6-BA+0.1 mg/L NAA,诱导率可达100.00%;下胚轴愈伤组织诱导的最佳培养基为MS+0.3 mg/L 2,4-D+1.5 mg/L 6-BA+1.5 mg/L NAA,诱导率可达100.00%。(3)根系和下胚轴的最佳愈伤组织分化培养基均为MS+0.5 mg/L 6-BA+0.3 mg/L NAA,分化率分别为100.00%和86.66%。(4)最佳生根培养基为MS+0.5 mg/L IBA,生根率可达50%以上。(5)将组培苗移至土壤后,在光照培养室培养15 d,其存活率可达83.33%。【结论】建立的扭果花旗杆组培体系可获得与实生苗形态一致的组培苗,为扭果花旗杆遗传转化体系的建立奠定了技术基础。

小叶栀子再生体系的建立研究

小叶栀子(Gardenia jasminoides Ellis.var.radicans)是优良的园林绿化景观植物,应用前景广阔.研究以小叶栀子的无菌叶片、顶芽为外植体,通过添加不同种类和质量浓度的植物生长调节剂进行愈伤组织的诱导、分化和生根培养,以期建立小叶栀子的再生体系.结果表明:外植体启动培养阶段,抑制杂菌污染的最佳抗生素组合为50 mg/L氨苄西林钠+50 mg/L硫酸链霉素的1:1混合溶液,污染率为25%,优于200 mg/L头孢霉素溶液和对照组;愈伤组织诱导阶段,两种外植体(无菌叶片和无菌顶芽)最早出伤时间分别为接种后10 d和18 d,最佳方案皆为MS培养基+2.0 mg/L 2,4-D+0.5 mg/L 6-BA,诱导率分别达到90%和93.3%;愈伤组织分化阶段,在MS培养基中添加0.05 mg/L的NAA和1.5~1.8 mg/L 6-BA,愈伤组织分化率普遍较高,其最佳植物生长调节剂配比为0.05 mg/L NAA+1.8 mg/L 6-BA,分化率达到96.7%;生根培养阶段,1/2 MS培养基+0.1 mg/L NAA+0.5 mg/L IBA为最佳方案,生根率达26.67%.研究成功建立了一套高效的小叶栀子再生体系,为大规模扩繁小叶栀子幼苗提供了相应的技术支持,也为其广泛的推广应用创造了有利条件.

茉莉花节间茎段的离体培养和植株再生

【目的】通过双瓣茉莉(Jasminum sambac)节间茎段离体培养获得再生植株,填补通过愈伤组织获得再生植株报道的空白。【方法】以双瓣茉莉节间茎段为外植体,设置不同84消毒液和Tween-20浓度组合筛选适宜的外植体消毒方式,并在此基础上设置9种不同的6-BA和NAA浓度组合筛选适宜的培养基进行愈伤组织诱导、分化及完整植株再生。【结果】茉莉花节间茎段经20%的84消毒液中滴加0.1 mL Tween-20消毒12 min时,污染率低且成活率高;在MS+1.5 mg/L 6-BA+1.5 mg/L NAA+30 g/L蔗糖+6 g/L琼脂培养基上愈伤组织诱导率达100%。在此培养基生长2个月后,茎段产生的愈伤组织开始分化形成芽和根;培养至第6个月,开始形成茎和叶;培养至第8个月,开始形成完整植株;培养至第12个月时,愈伤组织开始逐渐褐化,植株的生长放缓,部分叶片出现枯焦。【结论】茉莉花节间茎段诱导愈伤组织可在不继代的情况下再生为完整植株,为茉莉花遗传转化获得转基因植株提供了途径。

粉葛再生体系建立的研究

为建立高效粉葛组织培养再生体系,以不同消毒方式、基本培养基、蔗糖浓度、外植体等处理,研究其对粉葛初代培养、继代培养和生根培养的影响。结果表明,以75%酒精30 s+0.1%升汞+吐温-80消毒8 min时,外植体的成活率最高,可达83.33%。外植体的最佳取材部位为带芽茎段,其平均株高可达2.13 cm,繁殖系数为2。MS为最佳的基本培养基,繁殖系数为1.26;蔗糖浓度以30 g/L为最佳,其平均株高为1.69 cm,繁殖系数为1.55;最佳初代培养基为MS+琼脂5.5 g/L+蔗糖30 g/L+6-BA 0.2 mg/L+IBA 0.6 mg/L,其平均株高为3.13 cm,繁殖系数为3.17;最佳的继代培养基为MS+琼脂5.5 g/L+蔗糖30 g/L+6-BA 0.6 mg/L+IBA 0.8 mg/L+KT 0.2 mg/L,其平均株高为1.86 cm,繁殖系数为3.44;最佳的生根培养基为MS+琼脂5.5 g/L+蔗糖30 g/L+IBA 1.0 mg/L,其生根率为100%,平均主根数达7.1根。通过组培条件优化,初步建立粉葛的再生体系。

豆科植物组织培养的研究

本文采用A和B两种基本培养基,对15属31个种的豆科植物进行组织培养并得到再生植株。同时,探讨了培养基成分(大量元素、微量元素、有机成分和激素)、光照、温度及种子萌发率对愈伤组织分化的影响。对于多数豆科植物来说,A和B两种培养基的大量元素,B5培养基的微量元素和有机成分,1500—2500 lx自然光为主的光照和18—27℃的变温条件,以及当年采集的豆科植物种子,比较适合于愈伤组织的分化。根据豆科植物愈伤组织分化时形态结构特征上的变化,可将其分为a、b、c、d 4种基本类型,同属植物的愈伤组织基本上属于同一类型,不同属植物的愈伤组织往往属于不同类型。

辣椒子叶离体培养和植株再生体系的建立

选用辣椒 (CapsicumannuumL .) 4个栽培品种和 2个杂交亲本的子叶进行诱导不定芽分化、生长及生根成苗试验 ,从中筛选出 3个较好的激素配方 ,建立了辣椒栽培品种的高效快速离体植株再生体系。外植体分化频率可达 10 0 % ,每外植体平均分化不定芽数10 .9个 ,出苗 8～ 10株。发现添加氨基酸混合物 (LY)对不定芽的诱导分化及生长有较强的促进作用。从子叶外植体培养起至再生苗出瓶需 5 0～ 5 5d ,最快的 1批仅 44d。

蕨菜愈伤组织高效再生体系的建立

报告了蕨菜不同外植体在含有不同激素的培养基上诱导、分化及生根情况 ,建立起高效再生体系。诱导愈伤组织的最适培养基为 1/ 2MS +BA 0 .2mg/L +IBA 0 .4mg/L +NH4 H2 PO4 2 0 0mg/L ;诱导愈伤组织芽分化的最适培养基为 1/ 2MS ,芽分化率为 10 0 %;不定苗在 1/ 2MS +IAA 0 .2mg/L培养基上生根率为 10 0 %,同时可以形成健壮根茎 ;具有根茎的试管苗移栽成活率达 94%。

外部因子对蝴蝶兰叶片原球茎状体发生的影响

对影响蝴蝶兰叶片原球茎状体 (PL B,Protocorm - like- body)发生和植株再生的外部因子进行了研究。结果表明 :BA是决定原球茎状体发生的主要因子 ;苹果汁、香蕉汁和椰子汁明显促进原球茎状体的形成 ;在 MS+BA5mg/L+椰子汁 15%的培养基中 ,蝴蝶兰叶片原球茎状体的诱导率可达 6 0 %以上 ;活性炭可有效防止外植体叶块变褐死亡 ;多效唑 1.5mg/L可促进再生植株根的形成 ,使叶片变厚变短变绿 ,使试管苗移栽成活率达

植物组织培养再生相关基因鉴定、克隆和应用研究进展

离体植物组织体细胞胚胎发生是一个复杂的无性繁殖过程,依次经历外源植物激素信号应答、已分化细胞的脱分化、静止细胞的再分裂以及特定组织、器官原基或分生组织的形成等,是多个基因在外界因素刺激下协调、有序表达和互作的结果,不但受培养基中植物激素和营养成分的影响,也与外植体的生理状态关系密切。本文综述了外源激素和内源激素在植物组织培养中的作用,以及外源激素对内源激素的调节功能;重点介绍了5类与植物体细胞胚胎发生有关的候选基因,包括体细胞胚胎发生相关类受体蛋白激酶、阿拉伯葡聚糖酶、亚硝酸还原酶、生长素结合蛋白和抗氧化酶。再生相关基因的利用不但有助于提高植物组织培养植株再生率和遗传转化率,而且有助于获得安全型转基因植物,在基因工程育种中具有潜在应用前景。不同植物和同种植物不同外植体组织培养中调控体细胞胚胎发生的主效基因可能不同,关键再生相关基因的克隆和功能鉴定是今后需要加强的方向。

青天葵组织培养及植株再生的研究

目的:对青天葵进行组织培养并获得再生植株。方法:考察不同外植体、植物生长调节剂、添加物等对根状茎及再生植株生成的影响。结果:以球茎为外植体效果最好,6-BA2mg·L-1诱导球茎生成根状茎的效果优于6-BA1mg·L-1,椰汁、活性炭对根状茎的生长有促进作用。结论:青天葵球茎接种于1/2MS+6-BA2mg·L-1培养基上,能够诱导出芽,芽接种于添加了10%椰汁和1‰活性炭的培养基中能够生成大量的根状茎,将白色的根状茎接种于1/2MS+1‰活性炭的培养基中能够先形成球茎,并进一步形成再生植株;将绿色的根状茎接种于1/2MS+6-BA2mg·L-1+NAA2mg·L-1的培养基上能够直接形成再生植株。