新疆雪莲体细胞胚胎发生

通过体细胞胚胎发生途径实现了新疆雪莲（Saussurea involucrata Kar．et Kir．）的植株再生。选用新疆雪莲子叶为外植体,接种于MS＋0．5mg·L^-12,4-D＋0．05—1mg·L^-1BA的固体培养基上,进行愈伤组织的诱导。从第1次继代培养的愈伤组织中挑选出黄绿色、颗粒状、质地致密的腔陛愈伤组织,转移到含0．05—0．1mg·L^-1 2,4-D的MS液体培养基中进行悬浮培养,20天后可分化产生大量球形胚。继代过程中相继加入PEG和GA3,可以促进体细胞胚的分化和生长。体细胞胚在含有5mg·L^-1 GA3的MS固体培养基上,可发育成完整的植株。     

Rapid in vitro propagation of Saussurea lappa, an endangered medicinal plant, through multiple shoot cultures

Summary Rapid micropropagation of Saussurea lappa C. B. Clarke, an endangered medicinal plant endemic in the valley of Kashmir and western Himalayas of northern India, was achieved by culturing shoot tips (0.5–1 cm) of 2-wk-old seedlings on Murashige and Skoog’s medium (MS) supplemented with thidiazuron (TDZ, 0.45 μM). Although callus-free multiple shoots were obtained both on N⁶-benzyladenine- (BA) and TDZ-containing media, TDZ was most effective (90%) in inducing multiple shoots. Shoot tips containing proliferative buds were divided into equal halves and subcultured on MS liquid medium containing 0.225 μM TDZ for further multiplication and elongation. Multiplication of induced shoot buds was more effective when cultured in liquid medium than on agar-solidified medium. Shoots (8–10 cm) developed were rooted in MS medium containing naphthaleneacetic acid (NAA, 1.07 μM). Micropropagated plantlets were successfully transferred to soil after hardening.     

秋水仙素诱导天山雪莲四倍体的研究

利用组织培养附加秋水仙素的方法对濒危药用植物天山雪莲(Saussurea involucrata Kar.et Kir.)进行多倍体诱导,结果表明:(1)与种子作材料相比,采用秋水仙素处理无菌苗是获得天山雪莲多倍体植株的最佳途径.(2)二倍体植株的叶片薄、平展、较细长、色绿、尖齿不明显、根细长;经秋水仙素处理诱导得到的植株叶片变宽、变肥厚、叶色深绿,尖齿明显、根短粗.(3)染色体计数、气孔分析及流式细胞仪倍性综合检测表明,诱导得到的多倍体变异苗为纯合四倍体.     

新疆雪莲DFR基因的分离及同源遗传转化

二氢黄酮醇4-还原酶（dihydroflavonol 4-reductase,DFR）是植物重要的次生代谢产物花青素生物合成途径中的关键酶。运用RT-PCR和RACE技术从新疆雪莲(Saussurea involucrata Kar.et Kir.)中克隆得到DFR基因(GenBank登录号为JN092126)。DFR基因的cDNA全长序列含有1个1 029 bp的开放阅读框(ORF),编码343个氨基酸,该基因推断的蛋白与水母雪莲DFR基因推断的蛋白高度同源,相似性达到92%;以不同物种中DFR氨基酸序列进行比对分析,推断的蛋白含有与NADPH特异结合的结构域。将该基因运用农杆菌介导的叶片转化法进行同源转化,将含有转DFR基因的愈伤组织进行悬浮培养,紫外分光光度法测定愈伤组织的总黄酮含量,结果表明转基因愈伤组织的总黄酮含量明显高于非转基因愈伤组织的含量。该研究为提高新疆雪莲药用化学成分黄酮类物质及实现新疆雪莲花青素的人工生物合成的研究奠定基础,对解决天山雪莲资源匮乏提供参考。     

新疆雪莲体细胞胚胎发生

通过体细胞胚胎发生途径实现了新疆雪莲(Saussurea involucrata Kar.et Ki r.)的植株再生。选用新疆雪莲子叶为外植体, 接种于MS+0.5 mg.L-1 2,4-D+0.05-1 mg.L-1 BA的固体培养基上, 进行愈伤组织的诱导。从第1次继代培养的愈伤组织中挑选出黄绿色、颗粒状、质地致密的胚性愈伤组织, 转移到含0.05-0.1 mg.L-1 2,4-D 的MS液体培养基中进行悬浮培养,20天后可分化产生大量球形胚。继代过程中相继加入PEG和GA3 , 可以促进体细胞胚的分化和生长。体细胞胚在含有5 mg.L-1 GA3 的MS固体培养基上, 可发育成完整的植株。     

天山雪莲LEA14基因克隆及功能分析

从天山雪莲叶片低温诱导的EST文库中获得了1个胚胎发育晚期丰富蛋白基因(LEA)cDNA全长序列。序列分析表明,该基因含有1个468bp编码155个氨基酸的开放阅读框。NCBI保守域预测此蛋白属于LEA_2家族,命名为SiLEA14。系统进化分析表明,该蛋白与北柴胡的LEA-2蛋白亲缘关系最近。荧光定量PCR结果显示,SiLEA14表达量在低温、盐和干旱胁迫条件下迅速升高。亚细胞定位结果表明,SiLEA14蛋白定位于细胞核中。利用农杆菌介导法将该基因导入烟草,测定并分析转基因植株在冷冻和盐胁迫处理下的生理指标,结果表明,SiLEA14基因在烟草中的过量表达提高了烟草的抗冻和耐盐能力。     

天山雪莲未知蛋白基因的克隆及其耐铝特性分析

从天山雪莲叶片低温诱导的cDNA文库中克隆了一个未知蛋白基因(SikSD-82)cDNA全长序列。生物信息学分析表明SikSD-82基因共编码262个氨基酸,具有小麦铝诱导蛋白Wali7的保守结构域,属Gn_AT_Ⅱ家族的可溶性蛋白;其氨基酸序列与蓖麻的茎部特异性表达蛋白相似性最高为60.89%;进化树分析显示,该蛋白与番茄的茎部特异性表达蛋白关系最近。亚细胞定位结果表明,SikSD-82蛋白主要定位于细胞核。在铝胁迫条件下,转SikSD-82烟草幼苗根的伸长明显优于野生型,根部铝离子和丙二醛含量均显著低于野生型烟草,转SikSD-82烟草显示出较强的耐铝性。     

A comparison between hairy root cultures and wild plants of Saussurea involucrata in phenylpropanoids production

Saussurea involucrata is an important medicinal plant that produces a few bioactive secondary metabolites, such as hispidulin, rutin, and syringin. Previously, we established a hairy root culture system for this species through Agrobacterium-mediated transformation. The present study addressed the issue as how hairy root cultures perform in phenylpronoid accumulation. From the ethanolic extract of a hairy root culture established for Saussurea involucrata, syringin, rutin and hispidulin, were isolated and their chemical structures were confirmed by HPLC-ESI-MS. A quantitative study of the compounds showed great levels of syringin and hispidulin (being 43.5±1.13 and 0.34±0.023 mg g⁻¹ dry weight, respectively), about 40 and 3 times, respectively, higher than those from wild plants. But, the levels of rutin from hairy roots were much lower (0.71±0.043 vs. 6.59±0.56 mg g⁻¹ dry weight). Compared with untransformed root cultures, syringin and hispidulin levels were also higher. An experiment on culture media showed that MS was superior to others for phenylpropanoids accumulation in hairy roots, a 28-day culture produced 405 mg l⁻¹ syringin.     

The promoting role of an isolate of dark-septate fungus on its host plant Saussurea involucrata Kar. et Kir.

A dark-septate endophytic (DSE) fungus EF-37 was isolated from the roots of Saussurea involucrata Kar. et Kir., an endangered Chinese medicinal plant. The molecular identification of the fungus was based on internal transcribed spacer regions and the result showed that EF-37 was congeneric to Mycocentrospora. This study was conducted to clarify the influence of the root endophyte EF-37 on the host plant S. involucrata using material grown in a sterile culture bottle. After cultivation for 40 days, fungal hyphae were found to be branching repeatedly and forming “hyphae nets” in the epidermal layers. Significant differences were detected between the study groups in plant dry weight, plant height, root dry weight, shoot dry weight, and the number of hair root tips. There was a positive effect of endophyte EF-37 on plant root development, with results showing that cortical cells dissolved and formed aerate structures. There was a positive effect of endophyte EF-37 on plant growth, but chlorophyll fluorescence analysis showed that there were no significant differences between the study groups. In addition, analysis of the chemical composition of seedlings showed that the level of rutin was higher in plants cultivated with the EF-37 fungus compared to the controls. This study helps to establish a basis for germplasm conservation and for further investigation of the interaction between dark-septate fungi and this alpine plant.     

Effect of Lanthanum on Rooting of In Vitro Regenerated Shoots of Saussurea involucrata Kar. et Kir

In present study, the effect of lanthanum (La) on the rooting of regenerated shoots of Saussurea involucrata Kar. et Kir was analyzed. Rooting occurred from regenerated shoots inoculated on a medium supplemented with La, the plant rooting hormone indole-3-acetic acid (IAA), or both La and IAA together. The highest rooting efficiency (96%), root number/shoot (8.5), and root length (63 mm) were recorded in shoots cultured on medium containing 2.5 μM IAA combined with 100 μM La(3+). In order to elucidate the mechanism of rooting enhancement by La, we examined dynamic changes in antioxidant enzyme activities in plant tissue over time in culture. We found that the activities of peroxidase (POX) and superoxide dismutase (SOD) were significantly higher in plant tissue cultured in IAA plus La than in La or IAA alone. At the same time, the highest H(2)O(2) content was detected in plant tissue in the presence of 2.5 μM IAA plus 100 μM La(3+). In light of these data and previous results, we speculate that La enhanced IAA-induced rooting by acting as a mild abiotic stress to stimulate POX and SOD activities in plant cells. Then, IAA reacted with oxygen and POX to form the ternary complex enzyme-IAA-O(2) that dissociated into IAA radicals and O(2)(-). Subsequently, IAA-induced O(2)(-) readily converted to hydroxyl radical (HO·) via SOD-catalyzed dismutation. Finally, cell wall loosening and cell elongation occurred as a consequence of HO-dependent scission of wall components, leading to root growth. The treatment of IAA combined with La resulted in the highest plantlet survival (80%) compared to single treatments with IAA or La alone. These data suggest that rare earth elements enhance root morphogenesis and the growth of S. involucrata.     

In vitro propagation of Rauwolfia micrantha, a rare medicinal plant

Shoot tip and single node explants from young shoots of 1-year old flowering plants of Rauwolfia micrantha Hook. f. were cultured on Murashige & Skoog (MS) medium variously supplemented with 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). A combination of 13.2 M BA and 2.68 M NAA induced high frequency (77%) formation of up to 3 shoots from each node in 8 weeks. The regeneration of shoot tips from the field-grown plants and in vitro shoots placed horizontally differed. Repeated subculturing of the shoot tips and single nodes at 6-week intervals for over a year in combination of 4.4 M BA and 0.27 M NAA enabled mass multiplication of shoots without any evidence of decline. Rooting of the excised shoots on medium containing 2.6 M NAA was preceded by callus formation. The rooted plants were removed off the callus, hardened off and 80% established in pots. Micropropagated plants displayed uniform morphological, growth, flowering, fruiting and seed germination characteristics.Abbreviations BA 6-benzyladenie - IAA indole-3-acetic acid - IBA indole-3-butyrie acid - 2-ip 2-isopentenyladenine - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid     

新疆雪莲新的内切几丁质酶类冷诱导基因的分离、克隆和测序

通过RT-PCR技术,从经低温驯化的新疆雪莲的叶片中分离克隆了1个新的基因.实验证明,此基因的转录是由低温诱导激活,且随着雪莲低温驯化时间的推移,转录本的量明显增加.测序结果显示,此基因全长643 bp,编码200个氨基酸.序列分析发现,此基因属于Ⅱ类内切几丁质酶基因.初步预测此基因的编码产物可能具有抗冻活性.     

转新疆雪莲去饱和酶基因sikSAD重组酵母低温和酒精耐受性分析

根据GenBank收录的sikSAD基因序列, 采用反转录PCR技术从新疆雪莲(Sasussured involucrata Kar. et Kir)中克隆了sikSAD基因, 并构建了pYES2-sikSAD大肠杆菌/酵母穿梭表达载体, 通过电击法转化酿酒酵母288C菌株, 并利用PCR和SDS-PAGE对转化酿酒酵母进行鉴定, 最后通过低温胁迫和酒精胁迫进行抗性初步分析及方差分析。结果表明: 低温胁迫实验中, 转sikSAD基因酿酒酵母在低温条件下仍能存活, 并且在温度恢复到28 °C时能迅速生长, 生长状态良好, 不饱和脂肪酸油酸的含量有明显的变化。酒精胁迫实验中, 其能耐受一定浓度的酒精, 并且耐受能力比非转基因酿酒酵母提高了十几个百分点。可见, 在低温胁迫和高浓度酒精条件下, 转新疆雪莲sikSAD基因酿酒酵母表现出了优良的活性和生长优势, 显示出较强的抗性特征, 用分子手段改造酿酒酵母, 为工业生产提供高质量的酿酒酵母奠定实验基础。     

In vitro propagation of the Egyptian medicinal plant,Echinops spinosissimus Turra

The in vitro propagation of medicinal species is proving to be one successful approach to addressing the issues raised by increasing global demand, overharvest of wild populations and variability in product quality. In this report we describe a protocol for establishing nodal cultures of an Egyptian medicinal plant, Echinops spinosis-simus Turra and for the induction of regeneration in epidermal peels and leaf explants obtained from the nodal cultures. Kinetin was found to be optimal for induction and proliferation of nodal cultures. Transverse slices of leaf blades produced regenerants resembling somatic embryos in the presence of thidiazuron while regeneration was observed on epidermal peels only when the medium contained elevated auxin concentrations.     

In vitro shoot regeneration from leaf explants of Echinops kebericho: an endangered endemic medicinal plant

Echinops kebericho is a critically endangered endemic medicinal plant of Ethiopia. It is threatened due to over harvesting of its roots for medicinal purposes and from poor seed viability. This study aimed to develop a protocol for in vitro shoot regeneration from leaf explants of E. kebericho. The seeds were sterilized using ethanol followed by Clorox or calcium hypochlorite. Shoots from the germinated seeds were cultured on Murashige and Skoog (MS) medium containing different concentrations of α-naphthalene acetic acid (NAA) and 6-benzyl amino purine (BAP). Young leaves were cultured on MS medium containing different concentrations of BAP and NAA for shoot regeneration. For shoot multiplication, shoots were excised and cultured on MS medium containing different concentrations of BAP or kinetin (KIN) and NAA. The highest mean number of initiated shoots (4.00 ± 0.57) with 100% shoot induction was obtained on medium containing 1.0 mg/L BAP and 0.2 mg/L NAA. The highest shoot regeneration (33%) and shoot number (2.13 ± 0.06) were obtained on MS medium containing 2.0 mg/L BAP and 0.5 mg/L NAA. Medium containing 1.0 mg/L KIN and 0.2 mg/L NAA produced the highest number of shoots (4.67 ± 0.33) per explant. This protocol can be used for genetic improvement and conservation of this endangered species.     

In vitro clonal propagation through mature nodes of Tinospora cordifolia (Willd.) Hook. F. & Thoms.: An important ayurvedic medicinal plant

Summary A protocol was developed for rapid clonal propagation of the important medicinal climber, Tinospora cordifolia, through in vitro culture of mature nodal explants. Shoots were initiated on both Murashige and Skoog (MS) medium and woody plant medium (WPM) supplemented with 2.32 μM kinetin (KIN). Of the two basal media tested, WPM was found to be superior to MS medium for the induction of multiple shoots. Among the cytokinins tested, N⁶-benzyladenine (BA) was more effective than KIN for axillary shoot proliferation. KIN was superior to BA in terms of shoot elongation. An average multiplication rate of 6.3 shoots per explant was obtained with WPM supplemented with 8.87 μM BA. Shoot clumps harvested from this medium were transferred to WPM supplemented with 2.22 μM BA and 4.65 μM KIN for shoot elongation. Elongated shoots were rooted in half-strength MS medium supplemented with 2.85 μM indole-3-acetic acid (IAA). Rooted plantlets were successfully transferred to sand and established with 80% survival.     

In vitro propagation of the medicinal plant Plumbago zeylanica L. Through nodal explants

Summary A protocol for rapid in vitro propagation using nodal explants obtained from 2-yr-old, field-grown medicinal plants of Plumbago zeylanica L. belonging to the family Plumbaginaceae is described. High frequency bud break and fast development of shoots were induced on Murashige and Skoog's basal medium supplemented with 27.2 μM adenine sulfate +2.46 μM indole-3-butyric acid (IBA). Induction of rooting was achieved by transferring the shoots to the same basal medium containing 4.92 μM IBA. Using our protocol from one twig of P. zeylanica (eight responsive nodes per explant shoot) within a period of 5 mo., eight plantlets could be raised. After a hardening period of 4 wk, there was a 90% transplantation success in the field compared to the 60–65% survival of plantlets recorded in the experiments of previous workers. The plantlets derived through in vitro propagation mimic the growth and morphological characteristics of the donor plants.     

In vitro propagation of Spilanthes mauritiana DC., an endangered medicinal herb, through axillary bud cultures

Summary Spilanthes mauritiana DC., (Compositae), a East African medicinal herb containing pharmaceutically promising secondary metabolites, has successfully been raised in vitro. We have developed a clonal propagation protocol that uses juvenile plants as starting material. The addition of benzylaminopurine (BA) (1.0 μM) and naphthaleneacetic acid (NAA) (0.1 μM) to the culture medium resulted in maximum shooting response with minimal callusing. Shoots rooted best in vitro in MS medium supplemented with indole-3-acetic acid (IAA; 0.2 μM), and plants that had already developed roots showed better growth, with maximum survival rate, in the greenhouse after an initial hardening.     

天山雪莲SiICE2基因克隆及抗寒性分析

该研究以天山雪莲(Saussurea involucrata)的转录组数据为基础,利用Premier 5.0设计1对特异性引物SiICE2-Up和SiICE2-Down,以天山雪莲cDNA为模板克隆得到天山雪莲SiICE2基因的开放阅读框(ORF),对其进行生物信息学分析;构建植物表达载体pCAMBIA2300-35S-SiICE2-Nos,利用农杆菌介导法导入番茄(Lycopersicon esculentum),通过PCR和RT-PCR对转化植株进行验证,qRT-PCR分析转SiICE2基因番茄株系SiICE2基因的相对表达量;将转SiICE2基因型和野生型番茄在0℃处理后,进行抗寒性分析。结果表明:(1)成功克隆得到天山雪莲SiICE2基因,其大小为462bp,共编码153个氨基酸,系统进化分析发现SiICE2蛋白与菜蓟(Cynara scolymus L)亲缘关系最近。(2)成功构建了植物表达载体pCAMBIA2300-35S-SiICE2-Nos,经农杆菌介导法侵染番茄,PCR鉴定表明共有9株为转SiICE2基因番茄植株。(3)膜生理指标测定结果显示,随着低温处理时间的增加,转SiICE2基因型番茄的相对电导率、丙二醛含量均显著低于野生型,在处理时间为24h时,转SiICE2基因型番茄相对电导率比野生型低31.7%,丙二醛含量比野生型低4.2μmol/g。(4)抗氧化酶活性测定结果显示,随着低温处理时间的增加,转SiICE2基因番茄植株的POD、CAT和SOD活性均呈现持续递增趋势,野生型呈先逐渐升高后降低的趋势,且各处理时间内转SiICE2基因番茄的POD、CAT和SOD活性均显著高于野生型。研究发现,天山雪莲SiICE2基因可以显著增强非低温驯化番茄的抗寒性。     

Overexpression of the Saussurea medusa chalcone isomerase gene in S. involucrata hairy root cultures enhances their biosynthesis of apigenin

Saussurea involucrata is a medicinal plant well known for its flavonoids, including apigenin, which has been shown to significantly inhibit tumorigenesis. Since naturally occurring apigenin is in very low abundance, we took a transgenic approach to increase apigenin production by engineering the flavonoid pathway. A construct was made to contain the complete cDNA sequence of the Saussurea medusa chalcone isomerase (CHI) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Using an Agrobacterium rhizogenes-mediated transformation system, the chi overexpression cassette was incorporated into the genome of S. involucrata, and transgenic hairy root lines were established. CHI converts naringenin chalcone into naringenin that is the precursor of apigenin. We observed that transgenic hairy root lines grew faster and produced higher levels of apigenin and total flavonoids than wild-type hairy roots did. Over a culture period of 5 weeks, the best-performing line (C46) accumulated 32.1 mgL(-1) apigenin and 647.8 mgL(-1) total flavonoids, or 12 and 4 times, respectively, higher than wild-type hairy roots did. The enhanced productivity corresponded to elevated CHI activity, confirming the key role that CHI played for total flavonoids and apigenin synthesis and the efficiency of the current metabolic engineering strategy.