Additional betalain accumulation by genetic engineering leads to a novel flower color in lisianthus (Eustoma grandiflorum)

Betalains, comprising violet betacyanins and yellow betaxanthins, are pigments found in plants belonging to the order Caryophyllales. In this study, we induced the accumulation of betalains in ornamental lisianthus (Eustoma grandiflorum) by genetic engineering. Three betalain biosynthetic genes encoding CYP76AD1, dihydroxyphenylalanine (DOPA) 4,5-dioxygenase (DOD), and cyclo-DOPA 5-O-glucosyltransferase (5GT) were expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter in lisianthus, in which anthocyanin pigments are responsible for the pink flower color. During the selection process on hygromycin-containing media, some shoots with red leaves were obtained. However, most red-colored shoots were suppressed root induction and incapable of further growth. Only clone #1 successfully acclimatized and bloomed, producing pinkish-red flowers, with a slightly greater intensity of red color than that in wild-type flowers. T₁ plants derived from clone #1 segregated into five typical flower color phenotypes: wine red, bright pink, pale pink, pale yellow, and salmon pink. Among these, line #1-1 showed high expression levels of all three transgenes and exhibited a novel wine-red flower color. In the flower petals of line #1-1, abundant betacyanins and low-level betaxanthins were coexistent with anthocyanins. In other lines, differences in the relative accumulation of betalain and anthocyanin pigments resulted in flower color variations, as described above. Thus, this study is the first to successfully produce novel flower color varieties in ornamental plants by controlling betalain accumulation through genetic engineering.     

Plant regeneration from mesophyll protoplasts of lisianthus (Eustoma grandiflorum) by adding activated charcoal into protoplast culture medium

Plant regeneration from isolated protoplasts of 8 cultivars of lisianthus, Eustoma grandiflorum (Griseb.) Schinners, has been established by using activated charcoal. Protoplasts were isolated from lisianthus leaves grown in vitro and started to divide within 3–4 days of culture, but successful colony formation was only achieved by adding gellan gum blocks containing 1% (w/v) activated charcoal immediately after culture. Colonies consisting of as many as 50–100 cells formed after 30 days of culture and were transferred to fresh medium for callus proliferation and shoot regeneration, respectively. These shoots rooted on MS medium containing 0.5 mg l^–1 indolebutyric acid(IBA) and the plantlets were finally transplanted to pots. Morphological characteristics, growth habit and pollen fertility of protoplast-derived plants of one cultivar were not different from those of seed-grown plants as control.Abbreviations BA 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - MS Murashige & Skoog (1962) medium - IBA indolebutyric acid - MES 2-N-morpholinoethane sulfonic acid     

Regulation of gene expression involved in flavonol and anthocyanin biosynthesis during petal development in lisianthus (Eustoma grandiflorum)

To elucidate gene regulation of flower colour formation, the gene expressions of the enzymes involved in flavonoid biosynthesis were investigated in correlation with their product during floral development in lisianthus. Full-length cDNA clones of major responsible genes in the central flavonoid biosynthetic pathway, including chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3',5'-hydroxylase (F3'5'H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS), were isolated and characterized. In lisianthus, the stage of the accumulation of flavonols and anthocyanins was shown to be divided clearly. The flavonol content increased prior to anthocyanin accumulation during floral development and declined when anthocyanin began to accumulate. CHS, CHI, and F3H were necessary for both flavonol and anthocyanin biosynthesis and were coordinately expressed throughout all stages of floral development; their expressions were activated independently at the stages corresponding to flavonol accumulation and anthocyanin accumulation, respectively. Consistent with flavonol and anthocyanin accumulation patterns, FLS, a key enzyme in flavonol biosynthesis, was expressed prior to the expression of the genes involved in anthocyanin biosynthesis. The genes encoding F3'5'H, DFR, and ANS were expressed at later stages, just before pigmentation. The genes responsible for the flavonoid pathways branching to anthocyanins and flavonols were strictly regulated and were coordinated temporally to correspond to the biosynthetic order of their respective enzymes in the pathways, as well as in specific organs. In lisianthus, FLS and DFR, at the position of branching to flavonols and anthocyanins, were supposed to play a critical role in regulation of each biosynthesis.     

洋桔梗的转基因研究

以叶盘为外植体,通过农杆菌菌株C58Cl(pPMP90)介导,开展了用植物表达载体pK2-35S-GFP(含GFP基因)转化洋桔梗(Eustoma russellianum)的研究。洋桔梗叶盘用农杆菌浸染15-20min,在共培养基上与农杆菌共培养2d后,转移到分化培养基上诱导愈伤组织。10d后选取部分叶盘在紫外灯下观察GFP的荧光亮点,结果80%以上的叶盘都有GFP的荧光亮点,说明农杆菌对叶盘的感染效率很高。在分化培养基上红花洋桔梗叶盘不定芽分化频率约为30%,把分化培养基上形成的小苗转移到含有Km(20mg/L)的生根培养基上,进行生根诱导筛选具有Km抗性的转基因植株,统计结果说明红花洋桔梗的转化效率为2.4%。随机选取4株具有Km抗性的洋桔梗植株进行PCR检测,结果均为阳性,证明GFP基因已整合到洋桔梗基因组中,选取经PCR检测为阳性的植株叶片,用激光共聚焦显微镜观察,结果发现有GFP的绿色荧光出现在叶片细胞质中,说明叶片中有GFP蛋白的表达,转化试验成功。     

Stable genetic transformation ofPicea mariana (black spruce) via particle bombardment

Stable genetic transformation ofPicea mariana (black spruce) was obtained via particle bombardment into two target tissues, mature cotyledonary somatic embryos and suspensions from embryonal masses, with the Biolistic PDS-1000/He device. Seven transgenic embryogenic cell line were obtained from the mature cotyledonary somatic embryos after secondary somatic embryogenesis from two different cell lines (R4F14 and 119794-014). The suspension culture from embryonal masses produced five transgenic cell lines from one cell line (R4F14). Expression of the introduced β-glucuronidase (GUS) and neomycin phosphotransferase II (NPT II) genes was detected by histochemistry and fluorometry, and by ELISA in 10 of the lines. Two lines showed only NPT II gene expression. Four of the five lines obtained after bombardment of suspensions of embryonal masses showed lower levels of expression of GUS and NPT II. The integration of the foreign genes was confirmed by polymerase chain reaction analyses and Southern hybridization for GUS and NPT II, and complex hybridization patterns were observed. The 12 transgenic lines obtained had a typical embryogenic morphology and were capable of maturation and germination. Over 40 transgenic trees were regenerated from one of the transgenic lines, and they have a normal phenotype.     

Modified ELISA for the detection of neomycin phosphotransferase II in transformed plant species

 Identifying transformed plant lines carrying the antibiotic resistance marker gene, neomycin phosphotransferase II, requires a more definitive test than the ability of the plant to grow on kanamycin. Although a number of alternative assays have been described, most are cumbersome, time consuming and/or require the use of radioisotopes. This report describes an ELISA for the detection of the neomycin phosphotransferase II enzyme in transformed plant tissue. The ELISA utilises commercially available antibodies and provides a number of advantages, including an extremely low background, a reduction in the amount of tissue required for testing, and semi-quantitative data on neomycin phosphotransferase II gene expression. This method has been applied successfully to a number of independently transformed lines in nine plant species. Received: 4 January 1999 / Accepted: 17 April 1999     

Transformation and expression of a gene for herbicide resistance in a Brazilian sugarcane

 Embryogenic calli of the Brazilian sugarcane (Saccharum officinarum L.) genotype SP80–180 were transformed with two plasmids containing genes coding for neomycin phosphotransferase (neo) and phosphinotrycin acetyltransferase (bar), by particle bombardment using an apparatus developed at Copersucar Technology Center. Transformed plants were initially selected on culture medium containing Geneticin, and resistance was confirmed by localized application of a kanamycin solution to leaves of hardened plants at the nursery. A commercial formulation of ammonium gluphosinate was sprayed twice on these antibiotic-resistant plants. The resistant plants were considered co-transformed, and Southern analysis confirmed stable integration of both bar and neo genes. In addition, phosphinotrycin acetyltransferase expression was supported by RT-PCR analysis and neomycin phosphotransferase presence was demonstrated by western blotting. Similar analyses were also performed with micropropagated transformants after three cycles of subculture. Received: 15 December 1999 / Revision received: 15 April 2000 / Accepted: 1 June 2000     

Protoplasts to plants of Gentianaceae. Regeneration of lisianthus (Eustoma grandiflorum) is affected by calcium ion preconditioning,osmolality and pH of the culture media

A reliable method has been developed for regeneration of whole plants from isolated protoplasts of five cultivars of lisianthus,Eustoma grandiflorum (Griseb.) Schinners (Gentianaceae). Protoplasts were isolated from either cotyledons or leaves and cultured in agarose beads surrounded by liquid V-KM media containing 5.37 µM 1-naphthyleneacetic acid (NAA) and 2.28 µM zeatin. When microcalli were approximately 1 mm in diameter, the agarose beads were transferred to shoot regeneration media containing 0.1 µM indolebutyric acid (IBA) and 4.44 µM 6-benzylaminopurine (BAP). Shoots were produced from the calli during several sub-culture periods. Protoplast viability and the subsequent regeneration of plants were dependent on calcium levels and growth regulator presence in thein vitro seed germination media, on the osmolality of the protoplast purification solution, and osmolality increase and pH of the culture media. Shoots were rooted in Murashige & Skoog (1962) media containing 5.71 µM indole-3-acetic acid (IAA). Plantlets derived from protoplasts of five lisianthus cultivars (Fresh White, Hakusen, Miss Lilac, Fresh Purple and Doremi Wine Red) have been successfully transferred to the glasshouse.     

草原龙胆MADS-box基因的克隆及表达分析

植物MADS-box基因家族编码高度保守的转录因子,参与了包括花发育在内的多种发育进程。为阐释双子叶植物草原龙胆（Eustoma grandiflorum）花器官发育的分子调控机制,根据MADS-box基因保守序列设计简并引物,用3＇-RACE方法从草原龙胆中克隆了4个花器官特异表达的MADS-box家族基因。序列和系统进化树分析表明,这4个基因分别与金鱼草DEF基因、矮牵牛FBP3基因和FBP6基因以及拟南芥SEP3基因具有很高的同源性,分别属DEF/GLO、AG-like和SEP-like亚家族。从而将这4个基因分别命名为EgDEF1、EgGLO1、EgPLE1和EgSEP3-1。推导的氨基酸序列显示,这些基因编码的蛋白质都包含高度保守的MADS结构域、I结构域和K结构域,每个基因均有其亚家族特异的C-末端功能域。基因特异性RT-PCR检测结果显示：EgDEF1在萼片、花瓣、雄蕊及胚珠中高丰度表达,在心皮中微量表达;而EgGLO1在花瓣和雄蕊中高丰度表达,在萼片中微量表达;在根、茎、叶等营养器官中均未检测到上述2个基因的表达。EgPLE1在雌蕊、心皮和胚珠中特异表达,但表达的丰度存在差异,在雄蕊中的表达有所减弱。SEP-like亚家族基因EgSEP3-1在四轮花器官和胚珠中均特异表达,且表达丰度相对一致。     

草原龙胆MADS-box基因的克隆及表达分析

植物MADS-box 基因家族编码高度保守的转录因子, 参与了包括花发育在内的多种发育进程。为阐释双子叶植物草原龙胆(Eustoma grandiflorum)花器官发育的分子调控机制, 根据MADS-box基因保守序列设计简并引物, 用3'-RACE方法从 草原龙胆中克隆了4个花器官特异表达的MADS-box家族基因。序列和系统进化树分析表明, 这4个基因分别与金鱼草DEF基因、矮牵牛FBP3基因和FBP6基因以及拟南芥SEP3基因具有很高的同源性, 分别属DEF/GLO、AG-like和SEP-l ike亚家族。从而将这4个基因分别命名为EgDEF1、EgGLO1、EgPLE1和EgSEP3-1。推导的氨基酸序列显示, 这些基因编码的蛋白质都包含高度保守的MADS结构域、I结构域和K结构域, 每个基因均有其亚家族特异的C-末端功能域。基因特异性RT-PCR检测结果显示: EgDEF1 在萼片、花瓣、雄蕊及胚珠中高丰度表达, 在心皮中微量表达; 而EgGLO1在花瓣和雄蕊中高丰度表达, 在萼片中微量表达; 在根、茎、叶等营养器官中均未检测到上述2个基因的表达。EgPLE1在雌蕊、心皮和胚珠中特异表达, 但表达的丰度存在差异, 在雄蕊中的表达有所减弱。SEP-like亚家族基因EgSEP3-1在四轮花器官和胚珠中均特异表达,且表达丰度相对一致。     

Identification and Characterization of Pothos latent virus Causing Necrotic Ringspots and Line Patterns on Lisianthus (Eustoma grandiflorum) in Taiwan

Lisianthus (Eustoma grandiflorum) grown in screenhouses in Taiwan showed ringspots and concentric line patterns on leaves. A virus having isometric particles approximately 30–32 nm in diameter was isolated from affected lisianthus. Combined results of biological, cytological, serological, molecular and phylogenetic analyses show that the virus can be identified as Pothos latent virus (PoLV), genus Aureusvirus, family Tombusviridae. Inoculating the virus on non‐infected lisianthus plants reproduced the symptoms previously observed in the field. So, this is the first report of PoLV causing disease in lisianthus and the first report of the virus in Taiwan.     

Transformation of arctic bramble (Rubus arcticus L.) by Agrobacterium tumefaciens

Genetic transformation of arctic bramble (Rubus arcticus L.) was achieved utilizing a Ti-plasmid vector system of Agrobacterium tumefaciens. Internodal stem segments were inoculated with Agrobacterium strain EHA101 carrying a T-DNA with the CaMV 35 S promoter-gus-int marker gene from which β-glucuronidase (GUS) is expressed only in plants. Regenerants were produced on Murashige and Skoog medium. Growth of Agrobacterium was inhibited with cefotaxime. Kanamycin was used as the selective agent for the transformants. Regenerants were assayed by histochemical GUS staining, and by Southern analysis using a gus-int probe. Transgenic arctic bramble plants containing gus-int and expressing GUS were recovered. Expression has been stable for 3 years in micropropagation. Received: 22 October 1997 / Revision received: 7 January 1998 / Accepted: 2 February 1998     

Agrobacterium-mediated transient transformation of marigold (Tagetes erecta)

Hypocotyls, roots, leaf sections and shoot tips from Tagetes erecta plantlets were inoculated with Agrobacterium tumefaciens, harboring the binary vector pCAMBIA2301, containing the β-glucuronidase gene. Histochemical GUS assays of infected tissues showed transient gus gene expression after 3 days.     

Stable transformation of Eustoma grandiflorum by particle bombardment

Explants (7.5±2.5 mm) cut from stems and roots of 3-week-old Eustoma grandiflorum Grise, (lisianthus) cv. Glory White seedlings were bombarded with plasmid pBI221, which harbors the uidA gene encoding β-glucuronidase (GUS) driven by the cauliflower mosaic virus (CaMV) 35S promoter. More than 800 blue spots of GUS-expressing cells were observed per 90 explants. Explants bombarded with pARK22 harboring the bar gene encoding phosphinothricin acetyltransferase driven by the CaMV 35S promoter were selected for bialaphos resistance. Putative transgenic plants were obtained about 3 months after bombardment. Southern blot analysis of putative transgenic plants revealed the presence of the bar gene in their genome. Received: 10 April 1996 / Revision received: 7 November 1997 / Accepted: 22 November 1997     

Production of transgenic gentian plants by particle bombardment of suspension-culture cells

 Cell suspension cultures were established from leaf explants of gentian (Gentiana triflora×G. scabra) for the generation of transgenic plants by particle bombardment. The parameters for the bombardment of suspension culture cells with a particle gun were examined by monitoring the transient expression of a gene for β-glucuronidase driven by the cauliflower mosaic virus (CaMV) 35S promoter. We found that prior culture of suspension culture cells for 5 days on solid medium was optimum for successful particle bombardment. Putative transformed calli were obtained from bombarded cells after a two-step selection procedure. Cells were cultured first with 30 mg l^–1 hygromycin in liquid MS medium that contained 10 mg l^–1 N-phenyl-N′-1,2,3-thiadiazol-5-yl urea, 1 mg/l 1-naphthaleneacetic acid and 30 g l^–1 sucrose and then on solid medium prepared from the same liquid medium plus 2 g l^–1 gellan gum. After 12 weeks of selection on solid medium that contained 30 mg l^–1 hygromycin, two transgenic gentian plants were regenerated from each selected callus. Analysis by the polymerase chain reaction and Southern blotting revealed the stable integration of transferred DNA. Received: 3 June 1999 / Revision received: 21 September 1999 / Accepted: 20 September 1999     

Transformation of betulin diacetate by the Prins reaction

The interaction of betulin diacetate with formaldehyde by the Prins reaction in various media was studied. As a result, 3β,28-di-O-acetyl-30-hydroxymethyl-(20)29-lupene, 3β-acetyl-28-hydroxy-(20)29-lupene, and 3β,28-di-O-acetoxy-19-(5′,6′-dihydro-2′H-pyran-4′-yl)-20,29,30-trinorlupane were obtained.     

Field performance of transgenic potato plants compared with controls regenerated from tuber discs and shoot cuttings

Summary The objective of this study was to separate and determine effects on the field performance of transgenic potatoes that originate from the tissue culture process of transformation and from the genes inserted. The constructs introduced contained the reporter gene for betaglucuronidase (GUS) under the control of the patatin promoter (four different constructs) and the neomycin phosphotransferase gene under the control of the nopaline synthase promoter. Both genes might be expected to have a neutral effect on plant phenotype. The field performance of transgenic plants (70 independent transformants) was compared with non-transgenic plants regenerated from tuber discs by adventitious shoot formation and from shoot cultures established from tuber nodal cuttings. Plants from all three treatments were grown in a field trial from previously field-grown tubers, and plant performance was measured in terms of plant height at flowering, weight of tubers, number of tubers, weight of large tubers and number of large tubers. There was evidence of somaclonal variation among the transgenic plants; mean values for all characters were significantly lower and variances generally higher than from plants derived from nodal shoot cultures. A similar change in means and variances was observed for the non-transgenic tuber-disc regenerants when compared with shoot culture plants. Plant height, tuber weight and tuber number were, however, significantly lower in transgenic plants than in tuber-disc regenerants, suggesting an effect on plant performance either of the tissue culture process used for transformation or of the genes inserted. There were significant differences between constructs for all five plant characters. The construct with the smallest segment of patatin promoter and the lowest level of tuber specificity for GUS expression had the lowest values for all five characters. It is proposed that the nature of GUS expression is influencing plant performance. There was no indication that the NPTII gene, used widely in plant transformation, has any substantial effect on plant performance in the field.     

Transformation of a Texas cotton cultivar by using Agrobacterium and the shoot apex

 Transgenic cotton (Gossypium hirsutum L.) plants of a Texas cultivar CUBQHRPIS were obtained using Agrobacterium-mediated transformation coupled with the use of shoot-apex explants. After inoculation with A. tumefaciens strain LBA 4404 containing the pBI121 plasmid, regeneration of primary plants was carried out in a medium containing kanamycin (100  mg l^-1). Progeny obtained by selfing were germinated in the greenhouse and selected for expression of the T-DNA marker gene encoding neomycin phospho-transferase II (NPTII) by painting kanamycin (2%) on the leaves. Plants that survived the leaf painting were analyzed by DNA blots. Evidence for integration of the transgene (GUS) was observed in two successive generations from the regenerants (T₀). The transformed plants appeared to have more than one copy of the T-DNA. Received: 1 June 1998 / Accepted: 28 July 1998     

Differential expression in vitro of heterosis in lisianthus (Eustoma Grandiflorum) at various benzylandenine and gibberellic acid levels

Summary The responses of two lisianthus (Eustoma grandiflorum) inbred lines and their heterotic (in vivo) F₁ hybrid to different levels of benzyladenine (BA) and gibberellic acid (GA₃) were studied under in vitro conditions. Number of axillary shoots developed (NAS), main shoot length (MSL), total fresh and dry weights, and extent of callus formation were measured. The two parents differed in their response to both hormones by all parameters measured. High-parent heterosis for NAS and MSL proved to be dependent on the specific BA and GA₃ levels. The required BA concentrations for maximum NAS and MSL for the F₁ were higher than those required for either parent; this may be defined as a heterosis in BA-requirement for maximal in vitro response. Adding 0.25 or 1 mg/liter GA₃ to the medium caused a considerable increase in NAS and in dry weight gain only when combined with BA. On the other hand, MSL was increased by GA₃ addition only at 0 or 0.1 mg/liter BA, but not at 0.5 mg/liter BA. Callus formation was greatly encouraged by raising the BA level from 0 to 0.1 or 0.5 mg/liter and by the addition of GA₃ at low BA level. Assumed differences in endogenous BA and GA levels among the genotypes studied seemed to be related to the expression of heterosis.