In Vitro Culture of Orobanche aegyptiaca

Calli derived from seedlings of the parasitic plant Orobancheaegyptiaca were grown on enriched nutrient agar. Growth wasenhanced by the addition of 0.1 mg 1^–1 NAA and 5 % coconutmilk. The calli developed two kinds of elongated protrusions:thin ones which resemble the young root radicle and thick oneswhich resemble the adventitious roots of Orobanche plants. Bothformed pad-like structures when touching the substrate. In liquidculture, some of the calli were observed to surround isolatedcultured tomato roots. Orobanche aegyptiaca Pers., in vitro culture, germination, coconut milk, root parasite     

Infection of tomato roots using callii of broomrape

Parasitic weeds like members of the genera Orobanche and Striga cause heavy damage to grain, vegetables and other field crops. The germinating seedlings of the parasites attack the roots of their hosts, following a series of recognition events that control germination, seedling growth and attachment to the host root. An in vitro system has been developed that allows the production of partially differentiated callii of Orobanche aegyptiaca Pers. Protrusions of subcultures of these callii behaved like radicles of germinating seeds. Using this system normal infection of tomato roots was obtained in vitro. The infection process included haustorium formation and the development of young Orobanche plants under aseptic conditions. The system developed should now permit a study of biochemical events during infection of the host by Orobanche .     

Reactions of Lotus japonicus ecotypes and mutants to root parasitic plants

Witchweeds (Striga spp.) and broomrapes (Orobanche spp.) are obligate root parasitic plants on economically important field and horticultural crops. The parasites' seeds are induced to germinate by root-derived chemical signals. The radicular end is transformed into a haustorium which attaches, penetrates the host root and establishes connection with the vascular system of the host. Reactions of Lotus japonicus, a model legume for functional genomics, were studied for furthering the understanding of host-parasite interactions. Lotus japonicus was compatible with Orobanche aegyptiaca, but not with Orobanche minor, Striga hermonthica and Striga gesnerioides. Orobanche minor successfully penetrated Lotus japonicus roots, but failed to establish connections with the vascular system. Haustoria in Striga hermonthica attached to the roots, but penetration and subsequent growth of the endophyte in the cortex were restricted. Striga gesnerioides did not parasitize Lotus japonicus. Among seven mutants of Lotus japonicus (castor-5, har1-5, alb1-1, ccamk-3, nup85-3, nfr1-3 and nsp2-1) with altered characteristics in relation to rhizobial nodulation and mycorrhizal colonization, castor-5 and har1-5 were parasitized by Orobanche aegyptiaca with higher frequency than the wild type. In contrast, Orobanche aegyptiaca tubercle development was delayed on the mutants nup85-3, nfr1-3 and nsp2-1. These results suggest that nodulation, mycorrhizal colonization and infection by root parasitic plants in Lotus japonicus may be modulated by similar mechanisms and that Lotus japonicus is a potential model legume for studying plant-plant parasitism.     

An extensive hairy root production precedes shoot regeneration in protoplast-derived calli of cauliflower (Brassica oleracea var. botrytis)

Summary Protoplasts isolated from mesophyll tissues of cauliflower (Brassica oleraceavar. botrytis) were induced to divide in culture, with 2% of them producing calli. Upon transfer to a regeneration medium containing a low auxin/cytokinin balance (0.02mg/l 1-naphthaleneacetic and 2mg/l 6-benzylaminopurine), they displayed an extensive production of hairy roots before the regeneration of shoots. Negative effects of root differentiation on the subsequent caulogenesis by such calli were not observed, since 97% of the calli regenerated hairy roots and 93% gave shoots.Abbreviations NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - 2,4 D 2,4-dichlorophenoxyacetic acid - GA3 gibberellic acid - MES 2-N-morpholinoethane sulfonate     

Regeneration of plantlets from the callus of stem segments of adult plants of Ficus religiosa L.

Stem segments of adult plants of Ficus religiosa L. cultured on MS medium containing 1.0 mg/l 2,4-D produced callus. Shoots were regenerated when the induced calli were transferred to medium supplemented with 0.05 to 2.0 mg/l BAP. Callus derived shoots produced roots and developed into plantlets when transferred to medium supplemented with 1.0 mg/l NAA.Abbreviations MS Murashige and Skoog (1962) - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Production of plantlets from Aegle marmelos nucellar callus

Techniques have been developed for the regeneration of Aegle marmelos from nucellar explants. Slow-growing calli were induced from nucellar explants excised from 90–120 d-old developing fruits. The medium consisted of Murashige and Skoog formulation containing 40 g/l sucrose, 400 mg/l casein hydrolysate, 5 mg/l 1-naphthaleneacetic acid and 1 mg/l kinetin. The basal medium with high concentration (1–5 mg/l) of N6-benzyladenine (BA) and low concentration (0.1 mg/l) of NAA was suitable for regeneration of shoots from 3-month-old calli. Addition of 1 mg/l gibberellic acid (GA3) favoured shoot growth. Callus-derived shoots produced roots and developed into plantlets when transferred to half-strength MS medium supplemented with 0.5 mg/l indole-3-butyric acid (IBA) and 0.5 mg/l NAA. Approximately 5 months were required for the full regenerative process.     

Plant Regeneration from Protoplasts of Ligusticurn wallichii Franch

The hypocotyls of the embryoid derived plantlets of Ligusticum wallichii Franch were used for protoplast preparation. Protoplasts were obtained with the enzyme mixture containing 1.5% Onozuka R-10, 0.3% Macerozyme R-10, 0.5% Snailase, 5 mmol/l CaCl2, 1 mmol/l KH2PO4, 0.6 mol/l manitol, at pH 5.6–5.8 and 27℃. Protoplasts were cultured in a modified MS liquid medium containing 1 mg/l 2,4-D + 0.5 mg/l 6- BA. The first divisions were found after twelve days, and the dividing cells formed cell colonies of 0.5–1 mm after about fourty days. When they were transferred to MS agar medium (with half quantity of macronutrients) supplemented with 2,4-D (0.5mg/l) and 6-BA(0.5mg/l), they grew into calli. At last, on the medium without any phytohormones, the growing calli differentiated embryoids which developed into plantlets with many green leaves and roots.     

Agrobacterium rhizogenes-mediated transformation and the regeneration of transformants in Alhagi pseudalhagi]

The regenerated shoot segments of Alhagi pseudalhagi were sliced and infected with Agrobacterium rhizogenes strain A4. The hairy roots and transformed calli were obtained through selection on hormone free MS medium. The transformants were cultured on MS medium with 2 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5-1 mg/L 6-benzylaminopurine (6-BA) to induce calli. 3 mg/L 6-BA and 0.5 mg/L naphthalene acetic acid (NAA) were applied for shoot differentiation. Shoots were planted on MS medium with 2 mg/L indole-3-butyric acid (IBA) and produced roots. Opine analysis proved the integration and expression of T-DNA in over 95% hairy roots, 75% transformed calli and transformed plantlets respectively. The 81% hairy root cells had normal chromosome numbers (2n = 18). The alterations of chromosome number were observed. After one year of subculturing, the regeneration ability of transformants was maintained.     

Cell wall degrading enzymes in Orobanche aegyptiaca and its host Brassica campestris

Activities of the cell-wall degrading enzymes cellulase (EC 3. 2. 1. 4). poly-galacturonase (EC 3. 2. 1. 15) and xylanase (EC 3. 2. 1. 8), and also that of protease (EC 3. 4. 21. 14) have been determined in the parasite Orobanche aegyiptiaca Pers. and in the roots of infected and control host plants of Brassica campestris L. var. rapa Hartm. Shoots, roots and root-freed tubers of Orobanche showed pronounced activities of these enzymes. When expressed in terms of specific activity, protease, cellulase and polygalacturonase activities were higher and xylanase lower in all parts of Orobanche than in the roots of infected or control host plants. Orobanche roots had higher activities of cellulase, polygalacturonase and protease than tuber or shoot. While xylanase activity was higher in the shoot than in roots or tuber. Infection by Orobanche significantly enhanced the cellulase activity of host roots. Cellulase and polygalacturonase in the Orobanche roots appear as the main enzymes involved in establishing haustorial connection with the host root. Protease might be involved in degrading proteins and lipoproteins of the cell walls and membranes of the host tissues.     

发根农杆菌对骆驼刺的转化及转化组织的形态发生

将骆驼刺离体再生苗的茎切段经发根农杆菌A4菌株感染后,在含500mg/L头孢霉素的MS无激素培养基上培养,产生了转化的发根和愈伤组织.转化根在附加2mg/L2,4-D和0.5-1mg/L6BA的MS培养基上培养后,亦可诱导出愈伤组织.在含3mg/L6BA和0.5mg/LNAA的培养基上诱导出了苗的分化.冠瘿碱分析表明,在95%以上的发根和75%的转化愈伤组织及再生植株中都显示了T-DNA的整合和表达.染色体检查发现,约81%的发根细胞具有正常染色体数(2n=18),其余则存在染色体数目的变化,在继代培养一年之后,转化体仍维持旺盛的再生能力.     

An interchangeable system of hairy root and cell suspension cultures ofCatharanthus roseus for indole alkaloid production

Hairy roots ofCatharanthus roseus obtained by co-cultivation of hypocotyl segments withAgrobacterium rhizogenes, and cultured in SH (Schenk and Hildebrandt) basal medium, formed two types of calli when subcultured in SH medium with 1 mg/1 -naphthaleneacetic acid and 0.1 mg/l kinetin. One of them, a compact callus, when re-subcultured in SH basal medium gave rise to hairy roots again. A rhizogenic cell suspension culture was established from this type of callus. When cultured in SH medium with growth regulators, the rhizogenic callus produced catharanthine at a level of 41% of the level in the initial hairy roots. Upon transfer to SH basal medium, regenerated hairy roots produced this alkaloid at the original level of 1.5 mg/g dry wt. Using this cell/hairy root interchange system a new management system for hairy root culture in bioreactors has been devised and examined involving production of biomass in the form of a cell suspension in medium supplemented with growth regulators, and catharanthine production by hairy roots regenerated from these cells in medium without growth regulators.Abbreviations NAA -naphthaleneacetic acid - SH Schenk and Hildebrandt - SHNK SH medium + 1 mg 1^–1 NAA + 0.1 mg 1^–1 kinetin     

Efficient genetic transformation and regeneration system from hairy root of <Emphasis Type="Italic">Origanum vulgare</Emphasis>

Origanum vulgare L is commonly known as a wild marjoram and winter sweet which has been used in the traditional medicine due to its therapeutic effects as stimulant, anticancer, antioxidant, antibacterial, anti-inflammatory and many other diseases. A reliable gene transfer system via Agrobacterium rhizogenes and plant regeneration via hairy roots was established in O. vulgare for the first time. The frequency of induced hairy roots was different by modification of the co-cultivation medium elements after infection by Agrobacterium rhizogenes strains K599 and ATCC15834. High transformation frequency (91.3 %) was achieved by co-cultivation of explants with A. rhizogenes on modified (MS) medium. The frequency of calli induction with an 81.5 % was achieved from hairy roots on MS medium with 0.25 mg/L^?1 2,4-D. For shoot induction, initiated calli was transferred into a medium containing various concentrations of BA (0.1, 0.25, 0.5, 0.75 and 1 mg/L^?1). The frequency of shoot generation (85.18 %) was achieved in medium fortified with 0.25 mg/L^?1 of BA. Shoots were placed on MS medium with 0.25 mg/l IBA for root induction. Roots appeared and induction rate was achieved after 15 days.     

寄生植物种子萌发特异性及其与寄主的识别机制

寄生植物广泛分布于不同的生态环境中,并具有不同的生育习性及与寄主识别特性.本文阐述了根寄生植物列当属和独脚金属种子萌发的特异性,以及目前已发现的寄生植物种子萌发的信号物质,并就不同萌发信号物质、植物激素、真菌代谢物在寄生植物种子识别寄主中的作用以及寄生植物种子预培养阶段的呼吸作用特性与萌发信号物质的活化机理等做了综述.探讨了各种列当不同分化类型的愈伤组织诱导、离体无菌侵染新系统及其在寄生植物与寄主互作识别研究中的应用,提出了寄生植物与寄主识别机理研究中存在的问题并对研究前景进行了展望.     

Plant regeneration from protoplasts of immature Vigna sinensis cotyledons via somatic embryogenesis

Summary Protoplasts were isolated from immature cotyledons of Vigna sinensis and cultured in a modified MS Liquid medium containing 0. 2 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D), 1 mg/l naphthaleneacetic acid (NAA) and 0. 5 mg/l 6-benzylaminopurine (BAP) in the dark at a density of 1 × 10⁵/ml. The protoplasts began to divide in 3–5 days. Sustained cell division resulted in formation of cell clusters and small calli, with the cell division frequency and plating efficiency of cell colonies reaching 27. 7% and 1. 7% respectively. When calli of 2 mm in size were transferred onto MSB medium (MS salts and B5 vitamins) containing 500mg/l NaCl, 500 mg/ 1 casein hydrolysate (CH), 2 mg/l 2,4-D and 0. 5 mg/l BAP for further growth, approximately 5% of the calli developed embryogenically. The embryogenic calli were selected and subcultured on the same composition of MSB medium and were able to maintain somatic embryogenesis capacity in subculture for a long time. When the calli were moved to MSB medium with 0. 1 mg/l indole-3-acetic acid (IAA), 0. 5mg/l kinetin(KT), 3–5% mannitol and 2% sucrose in the light, many somatic embryos formed from the calli. Only part of the embryoids developed further to the cotyledonary stage, and the others died at the globular, heart-shaped or torpedo stages. Finally, some cotyledonary embryoids germinated and developed into plants or shoots. The shoots were readily rooted on 1/2 strength MS medium with 0. 1–0.3 mg/l indole-3-butyric acid (IBA). The plants grew well in soil and were fertile.Abbreviations 2, 4-D dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - BAP 6-benzylaminopurine - IAA indole-3-acetic acid - KT kinetin - IBA indole-3-butyric acid - CH casein hydrolysate - CM coconut milk - ZT zeatin     

A role for IAA in the infection of Arabidopsis thaliana by Orobanche aegyptiaca

BACKGROUND: Vascular continuity is established between a host plant and the root parasite broomrape. It is generally accepted that the direction of vascular continuity results from polar flow of auxin. Our hypothesis was that chemical disruptions of auxin transport and activity could influence the infection of the host by the parasite. METHODS: A sterile system for the routine infection of Arabidopsis thaliana seedlings in Nunc cell culture plates by germinated seeds of Orobanche aegyptiaca was developed. This method permitted a quantitative assay of the rate of host infection. The three-dimensional structure of the vascular contacts was followed in cleared tissue. IAA (indole acetic acid) or substances that influence its activity and transport were applied locally to the host root. RESULTS: The orientation of the xylem contacts showed that broomrape grafts itself upon the host by acting hormonally as a root rather than a shoot. Local applications of IAA, PCIB (p-chlorophenoxyisobutyric acid) or NPA (naphthylphthalamic acid) all resulted in drastic reductions of Orobanche infection CONCLUSIONS: Broomrape manipulates the host by acting as a sink for auxin. Disruption of auxin action or auxin flow at the contact site could be a novel basis for controlling infection by Orobanche.     

An efficient and reproducible method for regeneration of whole plants from mature seeds of a high yielding Indica rice (Oryza sativa L.) variety PAU 201

Tissue culture is one of the tools necessary for genetic engineering and many other breeding programs. Moreover, selection of high regenerating rice varieties is a pre-requisite for success in rice biotechnology. In this report we established a reproducible plant regeneration system through somatic embryogenesis. The explants used for regeneration were embryogenic calli derived from mature seeds cultured on callus induction media. For callus induction mature seeds were cultured on MS medium containing 30 g/l sucrose combined with 560 mg/l proline and 1.5-3.5 mg/l 2,4-D and 0.5-1.5 mg/l Kin. For plant regeneration, embryogenic calli were transferred to MS medium containing 30 g/l sucrose, supplemented with 1.0-3.0 mg/l BAP, 0.5-1.5 mg/l Kin and 0.5-1.5 mg/l NAA. The highest frequency of callus induction (44.4%) was observed on the MS medium supplemented with 2.5 mg/l 2,4-D, 0.5 mg/l Kin, 560 mg/l proline and 30 g/l sucrose. The highest frequency of shoot regeneration (42.5%) was observed on the MS medium supplemented with 2.0 mg/l BAP, 0.5 mg/l NAA and 0.5 mg/l Kin. The plantlets were hardened and transferred to soil in earthen pots. The developed method was highly reproducible. The in vitro developed plants showed normal growth and flowering under glasshouse conditions.     

紫色大花矮牵牛组织培养与植株再生

矮牵牛叶片外植体在MS+6-BA 1.0mg/L+NAA 0.1mg/L培养基上培养3周后产生致密的浅绿色愈伤组织;转入芽分化培养基MS+6-BA 0.5mg/L+4-PU 0.5mg/L+NAA 0.1mg/L 1周后,从愈伤组织表面不断分化产生幼芽;待幼芽长至3cm时转接至生根培养基1/2MS+NAA 1.0 mg/L+GA₃0.5mg/L中生根,长成完整植株。     

Transformation of Solanum nigrum L. protoplasts by Agrobacterium rhizogenes

Summary Solanum nigrum protoplasts were co-cultivated with Agrobacterium rhizogenes harboring agropine-type Ri plasmid (pRi15834). A large number of transformed calli were obtained on Murashige and Shoog's (MS) medium lacking plant growth regulators. Frequency of transformation was about 3.5×10^–3. In most of the calli, hairy roots appeared on MS medium without plant growth regulator. When the hairy roots were cut into segments and subcultured on MS medium lacking plant growth regulators, calli were readily formed. Plantlets were regenerated by transferring those calli to MS medium supplemented with 1 mg/l zeatin and 0.2 mg/l naphthaleneacetic acid. Frequency of plant regeneration was about 70%.     

Somatic embryogenesis and plant regeneration from leaf callus of Ocimum basilicum L

A effective protocol for complete plant regeneration via somatic embryogenesis has been developed for Ocimum basilicum L. Callus was initiated from leaf explant of young plant on supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) 1.0 mg l(-1), 3% sucrose and 0.9% agar. The calli showed differentiation of globular structure embryos when transferred to MS medium containing 2,4-D 0.5 mg l(-1) and BAP 1.0 mg l(-1). The maximum globular structure embryos were further enlarged and produced somatic embryos in MS basal medium supplemented with BAP 1.0 mg l(-1)+NAA 1.0 mg l(-1) + KN 0.5 mg l(-1). Continued formation of globular embryo and germination of embryos occurred in this medium. Complete plantlets were transferred onto specially made plastic cup containing soilrite followed by their transfer to the garden soil. Survival rate of the plantlets under ex vitro condition was 80%.