Growth patterns and alkaloid accumulation in hairy root and untransformed root cultures of Datura stramonium

The aim of this work was to study the possible relationship between alkaloid production and growth measured as: biomass increase and cellular division frequency, in Datura stramonium in vitro root cultures (hairy root and normal cultures). A comparison of growth values on a fresh and dry weight basis showed that there were differences between transformed and non-transformed lines. The differential growth between lines occurred due to a real biomass increase and not because of water accumulation. On the other hand, the rate of cell division showed a similar pattern for all lines studied. Therefore, the differences in growth are not due to different cell division rates, nor to the presence of larger meristems, but to the development and growth of lateral roots and the presence of active intercalary meristematic zones in each line. The maximum alkaloid production occurred when the cultures were not growing. This suggests an inverse relationship. Finally, the data support a specific model of growth at the level of cell division in root cultures which has not been described before in the literature. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of plant growth regulators on growth and biosynthesis of phenolic compounds in genetically transformed hairy roots of<Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer

In this study, morphological alterations, biomass growth, and secondary metabolite production of genetically transformed hairy roots ofPanax ginseng C. A. Meyer, were evaluated after administration of plant growth regulators. The addition of benzylamino purine and kinetin to the culture media increased biomass formation and phenolic compound biosynthesis in the hairy roots. α-Naphthaleneacetic acid and indole-3-butyric acid inhibited hairy root growth, however, low concentrations of indole-3-acetic acid slightly increased hairy root growth. Low concentrations of 2,4-Dichlorophenoxyacetic acid profoundly inhibited growth of hairy roots. The addition of plant growth regulators, such as auxin, did not increase total phenolic compounds in hairy roots that did not contain gibberellic acid and cytokinins. Callus formation was induced in cultures suspended in liquid medium amended with benzylamino purine and kinetin. Hairy roots regenerated from these calluses exhibited an active growth pattern with extensive lateral branching in non-amended medium, similar to the growth pattern of the original hairy roots.     

Influence of Position and Number of Nodal Roots on Outgrowth of Axillary Buds and Development of Branches in Trifolium repens (L.)

The implications of the presence of a root, either at the parentnode or at neighbour nodes, on branch formation of Trifoliumrepens (white clover) was investigated. Plants were freely rootedor rooting was restricted to every sixth or every twelfth nodealong the parent axis. The absence of a root at the parent nodehad little influence on the probability of the subtending axillarybud forming a branch but, on average, delayed the outgrowthof the bud. The probability that an axillary bud, emerging froma non-rooted parent node, developed to a lateral branch (branchwith elongated internodes) decreased with decreasing proximityof the parent node to a rooted node. Lateral branches emergingfrom non-rooted parent nodes which were two nodes distal toa rooted node had a higher rate of node appearance, a greatermean internode length and area per leaf, and were more branchedthan lateral branches emerging from other non-rooted parentnodes. The dry mass of each single root and of branches grownat rooted parent nodes were significantly higher in plants withrestricted rooting than in freely rooted plants. Restrictionin the number of rooted nodes per plant increased the numberof inflorescences. It is concluded that the whole plant responseto restricted root formation was continuous growth of the parentaxis and compensatory growth of the branch at the rooted node.In general, growth was slow for axillary buds whose developmentwas dependent on the basipetal movement or cross-transport withinthe stolons of resources exported from roots. Trifolium repens (L.); white clover; axillary bud outgrowth; branch development; clonal growth; nodal root     

Infection of turnip and radish storage roots with Agrobacterium rhizogenes

Within about 10 days after inoculation with Agrobacterium rhizogenes, the vascular bundles of storage root disks of turnip or radish developed small outgrowths with numerous root hairs. Thereafter, adventitious roots (hairy roots) emerged extensively from these outgrowths. The hairy roots which emerged fully supported the growth of host plants, though they lacked geotropism. An excised hairy root could be subcultured as an axenic root culture in the absence of phytohormones. Hairy root cultures with extensive lateral branches grew much more rapidly than those with few lateral branches or ordinary roots. Calli were induced from hairy root cultures in the presence of 2,4-D, and root proliferation from these calli occurred in the absence of 2,4-D. Both the primary hairy roots and the roots which grew from them synthesized agropine and mannopine.     

桃树修剪分枝模式的模拟

在不同修剪手法下,对栽培桃树(Prunuspersica(L.)Batsch)不同母枝上的分枝模式进行了比较研究.从分枝模式来看:修剪后的母枝基本由3个不同的区域组成,基部是不萌发的潜伏芽形成的未分枝区域;中部是延迟分枝和多次分枝组成的分枝区域(主要的枝条类型有短枝、长枝和多次枝);顶部是被剪除的部分.我们通过隐式半马尔可夫模型来模拟这一分枝模式,主要是定量描述1次枝和多次枝在母枝上的数量及其分布状况.在上述模型中,未分枝区、延迟分枝区和多次分枝区称为瞬时态,被剪除的部分称为吸收态.模拟的结果与观察的结果进行对比后发现,两者具有很好的一致性.这说明隐式半马尔可夫模型是模拟植物分枝过程的一种有效方法,尽管隐式半马尔可夫链模型只是一个描述性的模型,但仍能对其所描述的生物现象进行解释,在预测修剪手法对母枝分枝模式影响方面比传统的方法具有明显的优势.本研究结果是建立三维虚拟桃树树冠分枝结构的基础.     

桃树修剪分枝模式的模拟

在不同修剪手法下,对栽培桃树(Prunuspersica(L.)Batsch)不同母枝上的分枝模式进行了比较研究。从分枝模式来看修剪后的母枝基本由3个不同的区域组成,基部是不萌发的潜伏芽形成的未分枝区域;中部是延迟分枝和多次分枝组成的分枝区域(主要的枝条类型有短枝、长枝和多次枝);顶部是被剪除的部分。我们通过隐式半马尔可夫模型来模拟这一分枝模式,主要是定量描述1次枝和多次枝在母枝上的数量及其分布状况。在上述模型中,未分枝区、延迟分枝区和多次分枝区称为瞬时态,被剪除的部分称为吸收态。模拟的结果与观察的结果进行对比后发现,两者具有很好的一致性。这说明隐式半马尔可夫模型是模拟植物分枝过程的一种有效方法,尽管隐式半马尔可夫链模型只是一个描述性的模型,但仍能对其所描述的生物现象进行解释,在预测修剪手法对母枝分枝模式影响方面比传统的方法具有明显的优势。本研究结果是建立三维虚拟桃树树冠分枝结构的基础。     

The effect of plant growth regulators on biomass formation and lobeline production of Lobelia inflata L. hairy root cultures

We have studied the biomass and alkaloid production of geneticallytransformed hairy root cultures of Lobelia inflata L. Thehairy root clone 8009/h7 transformed with Agrobacteriumrhizogenes strain R 1601 was cultivated on B5 solid media containingdifferent amounts of the growth regulators KIN, IAA or NAA. KIN significantlydecreased growth and lobeline production and strongly inhibited biomassformation at 5 mg/l. IAA and NAA had characteristic morphologicaleffects on growth, in increasing the number of the lateral roots. However theyrestricted linear growth. Addition of IAA or NAA into the culture mediumincreased the biomass formation and lobeline production of hairy roots. It wasfound that the greatest amount of lobeline was obtained at the 0.2mg/l IAA concentration, similar to the effect of NAA.     

Modeling and optimization of hairy root growth in fed‐batch process

This article proposes a feeding strategy based on a kinetic model to enhance hairy roots growth. A new approach for modeling hairy root growth is used, considering that there is no nutrient limitation thanks to an appropriate feeding, and the intracellular pools are supposed to be always saturated. Thus, the model describes the specific growth rate from extracellular concentration of the major nutrients and nutrient uptakes depend on biomass growth. An optimized feeding strategy was determined thanks to the model to maintain the major nutrient levels at their optimum assuming optimal initial concentrations. The optimal feed rate is computed in open loop using kinetic model prediction or in closed loop using conductivity measurements to estimate biomass growth. Datura innoxia was chosen as the model culture system. Shake flask cultures were used to calibrate the model. Finally, cultures in bioreactor were performed to validate the model and the control laws. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

新疆雪莲毛状根的诱导及其植株再生体系的建立

利用发根农杆菌R1601、R1000、LBA9402感染新疆雪莲的叶片、叶柄和根段外植体,诱导产生毛状根。毛状根接种量为2.8 g/L(FW)时,20d生长量可达66.7 g/L,黄酮含量达到干重的10.23%。冠瘿碱的检测和rolB基因的PCR分析表明,Ri质粒中的T_DNA片段已经整合到毛状根细胞的基因组中。预培养时间、外植体类型以及发根农杆菌的菌株属性对毛状根诱导有着重要的影响。其中预培养2 d的新疆雪莲根段外植体,经过R1601感染后,毛状根的诱导率可达100%。诱导产生的毛状根在附加生长素的液体培养基中,有少量愈伤组织产生。由毛状根再生的植株与雪莲外植体再生的植株在形态上无明显区别,但前者的黄酮含量仅为后者的53%。     

Distribution of Cell Adhesion Molecules (CAM) along the Branching Neurite Surface: Model-Inherited Effects of the Branch Diameters and Mode of CAM Transport

In many cases, an increase in the surface density of cell adhesion molecules (CAM) in the distal parts of a growing neurite is favorable for the neurite elongation. This increase is attained by exocytotic insertion of CAM-containing vesicles into the growth cones with subsequent redistribution of CAM along the cell surface due to lateral diffusion and endocytosis. Using a mathematical model describing these processes, we quantitatively describe conditions providing two qualitatively different profiles in a branching neurite: (i) the CAM surface density increases along both daughter branches, which would be in favor of further outgrowth of both branches, i.e., successful branching, or (ii) the CAM surface density increases along one daughter branch and decreases along another branch, which could lead to the retraction of the latter. The geometric factors and mechanisms underlying the intracellular CAM transport to the daughter growth cones were proved to determine the profile of CAM surface density. A similarity in the diameters of daughter branches, their short lengths, a high value of the lateral transfer constant, and partitioning of CAM transport at the branching point proportionally to the surface areas of daughter branches are in favor of an increase in the CAM surface density along both daughter branches. Asymmetric branching can lead to a decrease in the CAM surface density along the thinner or thicker daughter branch, if CAM trafficking was equally partitioned or was proportional to the branch cross-sectional areas, respectively. The proposed model helps to understand possible relationships between the intracellular CAM trafficking, CAM surface distribution, and geometry of branching of the neurites.     

Enhancement of growth and secondary metabolite biosynthesis: Effect of elicitors derived from plants and insects

Plant-derived natural products have been and will continue to be valuable sources. Elicitors have been employed to modify cell metabolism in order to enhance the productivity of useful metabolites in plant cell/tissue cultures. In this study, several elicitors were used to improve the productivity of useful metabolites and to reduce culture time for archiving high concentration inP. ginseng hairy root cultures. The addition of chitosan, chitosan oligosaccharide and alginate oligosaccharide to the culture ofP. ginseng hairy roots caused growth to be inhibited with the increase in elicitor concentration. The usage of the chitosan elicitor andd-glucosamine caused a slight decrease in hairy root growth, whereas total ginseng saponin accumulated slightly with the increase in elicitor concentration. When gel beads were added to the culture medium at the initial period, hairy root growth was enhanced. The maximum growth was 1.35 times higher than that of the control at 1% (w/v). Total ginseng saponin content decreased due to the addition of alginate beads. This would result in consistent diffusion of lower levels of calcium ions during the culture period that promotes biomass growth.     

Biotransformation studies using hairy root cultures - A review

Agrobacterium rhizogenes induced hairy root cultures are entering into a new juncture of functional research in generating pharmaceutical lead compounds by bringing about chemical transformations aided through its inherent enzyme resources. Rational utilization of hairy root cultures as highly effective biotransformation systems has come into existence in the last twenty years involving a wide range of plant systems as well as exogenous substrates and diverse chemical reactions. To date, hairy root cultures are preferred over plant cell/callus and suspension cultures as biocatalyst due to their genetic/biochemical stability, hormone-autotrophy, multi-enzyme biosynthetic potential mimicking that of the parent plants and relatively low-cost cultural requirements. The resultant biotransformed molecules, that are difficult to make by synthetic organic chemistry, can unearth notable practical efficacies by acquiring improved physico-chemical properties, bioavailability, lower toxicity and broader therapeutic properties. The present review summarizes the overall reported advances made in the area of hairy root mediated biotransformation of exogenous substrates with regard to their reaction types, plant systems associated, bacterial strains/molecules involved and final product recovery.     

Product enhancement and recovery from transformed root cultures of Nicotiana glauca

Transformed roots of Nicotiana glauce synthesize the alkaloids nicotine and anabasine at levels reflecting the parent plants. Media composition, strength, and pH were evaluated with respect to biomass yield and productivity. Full-strength Gamborg's B5 medium proved the best for biomass yield while half-strength, or low-salt, medium enhanced alkaloid accumulation. A detailed investigation of media nitrate levels demonstrated how these may be manipulated to promote growth and intracellular or extracellular alkaloid levels. High nitrate concentrations were found to significantly enhance media alkaloid levels at the end of the growth phase. Media pH is also important, although transformed roots will grow in Gamborg's B5 medium between pH 3 and 9, root biomass is favored by an increase in medium alkalinity, while alkaloid release is encouraged by mildly acidic pH.Transformed roots release a proportion of their secondary metabolites into the growth medium. By continually removing root products, any feedback inhibition on enzymatic reactions is reduced, as are the toxic effects resulting from product accumulation. In this article we describe the use of Amberlite resins (XAD-2 and XAD-4) to enhance alkaloid levels (nicotine and anabasine) of hairy root cultures of Nicotiana glauca by a factor of 10 with no adverse effect on root growth. The performance of the Amberlite columns was subsequently investigated with respect to alkaloid adsorption and desorption, including an evaluation of the effects of pH and loading capacity. The resins also adsorb media constituents which are identified and quantified as part of this work. Resulting nutritional stresses are thought to be partly responsible for enhancing secondary metabolism at the expense of biomass yield. However, the net effects of using Amberlite resins as a means of product removal significantly increases the overall product yield and the extent to which products are released into the growth medium.     

Lateral root bridging as a strategy to enhance L-DOPA production in Stizolobium hassjoo hairy root cultures by using a mesh hindrance mist trickling bioreactor

Stizolobium hassjoo hairy roots exhibited a lateral root bridging behavior, enabling not only root dry weight but enhancement of intracellular L-DOPA content. When a single root tip was exerted a proper hindrance, the primary root growth was inhibited while lateral roots were profusely induced. The hindrance-induced lateral roots from individual primary root could bridge together under appropriate inoculation densities, leading to high density hairy root cultures producing secondary metabolites. In the present paper, a novel bioreactor was proposed based on a strategy of lateral root bridging by utilizing mesh as a hindrance, called "mesh hindrance mist trickling bioreactor (MHMTB)". Significant improvements of dry weight and L-DOPA production by using MHMTB were 1.8 and 2.2-folds, respectively, higher than those in the control run without the mesh hindrance within the root bed.     

Development of a kinetic metabolic model: application to Catharanthus roseus hairy root

A kinetic metabolic model describing Catharanthus roseus hairy root growth and nutrition was developed. The metabolic network includes glycolysis, pentose-phosphate pathway, TCA cycle and the catabolic reactions leading to cell building blocks such as amino acids, organic acids, organic phosphates, lipids and structural hexoses. The central primary metabolic network was taken at pseudo-steady state and metabolic flux analysis technique allowed reducing from 31 metabolic fluxes to 20 independent pathways. Hairy root specific growth rate was described as a function of intracellular concentration in cell building blocks. Intracellular transport and accumulation kinetics for major nutrients were included. The model uses intracellular nutrients as well as energy shuttles to describe metabolic regulation. Model calibration was performed using experimental data obtained from batch and medium exchange liquid cultures of C. roseus hairy root using a minimal medium in Petri dish. The model is efficient in estimating the growth rate.     

Infection, propagation, distribution and stability of plant virus in hairy root cultures

Nicotiana benthamiana hairy root cultures were infected with tobacco mosaic virus (TMV) and used for in vitro plant virus propagation. The roots were infected with TMV by addition of virus to the medium at the same time as root inoculation. Viral accumulation in the biomass was 7-11-fold greater when the initial infection was carried out in B5 medium rather than sodium phosphate buffer; virus accumulation also increased with increasing viral inoculum concentration. The amount of TMV accumulated in the biomass was similar when virus was retained in the medium for the duration of the cultures and when the inoculum virus was removed 23h after addition to the roots. In roots with established infections, the concentration of virus remained relatively constant and did not increase with further root growth. The distribution of virus within individual root mats harvested from shake flasks was not uniform; there was also significant variability in viral accumulation between replicate hairy root cultures. The picture that emerges from this work is that in vitro viral accumulation in hairy root cultures depends strongly on the viral inoculum concentration applied and the initial level of primary infection achieved, even though primary infection by external virus occurs mainly within only the first few hours of exposure to the biomass and is followed by substantial secondary infection by viral progeny within the root tissue.     

A developmentally based categorization of branching in Trifolium repens L.: influence of nodal roots

This study describes the successive stages of development of branches from axillary buds in fully rooted plants of Trifolium repens grown in near optimal conditions, and the way in which this developmental pathway differs when nodal root formation is prevented as plants grow out from a rooted base. Cuttings of a single genotype were established in a glasshouse with nodal root systems on the two basal phytomers and grown on so that nodal rooting was either permitted (+R) or prevented (-R). In +R plants, axillary tissues could be assigned to one of four developmental categories: unemerged buds, emerged buds, unbranched lateral branches or secondarily branched lateral branches. In -R plants, branch development was retarded, with the retardation becoming increasingly pronounced as the number of -R phytomers on the primary stolon increased. Retarded elongation of the internodes of lateral shoots on -R plants resulted in the formation of a distinct fifth developmental category: short shoots (defined as branches with two or more leaves but with mean internode length equal to, or less than, 10% of that of the immediately proximal internode on the parent stolon) which had reduced phytomer appearance rates but retained the potential to develop into lateral branches. Transfer of +R plants to -R conditions, and vice versa, after 66 d demonstrated that subsequent branch development was wholly under the control of the youngest nodal root present, regardless of the age and number of root systems proximal to it.     

Hairy root culture for mass-production of high-value secondary metabolites

Plant cell cultivations are being considered as an alternative to agricultural processes for producing valuable phytochemicals. Since many of these products (secondary metabolites) are obtained by direct extraction from plants grown in natural habitat, several factors can alter their yield. The use of plant cell cultures has overcome several inconveniences for the production of these secondary metabolites. Organized cultures, and especially root cultures, can make a significant contribution in the production of secondary metabolites. Most of the research efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone free media. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Hairy root cultures offer promise for high production and productivity of valuable secondary metabolites (used as pharmaceuticals, pigments and flavors) in many plants. The main constraint for commercial exploitation of hairy root cultivations is the development and scaling up of appropriate reactor vessels (bioreactors) that permit the growth of interconnected tissues normally unevenly distributed throughout the vessel. Emphasis has focused on designing appropriate bioreactors suitable to culture the delicate and sensitive plant hairy roots. Recent reactors used for mass production of hairy roots can roughly be divided as liquid-phase, gas-phase, or hybrid reactors. The present review highlights the nature, applications, perspectives and scale up of hairy root cultures for the production of valuable secondary metabolites.     

Comparison of plant-based expression platforms for the heterologous production of geraniol

We compared the ability of different plant-based expression platforms to produce geraniol, a key metabolite in the monoterpenoid branch of the terpenoid indole alkaloid biosynthesis pathway. A geraniol synthase gene isolated from Valeriana officinalis (VoGES) was stably expressed in different tobacco systems. Intact plants were grown in vitro and in the greenhouse and were used to generate cell suspension and hairy root cultures. VoGES was also transiently expressed in N. benthamiana. The highest geraniol content was produced by intact transgenic plants grown in vitro (48 μg/g fresh weight, fw), followed by the transient expression system (27 μg/g fw), transgenic plants under hydroponic conditions in the greenhouse and cell suspension cultures (16 μg/g fw), and finally hairy root cultures (9 μg/g fw). Differences in biomass production and the duration of cultivation resulted in a spectrum of geraniol productivities. Cell suspension cultures achieved a geraniol production rate of 1.8 μg/g fresh biomass per day, whereas transient expression produced 5.9 μg/g fresh biomass per day (if cultivation prior to agroinfiltration is ignored) or 0.5 μg/g fresh biomass per day (if cultivation prior to agroinfiltration is included). The superior productivity, strict process control and simple handling procedures available for transgenic cell suspension cultures suggest that cells are the most promising system for further optimization and ultimately for the scaled-up production of geraniol.