Efficiency of neural network-based combinatorial model predicting optimal culture conditions for maximum biomass yields in hairy root cultures

Key message

ANN-based combinatorial model is proposed and its efficiency is assessed for the prediction of optimal culture conditions to achieve maximum productivity in a bioprocess in terms of high biomass.

Abstract

A neural network approach is utilized in combination with Hidden Markov concept to assess the optimal values of different environmental factors that result in maximum biomass productivity of cultured tissues after definite culture duration. Five hidden Markov models (HMMs) were derived for five test culture conditions, i.e. pH of liquid growth medium, volume of medium per culture vessel, sucrose concentration (%w/v) in growth medium, nitrate concentration (g/l) in the medium and finally the density of initial inoculum (g fresh weight) per culture vessel and their corresponding fresh weight biomass. The artificial neural network (ANN) model was represented as the function of these five Markov models, and the overall simulation of fresh weight biomass was done with this combinatorial ANN–HMM. The empirical results of Rauwolfia serpentina hairy roots were taken as model and compared with simulated results obtained from pure ANN and ANN–HMMs. The stochastic testing and Cronbach’s α-value of pure and combinatorial model revealed more internal consistency and skewed character (0.4635) in histogram of ANN–HMM compared to pure ANN (0.3804). The simulated results for optimal conditions of maximum fresh weight production obtained from ANN–HMM and ANN model closely resemble the experimentally optimized culture conditions based on which highest fresh weight was obtained. However, only 2.99 % deviation from the experimental values could be observed in the values obtained from combinatorial model when compared to the pure ANN model (5.44 %). This comparison showed 45 % better potential of combinatorial model for the prediction of optimal culture conditions for the best growth of hairy root cultures.     

Efficiency of neural networks for prediction of in vitro culture conditions and inoculum properties for optimum productivity

This study represents an ANN based computational scheming of physical, chemical and biological parameters at flask level for mass multiplication of plants through micropropagation using bioreactors of larger volumes. The optimal culture environment at small scale for Glycyrrhiza plant was predicted by using neural network approach in terms of pH and volume of growth medium per culture flask, incubation room temperature and month of inoculation along with inoculum properties in terms of inoculum size, fresh weight and number of explant per flask. This kind of study could be a model system in commercial propagation of various economically important plants in bioreactors using tissue culture technique. In present course of study the ANN was trained by implementing MATLAB neural network. A feed-forward back propagation type network was created for input vector (seven input elements), with single hidden layer (seven nodes) and one output unit in output layer. The ‘tansig’ and ‘purelin’ transfer functions were adapted for hidden and output layers respectively. The four training functions viz. traingda, trainrp, traincgf, traincgb were randomly selected to train four networks which further examined with available dataset. The efficiency of neural networks was concluded by the comparison of results obtained from this study with that of empirical data obtained from the detailed tissue culture experiments and designated as Target set (mean fresh weight biomass per culture flask after 40 days of in vitro culture duration). Efficiency of networks for better training initialization was judged on the basis of comparative analysis of ‘Mean Square Error at zero epoch’ for each network trained in which the least error at initial point was observed with trainrp followed by traincgb and traincgf. A comparative assessment between experimental target data range obtained from wet lab practice and all trained network output range for the efficiency of trained networks for least deviation from target range revealed the output range of network ‘trainrp’ was closest to the empirical target range while least comparison was worked out from network ‘traincgb’ which had output range more than the target decided and ultimately showed meaningless result.     

青藏高原高寒灌丛生态系统草本层生物量分配格局

青藏高原高寒灌丛生态系统生物量分配的研究相对较少,尤其是其草本层。为了探究高寒灌丛生态系统草本层生物量分配特征及其影响因素,分析了青藏高原东北部灌丛生态系统的49个高寒灌丛样地的草本层地上与地下生物量特征及其气候因子之间的关系。结果表明1)草本层地上生物量与地下生物量分别为121.1,342.8 g/m2均大于高寒草地的地上生物量与地下生物量。2)草本层的根冠比为3.6低于高寒草地的根冠比。3)地上生物量与地下生物量之间呈现幂函数的关系y=8.0x0.83(R2=0.48,P0.001)。4)根冠比与年均温度、年均降雨量之间没有显著的相关关系。     

Yield enhancement strategies for the production of picroliv from hairy root culture of Picrorhiza kurroa Royle ex Benth.

Fast-growing hairy root cultures of Picrorhiza kurroa induced by Agrobacterium rhizogenes offers a potential production system for iridoid glycosides. In present study we have investigated the effects of various nutrient medium formulations viz B5, MS, WP and NN, and sucrose concentrations (1–8%) on the biomass and glycoside production of selected clone (14-P) of P. kurroa hairy root. Full strength B5 medium was found to be most suitable for maximum biomass yield on the 40th day of culture (GI = 32.72 ± 0.44) followed by the NN medium of the same strength (GI = 22.9 ± 0.43). Secondary metabolite production was 1.1 and 1.3 times higher in half strength B5 medium respectively in comparison to MS medium. Maximum biomass accumulation along with the maximum picroliv content was achieved with 4% sucrose concentration in basal medium. RT vitamin and Thiamine-HCl effected the growth and secondary metabolite production of hairy roots growing on MS medium but did not show any effect on other media. The pH of the medium played significant role in growth and secondary metabolite production and was found to be highest at pH 6.0 while lowest at pH 3.0 and pH 8.0. To enhance the production of biomass and Picroliv 5 liter working capacity bioreactor was used, 27-fold (324 g FW) higher growth was observed in bioreactor than shake flask and secondary metabolite production was similarly enhanced.     

Efficiency of different Agrobacterium rhizogenes strains on hairy roots induction in Solanum mammosum

This article presents the abilities and efficiencies of five different strains of Agrobacterium rhizogenes (strain ATCC 31798, ATCC 43057, AR12, A4 and A13) to induce hairy roots on Solanum mammosum through genetic transformation. There is significant difference in the transformation efficiency (average number of days of hairy root induction) and transformation frequency for all strains of A. rhizogenes (P < 0.05). Both A. rhizogenes strain AR12 and A13 were able to induce hairy root at 6 days of co-cultivation, which were the fastest among those tested. However, the transformation frequencies of all five strains were below 30 %, with A. rhizogenes strain A4 and A13 showing the highest, which were 21.41 ± 10.60 % and 21.43 ± 8.13 % respectively. Subsequently, the cultures for five different hairy root lines generated by five different strains of bacteria were established. However, different hairy root lines showed different growth index under the same culture condition, with the hairy root lines induced by A. rhizogenes strain ATCC 31798 exhibited largest increase in fresh biomass at 45 days of culture under 16 h light/8 h dark photoperiod in half-strength MS medium. The slowest growing hairy root line, which was previously induced by A. rhizogenes strain A13, when cultured in optimized half-strength MS medium containing 1.5 times the standard amount of ammonium nitrate and potassium nitrate and 5 % (w/v) sucrose, had exhibited improvement in growth index, that is, the fresh biomass was almost double as compared to its initial growth in unmodified half-strength MS medium.     

Controllability of Deterministic Networks with the Identical Degree Sequence

Controlling complex network is an essential problem in network science and engineering. Recent advances indicate that the controllability of complex network is dependent on the network''s topology. Liu and Barabási, et.al speculated that the degree distribution was one of the most important factors affecting controllability for arbitrary complex directed network with random link weights. In this paper, we analysed the effect of degree distribution to the controllability for the deterministic networks with unweighted and undirected. We introduce a class of deterministic networks with identical degree sequence, called (x,y)-flower. We analysed controllability of the two deterministic networks ((1, 3)-flower and (2, 2)-flower) by exact controllability theory in detail and give accurate results of the minimum number of driver nodes for the two networks. In simulation, we compare the controllability of (x,y)-flower networks. Our results show that the family of (x,y)-flower networks have the same degree sequence, but their controllability is totally different. So the degree distribution itself is not sufficient to characterize the controllability of deterministic networks with unweighted and undirected.     

Establishment of Gymnema sylvestre hairy root cultures for the production of gymnemic acid

Gymnema sylvestre is an important medicinal plant that bears bioactive compound namely gymnemic acid. In the present study, G. sylvestre was transformed by Agrobacterium rhizogenes. Seedling explants namely roots, stems, hypocotyls, cotyledonary nodal segments, cotyledons and young leaves were inoculated with A. rhizogenes strain KCTC 2703. Transformed (hairy) roots were induced from cotyledons and leaf explants. Six transgenic clones of hairy roots were established and confirmed by polymerase chain reaction (PCR) and RT-PCR using rolC specific primers. Hairy roots cultured using MS liquid medium supplemented with 3 % sucrose showed highest accumulation of biomass (97.63 g l^?1 FM and 10.92 g l^?1 DM) at 25 days, whereas highest accumulation of gymnemic acid content (11.30 mg g^?1 DM) was observed at 20 days. Nearly 9.4-fold increment of biomass was evident in suspension cultures at 25 days of culture and hairy root biomass produced in suspension cultures possessed 4.7-fold higher gymnemic acid content when compared with the untransformed control roots. MS-based liquid medium was superior for the growth of hairy roots and production of gymnemic acid compared with other culture media evaluated (B5, NN and N6), with MS-based liquid medium supplemented with 3 % sucrose was optimal for secondary metabolite production. The current results showed great potentiality of hairy root cultures for the production of gymnemic acid.     

Parnassius apollo last-instar larvae development prediction by analysis of weather condition as a tool in the species’ conservation

Parnassius apollo (Lepidoptera: Papilionidae) has already disappeared or is under threat of extinction in many of its former habitats. It has been documented that weather conditions – anomalies in particular – contributed to this process. In this study, we combined developmental data obtained previously for the last-instar Apollo larvae (collected in 1996, 1997, and 2003) with corresponding meteorological data to assess the effects of ambient temperature and rainfall episodes on the duration and the completion of the instar. For comparing the temperature effect, we applied the degree-day concept. We found significant positive correlation between the number of rainy days during the instar development (x) and its duration time (y): y = 8.293 + 0.936x (± 2.813) (r = 0.662, P < 10⁻⁷). Logarithmic transformation of the growth curves of the last-instar Apollo larvae revealed that there was no difference in growth among females; however, there was slower growth of males in 2003 in comparison to 1996. Growth (y) of female Apollo larvae as a function of instar duration (x) can by described by one common equation, irrespectively of the year: y = 317.6 + 502.3 lnx (± 263.3) (r = 0.82, P < 10⁻⁴).     

Azadirachtin production by hairy root cultivation of Azadirachta indica in a modified stirred tank reactor

Present investigation involves hairy root cultivation of Azadirachta indica in a modified stirred tank reactor under optimized culture conditions for maximum volumetric productivity of azadirachtin. The selected hairy root line (Az-35) was induced via Agrobacterium rhizogenes LBA 920-mediated transformation of A. indica leaf explants (Coimbatore variety, India). Liquid culture of the hairy roots was developed in a modified Murashige and Skoog medium (MM2). To further enhance the productivity of azadirachtin, selected growth regulators (1.0?mg/l IAA and 0.025?mg/l GA₃), permeabilizing agent (0.5?% v/v DNBP), a biotic elicitor (1?% v/v Curvularia (culture filtrate)) and an indirectly linked biosynthetic precursor (50?mg/l cholesterol) were added in the growth medium on 15th day of the hairy root cultivation period in shake flask. Highest azadirachtin production (113?mg/l) was obtained on 25th day of the growth cycle with a biomass of 21?g/l DW. Further, batch cultivation of hairy roots was carried out in a novel liquid-phase bioreactor configuration (modified stirred tank reactor with polyurethane foam as root support) to investigate the possible scale-up of the established A. indica hairy root culture. A biomass production of 15.2?g/l with azadirachtin accumulation in the hairy roots of 6.4?mg/g (97.28?mg/l) could be achieved after 25?days of the batch cultivation period, which was ~27 and ~14?% less biomass and azadirachtin concentration obtained respectively, in shake flasks. An overall volumetric productivity of 3.89?mg/(l?day) of azadirachtin was obtained in the bioreactor.     

三种森林生物量估测模型的比较分析

森林生物量的定量估算为全球碳储量、碳循环研究提供了重要的参考依据。该研究采用黑龙江长白山地区的TM影像和133块森林资源一类清查样地的数据, 选取地学参数、遥感反演参数等71个自变量分别构建多元逐步回归模型、传统BP (back propagation)神经网络模型和基于高斯误差函数的BP神经网络改进模型(Gaussian error function, Erf-BP), 进而估算该地区的森林生物量, 并进行比较分析。结果表明, 多元逐步回归模型估测的森林生物量预测精度为75%, 均方根误差为26.87 t·m^-2; 传统BP神经网络模型估测森林生物量的预测精度为80.92%, 均方根误差为21.44 t·m^-2; Erf-BP估测森林生物量的预测精度为82.22%, 均方根误差为20.83 t·m^-2。可见, 改进后的Erf-BP能更好地模拟生物量与各个因子之间的关系, 估算精度更高。     

采伐对长白山阔叶红松林生态系统碳密度的影响

采伐对森林生态系统碳密度和固碳能力有重要的影响,且影响的程度因采伐强度和方式不同而有巨大差异。以长白山地区原始阔叶红松林在不同采伐方式、采伐强度干扰后形成的次生林为研究对象,通过对2007至2009年建立的11块1 hm2永久样地中植被层、凋落物层和土壤层碳密度在采伐前后变化特征的分析,研究了采伐强度与恢复时间对阔叶红松林生态系统碳密度的影响。结果表明:在短期内,采伐导致了植被层和土壤表层(0—20cm)碳密度值的减少,其中植被碳密度与采伐强度有显著的线性负相关关系(y=-0.9x+91.17,R2=0.626,P<0.01),而后,随着植被的恢复,生态系统碳密度增加,其中植被、土壤层碳密度呈显著线性正相关关系。根据植被碳密度与恢复时间之间的相关关系,确定以生态系统恢复、木材生产与固碳三者兼顾的合适采伐强度为30%,轮伐期为45a。     

Production and release of pigments by culture of transformed hairy root of red beet

Adventitious roots were induced from red beet (Beta vulgaris L. cv. Detroit dark red) by infecting the plant with a soil bacterium, Agrobacterium rhizogenes. Based on analysis of opines which are uniquely produced in transformed hairy roots, the established clone was proved to be a transformed hairy root. In a shake culture of the beet hairy root clone with a liquid medium, it was found that significant amounts of pigments, mainly betanin and vulgaxanthin-I, were released into the medium by the cessation of culture shaking (temporary limitation of oxygen supply). The hairy root cells were capable of propagation even after the cells were subjected to shaking cessation. Repeated-batch culture of the beet hairy root was performed with the cell growth phases for 9 or 10 d and with pigment leakage phases during shaking cessation for 2 d, and more than 20% of the total intracellular pigments were recovered from the culture broth at a culture time of 35 d. The released pigments were confirmed to be substantially identical to those extracted from the hairy root and original plant cells of red beet.     

Characterization of fetal growth by repeated ultrasound measurements in the wild guinea pig (Cavia aperea)

Fetal growth during pregnancy has previously been studied in the domesticated guinea pig (Cavia aperea f. porcellus) after dissecting pregnant females, but there are no studies describing the fetal growth in their wild progenitor, the wild guinea pig (C aperea). In this study, 50 pregnancies of wild guinea pig sows were investigated using modern ultrasound technique. The two most common fetal growth parameters (biparietal diameter [BPD] and crown-rump-length [CRL]) and uterine position were measured. Data revealed similar fetal growth patterns in the wild guinea pig and domesticated guinea pig in the investigated gestation period, although they differ in reproductive milestones such as gestation length (average duration of pregnancy 68 days), average birth weight, and litter mass. In this study, pregnancy lasted on average 60.2 days with a variance of less than a day (0.96 days). The measured fetal growth parameters are strongly correlated with each (R = 0.91; P < 0.001) other and with gestational age (BPD regression equation y = 0.04x − 0.29; P < 0.001 and CRL regression equation y = 0.17x − 2.21; P < 0.01). Furthermore, fetuses in the most frequent uterine positions did not differ in their growth parameters and were not influenced by the mother ID. Our results imply that ultrasound measurement of a single fetal growth parameter is sufficient to reliably estimate gestational age in the wild guinea pig.     

Microbial biomass on particulate organic matter in seawater of the euphotic zone

Microbial biomass on suspended organic matter in seawater of the euphotic zone of Saanich Inlet was investigated. The viable microorganisms were measured by the glucose-uptake method. Microbial carbon on particulate organic matter in seawater was determined to be, on the average, 9.9 μg of C/liter, and there was a regression relationship as y = 0.0062 x − 1.79 with an unbiased variance V_yx^1/2 = 0.38, where x = particulate organic carbon in seawater (micrograms of C/liter) and y = logarithm of microbial carbon (micrograms of C/liter).     

金铁锁毛状根构型对其生长的影响

毛状根的构型是影响其生长速度和生物量积累的重要因素,为了规模化培养金铁锁毛状根,进一步解决金铁锁资源短缺问题,该研究以金铁锁毛状根为材料,通过改变培养基类型、碳源及碳源浓度,观察和分析了毛状根的生长状态,找出影响毛状根构型的因素。结果表明:最适合金铁锁毛状根生长的培养基为B5+蔗糖30 g·L~(-1),金铁锁毛状根主根长而粗壮,一级、二级侧根生长量大,根系表面积较大,生长效果最佳。经液体悬浮培养验证,测定毛状根的生长量,与在固体培养基培养的毛状根生长状态基本一致。通过该项研究,优化了培养基中营养成分的配比,实现了金铁锁毛状根的快速生长和生物量的积累。     

Induction of hairy roots in Arnica montana L. by Agrobacterium rhizogenes

The induction of hairy roots in Arnica montana L. by Agrobacterium rhizogenes mediated system was established. The frequency of genetic transformation varied from 4.8 to 12% depended on method of infection. The cefotaxime at concentration of 200 mg/l proved to suppress effectively the growth of A. rhizogenes after co-cultivation. Among the three tested nutrient media: Murashige and Skoog (MS), Gamborg’s (B5) and Schenk and Hildebrandt (SH), MS medium was superior for growth and high biomass production of transformed roots compared to other culture media. After culturing for 40 days the fresh weight of clone T4 increased 7.6 fold over the non-transformed roots. The transfer of rol A, rol B and rol C genes into Arnica genome was confirmed by PCR analysis. Established genetic transformation techniques in A. montana efficiently provided and generated a large number of transformed roots — an excellent system for studying gene function and could be used for the production of secondary metabolites synthesized in roots.     

Cognitron: A self-organizing multilayered neural network

A new hypothesis for the organization of synapses between neurons is proposed: “The synapse from neuron x to neuron y is reinforced when x fires provided that no neuron in the vicinity of y is firing stronger than y”. By introducing this hypothesis, a new algorithm with which a multilayered neural network is effectively organized can be deduced. A self-organizing multilayered neural network, which is named “cognitron”, is constructed following this algorithm, and is simulated on a digital computer. Unlike the organization of a usual brain models such as a three-layered perceptron, the self-organization of a cognitron progresses favorably without having a “teacher” which instructs in all particulars how the individual cells respond. After repetitive presentations of several stimulus patterns, the cognitron is self-organized in such a way that the receptive fields of the cells become relatively larger in a deeper layer. Each cell in the final layer integrates the information from whole parts of the first layer and selectively responds to a specific stimulus pattern or a feature.     

Hairy root cultures of Rehmannia glutinosa and production of iridoid and phenylethanoid glycosides

Hairy root lines through the infection of Agrobacterium rhizogenes strain (A4) were established from shoot tips and leaves of Rehmannia glutinosa Libosch. Ten lines of hairy roots were selected on the basis of biomass increase in half-strength Gamborg medium (¹/₂ B5). Transgenic status of the roots was confirmed by polymerase chain reaction using rolB and rolC specific primers. Iridoid glycosides (catalposide, loganin, aucubin and catalpol) and phenylethanoid glycosides (verbascoside and isoverbascoside) identified using HPLC?CESI?CMS, and their contents were compared with untransformed root culture and roots of 1-year-old field-grown plants of R. glutinosa by RP-HPLC. The growth and production of secondary metabolites in ten hairy root lines varied considerably as to the media. Woody plant (WP) medium displayed higher growth in terms of fresh (FW) and dry weights (DW) compared to ¹/₂ B5 medium. High-yielding hairy root lines produced higher amounts of loganin, catalposide, verbascoside and isoverbascoside in comparison to the untransformed root culture and roots of 1-year-old field-grown plants. The highest amounts of catalposide and loganin in transformed roots were 4.45?mg?g^?1 DW (RS-2 hairy root line) and 4.66?mg?g^?1 DW (RS-1 hairy root line), respectively. Aucubin and catalpol were detected in some lines in trace amounts. The highest amounts of verbascoside (16.9?mg?g^?1 DW) and isoverbascoside (3.46?mg?g^?1 DW) were achieved in RS-2 root line. The contents of catalposide, verbascoside and isoverbascoside in high-producing lines were several times higher than in untransformed root culture and roots of R. glutinosa plants grown in soil. Loganin and aucubin could not be detected in roots of field-grown plants. However, the levels of catalpol were much lower in the in vitro roots.