*3.2.4 Biotic stress resistance improvement*

Biotic stresses are one of the basic factor of crop losses in agriculture [47]. The main biotic stresses affecting crops include phytopathogens (viruses, bacteria, fungi), insect and pests (phytophagous insects, acari or nematodes). The strategy for dealing with biotic stresses is either in increasing of self defense mechanisms in plants or in introducing into the genome of constructs aimed against the pathogens [47, 48].

The producing of crops lines resistant to pathogens and pests using traditional breeding methods is based on increasing the own defense mechanisms in plants, but the introduction of constructs targeted pathogens into the genome is carried out by genetic engineering. The most of biotechnological crop lines resistant to biotic stresses created to date are obtained by transgenesis or RNA interference (RNAi) methods [47].

Nowadays, GE approaches are widely used to create new resistant lines [7–10, 44]. Herewith, it should be mentioned these methods makes possible to use both strategies to deal with biotic stresses [44, 47, 48]. Let us to consider some examples of the use several GE technologies to develop lines and varieties of crops resistant to biotic stresses.

*Targeting on plant susceptibility genes.* Plant susceptibility genes are essential to successful infection and development pathogens [49]. Thereat, affect these genes brings about development of plant resistance [7, 44, 49]. As targeting of TALENand CRISPR/Cas9-based *MLO* homologues providing resistance to powdery mildew allows obtaining resistant lines of barley, wheat, Arabidopsis, tomato and pea [7, 44, 49]. Additionally, the CRISPR/Cas9-mediated directed affecting of *DMR6* gene allows creating tomato lines resistant to *Pseudomonas syringae*, *Phytophthora capsici*, and *Xanthomonas* spp [49].

Targeting of disease susceptibility factors was also used for creation of virusresistant crops. As CRISPR/Cas9-based silencing of *eIF4e* factors associated with plant infection by positive sense RNA viruses allows creating virus-resistant plants. Herewith, CRISPR/Cas9-mediated disruption of *eIF4Es* gene function in *Arabidopsis* and cucumber promotes to develope potyvirus resistance [49]. Similarly, CRISPR/Cas9-based mutagenesis of *eIF4G* allows obtaining some rice lines resistant to rice tungro spherical virus (*RTSV*) [49].

To creation of crop lines resistant to biotic stress GE approaches are also used to impact on regulatory elements that can affect the process of pathogen proliferation [49]. So as, TALEN- and CRISPR/Cas9-mediated mutagenesis in effector binding site of promoter region of *OsSweet14* gene permits to develop *Xanthomonas oryzae* pv. *oryzae* resistant rice lines [7, 44, 49]. Also it was shown that Cas9/sgRNA-based targeting the effector binding element of *CsLOB1* gene in citrus provided the generation cancer-resistant citrus varieties [50].

*Targeting on genes of plant hormonal system.* It is known that hormonal signalling plays an important role in immune response of plants [51]. Herewith, salicylatemediated immune response develops in response to biotrophic and hemibiotrophic pathogens infection, and by jasmonate- and ethylene-mediated – against necrotrophic pathogens. Thus, directed effect on the genes of the hormonal response permit to create pathogen-resistant crop lines [49]. Recently this assumption has been confirmed experimentally. Herewith, CRISPR/Cas9-based targeting of the *SlJAZ2* gene in tomato resulted in genesis of resistance to *Pseudomonas syringae* pv. *tomato* (Pto) DC3000 [48].

*Targeting of pathogen genomes.* Another advanced strategy for pathogens defence is directed effect on pathogen genome. Currently, this strategy is mainly used against viral plant diseases [7, 44]. Thus, CRISPR/Cas9 system has been successfully applied to increase resistance to DNA-containing viruses, including tomato yellow leaf curl virus (*TYLCV*), beet root curl virus (*BCTV*), Merremia mosaic virus (*MeMV*), beans yellow dwarfism virus (*BeYDV*), cotton leaf curl virus and beet severe curly top virus (*BSCTV*) [44, 48]. It should be noted that one sgRNA designed to targeting a conserved region can mediate interference against a numerous DNA-containing viruses [8, 44]. Additionally, CRISPR/LshCas13a system capable to interfere of viral RNA turnip line is successfully applied to creation of resistant to RNA-containing turnip yellow mosaic virus (*TuMV*) [44].

#### *3.2.5 Abiotic stress resistance improvement*

Abiotic stresses are the main factors that negatively affect the yield of most crops [52]. In this regard, a creation of resistant to adverse environmental factors crop varieties is the urgent problem. However, the use of traditional breeding methods to develop such varieties is limited by the fact that the traits of resistance to abiotic stress are multigene controlled and have a complex inheritance type [52, 53]. The disadvantages of traditional breeding can be successfully overcome through the use of GE approaches. Herewith, a literature analysis has shown that the GE application

in various cultures allowed to increase their resistance to abiotic stresses [7, 44, 53, 54]. Let us consider detailed examples of the GE application to obtain varieties of crops resistant to abiotic stresses.

*Targeting of structural genes.* Structural genes are one of the most convenient targets to increase plant stress resistance. This class of genes can be divided into tolerance genes (T-genes) and sensitivity genes (S-genes). The T-genes encode enzymes of the antioxidant system, while S genes – negative regulators in plant defense mechanisms [54]. Therefore, "turn off" of S-genes allows to obtain drought-resistant crop varieties. This assumption was confirmed by CRISPR/Cas9 based targeting of *ARGOS8* (a negative regulator of ethylene response) in maize, that permit to obtain drought-resistant lines [53]. Similar results were obtained during directed mutagenesis of the *OST2* gene in *Arabidopsis*, *TaDREB2* gene in wheat (*Triticum aestivum*) and *OsDERFl*, *OsEPSPS* and *OsMSHl* genes in rice (*Oryza sativa* L.). In addition, CRISPR/Cas9-mediated targeting of *OsPDS*, *OsMPK2*, and *OsDEPl, OsAOXla*, *OsAOXlb*, *OsAOXlc* and *OsBEL* genes allows to develop rice lines resistant to a wide range of abiotic stresses [10, 44, 53, 54].

*Targeting of regulatory genes.* Regulatory genes, such as transcription factors, phosphatases and kinases are also involved in the regulation of intracellular signals during abiotic stresses and can be used as target for GE tools to create stress-resistant crop varieties [54]. Presently, CRISPR/Cas9-mediated silencing of *SlMAPK3* regulatory gene in tomato (*Solatium lycopersicum*), *TaERF3* gene in wheat, *OsSAPK2*, *OsPMS3*, *OsRAV2*, and *OsNAC.041* genes in rice permit to create lines of these crops that are resistant to abiotic stresses [44, 53].

Summarize the presented data, it should be noted that GE systems are successfully used to modify a wide range of economically valuable traits in main agricultural crops.


**13. Pyrimido[5,4-e[1,3,4]thiadiazolo[3,2-a]pyrimidines**

An efficient one-pot method for the synthesis of 2-methyl-5,7,9-triphenyl-8-thioxo-8,9-dihydro-5H-pyrimido[5,4-e[1,3,4]thiadiazolo[3,2-a] pyrimidin-6(7H)-one and its derivatives was reported. The present protocol is also extendable to a wide variety of substrates. The advantages of this protocol are the use of easily available catalyst, short reaction time, easy work-up, ease of product isolation, and high yield (**Figure 15**).

**Figure 15.** *Synthesis of Pyrimido[5,4-e[1,3,4]thiadiazolo[3,2-a]pyrimidines.*


**3. Results**

#### *3.1. Quantitative and Qualitative Evaluation of Results*

This section deals with quantitative and qualitative results obtained for the developed models. ANN and WANN trials were conducted depending on the different number of neurons in hidden layers. In contrast, SVM-LF and SVM-RF trials were performed by taking several values of SVM-g, SVM-c, and SVM-e parameters. These were represented in Tables 4–6 as a structure for the model.

#### *3.2. Comparison of Training and Testing Datasets for Scenario 1*

The training results obtained by ANN, Wavelet, and SVM have been shown in Table 4. As depicted in Table 4, for three developed ANN models, namely ANN-1, ANN-2, and ANN-3, ANN-1 has the highest PCC value of 0.832, the lowest RMSE value of 0.993, the highest NSE value of 0.685, and the highest WI value of 0.904.

Similarly, for the developed WANN model, WANN-1 has shown better performance, with a PCC value of 0.773. Furthermore, the WANN model also has the lowest RMSE value of 1.123, the highest NSE value of 0.597, and the highest WI value of 0.860. Furthermore, among developed SVM-RF and SVM-LF models, SVM-RF-3 has shown better performance than other developed models. The SVM-RF-3 model has the highest PCC value of 0.857; it has the lowest RMSE value of 0.956, the highest NSE value of 0.708, and the highest WI value of 0.895 during training datasets. The value of PCC, RMSE, NSE, and WI for MLR techniques was 0.695, 1.274, 0.483, and 0.800. Thus, it can be stated that SVM-RF has modeled the Epan most efficiently of all the machine learning algorithms developed for training.

**Table 4.** Results for ANN, WANN, SVM-RF, SVM-LF, and M.L.R. during the training and testing period for Scenario 1 (60–40: Training–Testing).

Among developed ANN models, ANN-1 has the highest PCC value of 0.589; it has the lowest RMSE value of 1.387 and the highest NSE value of 0.136. Similarly, for the WANN model, WANN-1 has shown better performance with a PCC value of 0.505, the lowest RMSE value of 1.394, the highest NSE value of 0.129, and a WI value of 0.676.

Furthermore, among developed SVM-RF and SVM-LF models, SVM-RF-3 has shown better performance than other developed models. The SVM-RF-3 model has the highest PCC value of 0.607, RMSE value of 1.349, NSE value of 0.183, and the highest WI value of 0.749 training datasets. The values of PCC, RMSE, NSE, and WI for MLR techniques were 0.587, 1.345, 0.188, and 0.725, respectively. The scatter plot and line diagram for the testing data set has been shown in Figure 6. From the line diagram, it can be observed that the obtained results were under-predicted for all models. The scatter plot shows that the highest value of the determination (R2) coefficients was obtained for the SVM-RF model. Thus, it can be suggested that SVM-RF has modeled the Epan most efficiently among all the machine learning algorithms developed for testing.


**8. References**

386 Current Frontiers in Cryopreservation

calluses (Jin et al., 1996; Kaur et al., 1987) and the correlation between pineapple variety and the toxicity of culture filtrates suggests that filtrates could be used to screen in vitro for disease resistance (Borras et al., 2001). Therefore, the cryopreservation of pineapple calluses can provide a means of effective source when in vitro screening of germplasm for fusariose disease would be attempted. Storing calluses in liquid nitrogen could preserve their regeneration capacity and limits the risk of somaclonal variation, which increases with

A simplified freezing protocol mentioned before for sugarcane embryogenic calluses (Martinez-Montero et al., 1998) was used for pineapple calluses of the genotypes "Smooth Cayenne" and "Perolera" (Martinez-Montero et al., 2005). For cryopreservation experiments, 15 to 20 day-old calluses, about 3 to 6 mm in diameter, were employed. Calluses were pretreated with a cryoprotective solution containing 5 to 20 % (v/v) dimethylsulfoxide (DMSO) and 0.5M sucrose for 1h at 0°C. After freezing calluses were transferred directly to recovery medium (MS medium supplemented with dicamba:BAP (2.5:0.5 mg.L-1) and citric acid (0.1 mg.L-1)). The survival, evaluated 45 days after thawing, corresponded to the

As results, the survival of calluses after pretreatment was high and similar for the two genotypes studied (Table 8). It decreased only with 20% of DMSO. After freezing in liquid nitrogen, survival was achieved with 10 and 15% DMSO only and was highest with 15% DMSO (57-67%). Re-growth of successfully cryopreserved calluses was very rapid and they increased in size during the recovery period. With this work, the application of a simplified freezing protocol achieved survival from used pineapple genotypes and this confirm that our previously simplified freezing protocol for sugarcane (Martinez-Montero et al., 1998)

(%) Sucrose (M) -LN +LN -LN +LN

10 818 a 253 b 856 a 304 b 15 7512 a 577 a 806 a 656 a 20 556 b 0 c 604 b 0 c Table 9. Effect of cryoprotective solution (DMSO + sucrose) on the survival rate (%) before (- LN) and after (+LN) application of cryopreservation protocol for calluses of two pineapple genotypes. *Values represent means of 50 samples from three replicate experiments, ± SE. Means within columns followed by the same letter are not significantly different (ANOVA p<0,05 Tukey,).*

For vegetatively propagated species, cryopreservation has a wide applicability both in terms of species coverage, since protocols have been successfully established for root and tubers, fruit trees, ornamentals and plantation crops, both from temperate and tropical origin and in

Survival (%) Smooth Cayenne Red Spanish

8511 a 0 c 876 a 0 c

*= 2 arcsine ((x/100)0,5) for*

percentage of calluses which had increased in size during the recovery period.

culture duration.

can be used wider to others species.

0.5

*Data were transformed for statistical analysis in accordance with x'*

DMSO

5

*percentage of survival.*

**6. Conclusions**

Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

varieties. *Cryo-Letters* 13, 239-52. ISSN: 0143-2044.

pp. 119-161. ISBN:<PHONE_NUMBER>, CABI, Wallingford.

*Cryobiology* 27(3), (June 1990), 247-68. ISSN: 0011-2240

IPGRI; ISBN 92-9043-428-7, Rome, Italy.

*Plant* 40, 427–433. ISSN 1054-5476

Science+Business Media, LLC 2011.

7, Rome, Italy.

1020-3362

0011-2240

Montpellier, pp. 15-17.

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 389

Eksomtramage T.; Paulet F.; Guiderdoni E.; Glaszmann J.C.; Engelmann F. (1992)

Engelmann, F. & Takagi, H. Eds. (2000) *Cryopreservation of tropical plant germplasm - Current*

Engelmann, F. (1997) Importance of desiccation for the cryopreservation of recalcitrant seed

Engelmann, F. (1997) *In vitro* conservation methods. In: *Biotechnology and Plant Genetic*

Engelmann, F. (2000) Importance of cryopreservation for the conservation of plant genetic

Engelmann, F. (2004) Plant cryopreservation: progress and prospects. *In Vitro Cell Dev Biol*

Engelmann, F. (2010) Use of biotechnologies for the conservation of plant biodiversity. *In Vitro Cell Dev Biol Plant* DOI 10.1007/s11627-010-9327-2, ISSN 1054-5476 Engelmann, F. (2011) Cryopreservation of Embryos: An Overview. In: *Plant Embryo Culture:*

Esterbauer, H.; Zollner, H.; Schauer, R.J. (1988) Hydroxyalkenals: Citotoxics products of

Fabre, J. & Dereuddre, J. (1990) Encapsulation-dehydration: A new approach to cryopreservation of *Solanum* shoot tips. *Cryo-Letters* 11, 413 – 426. ISSN: 0143-2044 Fahy, G.M.; Lilley, T.H.; Linsdell, H.; Douglas, M.S.J.; Meryman, H.T. (1990) Cryoprotectant

Fahy, G.M.; McFarlane, D.R.; Angell, C.A.; Meryman, H.T. (1984) Vitrification as an

Feher, A.; Pasternak, T.P.; Dudits, D. (2003) Transition of somatic cells to an embryogenic state. *Plant Cell, Tissue and Organ Culture* 74, 201-228. ISSN: 0167-6857 Feldmann, P.; Sapotille, J.; Gredoire, P.; Rott, P. (1994) Micropropagation of sugarcane. In: *In*

Finkle, B.J. & Ulrich, J.M. (1979) Effect of cryoprotectants in combination on the survival of

Finkle, B.J. & Ulrich, J.M. (1982) Cryoprotectant removal as a factor in the survival of frozen rice and sugarcane cells. *Cryobiology* 19(3), (June 1982) 329-335. ISSN: 0011-2240

frozen sugarcane cells. *Plant Physiology* 63, 598-604. ISSN: 0032-0889

Development of a cryopreservation process for embryogenic calluses of a commercial hybrid of sugarcane *(Saccharum* sp.) and application to different

*research progress and applications*. Tsukuba: JIRCAS; Rome: IPGRI; ISBN 92-9043-428-

and vegetatively propagated species. *Plant Genet. Res. Newsletter* 112, 9-18. ISSN:

*Resources Conservation and Use*. BV Ford-Lloyd, JH Newburry & JA Callow (Eds).

resources. In: *Cryopreservation of tropical plant germplasm - Current research progress and applications*. Engelmann, F.; Takagi, H., Eds. (8-20) Tsukuba: JIRCAS; Rome:

*Methods and Protocols, Methods in Molecular Biology.* Trevor, A.T. & Yeung, E.C. Eds. Vol. 710, DOI 10.1007/978-1-61737-988-8\_13, ISBN 978-1-61737-987-1, Springer

lipid peroxidtion. In: *ISI Atlas of Sciences: Biochemistry* 311-317. ISBN. 0-941708-00-4

toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms.

approach to cryopreservation. *Cryobiology*, 21(4), (August 1984), 407-426. ISSN:

*vitro culture of tropical plants*. C Teisson (ed). CIRAD, ISBN 2-87614-162-0,

Ashmore, S.E. (1997) *Status reports on the development and application of in vitro techniques for*

Ausborn, M.; Schreier, H.; Brezesinski, G.; Fabian, H.; Meyer, H.W.; Nuhn, P. (1994) The

Barraco, G.; Sylvestre, I.; Engelmann, F. (2011) Comparing encapsulation-dehydration and

Benson, E.E., Lynch, P.T., Jones, J. (1992) Variation in free radical damage in rice cell

Borras, O.; Santos, R.; Matos, A.; Cabral, R.; Arzola, M. (2001) A first attempt to use a

Côte, F.X.; Goué, O.; Domergue, R.; Panis, B.; Jenny, C. (2000) In-field behavior of banana

Cyr, D.R. (2000) Cryopreservation: roles in clonal propagation and germplasm conservation

Duan, X.Q.; Hall, J.A.; Nikaido, H.; Quicho, F.A. (2001) Crystal structures of the

Dumet, D.; Block,W.; Worland, R.; Reed, B.M.; Benson, E.E. 2000. Profiling cryopreservation

resistant to fusariose disease. *Plant Breed.* 120: 435-438. ISSN: 0179-9541 Chen, T.H.H.; Kartha, K.K.; Constable, F.; Gusta, L.V. (1984) Freezing characteristics of

*Scientia Horticulturae*, 130(1), (August 2011), 320-324. ISSN: 0304-4238 Benson, E.E. (2008a) Cryopreservation of phytodiversity: A critical appraisal of theory and practice. *Critical Reviews in Plant Sciences* 27(3), 141-219. ISSN: 0735-2689 Benson, E.E. (2008b) Cryopreservation theory. In: *Plant Cryopreservation: A Practical Guide.*

freeze-drying of liposomes. *J Control Release* 30, 105-116. ISSN: 0168-3659 Baier, S.K. & McClements, D.J. (2005) Influence of cosolvent systems on the gelation

Resources Institute, ISBN 92-9043-339-6, Rome, Italy.

43-54. ISSN: 1541-4337

*Genet* 86, 181-188. ISSN: 0040-5752

IPGRI. ISBN 92-9043-428-7, Rome, Italy.

(9 March 2001), 1115-1126. ISSN: 0022-2836

367–378. ISSN: 0143-2044

suspensions. *Cryo-Letters* 21, 19-24. ISSN: 0143-2044

USA.

0935

*the conservation and use of plant genetic resources.* International Plant Genetic

protective effect of free and membrane bound cryoprotectants during freezing and

mechanism of globular protein: Thermodynamic, kinetic, and structural aspects of globular protein gelation. *Comprehensive Reviews in Food Science and Food Safety* 4,

droplet-vitrification for cryopreservation of sugarcane (*Saccharum* spp.) shoot tips.

Reed, B.M. Ed., pp. 15–32. Chapt. 2. Springer, ISBN 978-0-387-72275-7, New York,

suspensions with different embryogenic potentials. *Planta* 188: 296–305. ISSN: 0032-

*Fusarium subglutinans* culture filtrate for the selection of pineapple cultivars

cultured *Catharanthus roseus* (L.) G. Don cells trated with dimethylsulfoxide and sorbitol in relation to cryopreservation. *Plant Physiol.* 75, 720-725. ISSN: 0032-0889 Chowdhury, M.K.U. & Vasil, I.K. (1993) Molecular analysis of plant regenerated from

embryogenic cultures of hybrid sugarcane cultivars (*Saccharum* spp.). *Theor. Appl.*

plants (*Musa* spp.) obtained after regeneration of cryopreserved embryogenic cell

of conifers. In: *Cryopreservation of tropical plant germplasm - Current research progress and applications.* Engelmann, F.; Takagi, H., Eds. (261-268) Tsukuba: JIRCAS; Rome:

maltodextrin/maltose-binding protein complexed with reduced oligosaccharides: flexibility of tertiary structure and ligand binding. *Journal of Molecular Biology* 306:5,

protocols for *Ribes ciliatum* using differential scanning calorimetry. *Cryo-Letters* 21:

Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

9043-428-7, Rome, Italy.

*Dis.* 80: 922-927. ISSN: 0191-2917

7923-2493-5, Dordrecht.

0011-3891

ISSN 1991-8178

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 391

Harding, K.; Johnston, J.; Benson, E. E. (2005) Plant and algal cell cryopreservation: issues in

Jackson, W.; Wagnespack, H.; Richard, C.; Garrison, D.; Lester, W. (1990) CP65-357 Kleentek

Jian, L.C.; Sun, D.L.; Sun, L.H. (1987) Sugarcane callus cryopreservation. In: *Plant cold hardiness.* Li, P.H. Ed., 323-337, Alan R. Liss, Inc., ISBN 0-8451-1084-8, New York. Jiménez, E.; Pérez, J.; Gil, V.; Herrera, J.; García, Y.; Alfonso, E. (1995) Sistemas para la

Riego E, Limonta E, Tellez P, Fuentes J (Eds).. Elfos Scientiae. Cuba 3:11.2 Jin, H.; Hartman, G.L.; Nickell, C.D.; Widholm, J.M. (1996) Phytotoxicity of culture filtrate

Joyce, S.M.; Cassells A.C.; Jain, M.S. (2003) Stressand aberrant phenotypes *in vitro* culture.

Kaczmarczyk, A.; Shvachko, N.; Lupysheva, Y.; Hajirezaei, M.R.; Keller, E.R.J. (2008)

Kartha, K.K. & Engelmann F. (1994) Cryopreservation and germplasm storage. In: *Plant cell*

Kartha, K.K.; Fowke, L.C.; Leung, N.L.; Caswell, K.L.; Hakman I. (1988) Induction of somatic

Kaur, G.; Singh, U.S.; Garg, G.K. (1987) Mode of action of toxin isolated from *Fusarium*

Kinoshita, K.; Li, S.J.; Yamazaki, M. (2001) The mechanism of the stabilization of the

Klimazewska, K.; Ward, C.; Cheliak, W.M. (1992) Cryopreservation and plant regeneration

Krishnamurthy, K.V.; Kishnaraj, R.; Chozavendam, R.; Samuel, C.F. (2000) The programme

Krishnaraj, S. & Vasil, I.K. (1995) Somatic embryogenesis in herbaceous monocots. In: *In*

*oxysporum* f. sp. ciceri. *Indian Phytopathol* 40: 76-84. ISSN 0367-973X

dimethyl sulfoxide. *Eur Biophysics J* 30, 207-220. ISSN 0175-7571

*Plant Cell Tissue and Organ Culture* 74, 103-21. ISSN:0167-6857

shoot tips. *Plant Cell Rep.* 27: 1551-1558. ISSN: 0721-7714

*glauca*). *J. Plant Physiol* 132, 529-39. ISSN: 0176-1617

*mariana*). *J Exp Bot* 43, 73-79. ISSN: 0022-0957

genetic integrity, concepts in 'Cryobionomics' and current European applications. In: *Contributing to a Sustainable Future*. Benett, I.J.; Bunn, E.; Clarke, H.; McComb, J. A., Eds., (112–119) Proc. Australian Branch IAPTC & B, Perth, Western Australia. Ishikawa, M.; Ide H.; Price, W.S.; Arata, Y.; Kitashima, T. (2000) Freezing behaviors in plant

tissues as visualized by NMR microscopy and their regulatory mechanisms. In: *Cryopreservation of tropical plant germplasm - Current research progress and applications.* Engelmann, F.; Takagi, H., Eds. (22-35) Tsukuba: JIRCAS; Rome: IPGRI. ISBN 92-

test in Lousiana. *Journal of the American Society of Sugar Cane Technologist* 21, 10-13.

propagación de caña de azúcar. In: *Avances de la Biotecnología Moderna* Estrada M,

from *Fusarium solani*, the causal agent of Soybean Sudden Death Syndrome. *Plant*

Influence of altering temperature preculture on cryopreservation results for potato

*and tissue culture.* Vasil, I.K. & Thorpe, T.A. Eds. (195–230) Kluwer, ISBN 978-0-

embryos and plantlets from cryopreserved cell cultures of white spruce (*Picea*

hexagonal II (HII) phase in PE membranes in the presence of low concentrations of

from embryogenic cultures of larch (*Larix* x *eurolepis*) and black spruce (*Picea*

of cell death in plants and animals. A comparison. *Curr Sci* 79, 1169-1181. ISSN:

*vitro embryogenesis in plants. Current Plant Science and Biotechnology in Agriculture,*

Finkle, B.J. ; Zavala, M.E.; Ulrich, J.M. (1985) Cryoprotective compounds in the viable

Fleck, R.A.; Day, J.G.; Clarke, K.J.; Benson, E.E. (1999) Elucidation of the metabolic and

Flynn, L. & Anderlini, T. (1990) Disease incedence and yield performance of tissue culture-

Gnanapragasam, S. & Vasil, I.K. (1990) Plant regeneration from a cryopreserved

Gnanapragasam, S. & Vasil, I.K. (1992) Cryopreservation of immature embryos,

Gonzalez-Arnao, M.T.; Engelmann F.; Huet C.; Urra C (1993) Cryopreservation of

Gonzalez-Arnao, M.T.; Engelmann. F.; Urra-Villavicencio. C.; Morenza, M.; Rios, A. (1998a)

Gonzalez-Arnao, M.T.; Juarez, J.; Ortega, C.; Navarro, L.; Duran-Vila, N. (2003)

Gonzalez-Arnao, M.T.; Moreira, T.; Urra, C. (1996) Importance of pregrowth with sucrose

Gonzalez-Arnao, M.T.; Ravelo, M.M.; Urra-Villavicencio, C.; Martinez-Montero, M.M.;

Gonzalez-Arnao, M.T.; Urra, C.; Engelmann, F.; Ortiz, R.; de la Fe, C. (1999)

Grune, T.; Michel, P.; Sitte, N.; Eggert, W.; Albrecht-Nebe, H.; Esterbauer, H.; Siems, W.G.

Guy, C. (1999) Molecular responses of plants to cold shock and cold acclimation. *J Mol*

Harding, K. (1999) Stability assessments of conserved plant germplasm. In: *Plant*

Harding, K. (2004) Genetic integrity of cryopreserved plant cells: A review. *Cryo-Letters 25,*

dehydration technique. *Cryo-Letters* 24 (2), 85-94. ISSN: 0143-2044

and storage duration. *Cryo-Letters* 20, 347-352. ISSN: 0143-2044

dehydration. *Cryo-Letters* 27, 141-148. ISSN: 0143-2044

Ed., 243-267, ISBN 0-8493-6102-8, CRC Press, Boca Raton. USA.

*Xanthophyceae*. *Cryo-Letters* 20, 271–282. ISSN: 0143-2044

*Sugar Cane Technologists* 10, 113. ISSN 1991-8178

*Plant Cell Rep.* 9, 419-423. ISSN: 0721-7714

*Science* 83, 205-215. ISSN: 0168-9452

*Letters* 14, 303-308. ISSN: 0143-2044

*Cryo-Letters* 19,177-182. ISSN: 0143-2044

*Cryo-Letters* 19, 375-382. ISSN: 0143-2044

(November 1996), 357-360. ISSN: 0891-5849

*Microbiol Biotechnol.* 1(2), 231-242. ISSN: 1464-1801

Francis Ltd., ISBN 0-7484-0746-4, London, UK.

*3-22.* ISSN: 0143-2044

freezing of plant tissues. In: *Cryopreservation of plant cells and organs. K*artha, K.K.

structural basis for the cryopreservation recalcitrance of *Vaucheria sessilis*,

generated seed cane over the crop cycle in Lousiana. *Journal of American Society of*

embryogenic cell suspension of a commercial sugarcane hybrid (*Saccharum* sp.)

embryogenic callus and cell suspension cultures of gramineous species. *Plant*

encapsulated apices of sugarcane: Effect of freezing procedure and histology. *Cryo-*

Cryopreservation of citrus apices using the encapsulation-dehydration technique.

Cryopreservation of ovules and somatic embryos of citrus using the encapsulation-

and vitrification for the cryopreservation of sugarcane apices using encapsulation-

Engelmann, F. (1998b) Cryopreservation of pineapple (*Ananas comosus*) apices.

Cryopreservation of encapsulated sugarcane apices: Effect of storage temperature

(1997) Increased levels of 4-hydroxynonenal modified proteins in plasma of children with autoimmune diseases. *Free Radical Biology and Medicine* 23 (3),

*Conservation & Biotechnology*. Chapt. 7. Benson, E.E. Ed., pp. 97–107, Taylor and

Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

method. CryoLetters. 16: 189-196. ISSN: 0143-2044

method. *Cryo-Letters* 16, 189-196. ISSN: 0143-2044

7923-3149-4, Dordrecht, Netherlands.

28, 283-287. ISSN: 0114-0671

161-169. ISSN: 0001-5342

0167-6857

Rome, Italy.

0168-9452

IPGRI, Rome, Italy.

Press Inc., New York.

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 393

Matsumoto T, Sakai A, Takahashi C y Yamada K (1995) Cryopreservation of *in vitro*-grown

Matsumoto, T.; Sakai, A.; Takahashi, C.; Yamada, K. (1995) Cryopreservation of *in vitro*-

McGann, L.E. & Walterson, M.L. (1987) Cryoprotection by dimehyl sulfoxide and dimethyl

Merkle, S.A.; Parrot, W.A.; Flinn, B.S. (1995) Morphogenicaspects of somatic embryo-

Mittler, R.; Vanderauwera, S.; Gollery, M.; Van Breusegem, F. (2004) Reactive oxygen gene

Mix-Wagner, G.; Conner, A.J.; Cross, R.J. (2000) Survival and recovery of asparagus shoot

Murashige, T.; Skoog, F. (1962) A revised medium for rapid growth and bioassays with

Nieves, N.; Martinez-Montero, M.E.; Castillo, R.; Blanco, M.A.; González-Olmedo, J.L. (2001)

Niu, D.K.; Wang, M.G.; Wang, Y.F. (1997) Plant cellular osmotica. *Acta Biotheoretica* 45(2),

Panis, B. & Lambardi, M. (2006) Status of cryopreservation technologies in plants (crops and

Panis, B. & Thinh, N.T. (2001) *Cryopreservation of Musa germplasm*, INIBAP Technical

Panis, B.; Piette, B.; Swennen, R. (2005) Droplet vitrification of apical meristems: a

Parsegian, V.A.; Rand, R.P.; Rau, D.C. (1995) Macromolecules and water: Probing with

Parsegian, V.A.; Rand, R.P.; Rau, D.C. (2000) Osmotic stress, crowding, preferential

*Academy of Sciences USA*, 3987-3992. ISSN-0027-8424

sulfone. *Cryobiology* 24(1), (February 1987), 11-16. ISSN: 0011-2240

network of plants. *Trends Plant Sci.* 9, 490–498. ISSN: 1360-1385

tobacco tissue cultures. *Physiol. Plant.* 15: 473-497. ISSN: 0031-9317

apical meristems of wasabi (Wasabia japonica) by encapsulation-vitrification

grown apical meristems of wasabi (*Wasabia japonica*) by encapsulation-vitrification

genesis. In: *In vitro embryogenesis in plants. Current Plant Science and Biotechnology in Agriculture,* Thorpe, T.A. Ed. Kluwer Academic Publishers; 20, 155-204. ISBN: 0-

tips after cryopreservation using the ¨droplet¨ method. *New Zeal. J. Crop & Hort Sci.,*

Effect of abscisic acid and jasmonic acid on partial desiccation of encapsulated somatic embryos of sugarcane. *Plant Cell Tissue and Organ Culture* 65, 15-21. ISSN:

forest trees). In: *The role of Biotechnology in Exploring and Protecting Agricultural Genetic Resources*, Ruane, J. & Sonnino, A. Eds. (61-78) FAO, ISBN 92-5-105480-0,

Guideline 5. Escalant, J.V. & Sharrock, S. Eds., International Network for the Improvement of Banana and Plantain, ISBN: 2-910810-45-3, Montpellier, France.

cryopreservation protocol applicable to all *Musaceae*. *Plant Sci.* 168, 45–55. ISSN:

osmotic stress. In: *Methods in Enzymology*: *Energetics of Biological Macromolecules.* Johnson, M.L. & Ackers, G.K. Eds. 259, (43-94), ISBN: 978-0-12-182160-9, Academic

hydration, and binding: A comparison of perspectives. *Proceedings of the National*

Thorpe, T.A. Ed. Kluwer Academic Publishers; 20, 417-469. ISBN: 0-7923-3149-4, Dordrecht , Netherlands.

Kristensen, M.M.H.; Find, J.I.; Floto, F.; Moller, J.D.; Norgaard, J.V.N.; Krogstrup, P. (1994)

Leprince, O.; Hendry, G.A.F.; McKersie, B.D. (1993) The mechanisms of desiccation tolerance in developing seeds. *Seed Science Research*, 3: 231-246. ISSN: 0960-2585 Lorenzo, J.C.; Ojeda, E.; Espinosa, A.; Borroto, C. (2001) Field performance of temporary

Lourens, A.G. & Martin, F.A. (1987) Evaluation of *in vitro* propagated sugarcane hybrids for

Lunn, J.E. & MacRae, E. (2003) New complexities in the synthesis of sucrose. *Current Opinion*

Luo, J. & Reed, B.M. (1997) Abscisic acid-responsive protein bovine serum albumin and

Maddox, A.D.; Gonsalves, F.; Shields, R. (1983) Successful preservation of suspension

Martinez-Montero, M.E.; Gonzalez-Arnao, M.T.; Borroto-Nordelo, C.; Puentes-Diaz, C.;

Martínez-Montero, M.E.; Martínez, J.; Castillo, R.; Engelmann, F.; González-Arnao, M.T.;

Martinez-Montero, M.E.; Martinez, J.; Engelmann, F. (2008) Cryopreservation of sugarcane

Martinez-Montero, M.E.; Mora, N.; Quiñones, J.; Gonzalez-Arnao, M.T.; Engelmann, F.;

Martinez-Montero, M.E.; Ojeda, E.; Espinosa, A.; Sánchez, M.; Castillo, R.; Gonzalez-Arnao,

callus-derived sugarcane plants. *Cryo-Letters* 23(1), 21-26. ISSN: 0143-2044

calluses. *Acta Hort. (ISHS)* 666, 127-131. (ISSN 0567-7572

somatic embryos. *Cryo-Letters*, 29(3), 229-242. ISSN: 0143-2044

simplified freezing process. *Cryo-Letters*, 19, 171-176. ISSN: 0143-2044 Martinez-Montero, M.E.; Lorenzo, J.C.; Ojeda, E.; Quiñones, J.; Mora, N.; Sánchez, M.;

cryopreserved by vitrification. *Cryobiology* 34:240-250. ISSN: 0011-2240 Lynch, P.T.; Benson, E.E.; Harding, K. (2007) Climate change: the role of ex situ and cryo-

somaclonal variation. *Crops Science* 27, 793-796. ISSN: 0011-183X

*in Plant Biology* 6, 208-224. ISSN: 1369-5266

*Science Letters* 28, 157-162. ISSN: 0304-4211

23(4), 360-375. ISSN: 1027-2852

244. ISSN: 0143-2044

Dordrecht , Netherlands.

0033-183X

1054-5476

ISSN: 1462-0316

Thorpe, T.A. Ed. Kluwer Academic Publishers; 20, 417-469. ISBN: 0-7923-3149-4,

The origin and development of somatic embryos following cryopreservation of an embryogenic suspension culture of *Picea sitchensis*. *Protoplasma* 182, 65-70. ISSN:

bioreactor-derived sugarcane plants. *In Vitro Cell Dev. Biol.-Plant*. 37, 803-806. ISSN

proline pretreatments improve recovery of *in vitro* currant shoot tips and callus

conservation in the future security of economically important, vegetatively propagated plants. *Journal of Horticultural Science & Biotechnology* 82 (2), 157–160.

cultures of three *Nicotiana* species at the temperature of liquid nitrogen. *Plant*

Engelmann, F. (1998) Cryopreservation of sugarcane embryogenic callus using a

Iglesias, A.; Martínez, J.; Castillo, R. (2006) Methodology for the cryopreservation of calli with embryogenic structures for the culture of sugarcane. *Biotecnología Aplicada*

(2005) Cryopreservation of pineapple (*Ananas comosus* (L.) Merr) apices and

Lorenzo, J.C. (2002a) Effect of cryopreservation on the structural and functional integrity of cell membranes of sugarcane embryogenic calluses. *Cryo-Letters* 23, 237-

M.T.; Engelmann, F.; Lorenzo, J.C. (2002b) Field performance of cryopreserved

Cryopreservation of Tropical Plant Germplasm with Vegetative Propagation –

*tuberosus* L.). *Cryo-Letters* 20, 325-336. ISSN: 0143-2044

CP5243. *Biotecnología Aplicada* 16, 20-23. ISSN: 1027-2852

Agricultural University, The Nederlands. ISBN 90-5485-517-7

9780792308195, Dordrecht, Netherlands.

*Physiol. Biochem.* 37, 145-154. ISSN: 0981-9428

ISSN: 0006-3495

2044

0168-9452

*SugarCane 3*, 11-14.

Review of Sugarcane (*Saccharum spp.*) and Pineapple (*Ananas comusus* (L.) Merrill) Cases 395

Stanley, C.; Krueger, S.; Parsegian, V.A.; Rau, D.C. (2008) Protein structure and hydration

Sun, W.Q. (1999) State and phase transition behaviors of *Quercus rubra* seed axes and

of recalcitrant seeds. *Cryobiology*, 38(4), (June 1999), 372-385. ISSN: 0011-2240 Swan, T.W.; O´Hare, D.; Gill, R.A.; Lynch, P.T. (1999) Influence of preculture conditions on

Swartz, H.J. (1991) Post culture behaviour: genetic and epigenetic effects and related

Tapia, R.; Castillo, R.; Blanco, M.A.; González, J.L.; Sánchez, M.; Rodríguez, Y. (1999)

Taylor, P.; Ko, H.; Adkins, S.; Rathus, C.; Birch, R. (1992) Establishment of embriogenic

spp. hybrids) *Plant Cell Tissue and Organ Culture* 28: 69-78. ISSN: 0167-6857 Tetteroo, F.A.A. (1996) *Desiccation tolerance of somatic embryoids.* PhD Thesis, Wageningen

Thierry, C.; Florin, B.; Petiard, V. (1999) Changes in protein metabolism during the

Thinh, N.T.; Takagi, H.; Yashima, S. (1999) Cryopreservation of in vitro-grown shoot tips of

Thomashow, M.F. (1999) Plant cold acclimation: Freezing tolerance genes and regulatory mechanisms. *Annu. Rev. Plant Physiol. Plant Mol. Biol* 50, 571-599. ISSN:1040-2519 Timasheff, S.N. (1993) The Control of Protein Stability and Association by Weak Interactions

Turner, S.; Senaratna, T.; Touchell, D.; Bunn, E.; Dixon, K.; Tan, B. (2001) Stereochemical

Turner, S.R.; Krauss, S.L.; Bunn, E. Senaratna, T.; Dixon, K.W.; Tan, B.; Touchell, D.H. (2001)

storage and cryopreservation. *Plant Science* 161, 1099-1106. ISSN: 0168-9452 Tyler, N.; Stushnoff, C.; Gusta, L.V. (1988) Freezing of water in dormant vegetative apple buds in relation to cryopreservation. *Plant Physiol* 87, 201-205. ISSN: 0032-0889 Ulrich, J.M.; Finkle, B.J.; Moore, P.H. (1984) Frozen preservation of cultured sugarcane cells.

*and Biomolecular Structure* 22, 67-97, (June 1993), ISSN: 1056-8700

probed by SANS and osmotic stress. *Biophysical Journal* 94, 2777–2789, (April 2008),

cotyledonary tissues: relevance to the desiccation sensitivity and cryopreservation

the post-thaw recovery of suspension cultures of Jerusalem artichoke (*Helianthus*

problems. In*: Micropropagation. Technology and Application.* PC Debergh & RH Zimmermann (eds.). pp. 95-121, Kluwer Academic Publishers, ISBN:

Induction, maduration and encapsulation of sugarcane somatic (*Saccharum* spp) cv

callus and high protoplast yielding suspension cultures of sugarcane (*Saccharum*

acquisition of tolerance to cryopreservation of carrot somatic embryos. *Plant*

banana (*Musa* spp.) by vitrification method. *Cryo-Letters* 20(3), 163-174. ISSN: 0143-

with Water: How Do Solvents Affect These Processes? *Annual Review of Biophysical*

arrangement of hydroxyl groups in sugar and polyalcohol molecules as an important factor in effective cryopreservation. *Plant Science* 160, 489-497. ISSN:

Genetic fidelity and viability of *Anigozanthos viridis* following tissue culture, cold

Paulet, F.; Engelmann, F.; Glaszmann, J.C. (1993) Cryopreservation of apices of *in vitro*

Pena, M. & Stay, M. (1997) Ventajas de la propagacion in vitro. *Canaveral* 3(24), 473–497.

Pérez, J.N.; Jiménez, E.; Gómez, R. (1999) Mejora genética de la caña de azúcar mediante la

Rabe, E. and C. J. Lovatt (1984). De novo arginine biosynthesis in leaves of phosphorus-

Rasmussen, P.H.; Jorgensen, B. & Nielsen, J. (1997) Cryoprotective properties of proline in

Reed, B.M. (2001) Implementing cryogenic storage of clonally propagated plants. *Cryo-*

Reinhoud, P.J.; Van Iren, F.; Kijne, J.W. (2000) Cryopreservation of undifferentiated plant

Ruan, K.; Xu, Ch.; Li, T.; Li, J.; Lange, R.; Balny, C. (2003) The thermodynamic analysis of

Rudolph, A.S. & Crowe, J.H. (1985) Membrane stabilization during freezing: the role of two

Sakai, A. & Engelmann, F. (2007) Vitrification, encapsulation-vitrification and droplet-

Sakai, A.; Kobayashi, S.; Oiyama, I. (1990) Cryopreservation of nucellar cells of navel orange

Sakai, A.; Matsumoto, T.; Hirai, D.; Charoensub, R. (2002) Survival of tropical apices cooled

Schäfer-Menuhr, A.; Schumacher, H.M.; Mix-Wagner, G. (1997) Long-term storage of old

Semenova, M.G.; Antipova, A.S.; Belyakova, L.E. (2002) Food protein interactions in sugar solutions. *Current Opinions in Colloid Interface Science* 7, 438–444. ISSN: 1359-0294 Spiteller, G. (1996) Enzymic lipid peroxidation-α consequence of cell injury? *Free Radical*

vitrification: a review. *Cryo-Letters* 28, 151–172. ISSN: 0143-2044

*Plant Cell Rep.* 12, 525-529. ISSN: 0721-7714

ISSN: 1026-0781

ISSN: 0143-2044

*Letters* 22, 97-104. ISSN: 0143-2044

IPGRI. ISBN 92-9043-428-7, Rome, Italy.

Publishers, ISBN:<PHONE_NUMBER>, New York.

*Res. Newsletter* 111, 19-24. ISSN: 1020-3362

*Biology and Medicine* 21(7), 1003-1009. ISSN: 0891-5849

*Journal of Biochemistry* 270, 1654–1661. ISSN: 0014-2956

0889

2240

ISSN: 0721-7714

Elfos Scientiae, Cuba.

plantlets of sugarcane (*Saccharum* sp. hybrids) using encapsulation-dehydration.

inducción de mutaciones y la selección in vitro. In: *Biodiversidad y Biotecnología de la Caña de Azúcar.* A Arencibia & MT Cornide (eds.). pp. 79-92, ISBN: 959-235-015-9,

deficient Citrus and Poncirus species. *Plant Physiology* 76(3): 747-752. ISSN: 0032-

cod muscle studied by differential scanning calorimetry. *Cryo-Letters* 18, 293-300.

cells. In: *Cryopreservation of tropical plant germplasm - Current research progress and applications.* Engelmann, F.; Takagi, H., Eds. (91-102) Tsukuba: JIRCAS; Rome:

protein stabilization by sucrose and glycerol against pressure-induced unfolding: The typical example of the 33-kDa protein from spinach photosystem II. *European*

natural cryoprotectants, trehalose and proline. *Cryobiology* 22, 367-377. ISSN: 0011-

(*Citrus sinensis* Osb. var brasiliensis Tanaka) by vitrification. *Plant Cell Rep*. 9, 30-33.

to -196°C by vitrification. In: *Plant cold hardiness, gene regulation and genetic engineering.* Li, P.H.; Palva, E.T. Eds., 109-119, Kluwer Academic/Plenum

potato varieties by cryopreservation of shoot-tips in liquid nitrogen. *Plant Genet.*

**19**

*Portugal*

Conceição Santos

**Somatic Embryogenesis and Cryopreservation**

It is widely accepted that propagation and conservation strategies are crucial in any forest breeding program and that plant biotechnology has powerful tools to propagate and preserve selected genotypes (Jain, 1999; Park et al., 1988; Park, 2002). In particular, in vitro techniques have demonstrated to be essential in a large number of agricultural and forest breeding programs, allowing large scale clonal propagation of elite genotypes (Pinto et al., 2008) or of endangered forest species (Brito et al., 2009). In vitro techniques are also essential in breeding programs involving genetic improvement (e.g. using techniques such as somatic hybridization or genetic transformation) or involving long term germplasm conservation by

Somatic embryogenesis offers advantages in improving forest species over other in vitro propagation methods (Park, 2002), namely: a) somatic embryos simultaneously possess both the shoot and root meristems; so a distinct rooting stage, which usually involves stressing procedures, is not necessary; b) during somatic embryogenesis, embryos/clusters are often formed faster and potentially at extremely high numbers per explant; c) somatic embryogenesis is amenable to automation, which means it may become cheaper than other clonal propagation techniques (Pinto et al., 2002; 2008); d) finally, a robust and efficient protocol of somatic embryogenesis will allow that embryogenic clonal lines can be preserved for long periods (e.g., in liquid N2) while corresponding plants are transferred to

The combination of somatic embryogenesis and conservation strategies in breeding programs allows that one may develop high-value forest clonal varieties merely by recovering from N2 those genotypes/clones that showed best characteristics in the field. These selected genotypes can later be used for advanced breeding programs and

From the exposed advantages, it is generally accepted among breeders that breeding programs using in vitro clonal strategy require as a pre-requisite that a cloning technique (preferably somatic embryogenesis) is well established. Then it also requires the optimization of long-term genetic testing methodologies of clonal lines and long term

**1. Introduction**

**1.1 General concepts**

cryopreservation (Benson, 2008; Fernandes et al., 2008).

field conditions and are monitored for their characteristics.

commercial forestry (Park, 2002).

**in Forest Species: The Cork Oak Case Study**

*CESAM & Department of Biology, University of Aveiro, Aveiro*


**2. Theoretical models for surgical motor skill acquisition**

Advancement in surgical education research sheds light on the understanding of competency and assessment, in the sense that competency cannot be assumed when trainees can perform parts of a task or individual surgical skills, as many complex tasks require the integration of many skills. Literature showed that surgical trainees at both undergraduate [12] and postgraduate levels [13] do not feel competent or ready to operate independently at the end of their training. Competence is not equal to excellence. Due to the lack of time and opportunities to practice, the learning process focuses on competence rather than excellence [14]. For surgical motor skill acquisition, the following four theoretical models are commonly used (**Table 1**) - Fitts and Posner's 3-Stage Model of Motor Skills Acquisition, Bandura's Theory of Social Learning, Ericsson's Deliberate Practice Model, and Motor Simulation Theory of Jeannerod.

Mental practice (MP) is "the cognitive rehearsal of a task in the absence of overt physical movement" [19]. It has successfully improved psychomotor performance to enhance skill and performance in sports and music [20, 21]. Mental imagery (MI) has been shown to activate similar neural processes to those used in the actual performance of a given skill [22, 23]. MP has been used lately as an alternative strategy in surgical training. The current literature shows the successful use of MP in surgical training or enhancing surgical performance, but lacks methodological details for the development of educational resources incorporating MP.

#### **Table 1.**

*Summary of surgical training theories.*

## *Redefining Surgical Skill Acquisition DOI: http://dx.doi.org/10.5772/intechopen.99408*

In summary, the current body of knowledge on psychomotor skill acquisition to an expert level [24, 25] requires:

The current model of surgical training programmes for motor skill acquisition focuses on the direct supervision of the expert in the operating room, providing direct observation, guidance, and feedback, ensuring compliance to the preoperative plan. Graduated responsibility and progression in surgical performance are allowed based on the level of surgical performance and adherence to patient safety [26]. The emphasis has been the development of competencies which is a minimum level of skill to be demonstrated, rather than mastery associated with a higher level of proficiency. Competency requires much less training than mastery and is facilitated by the very structured and focused training. Hence, to overcome the reduced opportunity to learn and practice in the operating room, low fidelity bench models, basic surgical skills, surgical laboratory practice, and higher fidelity human cadaver models to live animal model practice have been utilised [27]. From simulation to gaming and robotics, technology has also become the enabler in surgical training [28, 29]. These strategies have implications in cost-effectiveness and universal accessibility, as they require resources - both physical and trainee/trainer time, as they are synchronous platforms for learning [30]. However, the current model is singularly deficient in that there is no universally accessible opportunity to practice the surgical procedure outside of the operating theatre and without access to physical simulators.

Based on the review of these theoretical frameworks, we propose a model for the expert acquisition of motor skills in **Figure 1**. This model incorporates the theoretical framework of Fitts and Posner, Bandura, Ericsson, and Jeannerod, and operationalises the steps described in the theories of skill acquisition and


**2. Shell Evolution Caused by the SDPF-MU Interaction**

#### *2.1. Monopole Matrix Elements*

The SDPF-MU interaction was constructed in Ref. [18] to describe the structure of neutron-rich nuclei around *N* = 28 whose Fermi surface is located in the *sd* shell for protons and the *p f* shell for neutrons. Hence, the proton–neutron cross-shell interaction, i.e., the part of the interaction that is relevant to both the sd shell and the pf shell is responsible for the shell evolution occurring in this region.

The cross-shell part of the SDPF-MU interaction is provided by a minor modification of the *V*MU interaction [13]. The *V*MU interaction was proposed to give a universal behavior of shell evolution over the nuclear chart, consisting of a Gaussian central force and a *π* + *ρ* meson exchange tensor force. In the SDPF-MU interaction, the following refinements to the original *V*MU interaction are introduced:

The central force of the shell-model effective interaction is subject to complicated renormalization and many-body effects. The basic strategy of *V*MU is to determine the central force so that its monopole matrix elements are close to those of a reliable effective interaction. Here, the monopole matrix element between the orbitals *j*1 and *j*2 is defined by

$$V\_{\mathcal{T}}^{\mathfrak{m}}(j\_1, j\_2) = \frac{\sum\_{\mathcal{I}} (2\mathcal{I} + 1) \left< j\_1 j\_2; \mathcal{I} \middle| \begin{array}{c} V \middle| j\_1 j\_2; \mathcal{I} \middle| \mathcal{T} \end{array} \right>}{\sum\_{\mathcal{I}} (2\mathcal{I} + 1)},\tag{1}$$

where *J* runs over all the possible angular-momentum coupling that the Pauli principle allows, and *T* is the isospin coupling. In constructing the original *V*MU interaction, the GXPF1A interaction [19] was used as a reference, and a reasonable but not perfect agreement was achieved. Namely, while most of the monopole matrix elements agree within 0.2 MeV, a few matrix elements differ by 0.5 MeV or more; see Figure 1 of Ref. [13]. To obtain a better result, the central force of the SDPF-MU interaction has the form of

$$N\_{\mathbb{C}}(1,2) = D(R) \sum\_{\mathbb{S},T} f\_{\mathbb{S},T} P\_{\mathbb{S},T} \exp\left(-\left(r/\mu\right)^2\right),\tag{2}$$

where *S* and *T* denote the spin and isospin coupling, respectively, and *PS*,*T* is the projection operator onto a given (*S*, *T*). The*r* and *R* are the relative and center-of-mass coordinates, respectively: *r* = *r*1 − *r*2 and *R* = (*r*1 + *r*2)/2. The *D*(*R*) is the density dependent part that was newly introduced in the refined *V*MU, and its form was taken from the FPD6 interaction [20] as

$$D(R) = 1 + A\_d \{ 1 + \exp(\left( R - R\_0 \right) / a) \}^{-1} \tag{3}$$

with *R*0 = 1.2*A*−1/3 MeV and *a* = 0.6 fm. The interaction, thus, has six free parameters, *fS*,*T*, *μ*, and *Ad*. They were chosen to be *f*0,0 = −140 MeV, *f*1,0 = 0, *f*0,1 = 0.6 *f*0,0, *f*1,1 = −0.6 *f*0,0, *μ* = 1.2 fm, and *Ad* = −0.4. The resulting agreement with the monopole matrix elements of the central force of GXPF1B is quite good, as illustrated in Figure 1 of Ref. [21].

The two-body spin–orbit force in the SDPF-MU interaction was taken from that of the M3Y (Michigan 3-range Yukawa) interaction [22]. The two-body spin-orbit force plays a minor role on shell evolution compared with the central and tensor forces, as far as a restricted region of the nuclear chart is considered: see Table 1 and discussion below. However, some specific evolutions of shell gaps are dominated by the two-body spin–orbit force, thus, included here for completeness.

The overall strength of the SDPF-MU interaction is scaled by a factor *A*−0.3 in the same way as the USD (Universal *sd*) [23] and GXPF1 [24] interactions.

**Table 1.** Proton–neutron monopole matrix elements between the *sd* and *p f* orbitals obtained by the SDPF-MU interaction for the atomic mass number *A* = 42. The second to the fifth columns list the central (C), tensor (T), spin–orbit (LS), and the total values (in MeV), respectively. The sixth to ninth columns indicate the hierarchy of theC+T monopole matrix elements. The texts in red (blue) are to highlight the correspondence between the most attractive matrix elements of the central (tensor) force and Δ*n* = 0 (spin direction). See text for details.

Table 1 presents the proton–neutron cross-shell monopole matrix elements, calculated with the SDPF-MU interaction, for central, tensor, and spin–orbit forces. The proton– neutron monopole matrix element for a pair with (*n*1, *l*1) = (*n*2, *l*2) is given by

$$V\_{pn}^{\rm m}(j\_1, j\_2) = \frac{1}{2} \{ V\_{T=0}^{\rm m}(j\_1, j\_2) + V\_{T=1}^{\rm m}(j\_1, j\_2) \}. \tag{4}$$

The second column of Table 1 indicates that the strengths of the central matrix elements can be grouped into two categories: one has ∼ −1.1 MeV, and the other has much weaker strengths. As explained [1,13], this difference occurs because two orbitals with the difference of the number of nodes, Δ*n* = 0, have a large spatial overlap, thus, gaining much attraction through short-range forces. Comparing the second and the sixth columns, one finds a good correspondence between Δ*n* and the strength of the central matrix elements.

The monopole matrix elements of the tensor force are characterized by the relative spin direction between the two orbitals considered, as pointed out in Ref. [12]. When the spins of two orbitals (with *l* > 0) are parallel, i.e., *j*> - *j* > or *j*< - *j* < (*j*> and *j*< stand for *j* = *l* + 1/2 and *j* = *l* − 1/2, respectively), the tensor monopole matrix element is positive and otherwise negative. The third and the seventh columns of Table 1 exactly point to this property. This fact is accepted now, [1,12], and quantitative aspects of the tensor monopole matrix elements are as follows.

From the point 2, one concludes that the tensor force plays a role as important as the central force in shell evolution.

On the basis of the above arguments, let us label the orbital pairs to simply estimate the strengths of the monopole matrix elements due to the central and tensor forces without numerical calculations.

These labels are listed in the eighth column of Table 1. The actual sum of the central and tensor monopole matrix elements shown in the ninth column of Table 1 rather well follows this ordering, except for a few cases with Δ*n* = 1 in which the tensor force is less dominant.

Next, the two-body spin–orbit force is examined whose monopole matrix elements are presented in the fourth column of Table 1. The strengths of the elements are usually rather weak (see details in Supplemental Material in Ref. [1]), and the typical order of the monopole matrix elements is ∼ 20*A*−5/3 MeV ≈ 0.04 MeV at *A* = 42. The signs of the elements are determined so that the inner nucleon (usually with lower orbital angular momentum, *l*) produces the normal spin–orbit splitting to the outer orbitals. Namely, when the inner and the outer orbitals are labeled *i* and *j*, respectively, their monopole matrix elements satisfy *V*m *pn*(*i*, *j*) < 0 for *j* = *l* + 1/2 and *V*m *pn*(*i*, *j*) > 0 for *j* = *l* − 1/2.

More specifically, when monopole matrix elements between the *sd* and 1 *f* orbitals are considered, the *sd* orbitals are located closer to the center and thus can be regarded as the inner orbitals. Hence, this rule causes negative and positive monopole matrix elements for the 1 *f*7/2 and 1 *f*5/2 orbitals, respectively. One can also find that the monopole matrix elements between the 2*s* and 2*p* orbitals are much larger than the others. This is because this pair, having a relative orbital angular momentum *L*rel = 1 alone, gains much energy due to the short-range nature of the two-body spin–orbit force.


**4. Discussion**

182 Studies on Environmental and Applied Geomorphology

(Heitmeyer and Westphal, 2007; Heitmeyer, 2008b). Each HGM evaluation is much more than simply combining GIS layers. An HGM evaluation reviews the physical setting, climate and hydrology, and the distribution and characteristics of presettlement habitats to establish a potential natural landscape. The HGM then reviews changes due to development and succession to make restoration and management decisions based on the likelihood of natural communities to recover from disturbance and in light of future disturbances. Potential vegetation maps assembled from hydrologic, geomorphic, and soils data are

simply tools to visualize and quantify landscape response to management actions.

Fig. 10. A portion of a HGM map for the St. Louis region.

The near term intent is to complete an initial set of potential natural vegetation maps to help inform forest and land management plans for the entire UMRS (National Great Rivers Research and Education Center, 2010). The hydrology and geomorphology base layers described above were an important precursor to the rapid completion of the project. When the initial potential vegetation maps are complete, or as project needs dictate, potential vegetation maps for alternative floodplain management plans can be modeled to estimate There are many environmental and economic management needs that can be addressed with ecosystem modeling. Hydraulic models have become so precise that their results are routinely used for engineering design to simulate alternative design features (Silberstein, 2006). We believe the HGM approach for potential vegetation community assessment can achieve a similar standard for ecosystem restoration alternative analysis. The methods are not precise to species levels, nor very small spatial scale, at this stage of development but they do match well with the scale of most wildlife refuges and management areas that are the focus of most natural resource management and restoration activity. They also scale nicely for landscape ecology metrics and regional ecosystem management (USACE, 2011). HGM models have been developed for many floodplain systems (Klimas et al. 2009; U.S. Army Corps of Engineers, 2010), and they gain wide agency acceptance when developed collaboratively between managers and scientists.

These HGM methods for the UMRS are still quite simple in their statistical capacity and ability to model land cover occurrence. Future work will explore more rigorous landscape metrics that examine adjacency of land cover classes and associations with physical landscape features. The fundamental premise of the Hydrogeomorphic Method (HGM) is that vegetative communities segregate according to a single, or some combination of landscape features (e.g. geomorphology, hydrology, soil type). Indeed floodplain topography influences the frequency and duration flooding, which both directly influences plants via control over the length of oxic and anoxic phases, and indirectly influences plant communities by changing the physical properties of the soil (e.g. texture, pH, fertility). However, few studies have quantified the degree to which different plant communities segregate along key environmental gradients. By quantifying nonrandom associations among hydrology, soils and vegetation, land managers can increase their odds of successfully matching species and community types to suitable site conditions, thereby improving the odds of successful restoration.

To test the hypothesis that various plant communities segregate according to a given landscape feature or some combination of landscape features, an electivity index can be used (Jacobs, 1974; Jenkins, 1979; Pastor and Broschart, 1990). An electivity index calculates the juxtaposition of one cover type from one GIS data layer with some other landscape feature in a separate data layer.

These methods allow one to empirically test the hypothesis that a particular vegetation cover class 'elects' for a given landscape feature. If a particular cover class indeed elects for a given landscape feature, then it provides land managers with a prescription of broad-scale conditions that may be required for successful establishment of a given plant community under a given set of environmental conditions (Dr. Nathan DeJager, U.S. Geological Survey, Upper Midwest Environmental Sciences Center, La Crosse, Wisconsin, contributed text).

Hydro-Geomorphic Classification

Department of Forest Ecology and Management.

Madison, Paul West – The Nature Conservancy.

Louis, St. Louis, MO, and St. Paul, St Paul, MN.

Great Plains, USA. *Geoarchaeology* 17:141-154.

River Valley. *Geomorphology* 101:362-377.

**6. References**

189:681–684.

*BioScience* 54:413-427.

Illinois. 39pp. + maps.

and Potential Vegetation Mapping for Upper Mississippi River Bottomland Restoration 185

investigations, flow frequency analyses, and flood risk assessments that provided data that were adapted for this study. The Upper Mississippi River Environmental Management Program administered by the U.S. Army Corps of Engineers and U.S. Geological Survey supported land cover mapping, future landscape analysis recommendations, and technical assistance. The U.S. Army Corps of Engineers, Navigation and Ecosystem Sustainability Program provided support to Dr. Theiling and Dr. Heitmeyer. Finally, The Nature Conservancy Great Rivers Partnership graciously supported development of the systemwide presettlement land cover data through a grant to the University of Wisconsin

Many individuals supported data development and analysis: John Burant – U.S. Army Corps of Engineers, Nathan DeJager – U.S. Geological Survey, Tim Fox – U.S. Geological Survey, Edwin Hajic – Illinois State Museum, Ken Lubinski – U.S. Geological Survey, David Mladenoff – University of Wisconsin - Madison, J.C. Nelson – U.S. Geological Survey, John Nelson – Illinois Nature Preserves Commission, Jim Ross – U.S. Army Corps of Engineers, Michael Reuter – The Nature Conservancy, Ted Sickley – University of Wisconsin -

Barko, J., Johnson, B., & Theiling, C. 2006. *Environmental science panel report: implementing*

Belt, C.B. 1975. The 1973 flood and man's constriction of the Mississippi River. *Science*

Benda, L., Poff, N.L., Miller, D., Dunne, T., Reeves, G., Press, G., & Pollock, M. 2004. The

Bettis, E.A. & Mandel, R.D. 2002. The effects of temporal and spatial patterns of Holocene

Bettis, E.A., Anderson, J.D., & Oliver, J.S. 1996. *Land Sediment Assemblage (LSA) Units in the*

Bettis, E.A., Benn, D.W., & Hajic, E.R. 2008. Landscape evolution, alluvial architecture,

Brinson, M. M. 1993. *A hydrogeomorphic classification for wetlands*. Army Engineer Waterways Experiment Station, Vicksburg, MS, USA. Technical Report WRP-DE-4. Carlander, H.B. 1954. *A history of fish and fishing in the Upper Mississippi*. Special Publication

Chen, Y.H. & Simmons, D.B. 1986. Hydrology, hydraulics, and geomorphology of the Upper

Christensen, N.L., Bartuska, A.M., Brown, J.H., Carpenter, S., D'Antonio, C., Francis, R.,

Service, Rock Island Field Office, Rock Island, Illinois. 96 pp.

Mississippi River System. *Hydrobiologia* 136:5-20.

*adaptive management.* U.S. Army Engineer Districts: Rock Island, Rock Island, IL; St.

network dynamics hypothesis: how channel networks structure riverine habitats.

erosion and alluviation on the archaeological record of the Central and Eastern

*Upper Mississippi River Valley, United States Army Corps of Engineers, Rock Island District, Volume 1*. Quaternary Studies Program, Illinois State Museum, Springfield,

environmental history, and the archeological record for the Upper Mississippi

of the Upper Mississippi River Conservation Committee, U.S. Fish and Wildlife

Franklin, J.F., MacMahon, J.A., Noss, R.F., Parsons, D.J., Peterson, C.H., Turner,

A multiple reference condition analysis has been proposed for UMRS ecosystem restoration planning (Nestler and Theiling, 2010). Sufficient data exist to evaluate hydrologic and geomorphic ecosystem drivers and land cover in presettlement, several historic snapshots, and contemporary conditions for nearly the entire 2.8 million acres. The virtual reference condition (i.e., simulated hydrology, potential vegetation, or geomorphic features), or plausible alternative future condition, is an important tool to estimate future without project condition and the response to alternative restoration plans (Figure 11; USACE, 2000). It is possible to simulate alternative floodplain management scenarios and extrapolate benefits as simple acreage estimates (Figure 11, bottom), potential vegetation (Heitmeyer, 2008; 2010), or any range of habitat suitability (USFWS, 1980) or ecosystem services metrics that can be attributed to potential land cover estimates.

Fig. 11. Examples for UMRS benefits that may be attained by alternative floodplain management plans. LTRM\_WTR = low flow surface water, WS\_2YR = 50 percent exceedence/2-year flood, Levee = leveed area, WS\_Pool = potential inundation under Pumps Off scenario.

#### **5. Acknowledgements**

An Army Corps of Engineers, Long Term Training Program grant from the Rock Island District and Headquarters supported much of Dr. Theiling's effort. The U.S. Army Corps of Engineers, St. Paul, Rock Island, and St. Louis Districts collaborated on archaeological investigations, flow frequency analyses, and flood risk assessments that provided data that were adapted for this study. The Upper Mississippi River Environmental Management Program administered by the U.S. Army Corps of Engineers and U.S. Geological Survey supported land cover mapping, future landscape analysis recommendations, and technical assistance. The U.S. Army Corps of Engineers, Navigation and Ecosystem Sustainability Program provided support to Dr. Theiling and Dr. Heitmeyer. Finally, The Nature Conservancy Great Rivers Partnership graciously supported development of the systemwide presettlement land cover data through a grant to the University of Wisconsin Department of Forest Ecology and Management.

Many individuals supported data development and analysis: John Burant – U.S. Army Corps of Engineers, Nathan DeJager – U.S. Geological Survey, Tim Fox – U.S. Geological Survey, Edwin Hajic – Illinois State Museum, Ken Lubinski – U.S. Geological Survey, David Mladenoff – University of Wisconsin - Madison, J.C. Nelson – U.S. Geological Survey, John Nelson – Illinois Nature Preserves Commission, Jim Ross – U.S. Army Corps of Engineers, Michael Reuter – The Nature Conservancy, Ted Sickley – University of Wisconsin - Madison, Paul West – The Nature Conservancy.


**Abstract**

Microencapsulation has been the most frequently used technique for several different disciplines such as cell-based therapies and/or transplantation. Technology is based on the idea of combining and coating a material or isolating from an external source. Microencapsulation may be performed with different materials and, among natural biocompatible materials, alginate-based microencapsulation technique is the most appropriate material for microencapsulation. The structural components of alginate materials are the derivatives of alginic acid, which is found in brown algae as an intercellular gel matrix. This alginate is preferred for clinical applications due to its safety in human studies. Therefore, the choice and the combined system need to be carefully optimized to achieve biocompatible application through cell microencapsulation especially for long term. Specifications of alginate such as primary source, isolation process, viscosity, and purity contribute to improve its biocompatibility. Clinically, cell microencapsulation is the major contribution to the field of transplantation by its technique and additionally provides local immune isolation. This chapter discusses the potential benefits of clinically suitable alginates and their applications. This promising technology may highlight its considerable potential for patients that require transplantation and/or replacement therapy in the future.

**Keywords:** microencaspulation, alginate, cell therapy, drug delivery, transplantation


4.3 Forward compatibility

Forward compatibility was a desired important new feature of cereal\_fwd, yet implementing it proved to be a demanding task. Some initial assumptions and design decisions were challenged during implementation and are described below.


**6. Pharmacokinetics and dose considerations for ivermectin as cancer therapy**

Due to its relatively long history of extensive use, the pharmacokinetics of ivermectin has been well studied. The oral route is the only approved for ivermectin administration in humans although it can be given subcutaneously and the intravenous route of administration has also been investigated. Ivermectin is a fat-soluble compound and reaches a peak concentration 4-5 hours after oral administration, and it has a half-life of approximately 19 hours. After administration, it is subsequently extensively metabolized in human liver microsomes by cytochrome P-4503A4, converting the drug to at least ten metabolites, most of them hydroxylated and demethylated derivatives. Its excretion is mainly by the fecal route, and only 1% is excreted in the urine [49]. In healthy individuals and patients infected with onchocerciasis treated with a dose of 0.150 mg /Kg of Ivermectin, significant variability in pharmacokinetic parameters such as absorption, distribution, metabolism, and excretion is not observed [49].

The therapeutic dose for ivermectin as an anti-parasitic compound for human use is is between 0.1 and 0.4 mg/ Kg [4–7], resulting in an AUC of 1,444 μg/h/ mL. This drug exposure, which translates to a plasma concentration of 1.65 μM, is however less than concentrations of 5 μM or greater that has been found necessary to inhibit tumor cells *in vitro* In a phase I pharmacokinetic study done in healthy volunteers, it was demonstrated that doses up to 2 mg/Kg which leads to an AUC of 4,547 μg/h/mL can translate into a plasma concentration of 5 μM [50], thus the recommended dose for cancer therapy should likely be 2 mg/kg or higher.


*Edited by Yu-Chih Shen*

Schizophrenia treatment has many facets. This book begins with the glutamatergic and GABAergic hypofunctioning contribute to the schizophrenic symptoms and their current targeted therapeutics. The genetic, epigenetic, and immune etiologies of schizophrenia and their potential targeted therapeutics as approached in this book are interesting. Understanding cognitive biases and delusional circuits in schizophrenia is important; several behavioral cognitive therapies working on the reduction and avoidance of these cognitive biases are demonstrating their effectiveness. Advances in schizophrenia treatment followed, including transcranial magnetic stimulation and special sport program, are presented at the book's end.

Photo by IG\_Royal / iStock

Schizophrenia Treatment - The New Facets

Schizophrenia Treatment

The New Facets

*Edited by Yu-Chih Shen*


**4.2. Main results concerning the corrosion resistance of MMC coatings**

The comparison between the anticorrosive performance of metallic and MMC coatings was studied for several metallic matrix/ceramic particles (or nanoparticles) systems using different corrosive media [26,37,40,45,47]. Most of the results affirm that the presence of the particles increases the corrosion resistance of the MMC coating/substrate system, although it is impor‐ tant to mention that the optimum values of particle content in the metallic matrix to produce a coating with good anticorrosive performance directly depend on the metal/particle system produced and on the deposition parameters used. Moreover, the corrosion mechanism of the metallic coatings containing these particles is not completely elucidated; the process is generally related to blocking effects or to the creation of a more difficult path to the electrolyte attack in the coating [47,58,75,87]. In addition, there are also inconclusive results, as the conditions used to produce the coatings were not always adequate or the used parameters were not always completely mentioned (for example, the kind of substrate or the stirring time). Finally, the results presented here also show that the deposition parameters earlier described may have synergistic and/or antagonistic effects on the coating deposition process and on the anticorrosive properties of the coatings.

#### *4.2.1. Ni and Ni-alloy matrix composite coatings*

most used are the corrosion potential (Ecorr), which shows the nobility of the coating compared to the substrate, and the corrosion current density (jcorr), which is related to the intensity of the corrosion process. Based on the jcorr values, it is possible to calculate the corrosion rate and the coating efficiency (EfCoat) [32,52] as well as to estimate the coating porosity [82]. It is also possible to determine the Tafel anodic and cathodic slopes (βa and βc, respectively), which are related to the kinetic aspects of the anodic and cathodic electrochemical reactions [32,79].

The anodic branch of the polarization curve can also be used to study the passivity of the coatings and evaluate parameters such as the passivation potential (Epassivation), the critical

LPR is a real-time technique regulated by the ASTM G59 standard method and is based on the potential variation around the open circuit potential (OCP; typical variations approximately ±10 mV [76] to ±20 mV are used) [83,84]. The current required to maintain a specific displace‐ ment of the resting potential is directly related to the corrosion process on the electrode surface. This technique is particularly useful in aqueous systems and is applicable to obtain the polarization resistance (Rp) [75]. It is also possible to calculate the current and the corrosion rate if the values of Rp and the Tafel slopes are known [32,83]. In addition, it is possible to evaluate the porosity of the coating by comparing the Rp values of the coating and of the

EIS is a technique that provides detailed information on the electrical characteristics of the electrode/solution interface. EIS is based on the application of a small sinusoidal signal of potential (or current) to the working electrode according to a particular desired frequency range. As a response, it obtained the impedance (Z), which can be related to the opposition to the current flux in the system [32]. Important information about the charge transfer kinetics, structure, and properties of the interface electrode/electrolyte can also be achieved. The frequency range usually used for disturbing the system varies from 100 kHz to 10 MHz, using the amplitude signal in the range of 5 to 50 mV rms, depending on the studied system [32,85].

The results obtained from the EIS measurements can be used to construct diagrams repre‐ senting the behavior of the electrode in a particular electrochemical process. One of these diagrams is the Nyquist diagram, in which the real (Z) and imaginary (Z) impedance data are represented in a complex plane. The real impedance (Z) incorporates the ohmic resistance (i.e., the pure resistance that is independent of the frequency). The imaginary impedance (Z) incorporates the capacity and/or inductive reactance (i.e., the resistance that is dependent on

The Bode and phase diagrams are also used to represent the EIS results. These diagrams show the variations of the impedance modulus (|Z|) or the phase angle with the frequency, respectively. An advantage of the Bode diagram is the possibility of observing the impedance

current density for passivation (jcrit), and the pitting potential (Epitting) [32].

*4.1.2. Linear polarization resistance*

168 Electrodeposition of Composite Materials

*4.1.3. Electrochemical impedance spectroscopy*

the frequency applied to the system) [32].

substrate [82].

Nickel is the most studied metallic matrix to produce composite coatings with improved corrosion properties, and different ceramic particles and nanoparticles were used to this purpose. The corrosion resistance of nickel matrix composite coatings using α-Al2O3 particles (smaller than 1 μm; 98% purity) as the second phase on steel substrate, compared to a standard nickel coating produced onto the same substrate, was studied [87]. The corrosion evaluation data of the coating/substrate systems (from polarization curves and electrochemical impe‐ dance experiments) were performed in 0.5 mol L-1 Na2SO4 solution with different exposure times. Despite the physical (defects and dislocations) and chemical heterogeneities (the presence of nickel oxides or impurities), Al2O3 particles in the nickel coatings disturbed the electrochemical electrode reactions. As a result, the corrosion process was enhanced in some parts of the coating, whereas the phenomenon was inhibited in other parts. Therefore, no significant differences were observed concerning the anticorrosive behavior of the standard nickel coating and of the composite coating for the data obtained in the first days of exposure. Although the corrosion rates of both coatings increased with the exposure time, the corrosion resistance of Ni/Al2O3 composite coatings was always higher than the standard nickel coating in Na2SO4. Because of the adsorption of water, OH ions, and oxygen, the surfaces of both coatings were covered with a thin layer of nickel oxides and hydroxides causing an increase in Rct during the first 3 days of the experiment [87].

The EIS measurements showed that this result was related to the fact that the resistance of the electrolyte in the pores of the composite coating was much lower than in the pores of the standard nickel coating. It means that the presence of Al2O3 particles in the voids of the coating increases its porosity and the discontinuity of the passive layer formed on its surface. In the cracks and pores of the passive layer, the electrolytic solution is in contact with either nickel or dielectric Al2O3 particles. However, the passive layer on the surface of the Ni/Al2O3 coating was tighter possibly due to the more finely crystalline structure of the composite coating in comparison with that of the nickel standard coating. As a result, the pores and cracks in the passive layer on the composite coating were sufficiently small to reduce the diffusion of metal corrosion products from their inside effectively, and they built up in the pores, blocking them and decreasing the corrosion process [87]. After 14 days of exposure in the corrosive medium, the corrosion rate of the Ni/Al2O3 composite coating was three times lower than that of the nickel coating.

Ni/CeO2 composite coatings were produced on steel from an acid electrolyte containing CeO2 nanoparticles using square-wave pulse current mode under different stirring speed conditions and at 45°C. The corrosion behavior of these coatings was evaluated in a 3.5 g L-1 NaCl solution and compared to that observed from a nickel coating in the same medium [47]. More positive corrosion potentials and higher Rp values were obtained for the composite coatings (ranging from 3.54×10³ to 9.772×10³ Ω) compared to those verified for the nickel coating (2.05×10³ Ω). Once more, the presence of the inert particles in the coating was consid‐ ered important in the improvement of the corrosion resistance of composite coating because they may have acted as a physical barrier to the propagation of defects. The dispersion of the nanoparticles in the metallic matrix formed corrosion microcells, which facilitated the anodic polarization, resulting in the inhibition of pitting corrosion and in the promotion of uniform corrosion of the coating. If the crystallites in the coating remained on their nanometer size, the corrosion process could be explained considering that the electrolyte must travel a tortuous path to reach the substrate and this path is longer in the Ni/CeO2 composite coatings than in the nickel coating [47].

The increase in the stirring speed until 450 rpm increased the CeO2 particles in the nickel matrix coating, as well as the Rp values measured for these coatings, indicating a relationship between the content of particles in coating and its anticorrosive performance. Further increase in stirring speed, however, decreased both the particle incorporation and the Rp value, showing that it must be an optimum stirring speed condition to produce coatings with high corrosion resistance [47]. However, the effects of the pulse plating and the deposition temperature on the corrosion performance of the coating/substrate system were not evaluated.

EIS analysis was performed in aqueous NaOH (1 mol L-1) and HNO3 (1 mol L-1) solutions to evaluate the anticorrosive properties of Ni/TiO2 nanocomposite coatings. The coatings containing different TiO2 contents, (A: Ni-3.9 wt% TiO2, B: Ni-6.5 wt% TiO2, and C: Ni-8.3 wt % TiO2) were produced onto steel from an acid Ni(II) bath. The charge transfer resistances of pure nickel and Ni-TiO2 coatings [49] are shown in Table 3.

nickel coating and of the composite coating for the data obtained in the first days of exposure. Although the corrosion rates of both coatings increased with the exposure time, the corrosion resistance of Ni/Al2O3 composite coatings was always higher than the standard nickel coating

coatings were covered with a thin layer of nickel oxides and hydroxides causing an increase

The EIS measurements showed that this result was related to the fact that the resistance of the electrolyte in the pores of the composite coating was much lower than in the pores of the standard nickel coating. It means that the presence of Al2O3 particles in the voids of the coating increases its porosity and the discontinuity of the passive layer formed on its surface. In the cracks and pores of the passive layer, the electrolytic solution is in contact with either nickel or dielectric Al2O3 particles. However, the passive layer on the surface of the Ni/Al2O3 coating was tighter possibly due to the more finely crystalline structure of the composite coating in comparison with that of the nickel standard coating. As a result, the pores and cracks in the passive layer on the composite coating were sufficiently small to reduce the diffusion of metal corrosion products from their inside effectively, and they built up in the pores, blocking them and decreasing the corrosion process [87]. After 14 days of exposure in the corrosive medium, the corrosion rate of the Ni/Al2O3 composite coating was three times lower than that of the

Ni/CeO2 composite coatings were produced on steel from an acid electrolyte containing CeO2 nanoparticles using square-wave pulse current mode under different stirring speed conditions and at 45°C. The corrosion behavior of these coatings was evaluated in a 3.5 g L-1 NaCl solution and compared to that observed from a nickel coating in the same medium [47]. More positive corrosion potentials and higher Rp values were obtained for the composite coatings (ranging from 3.54×10³ to 9.772×10³ Ω) compared to those verified for the nickel coating (2.05×10³ Ω). Once more, the presence of the inert particles in the coating was consid‐ ered important in the improvement of the corrosion resistance of composite coating because they may have acted as a physical barrier to the propagation of defects. The dispersion of the nanoparticles in the metallic matrix formed corrosion microcells, which facilitated the anodic polarization, resulting in the inhibition of pitting corrosion and in the promotion of uniform corrosion of the coating. If the crystallites in the coating remained on their nanometer size, the corrosion process could be explained considering that the electrolyte must travel a tortuous path to reach the substrate and this path is longer in the Ni/CeO2 composite coatings than in

The increase in the stirring speed until 450 rpm increased the CeO2 particles in the nickel matrix coating, as well as the Rp values measured for these coatings, indicating a relationship between the content of particles in coating and its anticorrosive performance. Further increase in stirring speed, however, decreased both the particle incorporation and the Rp value, showing that it must be an optimum stirring speed condition to produce coatings with high corrosion resistance [47]. However, the effects of the pulse plating and the deposition temperature on

the corrosion performance of the coating/substrate system were not evaluated.

ions, and oxygen, the surfaces of both

in Na2SO4. Because of the adsorption of water, OH-

170 Electrodeposition of Composite Materials

in Rct during the first 3 days of the experiment [87].

nickel coating.

the nickel coating [47].

**Table 3.** Charge transfer resistance for pure nickel coating and Ni-TiO2 nanocomposite coatings (A–C) obtained from EIS data [49] after fitting by the equivalent circuit model

The Nyquist diagrams obtained from the experiments carried out in NaOH showed a capac‐ itive loop at high frequencies followed by an almost straight line at low frequencies, suggesting that the corrosion mechanism was controlled by both charge transfer and diffusion processes. The corrosion rate was affected by the interdiffusion of Ni2+ and OH- ions, confirming that there should be an effect related to the diffusion of these ions in the coating. The Rct values for the composite coatings exposed in this medium were improved with the increase of the TiO2 nanoparticle content in the coating, because they probably decreased the diffusion process [49].

In the experiments performed in HNO3, there was an observed discrete capacitive loop at high frequencies of the Nyquist diagram. An inductive loop was also observed at low frequencies, which was attributed to the relaxation process of H+ and NO3- species adsorbed on the electrode surface [49]. As observed in the experiments carried out in NaOH medium, the increase in the TiO2 content in the coating caused an increase in the charge transfer resistance of the composite coating. However, the Rct values obtained in acidic medium were significantly smaller than those verified in alkaline medium [49]. These results suggested that the acidic medium may have damaged the coating or that the corrosion mechanism has changed in this medium.

The presence of different concentrations of sodium dodecyl sulfate (SDS) in the electrodepo‐ sition bath to produce nickel/α-Al2O3 and its influence on the anticorrosive performance of the coating produced onto steel in NaCl (3.5% m/v) was also studied [8]. Although the use of a surfactant may enhance the incorporation of inert particle in the metallic matrix, it also has some disadvantages, as they can be incorporated in the metallic matrix and change the mechanical properties of the deposit (e.g., internal stress and fragility). This is why the concentration of surfactants in the deposition bath should be very well controlled [88]. The nanocomposite coatings produced using the optimum SDS concentration (125 g L-1) contained higher levels of alumina nanoparticles in the metallic matrix. This coating presented a more positive corrosion potential (Ecorr = -0.209 VAg/AgCl) and a lower corrosion current density (jcorr = 1.141×10-7 A cm- ²) than the bare substrate (Ecorr = -0.488 and jcorr = 4.832×10-6 A cm-2). It was considered that the incorporation of such low reactive nanometer alumina particles played an important role in improving the corrosion resistance of the coating/substrate system, as they apparently filled the pores and micropores of the coating and thereby reduced the corrosion process [8].

The corrosion performance of Ni-Co alloy matrix composite coatings containing different contents of SiC nanoparticles was evaluated [37]. The coatings were produced onto copper substrate from acid Ni(II) and Co(II) sulfamate solutions using DC and different stirring speeds. The polarization curves of both the composite coatings and the Ni-Co alloy coatings were performed in a 0.5 mol L-1 NaCl solution. It was found that the composite coating produced at 0.03 A cm-2 contained 3.2 wt% SiC and that this coating showed a lower corrosion current density and a more positive corrosion potential (Ecorr = -0.33 VSCE, jcorr = 7.9×10-3 A cm-2) compared to the results of a Ni-Co alloy coating (Ecorr = -0.39 VSCE, jcorr = 3.98×10-2 A cm-2). The better result obtained by this nanocomposite coating was attributed to a decrease in the size of the defects related to the incorporation of SiC nanoparticles, which is useful for creating a tortuous path to the corrosive medium attack the substrate, preventing pitting corrosion and enhancing the corrosion resistance of these nanocomposite coatings [37]. However, the results concerning Ni-Co/SiC coatings containing other SiC contents were not mentioned in the work.

Composite coatings of Ni-Zn matrix containing TiO2 nanoparticles (Degussa P-25 anatase, 25 nm) as the strengthening phase were produced onto a mild steel (DIN C25-AISI 1025) substrate using ultrasonic-assisted electrodeposition process [89]. These coatings were electrochemically evaluated in a natural 3.5 wt% NaCl solution using EIS measurements at the OCP. The results showed that higher Rct values were obtained for samples produced using 108 and 216 W cm-2 of ultrasonic powder densities, indicating a better anticorrosion performance of these coatings. On the contrary, the Rct values of coatings produced without using the ultrasonicassisted process were smaller than that obtained for the alloy Ni-Zn coating. The authors claim that the use of ultrasonic vibration together with electrodeposition might have improved the coating nanoparticles uniform dispersion and hence improved the anticorrosive ability of the composite coatings [89].


4.2.5. Electrode Spacing

To understand the influence between the electrode spacing and the boundary effect, and if the electrode spacing could be a normalization parameter, or whether its effect was a fixed distance regardless of the electrode rod spacing, this study fixed the medium depth and size and set the electrode spacing as 2 m and 4 m, respectively.

When the resistivity ratio value n was 0.1 and the electrode spacing *dx* was 2, the influence distance of the boundary effect was 8 m; moreover, when the electrode spacing *dx* was 4, the influence distance of the boundary effect was also 8 m. Although the use of a smaller electrode spacing had better resolution, the influence distances were the same. Therefore, it is not appropriate to use the electrode spacing as the normalization parameter of the influence space of the boundary effect as the influence distance does not become smaller as the electrode spacing becomes smaller. This result is similar to the 3D effect of the pipeline stratum, where neither were affected by the electrode spacing.