[106] Sarmento D, Martins M, Oliveira MM. Evaluation of somaclonal variation in almond

[107] Gribaudo I, Torello Marinoni D, Gambino G, Mannini F, Akkak A, Botta R. Assessment of genetic fidelity in regenerants from two *Vitis vinifera* cultivars Acta

[108] Bonga JM, Klimaszewska KK, von Aderkas P. Recalcitrance in clonal propagation, in

[109] Schaeffer WI. Terminology associated with cell, tissue and organ culture, molecular biology and molecular genetics. In Vitro Cellular Developmental Biology Plant 1990; 26:

[110] Grossoni P. Problems concerning the in vitro culture of *Olea europaea* L. Giornale

[111] Rugini E. *In vitro* propagation of some (*Olea europaea* L.) cultivars with different rootability and medium development using analytical data from developing shoots and

[112] Fiorino P, Leva AR. Investigations on the micropropagation of the olive (*Olea europaea*  L.) and influence of some mineral elements on the proliferation and rooting of explants.

[113] Leva AR, Petruccelli R, Muleo R, Goretti R, Bartolini G. Influenza di fattori trofici, regolativi e condizioni di coltura in vitro di diverse cultivar di olivo. Atti Convegno "L'Olivicoltura Mediterranea: Stato e Prospettive della Coltura e della Ricerca. Rende

[114] Grigoriadou K, Vasilakakis M, Eleftheriou EP *In vitro* propagation of the Greek olive cultivar "Chondrolia Chalkidikis". Plant Cell Tissue Organ Culture 2002; 71: 47-54. [115] Leva AR, Sadeghi H, Petruccelli R. Carbohydrates Modulate the In Vitro Growth of Olive Microshoots. I. The Analysis of Shoot Growth and Branching Patterns. Journal of

[116] Leva AR. Innovative protocol for "ex vitro rooting" on olive micropropagation Central

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[118] Leva AR, Muleo R, Petruccelli R Long-term somatic embryogenesis from immature

[119] Rugini E, Caricato G. Somatic embryogenesis and recovery from mature tissues of olive cultivars (*Olea europaea* L.) "Canino" and "Moraiolo". Plant Cell Report 1995; 14 :

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[92] Khan EU, Fu XZ, Wang J, Fan QJ, Huang XS, Zhang GN, Shi J, Liu JH Regeneration and characterization of plants derived from leaf in vitro culture of two sweet orange (*Citrus*

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[98] Prado MJ, Herrera MT, Vázquez RA, Romo S, González MV. Micropropagation of two selected male kiwifruit and analysis of genetic variation with AFLP markers.

[99] Prado MJ, Gonzalez MV, Romo S, Herrera MT. Adventitious plant regeneration on leaf explants from adult male kiwifruit and AFLP analysis of genetic variation. Plant Cell

[100] Popescu C F, Flak A, Glimelius K. Application of AFLPs to characterize somaclonal

[101] Xiangqian Li, Mingliang Xu, Schuyler S. Korban DNA methylation profiles differ between field- and *in vitro*-grown leaves of apple. Journal Plant Physiology 2002; 159:

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[121] Trabelsi EB, Bouzid S, Bouzid M, EIIoumi N, Belfeleh Z, Benabdallah A, and Ghezel R In Vitro Regeneration of Olive Tree by Somatic Embryogenesis Journal of Plant Biology 2003; 46: 173-180.

**Chapter 8**

© 2012 Pasqual et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Pasqual et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Tissue Culture Techniques for**

**Native Amazonian Fruit Trees**

Joyce Dória Rodrigues Soares and Filipe Almendagna Rodrigues

The fruits of the Amazon have attracted great interest in recent years, both nationally and internationally, according to its exotic flavors and pleasant and varied ways to use its pulp by agribusiness [1], pharmaceutical industry [2], high vitamin and antioxidant content [3].

In recent decades, the production of native fruits of the Amazon showed significant growth, mainly due to expansion of area for fruit production. It is noteworthy that this activity has had little impact on native vegetation, since most of the orchards were planted in areas previously occupied by other crops for market problems or environmental issues and

The Amazon forest has large number of non-domesticated fruit species and a minority being exploited through crop in place of natural occurrence [5]. According to the Brazilian Yearbook of Fruit [6], explored the country are 500 varieties of edible fruit-producing species native and exotic, and of these, 220 are still as untamed. The high rate of destruction of biomes, together with the predatory extraction, result in loss of genetic material of desirable characteristics, [7], with potential for use in food, as an ornamental or in pharmaceutical production can never be known. It is therefore essential to know these species and their growing needs for exploitation on a commercial scale, a rational and

However, little efficient production technology and knowledge of native Amazonian fruit tree species exist. Low orchard productivity indicates that Amazonian fruit is underused; underuse, in turn, has hindered its cultivation. In [8] notes that many of the current fruit production systems were developed empirically, which required technology that ensured greater productivity, sustainability, and profitability than the older production systems.

pressure for sustainable agriculture, ceased to be interesting for farmers [4].

Moacir Pasqual, Edvan Alves Chagas,

http://dx.doi.org/10.5772/52211

**1. Introduction**

sustainable.

Additional information is available at the end of the chapter

**Chapter 8**

## **Tissue Culture Techniques for Native Amazonian Fruit Trees**

Moacir Pasqual, Edvan Alves Chagas, Joyce Dória Rodrigues Soares and Filipe Almendagna Rodrigues

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52211

## **1. Introduction**

150 Recent Advances in Plant *in vitro* Culture

2003; 46: 173-180.

867-870.

2002; 586: 891-894.

2009; 3 (3): 37-43.

[121] Trabelsi EB, Bouzid S, Bouzid M, EIIoumi N, Belfeleh Z, Benabdallah A, and Ghezel R In Vitro Regeneration of Olive Tree by Somatic Embryogenesis Journal of Plant Biology

[122] Peyvandi M, Nemat Farahzadi H, Arbabian S, Noormohammadi Z, Hosseini-Mazinani M. Somaclonal Variation Among Somatic-Embryo Derived Plants of *Olea europaea L* "*cv. Kroneiki*". Journal of Sciences, Islamic Republic of Iran 2010; 21 (1): 7-14. [123] Capelo AM, Silvia S, Brito G, Santos C. Somatic embryogenesis induction in leaves and petioles of a mature wild olive. Plant Cell Tissue Organ Culture 2010; 103:237-242. [124] Mazri MA, Elbakkali A, Belkoura M, Belkoura I. Embryogenic competence of calli and embryos regeneration from various explants of Dahbia cv, a moroccan olive tree ( *Olea*

[125] Trabelsi EB, Naija S, Elloumi N, Belfeleh Z, Msellem M, Ghezel R, Bouzid S. Somatic embryogenesis in cell suspension cultures of olive *Olea europaea* L. 'Chetoui' Acta

[126] Briccoli Bati C, Godini G, Nuzzo V. Preliminar agronomic evaluation of two cultivars of olive trees obtained from micropropagation method. Acta Horticulture 2002; 586:

[127] Leva AR, Petruccelli R, Montagni G, Muleo R. Field performance of micropropagated olive plants (cv Maurino): morphological and molecular features. Acta Horticulture

[128] Leva AR. Morphological evaluation of olive plants propagated *in vitro* through axillary buds and somatic embryogenesis methods. African Journal of Plant Science

[129] Roselli G, Mariotti P, Tessa A. Caratterizzazione di progenie di olivo mediante analisi chimica e sensoriale degli olii. Proceedings del Simposio Nazionale : "Germoplasma

[131] Rotondi A*,* Magli M, Ricciolini C, Baldoni L. Morphological and molecular analyses for the characterization of a group of Italian olive cultivars. Euphytica 2003; 132: 129–137. [132] Leva AR, Petruccelli R, Polsinelli L. In vitro olive propagation: from the laboratory to

[133] Gucci R, Cantini C. Pruning and Training Systems for Modern Olive Growing. CSIRO

[134] Leva AR, Petruccelli R. Monitoring of cultivar identity in micropropagated olive plants using RAPD and ISSR markers. Biologia Plantarum 2012; 56 (2): 373-376. [135] Mencuccini M.Produzione di somacloni da cultivar di olivo e prime osservazioni in

[136] Leva AR, Muleo R, Petruccelli R. Stabilità in campo di diversi habitus vegetativi in individui di *Olea europaea* L (cv Frangivento) derivati da embriogenesi somatica.Proceedings of the National Congress : Biodiversità-Opportunità di Sviluppo Sostenibile. Istituto Agronomico Mediterraneo, Bari, Italy. 2001.Volume II. 407-414. [137] Leva AR, Petruccelli R. Dwarf olive trees for ornamental use: a morphological evaluation Journal of Horticultural Science & Biotechnology 2011; 86 (3) 217–220.

olivicolo e tipicità dell'olio" tenuto a Perugia 5 dicembre, 2003. pp 278-283 [130] Barranco D, Rallo L. Olive cultivars in Spain. HorTechnology 2000; 10:107–110.

*europaea* L.). African Journal of Biotechnology 2011; 10 (82):19089-19095.

Physiologiae Plantarum 2011; 33:319-324

the production line. OLIVAE 2004; 101: 18–26.

campo Acta Italus Hortus 2011; 1: 113-116.

Publishing, Australia 2000.

The fruits of the Amazon have attracted great interest in recent years, both nationally and internationally, according to its exotic flavors and pleasant and varied ways to use its pulp by agribusiness [1], pharmaceutical industry [2], high vitamin and antioxidant content [3].

In recent decades, the production of native fruits of the Amazon showed significant growth, mainly due to expansion of area for fruit production. It is noteworthy that this activity has had little impact on native vegetation, since most of the orchards were planted in areas previously occupied by other crops for market problems or environmental issues and pressure for sustainable agriculture, ceased to be interesting for farmers [4].

The Amazon forest has large number of non-domesticated fruit species and a minority being exploited through crop in place of natural occurrence [5]. According to the Brazilian Yearbook of Fruit [6], explored the country are 500 varieties of edible fruit-producing species native and exotic, and of these, 220 are still as untamed. The high rate of destruction of biomes, together with the predatory extraction, result in loss of genetic material of desirable characteristics, [7], with potential for use in food, as an ornamental or in pharmaceutical production can never be known. It is therefore essential to know these species and their growing needs for exploitation on a commercial scale, a rational and sustainable.

However, little efficient production technology and knowledge of native Amazonian fruit tree species exist. Low orchard productivity indicates that Amazonian fruit is underused; underuse, in turn, has hindered its cultivation. In [8] notes that many of the current fruit production systems were developed empirically, which required technology that ensured greater productivity, sustainability, and profitability than the older production systems.

© 2012 Pasqual et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Pasqual et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Such technology involves crop management, production of reproducible seedlings, and distribution of the seedlings to farmers. Therefore, the first step in domesticating native fruit species and in introducing them to commercial cultivation is developing seedling propagation techniques.

Tissue Culture Techniques for Native Amazonian Fruit Trees 153

[21], [22], [23]

[9]

are in an advanced stage, such as *Theobroma cacao* and *T. grandiflorum*, for most species have been developed with the aim of improving methods for initial establishment of cultures in

**Species Purpose of the study Author**

Effect of culture media on morphogenetic responses

embryogenesis

Micropropagation is a high-impact plant tissue culture technique that is more consistent than other tissue culture methods. Micropropagation involves a high reproduction rate over a short time period, which produces plants with excellent phytosanitary quality. The technique involves various steps, from aseptic *in vitro* culture establishment to rooting,

Most studies on the tissue culture of native Amazonian plant species have sought to improve methodologies for initial culture establishment. Disinfection plays a critical role in culture establishment because explant contamination during tissue culturing is

**Murmuru** In vitro propagation and somatic

**3.** *In vitro* **establishment and micropropagation**

culminating in seedling acclimatization [32].

Culture of embryos [12]

Establishment of culture [13]

Establishment of culture [14], [15]

somatic embryogenesis [16], [17] Multiplication of shoots [18] In vitro establishment [19], [14]

In vitro establishment [24], [25]

somatic embryogenesis [28], [29]

Establishment of culture [30]

direct organogenesis [7]

Cultivation of embryos [31]

Callus [26], [27]

Culture of embryos and callus [20]

vitro (Table 1).

**Açai**  (*Euterpe oleracea*.)

> **Araça**  (*Psidium spp.*)

**Bacuri**  (*Platonia insignis*)

**Cacau**  (*Theobroma cacao*)

**Caja**  (*Spondias mombin* )

**Camu-camu** (*Myrciaria dubia*)

> **Cupuaçu** (*Theobroma grandiflorum*)

> > **Inga**  (*Inga vera*)

**Murici**  (*Byrsonima basiloba*)

**Pupunha** (*Bactris gasipaes*)

**Table 1.**

The sexual propagation of native plants for use in horticulture, is not advantageous because it results in populations with wide variation in the period of maturation. In addition, some species have seeds with dormancy, which compromises the germination and seedling production on a commercial scale, or the recalcitrance of the seeds, preventing their storage for extended periods.

The method of propagation of fruit species most commonly used grafting. This technique, like other methods of vegetative propagation allow the cloning of selected plants directly from nature or from artificial hybridizations, maintaining their desirable traits. Vegetative propagation results in high quality seedlings in orchards and more uniform and earlier, with higher productivity and better quality of fruits [9]. Besides these advantages, the graft is also advantageous because it allows greater control of plant height facilitating the management and harvesting, and the formation of plants with resistance to pathogens in soils and drought tolerant.

## **2. Tissue culture techniques for native Amazonian fruit trees**

For decades numerous studies have been developed with the objective of establishing protocols for micropropagation of fruit species for clonal multiplication of superior individuals, in view of the numerous advantages it offers in vitro propagation.

Besides the application of tissue culture techniques for plant propagation of high agronomic value carriers or rare genes and those at risk of extinction, the technique is also used for cleaning clonal plants through tissue culture. In plant breeding programs, the tissue culture can be used to reduce the time for development of new cultivars and expansion of genetic variability. Among the tissue culture techniques most widely used in plant breeding, there may be mentioned: in vitro selection of resistant /tolerant to various stress factors of the genetic variability from pre-existing or induced somaclonal variation or by use of mutagenic, haploidization, somatic hybridization, and rescue of zygotic embryos obtained from crosses between different species or genera of plants [10]. Tissue culture also stands as an adjuvant in introgression of genes of agronomic interest through genetic engineering.

An application of tissue culture which is very important, especially for native plants is the maintenance and storage of germplasm. However, in many other species, tissue culture techniques have been also widely used for studying the metabolism, physiology, development and reproduction of plants with desirable commercial property of interest [11], like the nutraceutical and pharmaceutical products.

Studies on the application of tissue culture techniques in fruit tree species native to the Amazon, are still incipient. Except for a few species in which native tissue culture studies are in an advanced stage, such as *Theobroma cacao* and *T. grandiflorum*, for most species have been developed with the aim of improving methods for initial establishment of cultures in vitro (Table 1).

#### **Table 1.**

152 Recent Advances in Plant in vitro Culture

propagation techniques.

for extended periods.

soils and drought tolerant.

Such technology involves crop management, production of reproducible seedlings, and distribution of the seedlings to farmers. Therefore, the first step in domesticating native fruit species and in introducing them to commercial cultivation is developing seedling

The sexual propagation of native plants for use in horticulture, is not advantageous because it results in populations with wide variation in the period of maturation. In addition, some species have seeds with dormancy, which compromises the germination and seedling production on a commercial scale, or the recalcitrance of the seeds, preventing their storage

The method of propagation of fruit species most commonly used grafting. This technique, like other methods of vegetative propagation allow the cloning of selected plants directly from nature or from artificial hybridizations, maintaining their desirable traits. Vegetative propagation results in high quality seedlings in orchards and more uniform and earlier, with higher productivity and better quality of fruits [9]. Besides these advantages, the graft is also advantageous because it allows greater control of plant height facilitating the management and harvesting, and the formation of plants with resistance to pathogens in

For decades numerous studies have been developed with the objective of establishing protocols for micropropagation of fruit species for clonal multiplication of superior

Besides the application of tissue culture techniques for plant propagation of high agronomic value carriers or rare genes and those at risk of extinction, the technique is also used for cleaning clonal plants through tissue culture. In plant breeding programs, the tissue culture can be used to reduce the time for development of new cultivars and expansion of genetic variability. Among the tissue culture techniques most widely used in plant breeding, there may be mentioned: in vitro selection of resistant /tolerant to various stress factors of the genetic variability from pre-existing or induced somaclonal variation or by use of mutagenic, haploidization, somatic hybridization, and rescue of zygotic embryos obtained from crosses between different species or genera of plants [10]. Tissue culture also stands as an adjuvant in introgression of genes of agronomic interest through genetic engineering.

An application of tissue culture which is very important, especially for native plants is the maintenance and storage of germplasm. However, in many other species, tissue culture techniques have been also widely used for studying the metabolism, physiology, development and reproduction of plants with desirable commercial property of interest [11],

Studies on the application of tissue culture techniques in fruit tree species native to the Amazon, are still incipient. Except for a few species in which native tissue culture studies

like the nutraceutical and pharmaceutical products.

**2. Tissue culture techniques for native Amazonian fruit trees**

individuals, in view of the numerous advantages it offers in vitro propagation.

## **3.** *In vitro* **establishment and micropropagation**

Micropropagation is a high-impact plant tissue culture technique that is more consistent than other tissue culture methods. Micropropagation involves a high reproduction rate over a short time period, which produces plants with excellent phytosanitary quality. The technique involves various steps, from aseptic *in vitro* culture establishment to rooting, culminating in seedling acclimatization [32].

Most studies on the tissue culture of native Amazonian plant species have sought to improve methodologies for initial culture establishment. Disinfection plays a critical role in culture establishment because explant contamination during tissue culturing is extremely problematic. Explant contamination is most severe in woody species, which include all Amazonian fruit species. The potential for contamination is greatest when the explants are taken directly from the field. Although plants maintained under greenhouse conditions are easily controlled, explants derived from them also have a high potential for contamination. However, even plants maintained in nurseries or greenhouses and subjected to rigorous phytosanitary control can harbor microorganisms, which may limit *in vitro* culture procedures [20]. In [10] cautions that, even before harvesting material from the field, it is essential to take some measures for each step in the culture process, from the laboratory to acclimatization and plant development in greenhouses. The phytosanitary conditions of the mother plant help determine the ease of explant sterilization during isolation.

Tissue Culture Techniques for Native Amazonian Fruit Trees 155

authors observed that the lowest rate of contamination occurred when the explants were immersed in a 0.5% sodium hypochlorite solution for 10 minutes, but the rate of oxidation in the tissues was high for all treatments. Similarly, [21] found that immersing the explants in a 2% sodium hypochlorite solution for 10 to 20 minutes provided the best rate of disinfection

**Figure 1.** *In vitro* establishment of camu-camu held in Biofactory (UFRR) by the team of Fruits and

In evaluating the effect of a light regime on the mother plant and the branch type used for the *in vitro* establishment of araça (*Psidium* spp.), [13] observed lower rates of contamination when using nodal segment explants of herbaceous branches, and obtained the highest survival rates when the mother plants were kept in darkness. These results are likely due to the reduced exposure of younger branches to contamination in the environment from which

Another way to control explant oxidation during *in vitro* establishment is to reduce the concentrations of some components of the culture medium. In [33] found that reducing the concentrations of NH4NO3 and KNO3 macronutrients, sucrose, and agar by 50% in MS culture medium [36] controlled phenolic oxidation and the proliferation of peach palm

Tissue Culture of Embrapa Roraima and UFRR. Boa Vista, RR, 2010.

the explants were harvested.

explant shoots.

in camu-camu seeds. However, these treatments killed the seeds.

Several substances have been tested to minimize contamination and facilitate *in vitro*  establishment, and chlorine and ethanol-based compounds are now standard disinfection tools (Silva et al., 2005). In some cases, antibiotics are added to the culture medium [29]. Research has demonstrated the success of these active ingredients in disinfecting explants of native fruit tree species when establishing plant tissues *in vitro* for use in studies on reproduction. The active ingredients in disinfectant solutions and their exposure durations vary greatly.

In studying decontamination of floral explants from cupuaçu, [29] recommended immersing the explants in a 0.25% sodium hypochlorite solution for 20 minutes. The authors also observed that adding cefatoxime antibiotic to the culture medium was vital in controlling contamination in these explants.

In studying decontamination of explants from bacuri trees, [29] and his team found that pretreating the explants in an antifungal solution of carboxin (0.067% p/v), thiram (0.067% w/v), carbendazim (0.17% w/v), chlorothalonil (0.17% w/v), and thiophanate-methyl (0.067% w/v) [15], and immersing them in a 1.75% sodium hypochlorite solution for 30 minutes produced the best results.

In [31] found that the most efficient method for disinfecting the apices of peach palms was to immerse the peach palm explants in a 1.25% sodium hypochlorite solution for 20 minutes. The authors reported that 90% of these explants avoided contamination during *in vitro* culture. In [33] obtained the most promising results for the disinfection of explants from peach palm shoots; he first immersed the shoots in a 50% ethanol solution for 1 minute, and then in a 0.5% sodium hypochlorite solution for 5 minutes.

In Figure 1 you can see the success obtained in vitro establishment of camu-camu (*Myrciaria dubious*) conducted by staff of the Tissue Culture and Fruits Embrapa Roraima and UFRR.

In [34] and [35] note that explant oxidation during the *in vitro* establishment of native plants is common; in camu-camu, explant oxidation is a major obstacle to *in vitro* establishment. In [23] evaluated the efficiency of different concentrations of sodium hypochlorite solutions at different immersion durations in the disinfection of nodal segments of camu-camu. The authors observed that the lowest rate of contamination occurred when the explants were immersed in a 0.5% sodium hypochlorite solution for 10 minutes, but the rate of oxidation in the tissues was high for all treatments. Similarly, [21] found that immersing the explants in a 2% sodium hypochlorite solution for 10 to 20 minutes provided the best rate of disinfection in camu-camu seeds. However, these treatments killed the seeds.

154 Recent Advances in Plant in vitro Culture

isolation.

vary greatly.

the best results.

contamination in these explants.

extremely problematic. Explant contamination is most severe in woody species, which include all Amazonian fruit species. The potential for contamination is greatest when the explants are taken directly from the field. Although plants maintained under greenhouse conditions are easily controlled, explants derived from them also have a high potential for contamination. However, even plants maintained in nurseries or greenhouses and subjected to rigorous phytosanitary control can harbor microorganisms, which may limit *in vitro* culture procedures [20]. In [10] cautions that, even before harvesting material from the field, it is essential to take some measures for each step in the culture process, from the laboratory to acclimatization and plant development in greenhouses. The phytosanitary conditions of the mother plant help determine the ease of explant sterilization during

Several substances have been tested to minimize contamination and facilitate *in vitro*  establishment, and chlorine and ethanol-based compounds are now standard disinfection tools (Silva et al., 2005). In some cases, antibiotics are added to the culture medium [29]. Research has demonstrated the success of these active ingredients in disinfecting explants of native fruit tree species when establishing plant tissues *in vitro* for use in studies on reproduction. The active ingredients in disinfectant solutions and their exposure durations

In studying decontamination of floral explants from cupuaçu, [29] recommended immersing the explants in a 0.25% sodium hypochlorite solution for 20 minutes. The authors also observed that adding cefatoxime antibiotic to the culture medium was vital in controlling

In studying decontamination of explants from bacuri trees, [29] and his team found that pretreating the explants in an antifungal solution of carboxin (0.067% p/v), thiram (0.067% w/v), carbendazim (0.17% w/v), chlorothalonil (0.17% w/v), and thiophanate-methyl (0.067% w/v) [15], and immersing them in a 1.75% sodium hypochlorite solution for 30 minutes produced

In [31] found that the most efficient method for disinfecting the apices of peach palms was to immerse the peach palm explants in a 1.25% sodium hypochlorite solution for 20 minutes. The authors reported that 90% of these explants avoided contamination during *in vitro* culture. In [33] obtained the most promising results for the disinfection of explants from peach palm shoots; he first immersed the shoots in a 50% ethanol solution for 1 minute, and

In Figure 1 you can see the success obtained in vitro establishment of camu-camu (*Myrciaria dubious*) conducted by staff of the Tissue Culture and Fruits Embrapa Roraima and UFRR.

In [34] and [35] note that explant oxidation during the *in vitro* establishment of native plants is common; in camu-camu, explant oxidation is a major obstacle to *in vitro* establishment. In [23] evaluated the efficiency of different concentrations of sodium hypochlorite solutions at different immersion durations in the disinfection of nodal segments of camu-camu. The

then in a 0.5% sodium hypochlorite solution for 5 minutes.

**Figure 1.** *In vitro* establishment of camu-camu held in Biofactory (UFRR) by the team of Fruits and Tissue Culture of Embrapa Roraima and UFRR. Boa Vista, RR, 2010.

In evaluating the effect of a light regime on the mother plant and the branch type used for the *in vitro* establishment of araça (*Psidium* spp.), [13] observed lower rates of contamination when using nodal segment explants of herbaceous branches, and obtained the highest survival rates when the mother plants were kept in darkness. These results are likely due to the reduced exposure of younger branches to contamination in the environment from which the explants were harvested.

Another way to control explant oxidation during *in vitro* establishment is to reduce the concentrations of some components of the culture medium. In [33] found that reducing the concentrations of NH4NO3 and KNO3 macronutrients, sucrose, and agar by 50% in MS culture medium [36] controlled phenolic oxidation and the proliferation of peach palm explant shoots.

During the reproductive stage of shoots *in vitro*, the culture medium's composition varies with the species and the type of tissue or organ used as an explant. MS is most commonly used in the micropropagation of plant species. However, the Woody Plant Medium (WPM) [37] has been used successfully in woody species, including native Amazonian fruit tree species.

Tissue Culture Techniques for Native Amazonian Fruit Trees 157

shown as a great source of explants for obtaining callus, and the PVP the best alternative to control the oxidation of explants cupuaçu. On the other hand, [41] did not succeed in callus formation using nodal segments cupuaçu in medium supplemented with different concentrations of 2,4 D. Similar results were obtained by [28]. The authors assessed the responses of different morphogenetic cupuaçu explants subjected to various culture conditions in vitro and argue that the absence of induction of somatic embryogenesis observed in culture may be related to several factors such as type and stage of development

In [25] undertook a study of the induction of callus formation of the hybrid *Theobroma grandiflorum* x *T.obovatum*, which has resistance to disease witches' broom (*Crinipellis perniciosa*), a disease that greatly affects native Amazonian species of the genus Theobroma. Among the explants, the cotyledons were produced more callus tissues in culture medium. For açaizeiro, [12] reported the conversion can be in vitro isolated zygotic embryos from mature seeds and seedlings complete normal requiring the presence of NAA and BA in the culture medium. The concentration of 2.68 mM NAA combined with 1.11, 1.55 or 2.22 mM BAP promoted the best growth of the seedling shoot. The authors also found that required the presence of NAA and BA in the culture medium for the conversion of zygotic embryos and the early growth of seedlings grown in vitro. The same authors in studies on direct somatic embryogenesis using zygotic embryos of açai, satisfactory results, with a halving of

*In vitro* plants require a source of exogenous energy for carrying out the photosynthesis. Sucrose has been used more carbon source being present in fruit culture media at concentrations ranging native 20-40 g L-1 [26]. In [9] found that immature embryos murmuru showed higher germination in medium supplemented with 30 g L-1 sucrose. For embryos obtained from ripe fruits, 15 g L-1 sucrose in the medium was sufficient for it to reach the best germination rates. These results confirm the hypothesis that depending on the species and stage of development of embryos, the presence of carbohydrate in the medium may be in minute concentrations, or even not necessary because of embryos of many species use the

energy required for germination in vitro from their own reserves of the embryo [42].

Embryos younger typically require higher concentrations of carbohydrates in the culture medium to sustain germination [43, 44]. In [9] have obtained best results using 3% sucrose in the development of embryonic axes cupuaçu, in accordance with [16, 18, 19 and 45] in

One of the techniques used to obtain new varieties is controlled hybridization in the field and subsequent evaluation of the progenies. Thus, to obtain success in breeding is important

of the explants, using culture type and concentration of plant growth regulators.

the amount of nutrients in MS medium in the absence of fitoregulators.

**5.** *In vitro* **zygotic embryo culture**

studies performed with *Theobroma cacao*.

**6.** *In vitro* **germination of pollen grains**

When using growth regulators to induce new shoot growth, native plants generally respond better to a culture medium supplemented with low doses of auxins and/or cytokinins. In [38] obtained excellent results in nance trees using different benzyladenine (BAP) doses for micropropagation. However, high BAP concentrations (above 4.0 mg L-1) were inefficient in inducing the growth of axillary shoots in nodal segments. In [30] found that BAP reduced the number of shoots and leaves in Inga tree explants.

## **4. Somatic embryogenesis**

In somatic embryogenesis, somatic cells or tissues to develop the formation of a complete plant, through a series of stages similar to zygotic embryogenesis.

Most systems via somatic embryogenesis is indirect, in which somatic embryogenesis is induced and maintained through the multiplication. The advantage of this method is that large quantities of somatic embryos can be formed with minimal manipulation and laboratory space.

This process of plant regeneration was successfully achieved in cocoa [16, 17], cupuaçu [26,27] and murici [30].

In indirect somatic embryogenesis, the formation of pro-embryogenic callus is the first step in obtaining somatic embryos and is usually obtained with the culture of embryonic or juvenile explants in medium supplemented with auxin, especially 2,4-D and TDZ [39].

In [31] can be observed callus induction in shoot apices of peach palm. The highest percentage of induction was 60%, obtained by combining 10.0 mg L-1 2,4-D and 3.0 mg L-1 BAP.

Some studies have shown the ability of different cupuaçu explants to form callus. In [27] studied the induction of somatic embryogenesis in explants of cupuaçu, concluded that the hypocotyl region proved to be the most responsive of the embryonic axis, forming calluslooking white and bright. The MS medium supplemented with 2,4-D promoted the formation of large callus. Similar results were found by [24], studying the effect of auxin concentration and liquid medium on the development of calluses cupuaçu. The authors observed that the combination of NAA and 2,4-D induced callus formation and root formation in hypocotyl segments, and coconut water in medium without growth regulators favored the rooting and callus formation.

In [40] also studied the induction of somatic embryogenesis in explants cupuaçu and observed that the callus appeared as a process influenced by genotype. The staminode is shown as a great source of explants for obtaining callus, and the PVP the best alternative to control the oxidation of explants cupuaçu. On the other hand, [41] did not succeed in callus formation using nodal segments cupuaçu in medium supplemented with different concentrations of 2,4 D. Similar results were obtained by [28]. The authors assessed the responses of different morphogenetic cupuaçu explants subjected to various culture conditions in vitro and argue that the absence of induction of somatic embryogenesis observed in culture may be related to several factors such as type and stage of development of the explants, using culture type and concentration of plant growth regulators.

In [25] undertook a study of the induction of callus formation of the hybrid *Theobroma grandiflorum* x *T.obovatum*, which has resistance to disease witches' broom (*Crinipellis perniciosa*), a disease that greatly affects native Amazonian species of the genus Theobroma. Among the explants, the cotyledons were produced more callus tissues in culture medium.

For açaizeiro, [12] reported the conversion can be in vitro isolated zygotic embryos from mature seeds and seedlings complete normal requiring the presence of NAA and BA in the culture medium. The concentration of 2.68 mM NAA combined with 1.11, 1.55 or 2.22 mM BAP promoted the best growth of the seedling shoot. The authors also found that required the presence of NAA and BA in the culture medium for the conversion of zygotic embryos and the early growth of seedlings grown in vitro. The same authors in studies on direct somatic embryogenesis using zygotic embryos of açai, satisfactory results, with a halving of the amount of nutrients in MS medium in the absence of fitoregulators.

## **5.** *In vitro* **zygotic embryo culture**

156 Recent Advances in Plant in vitro Culture

**4. Somatic embryogenesis**

laboratory space.

BAP.

[26,27] and murici [30].

favored the rooting and callus formation.

the number of shoots and leaves in Inga tree explants.

plant, through a series of stages similar to zygotic embryogenesis.

species.

During the reproductive stage of shoots *in vitro*, the culture medium's composition varies with the species and the type of tissue or organ used as an explant. MS is most commonly used in the micropropagation of plant species. However, the Woody Plant Medium (WPM) [37] has been used successfully in woody species, including native Amazonian fruit tree

When using growth regulators to induce new shoot growth, native plants generally respond better to a culture medium supplemented with low doses of auxins and/or cytokinins. In [38] obtained excellent results in nance trees using different benzyladenine (BAP) doses for micropropagation. However, high BAP concentrations (above 4.0 mg L-1) were inefficient in inducing the growth of axillary shoots in nodal segments. In [30] found that BAP reduced

In somatic embryogenesis, somatic cells or tissues to develop the formation of a complete

Most systems via somatic embryogenesis is indirect, in which somatic embryogenesis is induced and maintained through the multiplication. The advantage of this method is that large quantities of somatic embryos can be formed with minimal manipulation and

This process of plant regeneration was successfully achieved in cocoa [16, 17], cupuaçu

In indirect somatic embryogenesis, the formation of pro-embryogenic callus is the first step in obtaining somatic embryos and is usually obtained with the culture of embryonic or juvenile explants in medium supplemented with auxin, especially 2,4-D and TDZ [39].

In [31] can be observed callus induction in shoot apices of peach palm. The highest percentage of induction was 60%, obtained by combining 10.0 mg L-1 2,4-D and 3.0 mg L-1

Some studies have shown the ability of different cupuaçu explants to form callus. In [27] studied the induction of somatic embryogenesis in explants of cupuaçu, concluded that the hypocotyl region proved to be the most responsive of the embryonic axis, forming calluslooking white and bright. The MS medium supplemented with 2,4-D promoted the formation of large callus. Similar results were found by [24], studying the effect of auxin concentration and liquid medium on the development of calluses cupuaçu. The authors observed that the combination of NAA and 2,4-D induced callus formation and root formation in hypocotyl segments, and coconut water in medium without growth regulators

In [40] also studied the induction of somatic embryogenesis in explants cupuaçu and observed that the callus appeared as a process influenced by genotype. The staminode is *In vitro* plants require a source of exogenous energy for carrying out the photosynthesis. Sucrose has been used more carbon source being present in fruit culture media at concentrations ranging native 20-40 g L-1 [26]. In [9] found that immature embryos murmuru showed higher germination in medium supplemented with 30 g L-1 sucrose. For embryos obtained from ripe fruits, 15 g L-1 sucrose in the medium was sufficient for it to reach the best germination rates. These results confirm the hypothesis that depending on the species and stage of development of embryos, the presence of carbohydrate in the medium may be in minute concentrations, or even not necessary because of embryos of many species use the energy required for germination in vitro from their own reserves of the embryo [42].

Embryos younger typically require higher concentrations of carbohydrates in the culture medium to sustain germination [43, 44]. In [9] have obtained best results using 3% sucrose in the development of embryonic axes cupuaçu, in accordance with [16, 18, 19 and 45] in studies performed with *Theobroma cacao*.

## **6.** *In vitro* **germination of pollen grains**

One of the techniques used to obtain new varieties is controlled hybridization in the field and subsequent evaluation of the progenies. Thus, to obtain success in breeding is important to catch up before going to the field the viability of pollen grains [46]. Thus, for the application of artificial pollination techniques, knowledge of the time of pollen viability and stigma receptivity is crucial for a successful fertilization of the flower [47]. However, the germination of the pollen grain depends on several factors, such as osmotic pressure, concentration and type of sugar, consistency, temperature, humidity, presence of enzymes and phytohormones in the middle [48, 46]. In native fruits, especially those species whose domestication process is more advanced, there are few studies in this line of research.

Tissue Culture Techniques for Native Amazonian Fruit Trees 159

recalcitrant seeds, low germination potential, and exclusively vegetative propagation, as

Ovary cultures are important in selective plant breeding. Obtaining plants from haploid tissues and subsequently doubling the chromosome number through colchicine treatment produces homozygous strains quickly, eliminating the multiple generations of selfing

Producing synthetic seeds from somatic embryos offers various advantages for propagating native fruit tree species, including year-round production, while avoiding the risk of losses from adverse weather, biomass degradation, pests, disease, and low production years. Moreover, synthetic seed technology enables more secure maintenance of the clonal identity of the material under laboratory conditions. Synthetic seeds can be sown directly into the

Protoplast fusion in somatic plant cells has great potential for combining genomes from sexually incompatible species. Somatic hybrids (artificial polyploids) of different species or genera are obtained through protoplast fusion and the subsequent regeneration of plants.

Although there are eminent importance of the native fruits of the Amazon, it appears that only a few studies with applications of tissue culture techniques in the domestication and breeding of these species. The works are practically concentrated in developing protocols for the establishment of *in vitro* plants, plus a few studies with somatic embryogenesis, immature embryo rescue protocols and studies of the germination of pollen grains to

Despite the reality and effectiveness of the techniques of tissue culture, it is important that some basic difficulties must be overcome to implement satisfactory in woody fruit trees, as is the case of native Amazon. The phenolic oxidation disinfestation and represent the most serious problems during the establishment of in vitro culture of explants woody

Suitable types of explants removed at appropriate times can help by having a lower content of endogenous phenols in tissues and, thus, less oxidation. Likewise, minor physical and chemical damage at the time of excision and pest may help reduce the problem of oxidation. Furthermore, the addition of antioxidant compounds as cysteine, ascorbic acid and adsorbents such as activated carbon and PVP can be critical in preventing oxidation, which is more pronounced in the initial stages of cultivation [53]. Additonally, with cultivation at low light intensities, and frequent replacement of the culture medium, the chances of success in the establishment and cultivation of explants of woody species are

field, thus eliminating the need for acclimatization structures, sowing, and nurseries.

These polyploids can be used as rootstocks in fruit production.

well as in endangered species [51, 52].

**8. Final thoughts**

support breeding programs.

species.

quite high.

required in conventional selective breeding.

In [49] Oliveira et al. (2001), studying the viability of pollen *in vivo* and *in vitro* staining method in genotypes of açai, the success observed in *in vitro* pollen germination of different cultivars. The authors found that pollen grains in vivo assai, drawn from floral buds and newly opened, exhibit high viability, being higher in the second stage of evaluation. With respect to time of storage at -100ºC, in vitro pollen showed a reduction in viability with increasing storage time. We also found that the viability of pollen grains varied with genotype and stage evaluated. With this study, the authors still recommend that the pollen of genotypes can be used for controlled pollination without fertilization damages, including, may remain stored for up to one year of storage under the conditions tested in that work.

In [50] tested four concentrations of galactose, glucose, lactose and sucrose, with or without boric acid, for the germination of pollen from cubiuzeiro (*Solanum topiro* Humb. & Bonpl.) and cupuaçu (*Theobroma grandiflorum* Willd. Ex. Spreng. Schummann). The authors found that higher germination rates occurred in 10, 15 and 20% sucrose after 25 hours, no effect of boric acid for the two species.

In [48] in their studies to obtain high levels of pollen from *Theobroma grandiflorum* found that pollen from one flower is not uniform. Therefore, it is recommended to use all anthers of a flower to prepare a sample. It is not necessary to have more than 300 pollen grains per sample. The stage at which the button is to be collected for the purpose of artificial pollination or germination and when all are detached sepals, petals with the extended length, style, stigma and exposed about two hours after the onset of anthesis (Step E) The pollen collected should be used, preferably up to two hours after it is collected, where there was more viable. The author also recommends the optimal medium for pollen germination cupuaçu should consist of 5% lactose, 0.01 H3BO3 and 1% agar, pH 6.1. The pollen collected cupuaçuazeiro button at the stadium and can remain viable up to 72 hours after the plant collected and stored at ambient conditions on the button, but at the end of this period, viability is very low, about 5%.

## **7. Possible tissue culture techniques for native Amazonian fruit trees**

There is a myriad of tissue culture techniques for native Amazonian fruit trees. However, this important technology has been seldom used.

Tissue culture techniques can be used for *in vitro* conservation, reproduction, exchange, and genetic resource conservation. These techniques are particularly useful in species that have recalcitrant seeds, low germination potential, and exclusively vegetative propagation, as well as in endangered species [51, 52].

Ovary cultures are important in selective plant breeding. Obtaining plants from haploid tissues and subsequently doubling the chromosome number through colchicine treatment produces homozygous strains quickly, eliminating the multiple generations of selfing required in conventional selective breeding.

Producing synthetic seeds from somatic embryos offers various advantages for propagating native fruit tree species, including year-round production, while avoiding the risk of losses from adverse weather, biomass degradation, pests, disease, and low production years. Moreover, synthetic seed technology enables more secure maintenance of the clonal identity of the material under laboratory conditions. Synthetic seeds can be sown directly into the field, thus eliminating the need for acclimatization structures, sowing, and nurseries.

Protoplast fusion in somatic plant cells has great potential for combining genomes from sexually incompatible species. Somatic hybrids (artificial polyploids) of different species or genera are obtained through protoplast fusion and the subsequent regeneration of plants. These polyploids can be used as rootstocks in fruit production.

## **8. Final thoughts**

158 Recent Advances in Plant in vitro Culture

boric acid for the two species.

viability is very low, about 5%.

this important technology has been seldom used.

to catch up before going to the field the viability of pollen grains [46]. Thus, for the application of artificial pollination techniques, knowledge of the time of pollen viability and stigma receptivity is crucial for a successful fertilization of the flower [47]. However, the germination of the pollen grain depends on several factors, such as osmotic pressure, concentration and type of sugar, consistency, temperature, humidity, presence of enzymes and phytohormones in the middle [48, 46]. In native fruits, especially those species whose

domestication process is more advanced, there are few studies in this line of research.

In [49] Oliveira et al. (2001), studying the viability of pollen *in vivo* and *in vitro* staining method in genotypes of açai, the success observed in *in vitro* pollen germination of different cultivars. The authors found that pollen grains in vivo assai, drawn from floral buds and newly opened, exhibit high viability, being higher in the second stage of evaluation. With respect to time of storage at -100ºC, in vitro pollen showed a reduction in viability with increasing storage time. We also found that the viability of pollen grains varied with genotype and stage evaluated. With this study, the authors still recommend that the pollen of genotypes can be used for controlled pollination without fertilization damages, including, may remain stored for up to one year of storage under the conditions tested in that work.

In [50] tested four concentrations of galactose, glucose, lactose and sucrose, with or without boric acid, for the germination of pollen from cubiuzeiro (*Solanum topiro* Humb. & Bonpl.) and cupuaçu (*Theobroma grandiflorum* Willd. Ex. Spreng. Schummann). The authors found that higher germination rates occurred in 10, 15 and 20% sucrose after 25 hours, no effect of

In [48] in their studies to obtain high levels of pollen from *Theobroma grandiflorum* found that pollen from one flower is not uniform. Therefore, it is recommended to use all anthers of a flower to prepare a sample. It is not necessary to have more than 300 pollen grains per sample. The stage at which the button is to be collected for the purpose of artificial pollination or germination and when all are detached sepals, petals with the extended length, style, stigma and exposed about two hours after the onset of anthesis (Step E) The pollen collected should be used, preferably up to two hours after it is collected, where there was more viable. The author also recommends the optimal medium for pollen germination cupuaçu should consist of 5% lactose, 0.01 H3BO3 and 1% agar, pH 6.1. The pollen collected cupuaçuazeiro button at the stadium and can remain viable up to 72 hours after the plant collected and stored at ambient conditions on the button, but at the end of this period,

**7. Possible tissue culture techniques for native Amazonian fruit trees**

There is a myriad of tissue culture techniques for native Amazonian fruit trees. However,

Tissue culture techniques can be used for *in vitro* conservation, reproduction, exchange, and genetic resource conservation. These techniques are particularly useful in species that have Although there are eminent importance of the native fruits of the Amazon, it appears that only a few studies with applications of tissue culture techniques in the domestication and breeding of these species. The works are practically concentrated in developing protocols for the establishment of *in vitro* plants, plus a few studies with somatic embryogenesis, immature embryo rescue protocols and studies of the germination of pollen grains to support breeding programs.

Despite the reality and effectiveness of the techniques of tissue culture, it is important that some basic difficulties must be overcome to implement satisfactory in woody fruit trees, as is the case of native Amazon. The phenolic oxidation disinfestation and represent the most serious problems during the establishment of in vitro culture of explants woody species.

Suitable types of explants removed at appropriate times can help by having a lower content of endogenous phenols in tissues and, thus, less oxidation. Likewise, minor physical and chemical damage at the time of excision and pest may help reduce the problem of oxidation. Furthermore, the addition of antioxidant compounds as cysteine, ascorbic acid and adsorbents such as activated carbon and PVP can be critical in preventing oxidation, which is more pronounced in the initial stages of cultivation [53]. Additonally, with cultivation at low light intensities, and frequent replacement of the culture medium, the chances of success in the establishment and cultivation of explants of woody species are quite high.

Another important aspect to be highlighted concerns the small number of fruit species native to the Amazon that has been the subject of study and application of techniques of tissue culture. These species can be summed up pretty much to those whose breeding programs are in advanced stage. Thus, it appears that there is still vast field of study to be explored and a significant deficiency of techniques and information that will enable progress in the domestication and breeding of these species.

Tissue Culture Techniques for Native Amazonian Fruit Trees 161

[7] Luis ZG. Propagação in vitro e caracterização anatômica de gemas adventíceas e embriões somáticos de murici (Byrsonima basiloba Juss., Malpighiaceae). Dissertation.

[8] Sudam DRN, Genamaz CT. Workshop biodiversidade de fruteiras com potencial sócio-

[9] Pereira JES, Maciel TMS, Costa FHS, Pereira MAA. Germinação *in vitro* de embriões zigóticos de murmuru (*Astrocaryum ulei*). Ciência Agrotécnica 2006; 30(2): 251-256. [10] Pasqual, M. Introdução à cultura de tecidos: fundamentos básicos*.* Lavras:

[11] Borém, A. Biotecnologia Aplicada ao Melhoramento de Espécies Silvestres. In: BORÉM, A.; LOPES, M. T. G.; CLEMENT, C. R. Domesticação e Melhoramento de Espécies

[12] Ledo AS, Lameira AO, Benbadis AK, Menezes IC, Ledo CAS, Oliveira MSP. Cultura *in vitro* de embriões zigóticos de açaizeiro. Revista Brasileira de Fruticultura 2001;

[13] Souza JÁ. Efeito do tipo de ramo e do regime de luz fornecido à planta matriz no estabelecimento *in vitro* de araçazeiro cv. "Irapuã". Ciência Rural 2006; 36(6):1920-

[14] Rodrigues EF. Desenvolvimento do eixo embrionário in vitro e calogênese de cupuaçu (*Theobroma grandiflorum* (Willd. ex Spreng.) Schum.) e estabelecimento do ápice caulinar de bacuri (Platonia insignis Martius). Thesis. Universidade Estadual

[15] Ferreira MGR, Santos MRA, Santos ER, Rocha JF, Correia AO. Desinfestação de explantes radiculares de bacurizeiro (*Platonia insignis* Mart.). Saber Científico 2009b;

[16] Duhem K, Le Mercier N. Données nouvelles surl' induction et le développement d' embryons somatiques chez *Theobroma cacao* L. Café Cacao Thé 1989; 33(1):9-14. [17] Söndahl MR, Liu S, Bellato C, Bragin A. Cacao somatic embryogenesis. Acta

[18] Janick J, Whipkey A. Axillary proliferation of shoots from cotyledonary nodal tissue of

[19] Kononowicz AK, Janick J. *In vitro* development of zygotic embryos of *Theobroma cacao*. Journal of the American Society of Horticultural Science 1984; 109(2):266-269. [20] Medeiros CPC. Indução *in vitro* de respostas morfogenéticas em explantes nodais de cajazeira (*Spondias mombin* L.). Dissertation, Universidade Federal do Ceará;

[21] Kikuchi TYP, Nunes HCB, Mota MGC, Vieira IMS, Ribeiro SI, Corrêa MLP. Assepsia para sementes de camu-camu (*Myrciaria dubia* (H.B.K) Mc vaugh) cultivadas *in vitro*. In:

Anais do IV Encuentro Latinoamericano de Biotecnologia Vegetal; 2001.

econômico na Amazônia: 24-26 August 1999; relatório final. Belém; 2000.

Amazônicas. Ed. UFV. Viçosa: UFV; 2009, p117-163.

Universidade de Brasília; 2008.

UFLA/FAEPE; 2001.

23(3):468-472.

Paulista; 2000.

Horticulturae 1993; 336:245-248.

cacao. Revista *Theobroma* 1985; 15:125-131.

2(2):56-62.

1999.

1922.

## **9. Conclusion**

Although native Amazonian fruit tree species are important, few studies on the use of tissue culture techniques for domesticating and improving these species exist. Research has instead focused on developing protocols for the *in vitro* establishment of plants, and some studies have examined somatic embryogenesis, immature embryo rescue, and germination protocols for pollen grains as a basis for selective breeding programs.

## **Author details**

Moacir Pasqual, Joyce Dória Rodrigues Soares and Filipe Almendagna Rodrigues *Federal University of Lavras (UFLA), Department of Agriculture, Lavras/MG, Brasil*

Edvan Alves Chagas *Brazilian Agricultural Research Corporation (EMBRAPA), Distrito industrial, Boa Vista-RR, Brasil*

## **10. References**

[7] Luis ZG. Propagação in vitro e caracterização anatômica de gemas adventíceas e embriões somáticos de murici (Byrsonima basiloba Juss., Malpighiaceae). Dissertation. Universidade de Brasília; 2008.

160 Recent Advances in Plant in vitro Culture

**9. Conclusion**

**Author details**

Edvan Alves Chagas

**10. References**

9(2):221-230.

Universidade Federal de Pelotas; 2004.

Horticulture, 12-17 October 2008, Vitória; 2008.

Another important aspect to be highlighted concerns the small number of fruit species native to the Amazon that has been the subject of study and application of techniques of tissue culture. These species can be summed up pretty much to those whose breeding programs are in advanced stage. Thus, it appears that there is still vast field of study to be explored and a significant deficiency of techniques and information that will enable

Although native Amazonian fruit tree species are important, few studies on the use of tissue culture techniques for domesticating and improving these species exist. Research has instead focused on developing protocols for the *in vitro* establishment of plants, and some studies have examined somatic embryogenesis, immature embryo rescue, and germination

*Brazilian Agricultural Research Corporation (EMBRAPA), Distrito industrial, Boa Vista-RR, Brasil*

[1] Fernandes ARF, Carvalho J G, Melo PC. Efeito do fósforo e do zinco sobre o crescimento de mudas do cupuaçuzeiro (*Theobroma Grandiflorum Schum*.) Cerne 2003;

[2] Franzon RC. Caracterização de mirtáceas nativas do sul do Brasil. Dissertation.

[3] Auler AS, Wang X, Edwards RL, Cheng H, Cristalli OS, Smart PL, Richards DA. Palaeoenvironments in semi-arid northeastern Brazil inferred from high precision mass spectrometric speleothem and travertine ages and the dynamics of South American

[4] Rebouças FS, Carmo DO, Almeirda NS, Dantas ACVL, Costa MAPC, Almeirda WAB. Germinação de cupuaçuzeiro *in vivo* e *in vitro*. In: XX Congresso Brasileiro de Fruticultura 54th Annual Meeting of the Interamerican Society for Tropical

rainforests. Speleogenesis and Evolution of Karst Aquifers 2004; 2:1-4.

[5] Mitra SK. Important *Myrtaceae* Fruit Crops. Acta Horticulturae 2010; 849:33-38.

[6] Anuário brasileiro de fruticultura. Panorama. Editora Gazeta; 2008.

progress in the domestication and breeding of these species.

protocols for pollen grains as a basis for selective breeding programs.

Moacir Pasqual, Joyce Dória Rodrigues Soares and Filipe Almendagna Rodrigues *Federal University of Lavras (UFLA), Department of Agriculture, Lavras/MG, Brasil*

[22] Gutiérrez-Rosati A, Rojas EI, Micky M, Rodriguez M. Avances em la introducción de genotipos de camu camu (*Myrciaria dubia* H.B.K. Mc Vaugh). Folia Amazônica 2006; p.57-60,

Tissue Culture Techniques for Native Amazonian Fruit Trees 163

[38] Nogueira RC. Indução de calos em explantes foliares de murici-pequeno. Ciência e

[39] Akram M, Aftab F. High frequency multiple shoot formation from nodal explants of teak (*Tectona grandis* L.) induced by thidiazuron. Propagation of Ornamental Plants

[40] Almeida CF, Rodrigues SM, Lemos OF. INDUÇÃO DE CALOS EMBRIOGÊNICOS EM EXPLANTES DE CUPUAÇU. In: XIV Seminário de Iniciação Científica da EMBRAPA.

[41] Roberts SA, Cameron RE. The effects of concentration and sodium hydroxide on the rheological properties of potato starch gelatinization. Carbohydrate Polymers 2002; 50:

[42] García JL, Troncoso J, Sarmiento R, Troncoso A. Influence of carbon source and concentration on the in vitro development of olive zygotic embryos and explants raised

[43] Chagas EA, Pasqual M, Dutra LF, Silva AB, Cazetta JO, Santos FC, Cardoso P. Desempenho de diferentes estádios embrionários no cultivo in vitro de embriões de

[44] Chagas EA, Pasqual M, Ramos JD, Cardoso P, Cazetta JO, Figueiredo MA. Development of globular embryos from the hybridization between 'Pêra Rio' sweet orange and 'Poncã' mandarin. Revista Brasileira de Fruticultura 2003b; 25(3):483-

[45] Söndahl MR, Liu S, Bellato C, Bragin A Cacao somatic embryogenesis. Acta

[46] Chagas EA, Pio R, Chagas PC, Pasqual M, Neto JEB. Composição do meio de cultura e condições ambientais para a germinação de porta-enxertos de pereira. Ciência Rural

[47] Ramos JD, Pasqual M, Pio LAS, Chagas EA, Pio R. Stigma receptivity and in vitro citrus pollen grains germination protocol and adjustment. Interciência 2008; 33:51-55. [48] Antonio IC. *In vitro* germination of cupuassu [Theobroma grandiflorum (Willdenow ex

[49] Oliveira MSP, Maués MM, Kalume MAA. Viabilidade de pólen in vivo e in vitro em

[50] Neves TS, Machado GME, Oliveira RP. Efeito do tipo e concentração de carboidratos e ácido bórico na germinação de grãos de pólen de cubiuzeiro e cupuaçuzeiro. In: Congresso brasileiro de fruticultura, 14.; reunião interamericana de horticultura tropical, 42.; Simpósio internacional de mirtáceas, 1., 1996, Curitiba. Resumos...

[51] Andrade SEM. Princípios da cultura de tecidos vegetais. Planaltina: Embrapa Cerrados;

Sprengel) Schumann] pollen grain. Científica 2004; 32(2):101-106.

genótipos de açaizeiro. Acta Botânica Brasileira 2001; 15(1):27-33.

Curitiba: Sociedade Brasileira de Fruticultura, 1996. p.213.

from them. Plant Cell, Tissue and Organ Culture, Dordrecht, 69:95-100, 2002.

'Pêra Rio' x 'Poncã'. Revista Brasileira de Fruticultura 2003a; 25(3): 523-525.

Agrotecnologia 2007; 31(2):366-370.

Embrapa Amazônia Oriental, Belém; 2010.

Horticulturae, Wageningen, 336:245-248, 1993.

2008; 8(2):72-75.

2446-2455.

488.

2002.

2010; 40: 261-266.

[38] Nogueira RC. Indução de calos em explantes foliares de murici-pequeno. Ciência e Agrotecnologia 2007; 31(2):366-370.

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p.57-60,

[22] Gutiérrez-Rosati A, Rojas EI, Micky M, Rodriguez M. Avances em la introducción de genotipos de camu camu (*Myrciaria dubia* H.B.K. Mc Vaugh).  Desinfestação *in vitro* de segmentos nodais de camu-camu. in: XXI

[24] Ferreira MGR, Cárdenas FEN, Carvalho CHS, Carneiro AA, Damião Filho CF. Desenvolvimento de calos em explantes de cupuaçuzeiro (*Theobroma grandiflorum* Schum) em função da concentração de auxinas e do meio líquido. Revista Brasileira de

[25] Venturieri GA, Venturieri GC. Calogênese do híbrido *Theobroma grandiflorum* x

[26] Ferreira MGR, Cárdenas FHN, Carvalho CHSC, Carneiro AA, Dantas Filho CF. Resposta de eixos embrionários de cupuaçu (*Theobroma grandiflorum* Schum.) à concentração de sais, doses de sacarose e renovação do meio de cultivo. Revista

[27] Ferreira MGR, Cárdenas FEN, Carvalho CHS, Carneiro AA, Filho CFD. Indução de calos embriogênicos em explantes de cupuaçuzeiro. Revista Brasileira de Fruticultura

[28] Ledo AS, Lameira AO, Benbadis AK. Explantes de cupuaçuzeiro submetidos a diferentes condições de cultura *in vitro*. Revista Brasileira de Fruticultura 2002.

[29] Ferreira MGR, Santos MRA, Bragado ACR. Propagação *in vitro* de cupuaçuzeiro:

[30] Stein VC. Organogênese direta em explantes de ingazeiro (*Inga vera* Willd. subsp. *affinis*

[31] Santos MRA, Ferreira MGR, Correira AOC, Rocha JF. *In vitro* establishment and

[32] Bastos LP. Cultivo *in vitro* de mangabeira (*Hancornia speciosa*). Revista Brasileira de

[33] Teixeira JB. Limitações ao processo de cultivo *in vitro* de espécies lenhosas. Brasília:

[34] Sato AY, Dias HCT, Andrade LA, Souza VC. Micropropagação de Celtis sp: controle da

[35] Erig CA, Schuch MW. Estabelecimento *in vitro* de plantas de marmeleiro (*Cydonia oblonga* Mill.) cultivares MC, Adans e Portugal. Ciência Rural 2003; 8(2):107-115. [36] Murashige T, Skoog FA. revised medium for rapid growth and bioassays with tabacco

[37] Lloyd G, Mccown B. Commercially feasible micropropagation of montain laurel, *Kalmia latifolia*, by use of shoot tip culture. Com. Proc. Int. Plant Prop. Soc. 1981; 30:421-327.

desinfestação de explantes florais. Saber Científico 2009a; 2(2):37-44.

(DC.) T.D. Penn.). Revista Brasileira de Biociências 2007; 5(2):723-725.

callogenesis in shoot tips of peach palm. Revista Caatinga 2010; 23(1):40-44.

*T.obovatum* (Sterculiaceae). Acta Amazônica 2004; 34(4):507–511.

Congresso Brasileiro de Fruticultura, Natal; 2010.

Brasileira de Fruticultura 2002; 24(1):246-248.

Fruticultura 2001; 23(1):473-476.

Biociências 2007; 5(2):1122-1124.

Embrapa-Recursos Genéticos e Biotecnologia; 2001.

cantaminação e oxidação. Cerne 2001; 7(2):117–123.

tissue culture. Physiologia Plantarum 1962; 15:473-497.

2006; 26(2):372-374.

24(3):604-607.

[52] Engelmann F, Engels JMM. Technologies and Strategies for *ex situ* conservation. In: ENGELS, J. M. M. et al. (Eds.). Managing Plant Genetic Diversity. Oxford: IPGRI; 2002, p89-103.

**Chapter 9**

© 2012 Lima et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Lima et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Polyamines, Gelling Agents**

Additional information is available at the end of the chapter

the first practical results reported by White in 1934 [1].

http://dx.doi.org/10.5772/51028

**1. Introduction**

of a new individual.

**in Tissue Culture, Micropropagation**

**of Medicinal Plants and Bioreactors**

Giuseppina Pace Pereira Lima, Renê Arnoux da Silva Campos, Lilia Gomes Willadino, Terezinha J.R. Câmara and Fabio Vianello

Currently, tissue cultures of species of agricultural importance have wide applicability in industrial production processes. Tissue culture is a name given to a set of techniques that allow the regeneration of cells, tissues and organs of plants, from segments of plant organs or tissues, using nutrient solutions in aseptic and controlled environment. This regeneration is based on the totipotency of plant cells. Totipotency is a capability indicating that plant cells, in different times, may express the potential to form a new multicellular individual. Tissue culture appears to be a good alternative to conventional propagation, requiring less physical space, with high multiplication rate, without incidence of pests and diseases during cultivation, and enabling higher control of the variables involved. Thus, in the *in vitro* environment, with the required stimuli and appropriate conditions, different cell types express different behaviors, possibly leading to cell multiplication and differentiation into a specific tissue, characterized by a form and a function, which may lead to the regeneration

The discovery of this feature in plant cells is indistinguishable from the first studies on tissue culture in the early twentieth century by Heberlandt in 1902, which were followed by

Over the years, various tissue culture techniques were developed, being micropropagation, meristem culture and somatic embryogenesis, the most used. The degree of success of any technology employing cultured cells, plant tissues or organs, is mainly dependent on the choice of the nutritional components and growth regulators which control, in a large extent, the developmental *in vitro* pattern. Therefore, it is crucial to evaluate the nutritional and

[53] Santos RB, Paiva R, Paiva PDO, Santana JRF Problemas no cultivo *in vitro*: cultura de tecidos. Paiva e Paiva, UFLA, Lavras, MG. 9:73-79, 2001.

## **Polyamines, Gelling Agents in Tissue Culture, Micropropagation of Medicinal Plants and Bioreactors**

Giuseppina Pace Pereira Lima, Renê Arnoux da Silva Campos, Lilia Gomes Willadino, Terezinha J.R. Câmara and Fabio Vianello

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51028

## **1. Introduction**

164 Recent Advances in Plant in vitro Culture

p89-103.

[52] Engelmann F, Engels JMM. Technologies and Strategies for *ex situ* conservation. In: ENGELS, J. M. M. et al. (Eds.). Managing Plant Genetic Diversity. Oxford: IPGRI; 2002,

[53] Santos RB, Paiva R, Paiva PDO, Santana JRF Problemas no cultivo *in vitro*: cultura de

tecidos. Paiva e Paiva, UFLA, Lavras, MG. 9:73-79, 2001.

Currently, tissue cultures of species of agricultural importance have wide applicability in industrial production processes. Tissue culture is a name given to a set of techniques that allow the regeneration of cells, tissues and organs of plants, from segments of plant organs or tissues, using nutrient solutions in aseptic and controlled environment. This regeneration is based on the totipotency of plant cells. Totipotency is a capability indicating that plant cells, in different times, may express the potential to form a new multicellular individual. Tissue culture appears to be a good alternative to conventional propagation, requiring less physical space, with high multiplication rate, without incidence of pests and diseases during cultivation, and enabling higher control of the variables involved. Thus, in the *in vitro* environment, with the required stimuli and appropriate conditions, different cell types express different behaviors, possibly leading to cell multiplication and differentiation into a specific tissue, characterized by a form and a function, which may lead to the regeneration of a new individual.

The discovery of this feature in plant cells is indistinguishable from the first studies on tissue culture in the early twentieth century by Heberlandt in 1902, which were followed by the first practical results reported by White in 1934 [1].

Over the years, various tissue culture techniques were developed, being micropropagation, meristem culture and somatic embryogenesis, the most used. The degree of success of any technology employing cultured cells, plant tissues or organs, is mainly dependent on the choice of the nutritional components and growth regulators which control, in a large extent, the developmental *in vitro* pattern. Therefore, it is crucial to evaluate the nutritional and

© 2012 Lima et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Lima et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

metabolic needs of cells and tissues of each species to be cultivated. In general, the choice of the medium is carried out taking into account, in addition to these needs, the purpose of the *in vitro* cultivation, maximizing plant development.

Polyamines, Gelling Agents in Tissue Culture, Micropropagation of Medicinal Plants and Bioreactors 167

High levels of free putrescine were correlated with the ease of cultures to reach stabilization, as observed with juvenile tissues. Adult tissues, containing low levels of putrescine, were difficult to stabilize in culture [10]. Furthermore, it has been reported [11] that the application of putrescine may reduce the production of unwanted ethylene and can enhance

The exogenous application of polyamines has shown positive effect in the micropropagation of several species, for example, in buds from newly developed shoots, obtained from forced outgrowth of mature field-grown hybrids of hazelnut trees (*Corylus avellana* L.), cultured *in vitro* on MS medium [3] and on a modified Driver and Kuniyuki medium [12] [DKW] DKW/Juglans containing 6.7 μM, 11.1 μM or 15.5 μM N-6-benzyladenine (BA), supplemented with or without a combination of polyamines (0.2 mM putrescine + 0.2 mM spermidine + 0.05 mM spermine). The effects of culture medium and BA were found to be insignificant on explant response. Polyamines were found to have a strong effect on both shoot elongation and on the number of buds per shoot. Polyamines increased both the mean shoot elongation by 83% and the number of buds per shoot by 41%, compared to controls. In the presence of polyamines, shoot elongation continued up to 4.0 cm, while in the absence, the shoot elongation reached only 2.0 cm. Moreover, the results indicate that polyamines added to culture medium can improve the stabilization of cultures and enhance the

The effects of exogenous polyamines on somatic embryo formation in carrot (*Daucus carota* L.) cells were investigated [14]. The results showed an enhancement of somatic embryo formation following the addition of spermine. However, this effect was not always reproducible. Spm addition increased the DNA content in cells, regardless the development and the lag time of

Many studies have shown the beneficial effects of applying various polyamines on the rooting process. Polyamines have been shown to be key factors, in conjunction with auxins, in the process of adventitious rooting production [15,16]. The *in vitro* rooting of wild-type tobacco (*Nicotiana tabacum* cv. Xanthi) shoots was promoted by polyamines in the absence of any other growth regulator and was inhibited by two inhibitors of polyamine metabolism [17]. According to authors, some arguments causally implicated Put in the inductive rooting phase of poplar: i) the transient increase of Put did not occur in the non-rooting cuttings, ii) it was observed only in the basal rooting zone, iii) inhibitors of Put biosynthesis, such as DFMO and DFMA (α-α-difluoromethylornithine and difluoromethylarginine, respectively) applied prior to, or at the beginning of, the inductive phase, inhibited rooting [18], iv) an inhibitor of Put conversion into Spd and Spm, CHA (cyclohexylamine, an inhibitor of Spd synthase), promoted the accumulation of endogenous Put and favored rooting in the absence of auxin, v) the administration of Put, prior to, or at the beginning of, the inductive phase, stimulated rooting [19]. Some studies correlated this effect with PAs antioxidant properties, such as during the morphogenesis of *Hemerocallis sp*. It was verified that exogenous Put, alone (10 μM Put), with Spd (10 μM Spd + 10 μM Put) or with Spm (10 μM Spm + 10 μM Put), induced the best results, regarding both the number and the height of

somatic embryo formation, and suppressed the protein secretion from cells.

morphogenesis.

morphogenic capacity of mature explants [13].

In general, the culture medium is composed of inorganic salts, reduced nitrogen compounds, a carbon source, vitamins and amino acids. Other compounds may be added for specific purposes, such as plant growth regulators, gelling agents, organic nitrogen compounds, organic acids and plant extracts.

Throughout the history of tissue culture, various kinds of culture media have been developed. However, the MS (Murashige & Skoog) medium [2] is the most widely used for the regeneration of dicots, and therefore it has a great importance in the applications of tissue culture in agriculture.

## **2. The use of polyamines as growth regulators**

Many plant growth regulators have been used in vitro. Generally, literature reports on the use of auxins, cytokinins and gibberellins and different balances of auxins and cytokinins to encourage the development of specific organs. An auxin/cytokinin ratio of 10 induces the rapid growth of undifferentiated callus, a ratio of 100 leads to root development and a ratio of 4 favors development of shoots [3].

However, some studies report on the use of polyamines, as growth regulators. Polyamines (PAs) are low molecular weight aliphatic amines, implicated in various physiological and developmental processes in plants [4], such as growth regulation, cell division and differentiation, and also in the plant response to various sources of stresses. The exogenous application of PAs has been used by many researchers to provide or enhance growth and cell division [4]. The most common types of PAs are spermidine (SPD), spermine (SPM), and their diamine precursor, putrescine (PUT).

In plants, the mechanisms regulating both the biosynthesis and degradation of polyamines are less studied than in other organisms. Some papers proposed the use of exogenous polyamines during the processes leading to *in vitro* plant and callus formation [5,6] and endogenous PAs concentration has been related to several organo-genetic processes[7].

Beneficial effects of polyamines on *in vitro* regeneration and somatic embryogenesis were documented in several crops. Promoter effect of PAs in the conversion of somatic embryos or shoot regeneration has been evidenced in several plant species. The exogenous addition of PAs, mainly SPD or PUT at 2.0 mM concentration, not only elevated the endogenous levels of PAs but also enhanced the frequency of conversion of protocorm-like-bodied (PLBs) to shoots in *Dendrobium huoshanense* [8]. On the other hand, spermidine showed no effect on *Dendrobium* "Sonia"*.* Among various tested polyamines, the maximum number of PLBs was produced with 0.4 mM putrescine treatment. The increase (1.0 mM) or decrease (0.2 mM) of SPD concentration caused a decrease in the production of PLBs. All treatments with spermidine and spermine resulted in the production of less number of PLBs than control. Thus, different responses to the application of different polyamines may occur [9]*.*

High levels of free putrescine were correlated with the ease of cultures to reach stabilization, as observed with juvenile tissues. Adult tissues, containing low levels of putrescine, were difficult to stabilize in culture [10]. Furthermore, it has been reported [11] that the application of putrescine may reduce the production of unwanted ethylene and can enhance morphogenesis.

166 Recent Advances in Plant in vitro Culture

tissue culture in agriculture.

of 4 favors development of shoots [3].

their diamine precursor, putrescine (PUT).

*in vitro* cultivation, maximizing plant development.

**2. The use of polyamines as growth regulators**

compounds, organic acids and plant extracts.

metabolic needs of cells and tissues of each species to be cultivated. In general, the choice of the medium is carried out taking into account, in addition to these needs, the purpose of the

In general, the culture medium is composed of inorganic salts, reduced nitrogen compounds, a carbon source, vitamins and amino acids. Other compounds may be added for specific purposes, such as plant growth regulators, gelling agents, organic nitrogen

Throughout the history of tissue culture, various kinds of culture media have been developed. However, the MS (Murashige & Skoog) medium [2] is the most widely used for the regeneration of dicots, and therefore it has a great importance in the applications of

Many plant growth regulators have been used in vitro. Generally, literature reports on the use of auxins, cytokinins and gibberellins and different balances of auxins and cytokinins to encourage the development of specific organs. An auxin/cytokinin ratio of 10 induces the rapid growth of undifferentiated callus, a ratio of 100 leads to root development and a ratio

However, some studies report on the use of polyamines, as growth regulators. Polyamines (PAs) are low molecular weight aliphatic amines, implicated in various physiological and developmental processes in plants [4], such as growth regulation, cell division and differentiation, and also in the plant response to various sources of stresses. The exogenous application of PAs has been used by many researchers to provide or enhance growth and cell division [4]. The most common types of PAs are spermidine (SPD), spermine (SPM), and

In plants, the mechanisms regulating both the biosynthesis and degradation of polyamines are less studied than in other organisms. Some papers proposed the use of exogenous polyamines during the processes leading to *in vitro* plant and callus formation [5,6] and endogenous PAs concentration has been related to several organo-genetic processes[7].

Beneficial effects of polyamines on *in vitro* regeneration and somatic embryogenesis were documented in several crops. Promoter effect of PAs in the conversion of somatic embryos or shoot regeneration has been evidenced in several plant species. The exogenous addition of PAs, mainly SPD or PUT at 2.0 mM concentration, not only elevated the endogenous levels of PAs but also enhanced the frequency of conversion of protocorm-like-bodied (PLBs) to shoots in *Dendrobium huoshanense* [8]. On the other hand, spermidine showed no effect on *Dendrobium* "Sonia"*.* Among various tested polyamines, the maximum number of PLBs was produced with 0.4 mM putrescine treatment. The increase (1.0 mM) or decrease (0.2 mM) of SPD concentration caused a decrease in the production of PLBs. All treatments with spermidine and spermine resulted in the production of less number of PLBs than control. Thus, different responses to the application of different polyamines may occur [9]*.*

The exogenous application of polyamines has shown positive effect in the micropropagation of several species, for example, in buds from newly developed shoots, obtained from forced outgrowth of mature field-grown hybrids of hazelnut trees (*Corylus avellana* L.), cultured *in vitro* on MS medium [3] and on a modified Driver and Kuniyuki medium [12] [DKW] DKW/Juglans containing 6.7 μM, 11.1 μM or 15.5 μM N-6-benzyladenine (BA), supplemented with or without a combination of polyamines (0.2 mM putrescine + 0.2 mM spermidine + 0.05 mM spermine). The effects of culture medium and BA were found to be insignificant on explant response. Polyamines were found to have a strong effect on both shoot elongation and on the number of buds per shoot. Polyamines increased both the mean shoot elongation by 83% and the number of buds per shoot by 41%, compared to controls. In the presence of polyamines, shoot elongation continued up to 4.0 cm, while in the absence, the shoot elongation reached only 2.0 cm. Moreover, the results indicate that polyamines added to culture medium can improve the stabilization of cultures and enhance the morphogenic capacity of mature explants [13].

The effects of exogenous polyamines on somatic embryo formation in carrot (*Daucus carota* L.) cells were investigated [14]. The results showed an enhancement of somatic embryo formation following the addition of spermine. However, this effect was not always reproducible. Spm addition increased the DNA content in cells, regardless the development and the lag time of somatic embryo formation, and suppressed the protein secretion from cells.

Many studies have shown the beneficial effects of applying various polyamines on the rooting process. Polyamines have been shown to be key factors, in conjunction with auxins, in the process of adventitious rooting production [15,16]. The *in vitro* rooting of wild-type tobacco (*Nicotiana tabacum* cv. Xanthi) shoots was promoted by polyamines in the absence of any other growth regulator and was inhibited by two inhibitors of polyamine metabolism [17]. According to authors, some arguments causally implicated Put in the inductive rooting phase of poplar: i) the transient increase of Put did not occur in the non-rooting cuttings, ii) it was observed only in the basal rooting zone, iii) inhibitors of Put biosynthesis, such as DFMO and DFMA (α-α-difluoromethylornithine and difluoromethylarginine, respectively) applied prior to, or at the beginning of, the inductive phase, inhibited rooting [18], iv) an inhibitor of Put conversion into Spd and Spm, CHA (cyclohexylamine, an inhibitor of Spd synthase), promoted the accumulation of endogenous Put and favored rooting in the absence of auxin, v) the administration of Put, prior to, or at the beginning of, the inductive phase, stimulated rooting [19]. Some studies correlated this effect with PAs antioxidant properties, such as during the morphogenesis of *Hemerocallis sp*. It was verified that exogenous Put, alone (10 μM Put), with Spd (10 μM Spd + 10 μM Put) or with Spm (10 μM Spm + 10 μM Put), induced the best results, regarding both the number and the height of differentiated shoots. The combination of the three polyamines in culture medium (10 μM SPD + 10 μM SPM + 10 μM PUT) induced the highest percentage of microplant formation, whereas the treatment with SPD and SPM (10 μM SPD + 10 μM SPM) led to the highest amount of necrotic tissue (65%). Spermine alone (10 μM Spm) was effective in controlling oxidative processes. In fact, oxidative stress during *in vitro* cultivation of plants occurs with high frequency [8].

Polyamines, Gelling Agents in Tissue Culture, Micropropagation of Medicinal Plants and Bioreactors 169

potato, cassava, maize and rice. Thus, the possibility to find native starch with specific

In early experiments, using maize starch as gelling agent, the growth and differentiation of cultured plant cells from tobacco and carrot have been increased. In a medium solidified with starch, cell dry weight increased more than three times with respect to cells grown in a

In India, several tissue culture studies were developed using gums and starches derived from tropical species, such as true sago palm (*Metroxylon sagu*) and "isubgol" (*Plantago ovata Forsk*.). Results revealed that these gelling agents were satisfactory in the micropropagation of chrysanthemum (*Dendrathema grandiflora* Tzvelev). Moreover, the cost of cultures was less than 5% of that of commercial agar, resulting in an alternative low-cost gelling agent for

Isubgol was also successfully used as gelling agent for culture media for *in vitro* germination, formation of aerial parts and roots of *Syzygium cuminii* and cultures of anthers in *Datura innoxia*. Cultures showed similar responses to those observed in medium solidified

Katira gum, derived from the bark of *Cochlospermum religiosum*, was used as gelling agent in the culture medium for micropropagation of *Syzygium cuminii* and for somatic embryogenesis of *Albizzia lebbeck*. Various combinations of agar (0.2-0.6%) and starch (1-3%)

Guar gum, derived from the endosperm of *Cyamopsis tetragonoloda* and locust bean gum from carob (*Ceratonia siliqua*) (composed of mannose:galactose ratios of 1.6:1 and 3.3:1, respectively) is commercially produced in great amounts. Others, such as tare and fenugreek gums, have also been used [28]. These gums were used as gelling agents for *in vitro* multiplication and regeneration as well for germination of *Linum usitatissimum* and *Brassica juncea*, for the multiplication of aerial parts of *Crateavea nurvala*, and their subsequent rooting, for the *in vitro* androgenesis of anthers on *Nicotiana tabacum*, and for somatic embryogenesis of callus cultures on *Calliandra twedii*. The media were gelled with 2, 3 or 4% gum, as compared to agar (0.9%), and for all species the morphogenetic responses

Xanthan gum has minimal change in viscosity over a wide temperature range, and presents good gelling ability in a broad pH range (3.2-9.8). Xanthan gum, a microbial desiccationresistant polysaccharide, is commercially produced by aerobic submerged fermentation of *Xanthomonas campestris*. It has a β-(1,4)-D-glucopyranose glucan backbone with side chains of (3,1)-α-linked D-mannopyranose-(2,1)-β-D-glucuronic acid-(4,1)-β-D-mannopyranose, on alternating residues. This gum, cream-colored, odorless, free flowing powder, hydrates rapidly in cold and hot water to give a reliable viscosity, even at low concentrations. It is highly resistant to enzymatic degradation, extremely stable over a wide pH range, and

properties is quite high in tropical regions.

medium gelled with agar [24].

with agar [26].

industrial scale micropropagation [25].

were added to increase the firmness of the medium [27].

were improved in the medium gelled with guar gum [29].

forms highly pseudoplastic aqueous solutions [30].

However, in some species, exogenous polyamines do not play a significant role on *in vitro* growth, such as in *Hancornia speciosa.* In this specie, the application of exogenous polyamines did not improve the growth of callus [20]. Conversely, in most studies it has been reported that the use of specific polyamines or polyamine combinations can enhance the performances of tissue culture in some plant species, especially in those with low rooting potential, browning and hyperhydricity.

## **3. Alternative gelling agents**

The *in vitro* cultivation of plant tissues is generally carried out in a solid or semi-solid nutrient medium, using gelling agents. Traditionally, agar is used, which is a polysaccharide extracted from seaweeds. This hydrocolloid is composed of agarobiose (3-β-Dgalactopyranosyl-(1,4)-3,6-anhydro-α-L-galactose) [21]. The main differences among different agar-products are due to the impurities, their level and composition, which can vary according to manufacturers. Agar has been widely used since it has convenient gelling properties and stability during tissue culture. In all media used for *in vitro* culture of plants, agar is the major source of unknown variations [22], besides it's a high costs.

Gums, such as gelan, produced by bacteria and commercialized under the name of Gel-Gro®, Gerlite® (Kelko, Merck) and Phyta-gel® (Sigma), are polysaccharides that do not contain contaminating materials. Moreover, these products are used in lesser amount per liter than agar, to obtain the same consistency. They are added to the medium at approximately one fourth the concentration of agar. Furthermore, they appear more transparent. Despite emerging as alternatives to agar, the high cost of these products still limits their use in commercial cultures. These polysaccharides are imported from North America and Europe and therefore this leads to increasing costs for further micropropagation applications.

An alternative for cost reduction is the partial replacement of some of these gelling agents with other polysaccharides. Starch is an inexpensive alternative among studied gelling agents, and its use may reduce the costs of tissue culture. Nevertheless, starch is hydrolyzed by plant amylolytic enzymes during the in vitro culture. To circumvent this occurrence, the increase of air exchange in the bottle and consequently the increase of evaporation of excess water, may reduce this drawback [23].

Tropical countries possess a lot of starchy, little studied, native species, whose characteristics could serve still unfilled market niches. It should be considered that, among five of the raw products used in world for starch production, four are of tropical origin: potato, cassava, maize and rice. Thus, the possibility to find native starch with specific properties is quite high in tropical regions.

168 Recent Advances in Plant in vitro Culture

potential, browning and hyperhydricity.

**3. Alternative gelling agents**

micropropagation applications.

water, may reduce this drawback [23].

high frequency [8].

differentiated shoots. The combination of the three polyamines in culture medium (10 μM SPD + 10 μM SPM + 10 μM PUT) induced the highest percentage of microplant formation, whereas the treatment with SPD and SPM (10 μM SPD + 10 μM SPM) led to the highest amount of necrotic tissue (65%). Spermine alone (10 μM Spm) was effective in controlling oxidative processes. In fact, oxidative stress during *in vitro* cultivation of plants occurs with

However, in some species, exogenous polyamines do not play a significant role on *in vitro* growth, such as in *Hancornia speciosa.* In this specie, the application of exogenous polyamines did not improve the growth of callus [20]. Conversely, in most studies it has been reported that the use of specific polyamines or polyamine combinations can enhance the performances of tissue culture in some plant species, especially in those with low rooting

The *in vitro* cultivation of plant tissues is generally carried out in a solid or semi-solid nutrient medium, using gelling agents. Traditionally, agar is used, which is a polysaccharide extracted from seaweeds. This hydrocolloid is composed of agarobiose (3-β-Dgalactopyranosyl-(1,4)-3,6-anhydro-α-L-galactose) [21]. The main differences among different agar-products are due to the impurities, their level and composition, which can vary according to manufacturers. Agar has been widely used since it has convenient gelling properties and stability during tissue culture. In all media used for *in vitro* culture of plants,

Gums, such as gelan, produced by bacteria and commercialized under the name of Gel-Gro®, Gerlite® (Kelko, Merck) and Phyta-gel® (Sigma), are polysaccharides that do not contain contaminating materials. Moreover, these products are used in lesser amount per liter than agar, to obtain the same consistency. They are added to the medium at approximately one fourth the concentration of agar. Furthermore, they appear more transparent. Despite emerging as alternatives to agar, the high cost of these products still limits their use in commercial cultures. These polysaccharides are imported from North America and Europe and therefore this leads to increasing costs for further

An alternative for cost reduction is the partial replacement of some of these gelling agents with other polysaccharides. Starch is an inexpensive alternative among studied gelling agents, and its use may reduce the costs of tissue culture. Nevertheless, starch is hydrolyzed by plant amylolytic enzymes during the in vitro culture. To circumvent this occurrence, the increase of air exchange in the bottle and consequently the increase of evaporation of excess

Tropical countries possess a lot of starchy, little studied, native species, whose characteristics could serve still unfilled market niches. It should be considered that, among five of the raw products used in world for starch production, four are of tropical origin:

agar is the major source of unknown variations [22], besides it's a high costs.

In early experiments, using maize starch as gelling agent, the growth and differentiation of cultured plant cells from tobacco and carrot have been increased. In a medium solidified with starch, cell dry weight increased more than three times with respect to cells grown in a medium gelled with agar [24].

In India, several tissue culture studies were developed using gums and starches derived from tropical species, such as true sago palm (*Metroxylon sagu*) and "isubgol" (*Plantago ovata Forsk*.). Results revealed that these gelling agents were satisfactory in the micropropagation of chrysanthemum (*Dendrathema grandiflora* Tzvelev). Moreover, the cost of cultures was less than 5% of that of commercial agar, resulting in an alternative low-cost gelling agent for industrial scale micropropagation [25].

Isubgol was also successfully used as gelling agent for culture media for *in vitro* germination, formation of aerial parts and roots of *Syzygium cuminii* and cultures of anthers in *Datura innoxia*. Cultures showed similar responses to those observed in medium solidified with agar [26].

Katira gum, derived from the bark of *Cochlospermum religiosum*, was used as gelling agent in the culture medium for micropropagation of *Syzygium cuminii* and for somatic embryogenesis of *Albizzia lebbeck*. Various combinations of agar (0.2-0.6%) and starch (1-3%) were added to increase the firmness of the medium [27].

Guar gum, derived from the endosperm of *Cyamopsis tetragonoloda* and locust bean gum from carob (*Ceratonia siliqua*) (composed of mannose:galactose ratios of 1.6:1 and 3.3:1, respectively) is commercially produced in great amounts. Others, such as tare and fenugreek gums, have also been used [28]. These gums were used as gelling agents for *in vitro* multiplication and regeneration as well for germination of *Linum usitatissimum* and *Brassica juncea*, for the multiplication of aerial parts of *Crateavea nurvala*, and their subsequent rooting, for the *in vitro* androgenesis of anthers on *Nicotiana tabacum*, and for somatic embryogenesis of callus cultures on *Calliandra twedii*. The media were gelled with 2, 3 or 4% gum, as compared to agar (0.9%), and for all species the morphogenetic responses were improved in the medium gelled with guar gum [29].

Xanthan gum has minimal change in viscosity over a wide temperature range, and presents good gelling ability in a broad pH range (3.2-9.8). Xanthan gum, a microbial desiccationresistant polysaccharide, is commercially produced by aerobic submerged fermentation of *Xanthomonas campestris*. It has a β-(1,4)-D-glucopyranose glucan backbone with side chains of (3,1)-α-linked D-mannopyranose-(2,1)-β-D-glucuronic acid-(4,1)-β-D-mannopyranose, on alternating residues. This gum, cream-colored, odorless, free flowing powder, hydrates rapidly in cold and hot water to give a reliable viscosity, even at low concentrations. It is highly resistant to enzymatic degradation, extremely stable over a wide pH range, and forms highly pseudoplastic aqueous solutions [30].

A gelling agent developed in Brazil (patent PI9003880-0 FAPESP/UNESP) was tested as an alternative to agar in the micropropagation of sweet potato (*Ipomoea batata*). The product consists of a mixture of starch from seeds of pigeonpea (*Cajanus cajan*) and cassava starch (*Manihot esculenta*) at a ratio of 2:3, being used as a gelling agent in the MS culture medium at a concentration of 7%. It was observed that this starch mixture increased the fresh weight of cultures when compared to microplants grown on agar. Thus, this starch mixture represents a good alternative for agar replacement in the micropropagation of sweet potato. Moreover, this substitution reduced by over 94% the final cost of the culture medium, demonstrating a high economic validity [25].

Polyamines, Gelling Agents in Tissue Culture, Micropropagation of Medicinal Plants and Bioreactors 171

In a study on the micropropagation of african violet, the cultivation of shoots in liquid medium, with cotton wad and different combinations of starch, semolina, potato powder as an alternative to agar, was tested. The highest frequency of regeneration was found in media containing agar (0.8%) or with a combination of starch, semolina, potato powder (2:1:1) and starch (6%) plus agar (0.4%). The maximum numbers shoots was produced in media containing agar (0.8%), the combination of starch (6%), plus agar (0.4%) and in liquid medium with cotton wad substrate. The best shoot proliferation took place in liquid medium with cotton substrate. The results showed that the combination of starch, semolina, potato powder (2:1:1) and 6% starch, plus 0.4% agar, can be suitable alternatives for agar alone in shoot regeneration step, but shoot number will result lower than in agar alone.

The type of medium (liquid or semi-solid) can directly affect the rooting process. Although the liquid medium positively influences the availability of water, nutrients, hormones and oxygen levels, the vast majority of protocols for micropropagation were established in semi-

Some other agar substitutes have been tested, such as corn starch mixtures (Gelrite®), used for shoot proliferation of apples, pears and red raspberries [34]. However, their use may induce some problems during micropropagation. Gelrite®, for example, causes hyperhydricity and, in some cases, vitrification on regenerated shoots [35]. Hyperhydric shoots are characterized by a translucent aspect due to a chlorophyll deficiency, a not very developed cell wall and high water content. The losses of up to 60% of cultured shoots or plantlets, due to hyperhydricity, have been reported in commercial plant micropropagation [36], reflecting the importance of this problem. The hyperhydricity of shoots was induced in *Prunus avium*, after four weeks multiplication cycles in Gelrite® [37]. This performance has been confirmed by other authors [38]. The vitrification symptoms (thick and translucent stems and leaves, wrinkled and curled leaves) were found on 100 % shoots, after 21 days

Besides the properties of these described substances, it is not always possible to use these agents. In studies with *Eucalyptus grandis* x *E. urophylla* in liquid medium containing aluminum, an acrylic blanket was used to support the shoots, because the addition of AlCl3.6H2O decreased medium pH (to around pH 3.5), affecting medium ionic balance, not

Medicinal and aromatic plants are of great importance for the pharmaceutical industry and traditional medicine in several countries. Just to emphasize the importance of medicinal plants in their various aspects for human health, data from the World Health Organization (WHO) indicated that 80% of the population uses them as basic drugs, in traditional

These options are cheaper than agar [32].

solid media [33].

culture.

allowing the gelation of agar [39].

**4. Micropropagation of medicinal species**

medicine, in the form of plant extracts or their bioactive compounds.

Another successful Brazilian experience deals with xyloglucans, extracted from the seeds of jatoba (*Hymenaea courbaril*) and mixed with agar to prepare a solid medium for the micropropagation of apples. The performance of this new mixture, composed of 0.4% agar and 0.2% xyloglucan (w/v), was verified on *Malus prunifolia Borkh* and cv. *Jonagored* (*Malus domestica*), and compared to the medium solidified with agar (0.6 %, w/v). The growth and the multiplication of shoots were higher in the modified medium. Furthermore, a lower incidence of hyperhydricity and a higher percentage of shoot rooting in the absence of auxin, were observed. When 0.25 mM indole-3-butyric acid (IBA) was added to both the media, the modified medium gave better results in terms of rooting percentage and root quality than the traditional agar medium [31].

The efficacy of the partial substitution of agar by galactomannans (GMs), obtained from seeds of *Cassia fastuosa* (cassia) and *Cyamopsis tetragonolobus* (guargum - a commercial GM), was tested in the micropropagation of strawberry (*Fragaria x ananassa* Duchesne cv. Pelican). GMs were mixed with agar in the proportion of 0.3/0.3 % (w/v) in MS medium, and the performances were compared with the behavior of the medium containing only agar (0.6% w/v). Strawberry shoots, grown in the modified medium, showed both an enhanced cell proliferation and a higher length of roots with respect to controls. These results showed that agar could be partially replaced by GMs, since experimental data confirmed a favorable interaction between the two polysaccharides.

Another study, involving guar gum extracted from the seeds of *Cassia fastuosa* or *Cyamopsis tetragonolobus,* mixed in equal proportions with agar to a final concentration of 0.3% (w/v) for each type of gelling agent, was carried out on the micropropagation of "Durondeau" pear (*Pyrus communis* L. cv. Durondeau). The production of multiple shoots and the formation of roots from shoots were compared with a control media, solidified with agar alone at a concentration of 0.6 % (w/v). In the medium solidified with the mixtures of agar/guar and agar/cassia GM, an increase in the number of regenerated shoots of 32 and 17%, respectively, was obtained. The modified media promoted both a higher number of roots and increased the rooting percentage. A maximum of 91% rooting was obtained in the medium solidified with the agar/cassia GM, containing 9.8 mM indole-3-butyric acid. With this medium, less callus formation at the base of the shoot was also observed [23].

In a study on the micropropagation of african violet, the cultivation of shoots in liquid medium, with cotton wad and different combinations of starch, semolina, potato powder as an alternative to agar, was tested. The highest frequency of regeneration was found in media containing agar (0.8%) or with a combination of starch, semolina, potato powder (2:1:1) and starch (6%) plus agar (0.4%). The maximum numbers shoots was produced in media containing agar (0.8%), the combination of starch (6%), plus agar (0.4%) and in liquid medium with cotton wad substrate. The best shoot proliferation took place in liquid medium with cotton substrate. The results showed that the combination of starch, semolina, potato powder (2:1:1) and 6% starch, plus 0.4% agar, can be suitable alternatives for agar alone in shoot regeneration step, but shoot number will result lower than in agar alone. These options are cheaper than agar [32].

The type of medium (liquid or semi-solid) can directly affect the rooting process. Although the liquid medium positively influences the availability of water, nutrients, hormones and oxygen levels, the vast majority of protocols for micropropagation were established in semisolid media [33].

Some other agar substitutes have been tested, such as corn starch mixtures (Gelrite®), used for shoot proliferation of apples, pears and red raspberries [34]. However, their use may induce some problems during micropropagation. Gelrite®, for example, causes hyperhydricity and, in some cases, vitrification on regenerated shoots [35]. Hyperhydric shoots are characterized by a translucent aspect due to a chlorophyll deficiency, a not very developed cell wall and high water content. The losses of up to 60% of cultured shoots or plantlets, due to hyperhydricity, have been reported in commercial plant micropropagation [36], reflecting the importance of this problem. The hyperhydricity of shoots was induced in *Prunus avium*, after four weeks multiplication cycles in Gelrite® [37]. This performance has been confirmed by other authors [38]. The vitrification symptoms (thick and translucent stems and leaves, wrinkled and curled leaves) were found on 100 % shoots, after 21 days culture.

Besides the properties of these described substances, it is not always possible to use these agents. In studies with *Eucalyptus grandis* x *E. urophylla* in liquid medium containing aluminum, an acrylic blanket was used to support the shoots, because the addition of AlCl3.6H2O decreased medium pH (to around pH 3.5), affecting medium ionic balance, not allowing the gelation of agar [39].

## **4. Micropropagation of medicinal species**

170 Recent Advances in Plant in vitro Culture

demonstrating a high economic validity [25].

quality than the traditional agar medium [31].

interaction between the two polysaccharides.

also observed [23].

A gelling agent developed in Brazil (patent PI9003880-0 FAPESP/UNESP) was tested as an alternative to agar in the micropropagation of sweet potato (*Ipomoea batata*). The product consists of a mixture of starch from seeds of pigeonpea (*Cajanus cajan*) and cassava starch (*Manihot esculenta*) at a ratio of 2:3, being used as a gelling agent in the MS culture medium at a concentration of 7%. It was observed that this starch mixture increased the fresh weight of cultures when compared to microplants grown on agar. Thus, this starch mixture represents a good alternative for agar replacement in the micropropagation of sweet potato. Moreover, this substitution reduced by over 94% the final cost of the culture medium,

Another successful Brazilian experience deals with xyloglucans, extracted from the seeds of jatoba (*Hymenaea courbaril*) and mixed with agar to prepare a solid medium for the micropropagation of apples. The performance of this new mixture, composed of 0.4% agar and 0.2% xyloglucan (w/v), was verified on *Malus prunifolia Borkh* and cv. *Jonagored* (*Malus domestica*), and compared to the medium solidified with agar (0.6 %, w/v). The growth and the multiplication of shoots were higher in the modified medium. Furthermore, a lower incidence of hyperhydricity and a higher percentage of shoot rooting in the absence of auxin, were observed. When 0.25 mM indole-3-butyric acid (IBA) was added to both the media, the modified medium gave better results in terms of rooting percentage and root

The efficacy of the partial substitution of agar by galactomannans (GMs), obtained from seeds of *Cassia fastuosa* (cassia) and *Cyamopsis tetragonolobus* (guargum - a commercial GM), was tested in the micropropagation of strawberry (*Fragaria x ananassa* Duchesne cv. Pelican). GMs were mixed with agar in the proportion of 0.3/0.3 % (w/v) in MS medium, and the performances were compared with the behavior of the medium containing only agar (0.6% w/v). Strawberry shoots, grown in the modified medium, showed both an enhanced cell proliferation and a higher length of roots with respect to controls. These results showed that agar could be partially replaced by GMs, since experimental data confirmed a favorable

Another study, involving guar gum extracted from the seeds of *Cassia fastuosa* or *Cyamopsis tetragonolobus,* mixed in equal proportions with agar to a final concentration of 0.3% (w/v) for each type of gelling agent, was carried out on the micropropagation of "Durondeau" pear (*Pyrus communis* L. cv. Durondeau). The production of multiple shoots and the formation of roots from shoots were compared with a control media, solidified with agar alone at a concentration of 0.6 % (w/v). In the medium solidified with the mixtures of agar/guar and agar/cassia GM, an increase in the number of regenerated shoots of 32 and 17%, respectively, was obtained. The modified media promoted both a higher number of roots and increased the rooting percentage. A maximum of 91% rooting was obtained in the medium solidified with the agar/cassia GM, containing 9.8 mM indole-3-butyric acid. With this medium, less callus formation at the base of the shoot was

Medicinal and aromatic plants are of great importance for the pharmaceutical industry and traditional medicine in several countries. Just to emphasize the importance of medicinal plants in their various aspects for human health, data from the World Health Organization (WHO) indicated that 80% of the population uses them as basic drugs, in traditional medicine, in the form of plant extracts or their bioactive compounds.

Micropropagation of some medicinal plants has been achieved through the rapid proliferation of shoot-tips and ancillary buds in culture. The success of micropropagation of medicinal species opens perspectives for the production of seedlings, followed by selection of superior genotypes, their clonal multiplication for obtaining genotypic uniformity within the plants to use in plantations at high productivity. Numerous limiting factors have been reported to influence the success of *in vitro* propagation of medicinal plants and, therefore, it is unwise to define a particular protocol for the micropropagation of these species [40].

Polyamines, Gelling Agents in Tissue Culture, Micropropagation of Medicinal Plants and Bioreactors 173

stages. As an example, *Jatropha elliptica*, an herbaceous native species in Brazilian cerrado with purgative properties, used in the treatment of severe itching, syphilis and treatment of snake bites, presents low seed germination potential, about only 6%. The developed micropropagation protocol consists of MS medium, supplemented with 0.5 µmol L-1 indolacetic acid (IAA) and 1 µmol L-1 BAP for the multiplication step, and 5 µmol L-1

An efficient micropropagation protocol has been proposed for *Stevia rebaudiana*, an important antidiabetic medicinal plant. The highest frequency (94.5%) of multiple shoot regeneration and maximum number of shoots (15.7 shoots per explant) were obtained on MS medium, supplemented with 1.0 mg L-1 BAP. Furthermore, the *in vitro* derived nodal explants, grown in the same nutritional conditions, produced a total of 123 shoots per explant after only three subcultures. The highest frequency of rooting (96%) was obtained on half-strength MS medium, in the presence of 0.4 mg L-1 NAA. The rooted plantlets were successfully transferred into plastic cups, containing sand and soil, in the ratio of 1:2, and

Some medicinal plants are "recalcitrant" to vegetative propagation, and in these cases, the technique of *in vitro* cultivation, and the use of different plant growth regulators, can synergistically help plant production. *Byrsonima intermedia* A. Juss is a shrub found in Brazilian cerrado, whose husk presents medicinal activities in diarrheas and dysenteries. The *Byrsonima* genus presents low germination rate and slow plantlet emergency, which makes sexual propagation difficult. The effect of different concentrations of 2,4 dichlorophenoxyacetic acid (2,4-D), thidiazuron (TDZ) and BAP in callus formation, was evaluated. The results demonstrated that there was no formation of callus in leaf explants maintained in the absence of 2,4-D, and that the addition of TDZ and BAP had no influence in the callogenetic process. For callus induction and proliferation, the results suggested the use of MS medium, supplemented with 1.0 mgL-1 2,4-D, maintaining the explants in the

Another species, presenting difficulties in spreading, is the Brazilian ginseng (*Pfaffia tuberosa*), whose roots are used to produce drugs and dietary supplements. A protocol for its micropropagation was developed using nodal explants cultivation in MS medium supplemented with 1 mmolL-1 thidiazuron (TDZ), followed by shoot subculture on the same medium, lacking of growth regulators. The methodology was proven to be valid, due to the high rate of multiplication, good development of shoots, roots and great adaptation to *ex*

The use of tissue culture techniques is also of great importance in the development of new plant products, by preserving germplasms. Micropropagation is a useful technique to preserve the gene pool, especially in cases where small amounts of viable seeds are produced. As an example, canopy (*Canopy macrospyphonia*), a medicinal species, considered highly vulnerable and widely used by traditional communities inhabiting Brazilian cerrado.

naphthalene acetic acid (NAA) for shoot rooting [45].

subsequently transferred in the greenhouse [46].

dark [47].

*vitro* conditions [48].

In fact, a large-scale application depends mainly on the development of an efficient protocol for proliferation, rooting and acclimatization of explants in *ex vitro* conditions.

Jaborandi plant (*Pilocarpus microphyllus*) is a tree, extensively used in the pharmaceutical industry for the manufacture of drugs, used for the treatment of glaucoma. The micropropagation of this specie was conducted on apical explants grown in MS medium, supplemented with 6.66 mM benzyl-aminopurine (BAP) [41]. *Echinodorus scaber* is an herbaceous water plant with cleansing, anti-ophidic, diuretic, anti-rheumatic and antiinflammatory effects, used in traditional medicine to normalize uric acid, treating gout and osteoarthritis, and also used for the manufacturing of soft drinks. The protocol for this specie consists in the inoculation of nodal explants in MS medium, supplemented with 1 mgL-1 BAP [42].

For the micropropagation of *Tournefortia paniculata*, a shrub whose leaves are used as decoction for diuretic and urinary infections, the shoots were grown on WPM (Lloyd & McCown) medium, supplemented with 1 mg L-1 BAP, and shoot rooting was induced in the same medium, without growth regulators. For acclimatization, microplants were placed in a commercial substrate, and cultured in a greenhouse, and then transplanted in the field [43].

*Lipia gracilis* plant is rich in essential oils, containing thymol and canvacrol, as main chemical compounds, responsible for the proven antimicrobial activity. A protocol for its micropropagation has been defined, using MS medium supplemented with 1 mg L-1 BAP [44].

The proliferation success, expressed as number of new produced shoots, is the most important parameter that should be optimized *in vitro,* since often the proliferation process is slow and, as consequence, the number of produced plants is limited. This feature can also be combined to obtain secondary metabolites. Micropropagation ensures a uniform production, which cannot be guaranteed by cultivation of seeds of allogamous plants, due to the high genetic variability of offsprings, which cannot guarantees the uniformity on types and levels of metabolites. Several protocols for medicinal plants have been described [42], but many species still require further studies, in particular, to increase or maintain the production of desirable metabolites.

The micropropagation of medicinal plants is particularly important if the plant produces few seeds, seeds with low germination potential and needs special care to reach the mature stages. As an example, *Jatropha elliptica*, an herbaceous native species in Brazilian cerrado with purgative properties, used in the treatment of severe itching, syphilis and treatment of snake bites, presents low seed germination potential, about only 6%. The developed micropropagation protocol consists of MS medium, supplemented with 0.5 µmol L-1 indolacetic acid (IAA) and 1 µmol L-1 BAP for the multiplication step, and 5 µmol L-1 naphthalene acetic acid (NAA) for shoot rooting [45].

172 Recent Advances in Plant in vitro Culture

of these species [40].

mgL-1 BAP [42].

[44].

production of desirable metabolites.

Micropropagation of some medicinal plants has been achieved through the rapid proliferation of shoot-tips and ancillary buds in culture. The success of micropropagation of medicinal species opens perspectives for the production of seedlings, followed by selection of superior genotypes, their clonal multiplication for obtaining genotypic uniformity within the plants to use in plantations at high productivity. Numerous limiting factors have been reported to influence the success of *in vitro* propagation of medicinal plants and, therefore, it is unwise to define a particular protocol for the micropropagation

In fact, a large-scale application depends mainly on the development of an efficient protocol

Jaborandi plant (*Pilocarpus microphyllus*) is a tree, extensively used in the pharmaceutical industry for the manufacture of drugs, used for the treatment of glaucoma. The micropropagation of this specie was conducted on apical explants grown in MS medium, supplemented with 6.66 mM benzyl-aminopurine (BAP) [41]. *Echinodorus scaber* is an herbaceous water plant with cleansing, anti-ophidic, diuretic, anti-rheumatic and antiinflammatory effects, used in traditional medicine to normalize uric acid, treating gout and osteoarthritis, and also used for the manufacturing of soft drinks. The protocol for this specie consists in the inoculation of nodal explants in MS medium, supplemented with 1

For the micropropagation of *Tournefortia paniculata*, a shrub whose leaves are used as decoction for diuretic and urinary infections, the shoots were grown on WPM (Lloyd & McCown) medium, supplemented with 1 mg L-1 BAP, and shoot rooting was induced in the same medium, without growth regulators. For acclimatization, microplants were placed in a commercial substrate, and cultured in a greenhouse, and then transplanted in the field [43].

*Lipia gracilis* plant is rich in essential oils, containing thymol and canvacrol, as main chemical compounds, responsible for the proven antimicrobial activity. A protocol for its micropropagation has been defined, using MS medium supplemented with 1 mg L-1 BAP

The proliferation success, expressed as number of new produced shoots, is the most important parameter that should be optimized *in vitro,* since often the proliferation process is slow and, as consequence, the number of produced plants is limited. This feature can also be combined to obtain secondary metabolites. Micropropagation ensures a uniform production, which cannot be guaranteed by cultivation of seeds of allogamous plants, due to the high genetic variability of offsprings, which cannot guarantees the uniformity on types and levels of metabolites. Several protocols for medicinal plants have been described [42], but many species still require further studies, in particular, to increase or maintain the

The micropropagation of medicinal plants is particularly important if the plant produces few seeds, seeds with low germination potential and needs special care to reach the mature

for proliferation, rooting and acclimatization of explants in *ex vitro* conditions.

An efficient micropropagation protocol has been proposed for *Stevia rebaudiana*, an important antidiabetic medicinal plant. The highest frequency (94.5%) of multiple shoot regeneration and maximum number of shoots (15.7 shoots per explant) were obtained on MS medium, supplemented with 1.0 mg L-1 BAP. Furthermore, the *in vitro* derived nodal explants, grown in the same nutritional conditions, produced a total of 123 shoots per explant after only three subcultures. The highest frequency of rooting (96%) was obtained on half-strength MS medium, in the presence of 0.4 mg L-1 NAA. The rooted plantlets were successfully transferred into plastic cups, containing sand and soil, in the ratio of 1:2, and subsequently transferred in the greenhouse [46].