Open access

In vitro Tissue Culture, a Tool for the Study and Breeding of Plants Subjected to Abiotic Stress Conditions

Written By

Rosa Ma Pérez-Clemente and Aurelio Gómez-Cadenas

Submitted: November 17th, 2011 Published: October 17th, 2012

DOI: 10.5772/50671

Chapter metrics overview

9,274 Chapter Downloads

View Full Metrics

1. Introduction

Abiotic stress factors are the main limitation to plant growth and yield in agriculture. Among them, drought stress caused by water deficit, is probably the most impacting adverse condition and the most widely encountered by plants, not only in crop fields but also in wild environments. According to published statistics, the percentage of drought-affected land area in the world in 2000 was double that of 1970 [1].

Another major environmental factor that limits crop productivity, mainly in arid and semi-arid regions is high salinity. Approximately 19.5% of the irrigated soils in the world have elevated concentrations of salts either in the soil or in the irrigation water [2], damaging both the economy and the environment [3, 4]. The deleterious effects of salinity on plant growth are associated with low osmotic potential of soil solution (water stress), nutritional imbalance, specific ion effect (salt stress), or a combination of these factors [5].

Abiotic stress leads to a series of morphological, physiological, biochemical, and molecular changes that adversely affect plant growth and productivity [6]. Drought, salinity, extreme temperatures, and oxidative stress are often interconnected, and may induce similar cellular damage (for more details see [7]).

During the course of its evolution, plants have developed mechanisms to cope with and adapt to different types of abiotic and biotic stress. Plants face adverse environmental conditions by regulating specific sets of genes in response to stress signals, which vary depending on factors such as the severity of stress conditions, other environmental factors, and the plant species [8].

The sensing of these stresses induces signaling events that activate ion channels, kinase cascades, production of reactive oxygen species, and accumulation of hormones [9].

These signals ultimately induce expression of specific genes that lead to the assembly of the overall defense reaction. In contrast to plant resistance to biotic stresses, which is mostly dependent on monogenic traits, the genetically complex responses to abiotic stresses are multigenic, and thus more difficult to control and engineer [10].

The conventional breeding programs are being used to integrate genes of interest from inter crossing genera and species into the crops to induce stress tolerance. However, in many cases, these conventional breeding methods have failed to provide desirable results [11].

In recent decades, the use of techniques based on in vitroplant tissue culture, has made possible the development of biotechnological tools for addressing the critical problems of crop improvement for sustainable agriculture. Among the available biotechnologicaltools for crop breeding, genetic engineering based on introgression of genes that are known to be involved in plant stress response and in vitroselection through the application of selective pressure in culture conditions, for developing stress tolerant plants, have proved to be the most effective approaches [12].

On the other hand, it is often difficult to analyze the response of plants to different abiotic stresses in the field or in greenhouse conditions, due to complex and variable nature of these stresses. In vitrotissue culture-based tools have also allowed a deeper understanding of the physiology and biochemistry in plants cultured under adverse environmental conditions [13].

In this work, the progress made towards the development of abiotic stress-tolerant plants through tissue culture-based approaches is described. The achievements in the better understanding of physiological and biochemical changes in plants under in vitrostress conditions are also reviewed.


2.Somaclonal variation

Somaclonal variation is defined as the genetic and phenotypic variation among clonally propagated plants of a single donor clone. It is well known that genetic variations occur in undifferentiated cells, isolated protoplasts, calli, tissues and morphological traits of regenerated plants. The cause of variation is mostly attributed to changes in the chromosome number and structure. Generally, the term somaclonal variation is used for genetic variability present among all kinds of cells/plants obtained from cells cultured in vitro[14].

Plants regenerated from tissue and cell cultures show heritable variation for both qualitative and quantitative traits. Somaclonal variation caused by the process of tissue culture is also called tissue culture-induced variation to more specifically define the inducing environment [15]. The occurrence of uncontrolled and spontaneous variation during the culture process is an unexpected and mostly undesired phenomenon when plants are micropropagated at the commercial scale [16]. However, apart from these negative effects, its usefulness in crop breeding through creation of novel variants has been extensively reported [17]. Induced somaclonal variation can be used for genetic manipulation of crops with polygenic traits [18]. The new varieties derived from in vitrotissue culture could exhibit disease resistance and improvement in quality as well as better yield [19].

Somaclonal variants can be detected using various techniques which are broadly categorized as morphological, physiological/biochemical and molecular detection techniques. There are two main approaches for the isolation of somaclonal variants: screening and cell selection.

Screening involves the observation of a large number of cells or regenerated plants for the detection of variant individuals. Mutants for several traits can be far more easily isolated from cell cultures than from whole plant populations. This is because a large number of cells can be easily and effectively screened for mutant traits. Screening of as many plants would be very difficult, ordinarily impossible [17]. Mutants can be effectively selected for disease resistance, improvement of nutritional quality, adaptation to stress conditions, e.g., saline, soils, low temperature, toxic metals, resistance to herbicides and to increase the biosynthesis of plant products used for medicinal or industrial purposes. Screening has been profitably and widely employed for the isolation of cell clones that produce higher quantities of certain biochemicals [20].

In the cell selection approach, a suitable pressure is applied to permit the preferential survival/growth of variant cells. Selection strategies have been successfully developed for the recovery of genotypes resistant to various toxins, herbicides, high salt concentration etc. [21]. When the selection pressure allows only the mutant cells to survive or divide, it is called positive selection. On the other hand, in the case of negative selection, the wild type cells divide normally and therefore are killed by a counter selection agent, e.g., 5-Bromodeoxyuridine, or arsenate. The mutant cells are unable to divide as a result of which they escape the counter selection agent. These cells are subsequently rescued by removal of the counter selection agent [11].


3. In vitroselection of plants tolerant to abiotic stress

Many studies have reported that the in vitroculture alone or combined with mutagenesis, induced with physicochemical or biological agents, can be exploited to increase genetic variability and mutants, as a potential source of new commercial cultivars [22]. In vitroculture environments can be mutagenic and plants regenerated from organ cultures, calli, protoplasts and via somatic embryogenesis sometimes exhibit phenotypic and/or genotypic variations [22].

It is important to point that tissue culture increases the efficiency of mutagenic treatments and allows handling of large populations and rapid cloning of selected variants [17]. The similarities of the effects induced by the stress in the plant cultured in vitroand in vivoconditions suggest that the in vitrosystem can be used as an alternative to field evaluations for studying the general effect of water-stress on plant growth and development.

The most widely used method for the selection of genotypes tolerant to abiotic stress is the in vitroselection pressure technique. This is based on the in vitroculture of plant cells, tissues or organs on a medium supplemented with selective agents, allowing selecting and regenerating plants with desirable characteristics. In table 1 a list of species in which this technique has been successfully applied to obtain genotypes with increased resistance to different abiotic stresses is shown.

Plant speciesstressReferences
Chrysanthemum morifolium(chrysanthemum)salt[66]
Brassica napus(rapeseed)salt[67]
Citrus aurantium(sour orange)salt[68]
Ipomoea batatas(sweet potato)salt[70]
Saccharumsp. (sugarcane)salt[71]
Solanumtuberosum(potato)salt[32], [72]
Triticumaestivum(wheat)salt[21], [73]
Brassica juncea(indian mustard)drought[75]
Prunusavium(colt cherry)drought[72]
Saccharumsp. (sugarcane)drought[76]
Glycine max(soybean)Al[78]
Setariaitalica (millet)Zn[79]

Table 1.

In vitroselection for increased resistance to abiotic stresses.

The most important successes on this respect are described below:

3.1.In. vitroselection of salt-tolerant plants

The problem of soil salinity has been aggravated during the last decades as a consequence of some agricultural practices such as irrigation and poor drainage systems. As described in the introduction, it has been estimated that around 20 % of the irrigated land in the world is affected by salinity, and it is expected that the increase of salinization in agricultural fields will reduce the land available for cultivation by 30% in the next 25 years and up to 50% by the year 2050 [23].

The in vitroselection pressure technique has been effectively utilized to induce tolerance to salt stress in plants through the use of salts as a selective agent, allowing the preferential survival and growth of desired genotypes. This approach has being done using a number of plant materials (callus, suspension cultures, somatic embryos, shoot cultures, etc.) which has been screened for variation in their ability to tolerate relatively high levels of salt in the culture media. In most of the studies, the salt used has been NaCl [24].

Several researchers have compared the response of other Cl- and SO42- salts including KCl, Na2SO4, and MgSO4 during in vitroscreening. This use of multiple salts as a selection pressure parallels the salinity under field conditions and may be a better choice [11].

3.2.In. vitroselection of drought-tolerant plants

Drought is a major abiotic stress which causes important agricultural losses, mainly in arid and semiarid areas. Drought stress causes moisture depletion in soil and water deficit with a decrease of water potential in plant tissues. In vitroculture has been used to obtain drought-tolerant plants assuming that there is a correlation between cellular and in vivoplant responses [25]. During the last years, in vitroselection for cells exhibiting increased tolerance to water or drought stress has been reported (Table 1).

Polyethylene glycol (PEG), sucrose, mannitol or sorbitol have been used by several workers as osmotic stress agents for in vitroselection [25; 26] However, PEG has been the most extensively used to stimulate water stress in plants. This compound of high molecular weight is a non-penetrating inert osmoticum that reduces water potential of nutrient solutions without being taken up by the plant or being phytotoxic [26]. Because PEG does not enter the apoplast, water is withdrawn not only from the cell but also from the cell wall. Therefore, PEG solutions mimic dry soil more closely than solutions of compounds with low molecular weights, which infiltrate the cell wall with solute [27].

Besides salt and drought, a few reports are also available for the development of plants tolerant to other abiotic stress (metal, chilling, UV and frost) through in vitroselection (Reviewed in [11]).


4. Characterization of salt- or drought-tolerant plants during in vitroselection

A second step in the process of obtaining genotypes more tolerant to a particular stress condition is the characterization of the regenerants that survive to the imposed pressure selection under in vitroconditions. Salinity and drought affect many physiological processes such as reductions of cell growth, leaf area, biomass and yield. The activation of the plant antioxidant defense system has been positively associated with salt and drought tolerance [11], and the same pattern has been confirmed on in vitrocultures [28]. Therefore, by measuring antioxidant activities in vitro, a rapid preliminary selection of tolerant genotypes could be performed. In fact, different authors have determined the main antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) [29].

Lipids play an important role as the structural constituent of most of the cellular membranes [30]. Moreover, often there is no need of intact plants to perform the initial selection, as in the case of callus culture that can be used as a plant material for the selection of tolerant genotypes. As an example it is well known that free radical-induced peroxidation of lipid membrane is a sign of stress-induced damage at cellular level. Therefore, the level of malonyldialdehyde, produced during peroxidation of membrane lipids, is often used as an indicator of oxidative damage [31]. It has been reported that selected callus lines of Solanumtuberosumsubjected to NaCl showed an increase in lipid peroxidation in comparison with salt tolerant lines [32].

For overcoming salt or drought stress, plants have developed protective mechanisms including osmotic adjustment that is usually accomplished by accumulation of compatible solutes such as proline, glycine betaine and polyols [33]. It has been also reported that proline levels increased in response to water stress in tomato calli [34].Taking into account the generated knowledge about plants responses to abiotic stress conditions, the determination of antioxidant enzyme activities, and levels of malonyldialdehyde and proline in plants recovered under selective conditions may help to isolated the most tolerant genotypes.

In recent years, both basic and applied research has led to understand the mechanisms underlying the stress response and the identification of the specific genes/metabolites that are responsible for tolerance phenotypes the “omics” approaches have had a significant development. Through the application of transcriptomics hundreds of genes have been linked with environmental stress responses and regulatory networks of gene expression have been delineated [35]. Moreover, plant tolerance to abiotic stress conditions has been associated with changes in proteome composition. Since proteins are directly involved in plant stress response, proteomics studies can significantly contribute to unravel the possible relationships between protein abundance and plant stress acclimation [36].

Relatively less is known about changes at the metabolomic level. Metabolome analysis has become a valuable tool to study plant metabolic changes that occur in response to abiotic stresses. This approach has already enabled to identify different compounds whose accumulation is affected by exposure to stress conditions. However, much work is still required to identify novel metabolites and pathways not yet linked to stress response and tolerance [37].

In this context, an integrated approach incorporating in vitroplant tissue culture to proteomics and metabolomics technique, can contribute to elucidate the metabolites involved in stress response and select desired genotypes at early stages of plant development or at callus stage [38].


5.Transgenesis for abiotic stress tolerance

Transgenic approaches are among the available tools for plant improvement programs based on biotechnological methodologies. Nowadays, many mechanisms and gene families, which confer improved productivity and adaptation to abiotic stresses, are known. These gene families can be manipulated into novel combinations, expressed ectopically, or transferred to species in which they do not naturally occur. Therefore, the possibility to transform the major crop species with genes from any biological source (plant, animal, microbial) is an extremely powerful tool for molecular plant breeding [39].

To date, successes in genetic improvement of environmental stress resistance have involved manipulation of a single or a few genes involved in signaling/regulatory pathways or that encode enzymes involved in these pathways (such as osmolytes/compatible solutes, antioxidants, molecular chaperones/osmoprotectants, and water and ion transporters [8]. The disadvantage of this approach is that there are numerous interacting genes involved, and efforts to improve crop drought tolerance through manipulation of one or a few of them is often associated with other, often undesirable, pleiotropic and phenotypic alterations [8].

The plant hormone abscisic acid (ABA) regulates the adaptive response of plants to environmental stresses such as drought, salinity, and chilling via diverse physiological and developmental processes [40, 41].

The ABA biosynthetic pathway has been deeply studied and many of the key enzymes involved in ABA synthesis have been used in transgenic plants in relation to improving abiotic stress tolerance [42]. Transgenic plants overexpressing the genes involved in ABA synthesis showed increased tolerance to drought and salinity stress [42, 43]. Similarly, many studies have illustrated the potential of manipulating CBF/DREB genes to confer improved drought tolerance [44, 45].

Another mechanism involved in plant protection to osmotic stress associated to drought and salinity involves the upregulation of compatible solutes that function primarily to maintain cell turgor, but are also involved in avoiding oxidative damage and chaperoning through direct stabilization of membranes and/or proteins [46; 47]. Many genes involved in the synthesis of these osmoprotectants have been explored for their potential in engineering plant abiotic stress tolerance [10, 47].

The cellular and metabolic processes involved in salt stress are similar to those occurring in drought-affected plants and are responses to the osmotic effect of salt [48, 49]. As described above, the use of genes related to osmoprotectant synthesis has been successfully used in developing drought-tolerant crops and the transfer of glycine betaine intermediates have improved the drought and salt tolerance of transgenic plants in many cases [50].

The amino acid proline is known to occur widely in higher plants and normally accumulates in large quantities in response to environmental stresses [51, 52]. The osmoprotectant role of proline has been verified in some crops by overexpressing genes involved in proline synthesis [53].

Other approaches successfully developed in a variety of crops to obtain abiotic-stress-tolerant plants by transgenesis, have been manipulation of transcription factors (TFs), late embryogenesis abundant (LEA) proteins, and antioxidant proteins [54].On the other hand, the use of genetic and genomic analysis to identify DNA molecular markers associated to stress resistance can facilitate breeding strategies for crop improvement. This approach is particularly useful when targeting characters controlled by several genes, as in the case of most abiotic stress.

The potential to map different Quantitative Trait Loci (QTL) contributing to an agronomical trait and to identify linked molecular markers opens up the possibility to transfer simultaneously several QTLs and to pyramid QTLs for several agronomical traits in one improved cultivar [55]. However, the application of molecular markers in breeding programs requires preliminary studies to identify and validate potential markers [55].

Although the use of Marker-Assisted Selection may be helpful for crop improvement, its practical application in genetic improvement of resistance or tolerance to stress has been limited since no many stress tolerance QTL have been identified [56]. For future biotechnology improvements such as tolerance to drought or nutrient limitation, forward breeding will be necessary to co-optimize transgenic expression and genetic background because endogenous genes and environmental factors may have the potential to influence the phenotypes resulting from transgenic modifications [57].

It is important to point that genetic modification of higher plants by introducing DNA into their cells is a highly complex process. Practically any plant transformation experiment relies at some point on cell and tissue culture. Although the development transformation methods that avoid plant tissue culture have been described for Arabidopsis, and have been extended to a few crops, the ability to regenerate plants from isolated cells or tissues in vitrois needed for most plant transformation systems. Not all plant tissue is suited to every plant

Plant speciesstressReferences
Ipomoea batatas(sweet potato)salt[16]
Brassica juncea(mustard)Cd[83]
Zea mays(maize)drought[89]
Pinustaeda(loblolly pine)salt[90]
Populustomentosa(chinese white poplar)salt[91]
Citrus sinensis x Poncirus trifoliate(Carrizo citrange)drought[92]
Petunia sp. (petunia)drought[93]
Brassica juncea(indian mustard)As and Cd[95]
Brassica napus(rape)freezing[96]
Pinusvirginiana(Virginia pine)metal[90]
Alyssum sp. (alyssum)Ni[98]

Table 2.

.Genetic transformation for increased resistance to abiotic stresses.

transformation method, and not all plant species can be regenerated by every method [58].There is, therefore, a need to find both a suitable plant tissue culture/regeneration regime and a compatible plant transformation methodology. Today, many agronomical and horticultural important species are routinely transformed, and the list of species that is susceptible to Agrobacterium-mediated transformation seems to grow daily (Table 2).


6.In vitrotissue culture as a tool for physiological and biochemical studies in plants

Because of the great interest for both basic and applied research, many scientific endeavours have long addressed the understanding of the mechanisms underlying the stress response and the identification of the specific genes/metabolites that are responsible for tolerance phenotypes.

In the last decades, in vitroculture of plants has become an integral part of advances in plant science research. Plant tissue culture techniques allow for close monitoring and precise manipulation of plant growth and development, indeed, the in vitrosystem offers the advantage that relatively little space is needed to culture plants and this system allows a rigorous control of physical environment and nutrient status parameters, which are difficult to regulate with traditional experimental system [59]. Furthermore, any complex organ-organ and plant-environment interaction can be controlled or removed, and the level of stress can be accurately and conveniently controlled [60]. All this together makes that some aspects of plant growth, that were barely understood before the advancement of the science of tissue culture, such as the metabolism and interaction of plant hormones, as well as their physiological effects can be deeply studied [61].

Shoot apex culture has been widely used to evaluate plant physiological responses to salinity and osmotic stress in various species, including apple [59], olive [62] and tomato [63]. With regard to the whole plant, a similar response to salt stress could be expected in plantlets grown through in vitroshoot apex culture [63], because such explants can be considered mini-replicas of a plant with anatomical organization and ability to root and grow into whole plant.

We have previously described the use of an in vitrotissue culture technique to study the performance of different citrus genotypes cultured under salt stress conditions, avoiding the effect of the root by culturing shoots without the root system. The method proved to be a good tool for studying biochemical processes involved in the response of citrus to salt stress [64]. Some citrus genotypes have been classified as relatively salt tolerant under field conditions due to their ability to restrict chloride ions to roots while others have proved to be more sensitive to salinity [65].

In vitrotissue culture approach allowed us to observe that when shoots are cultured without a root system, all genotypes accumulated the same chloride levels and exhibited similar leaf damage as a consequence of salt stress treatment. There was no increase of malonyldialdehydelevels in any genotype, and common patterns of hormonal signaling were observed among genotypes. On the view of these results we concluded that under the same salt conditions and with the same level of leaf chloride intoxication, no biochemical differences exist among tolerant and sensitive genotypes. This points to the roots as a key organ not only as a filter of chloride ions but also as a signalling system in citrus [64]. In vitrotissue culture provided the tools to perform this studies that it would be impossible to carry out with whole plants grown under field or greenhouse conditions.


7. Conclusion

Use of in vitrocell and tissue-based systems offers a remarkable tool for dissecting the physiological, biochemical and molecular regulation of plant development and stress response phenomena. In recent years, considerable progress has been made regarding the development and isolation of stress tolerant genotypes by using in vitrotechniques.

The most successful applied tools have been the induction of somaclonal variation and in vitroselection of plants tolerant to different abiotic stresses and the development of transgenic genotypes throughout different approaches.

In vitroselection makes possible to save the time required for developing disease resistant and abiotic stress tolerant lines of commercial crops and other plant species. However, in vitroselected variants should be finally field-tested to confirm the genetic stability of the selected traits under field conditions. The development of in vitroselection technology, together with molecular approaches and functional genomics will provide a new opportunity to improve stress tolerance in plants relevant to food production and environmental sustainability.

Development of transgenic plants using biotechnological tools has become important in plant-stress biology. Previous works on genetics and molecular approaches have shown that most of the abiotic stress tolerant traits are multigenic. Therefore, to improve stress tolerance several stress related genes need to be transfered. More recently manipulation of single transcription factors has provide the same effect as manipulation of multiple genes. This has become a promising approach to get abiotic stress tolerant crops.

A limiting factor for the widespread application of this technology is that, with few exceptions, genetic transformation protocols require plant regeneration of transformants using in vitroplant tissue culture tools. Although the list of species that are susceptible to Agrobacterium-mediated transformation has been increased recently, still are many genotypes for which regeneration protocols are not available.

On the other hand, plant tissue culture is also an invaluable laboratory tool to study basic aspects of plant growth and development, and to manipulate these processes since it makes possible to have a large number of plants in a small space, without the interference of other biotic or abiotic stress factors. It also allows growing plants in the same nutritional and environmental conditions all year around.



This work was supported by the Spanish Ministerio de Economía y competitividad (MINECO) and UniversitatJaume I/FundacióBancaixa through grants No. AGL2010-22195-C03-01/AGR and P11B2009-01, respectively.


  1. 1. IsendahlN.SchmidtG.Drought in the Mediterranean-WWF policy proposals; A. WWF Report, Madrid2006
  2. 2. JinT. C.ChangQ.LiW. F.YinD. X.LiZ. J.WangD. L.LiuB.LiuL. X.Stress-inducible expression of GmDREB1 conferred salt tolerance in transgenic alfalfaPlant CellTissue and Organ Culture2010100219
  3. 3. RengasamyP.Soil processes affecting crop production in salt-affected soilsFunctional Plant Biology201037255
  4. 4. YangY. L.ShiR. X.WeiX. L.FanQ.AnL. Z.Effect of salinity on antioxidant enzymes in calli of the halophyteNitrariatangutorumBobr. Plant Cell Tissue and Organ Culture2010102387
  5. 5. Gómez-CadenasA.ArbonaV.JacasJ.Primo-MilloE.TalonM.Abscisic acid reduces leaf abscission and increases salt tolerance in citrus plantsJournal of Plant Growth Regulation200321234
  6. 6. WangW. X.VinocurB.ShoseyovO.AltmanA.Biotechnology of plant osmotic stress tolerance: physiological and molecular considerationsActaHorticulturae2001560285
  7. 7. JewellM. C.CampbellB. C.GodwinI. D.transgenic plants for abiotic stress resistance. In: Transgenic Crop Plants. C. Kole et al. (eds.), pringer-Verlag Berlin Heidelberg.2010
  8. 8. WangW.VinocurB.AltmanA.Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance. Planta20032181
  9. 9. CheongY. H.ChangH. S.GuptaR.WangX.ZhuT.LuanS.Transcriptional profiling reveals novel interactions between wounding, pathogen, abiotic stress, and hormonal responses in Arabidopsis. Plant Physiology2002129661677
  10. 10. VinocurB.AltmanA.Recent advances in engineering plant tolerance to abiotic stress: achievements and limitationsCurrent Opinion in Biotechnology200516123
  11. 11. RaiM. K.KaliaR. K.SinghR.GangolaM. P.DhawanA. K.Developing stress tolerant plants through in vitro selection-An overview of the recent progressEnvironmental and Experimental Botany20117189
  12. 12. SakhanokhoH. F.KelleyR. Y.Influence of salicylic acid onin vitropropagation and salt tolerance in Hibiscus acetosella and Hibiscus moscheutos (cv ‘Luna Red’) African Journal of Biotechnology200981474
  13. 13. BenderradjiL.2012 ISRN Agronomy, Article ID 367851.
  14. 14. Lestari E.G.In vitroselection and somaclonal variation for biotic and abiotic stress tolerance. Biodiversitas20067297
  15. 15. KaepplerS. M.KaeplerH. F.RheeY.Epigenetic aspects of somaclonal variation in plants.Plant Molecular Biology200043179188
  16. 16. GaoX.YangD.CaoD.AoM.SuiX.WangQ.KimatuJ.WangL.In vitromicropropagation of Freesia hybrida and the assessment of genetic and epigenetic stability in regenerated plantlets. Journal of Plant Growth Regulation201029257
  17. 17. PredieriS.Mutation induction and tissue culture in improving fruitsPlant CellTissue and Organ Culture200164185
  18. 18. Jain S.M.Tissue culture-derived variation in crop improvementEuphytica2001118153
  19. 19. BiswasM. K.DuttM.RoyU. K.IslamR.HossainM.Development and evaluation ofin vitrosomaclonal variation in strawberry for improved horticultural traits. ScientiaHorticulturae2009122409
  20. 20. MatkowskiA.Plantin vitroculture for the production of antioxidants- A review. Biotechnology Advances200826584
  21. 21. ZairI.ChlyahA.SabounjiK.TittahsenM.ChlyahH.Salt tolerance improvement in some wheat cultivars after application of in vitro selection pressurePlant Cell Tissue and Organ Culture200373237
  22. 22. OrbovićV.ĆalovićM.ViloriaZ.NielsenB.GmitterF.CastleW.GrosserJ.Analysis of genetic variability in various tissue culture-derived lemon plant populations using RAPD and flow cytometry. Euphytican2008161329
  23. 23. RozemaJ.FlowersT.Crops for a salinized world.Science20083221478
  24. 24. WoodwardA. J.BennettI. J.The effect of salt stressandabscisicacidonproline production, chlorophyll content and growth ofin vitropropagated shoots of Eucalyptus camaldulensis. Plant Cell Tissue and Organ Culture200582189
  25. 25. Mohamed M.A.H., Harris, P.J.C., Henderson, J.In vitro selection and charac-terisation of a drought tolerant clone of Tagetesminuta.Plant Science2000159213
  26. 26. HassanN. M.SeragM. S.El -FekyF. M.Changes in nitrogen content and protein profiles followingin vitroselection of NaCl resistant mung bean and tomato. ActaPhysiologiaePlantarum200426165
  27. 27. VersluesP. E.OberE. S.SharpR. E.Root growth and oxygen relations at low water potentials. Impact of oxygen availability in polyethylene glycol solutions.Plant Physiology19981161403
  28. 28. Lascano H.R.Antioxidant system response of different wheat cultivars under drought: field and in vitro studiesAustralian Journal of Plant Physiology2001281095
  29. 29. Hossain Z., Mandal A.K.A., Datta S.K., Biswas A.K. Development of NaCl tolerant line in Chrysanthemum morifolium Ramat.through shoot organogenesis of selected callus line.Journal of Biotechnology2007129658
  30. 30. ParidaA. K.DasA. B.Salt tolerance and salinity effects on plants: a review.Ecotoxicology and Environmental Safety200560324
  31. 31. DemiralT.TurkanI.Comparativelipid.peroxidationantioxidant.defensesystems.prolinecontent.inroots.oftwo.ricecultivars.differingin.salttolerance.Environmental and Experimental Botany.200553247
  32. 32. QueirosF.FidalgoF.SantosI.SalemaR.In vitroselection of salt tolerant cell lines in Solanumtuberosum L. BiologiaPlantarum.200751728
  33. 33. GhoulamC.AhmedF.KhalidF.Effects of salt stress on growth, inorganic ions and proline accumulation in relation to osmotic adjustment in five sugar beet cultivarsEnvironmental and Experimental Botany200147139
  34. 34. Aazami M.A., Torabi M., ShekariF.Response of some tomato cultivars to sodium chloride stress under in vitro culture condition African Journal of Agricultural Research.201052589
  35. 35. TodakaD.NakashimaK.ShinozakiK.Yamaguchi-ShinozakiK.Toward understanding transcriptional regulatory networks in abiotic stress responses and tolerance in rice. Todaka et al. Rice2012
  36. 36. KosováK.VítámvásP.PrášilI. T.RenautJ.Plant proteome changes under abiotic stress- Contribution of proteomics studies to understanding plant stress response. Journal of Proteomics,20111551
  37. 37. Cevallos-CevallosJ. M.Reyes DeCorcuera. J. I.EtxeberriaE.DanylukM. D.RodrickJ. E.E.Metabolomic analysis in food science: a review. Trends in Food Science and Technology200920557
  38. 38. PalamaT.MenardP.FockI.BourdonE.Govinden-SoulangeJ.BahutM.PayetB.VerpoorteR.KodjaH.Shoot differentiation from protocorm callus cultures of Vanilla planifolia (Orchidaceae): proteomic and metabolic responses at early stageBMCP Plant Biology2010
  39. 39. MiflinB.Crop improvement in the 21st century. Journal of Experimental Botany2000511
  40. 40. Gómez-CadenasA.TadeoFr.Primo-MilloE.TalónM.Involvement of abscisic acid and ethylene in the response of citrus seedlings to salt shock.PhysiologiaPlantarum1998103475
  41. 41. ArbonaV.Gómez-CadenasA.Hormonal modulation of citrus responses to floodingJournal of Plant Growth Regulation200827241
  42. 42. SchwartzS. H.QinX.ZeevaartJ. A. D.Elucidation of the indirect pathway of abscisic acid biosynthesis by mutants, genes, and enzymesPlant Physiology20031311591
  43. 43. ParkH. Y.SeokH. Y.ParkB. K.KimS. H.GohC. H.BhL.LeeC. H.MoonY. H.Overexpression of Arabidopsis ZEP enhances tolerance to osmotic stressBiochemical and Biophysical Research Communications20083758085
  44. 44. Al-AbedD.MadasamyP.TallaR.GoldmanS.RudrabhatlaS.Genetic engineering of maize with the Arabidopsis DREB1A/CBF3 gene using split-seed explants.Crop Science2007472390
  45. 45. TrujilloL. E.SotolongoM.MenendezC.OchogaviaM. E.CollY.HernandezI.Borras-HidalgoO.ThommaB. P. H. J.VeraP.HernandezL.SodE. R. F.novela.sugarcaneethylene.responsivefactor. . E. R. F.enhancessalt.droughttolerance.whenoverexpressed.intobacco.plantsPlant and Cell Physiology200849512
  46. 46. Mc NeilS. D.NuccioM. L.HansonA. D.Betaines and related osmoprotectants. Targets for metabolic engineering of stress resistancePlant Physiology1999120945
  47. 47. ZhangY. Y.LiY.GaoT.ZhuH.WangD. J.ZhangH. W.NingY. S.LiuL. J.WuY. R.ChuC. C.GuoH. S.XieQ.2008Arabidopsis SDIR1 enhances drought tolerance in crop plants. BiosciBiotechnolBiochem7222512254
  48. 48. Yeo A.R., Lee K.S., Izard P., Boursier P.J., Flowers T.J. Short- and long-term effects of salinity on leaf growth in rice (Oryzasativa L.).Journal of Experimental Botany199142881
  49. 49. MunnsR.TesterM.Mechanisms of salinity tolerance.Annual Review of Plant Physiology2008;59651
  50. 50. WuW.SuQ.XiaX.WangY.LuanY.AnL.TheSuaedaliaotungensiskitagbetaine aldehyde dehydrogenase gene improves salt tolerance of transgenic maize mediated with minimum linear length of DNA fragment. Euphytica200815917
  51. 51. KaviKishore. P. B.SangamS.AmruthaR. N.LaxmiP. S.NaiduK. R.RaoK. R. S. S.RaoS.ReddyK. J.TheriappanP.SreenivasuluN.Regulation of proline biosynthesis, degradation, uptake and transport in higher plants: its implications in plant growth and abiotic stress tolerance. Current Science200588424
  52. 52. AshrafM.FooladM. R.Roles of glycine betaine and proline in improving plant abiotic stress resistanceEnvironmental and Experimental Botany200759206
  53. 53. Hmida-SayariA.Gargouri-BouzidR.BidaniA.JaouaL.SavoureA.JaouaS.Overexpression of D1pyrroline-5-carboxylate synthetase increases proline production and confers salt tolerance in transgenic potato plants. Plant Science2005
  54. 54. Umezawa T., Fujita M., Fujita Y., Yamaguchi-Shinozaki K., Shinozaki K., Engineering drought tolerance in plants: discovering and tailoring genes to unlock the future.Current Opinion in Biotechnology200617113
  55. 55. YuK. F.ParkS. J.ZhangB. L.HaffnerM.PoysaV.AnS. S. R.markerin.thenitrate.reductasegene.ofcommon.beanis.tightlylinked.toa.majorgene.conferringresistance.tocommon.bacterialblight.Euphytica20041388995
  56. 56. Foolad, M.R., Recent advances in genetics of salt tolerance tomato.Plant CellTissue and Organ Culture200476101119
  57. 57. Mumm R.H.Backcross versus forward breeding in the development of transgenic maize hybrids: theory and practiceCrop ScienceSuppl 3)200747164171
  58. 58. Benson E.E. Special symposium: in vitro plant recalcitranceIn vitroplant recalcitrance: an introduction. In Vitro Cellular and Developmental Biology Plant200036141148
  59. 59. ShibliR. A.SmithM. A. L.SpomerL. A.Osmotic adjustment and growth responses of threeChrysanthemum morifoliumRamat cultivars to osmotic stress induced in vitro. Journal of Plant Nutrition,1992151374
  60. 60. StephenR. G.ZengL.ShannonM. C.RobertsS. R.Rice is more sensitive to salinity than previously thoughtAnnual Report of U.S. Department of Agriculture’s Research Service,4812002
  61. 61. BairuM. W.KaneM. E.Physiological and developmental problems encountered by in vitro cultured plantsPlant Growth Regulation201163101
  62. 62. ShibliR. A.Al-JubooryK.Comparative response of ‘Nabali’ olive microshoot, callus and suspension cell cultures to salinity and water deficit.Journal of Plant Nutrition20022561
  63. 63. CanoE. A.Perez-AlfoceaF.MorenoV.CaroM.BolarinM. C.Evaluation of salt tolerance in cultivated and wild tomato species through in vitro shoot apex culturePlant CellTissue and Organ Culture19985319
  64. 64. MontoliuA.López-ClimentM. F.ArbonaV.Pérez-ClementeR. M.Gómez-CadenasA. A.novelin.vitrotissue.cultureapproach.tostudy.saltstress.responsesin.citrusPlant Growth Regulation200959179
  65. 65. López-ClimentM. F.ArbonaV.Pérez-ClementeR. M.Gómez-CadenasA.Relationship between salt tolerance and photosynthetic machinery performance in citrusEnvironmental and Experimental Botany200862176
  66. 66. Hossain Z., Mandal A.K.A., Datta S.K., Biswas A.K., Development of NaCl tolerant line in Chrysanthemum morifoliumRamat.through shoot organogenesis of selected callus line. Journal of Biotechnology.2007129658
  67. 67. RahmanM. H.KrishnarajS.ThorpeT. A.Selection for salt tolerance in vitro using microspore-derived embryos of Brassica napus cv. Topas, and the characterization of putative tolerant plants. In Vitro Cellular and Developmental Biology- Plant199531116
  68. 68. KocN. K.BasB.KocM.KusekM.Investigations of in vitro selection for salt tolerant lines in sour orange (Citrus aurantium L.)Biotechnology20098155
  69. 69. Kripkyy O., Kerkeb L., Molina A., Belver A., Rodrigues Rosales P., Donaire P.J., Effects of salt-adaptation and salt-stress on extracellular acidification and microsomephosphohydrolase activities in tomato cell suspensions.Plant Cell Tissue and Organ Culture.20016641
  70. 70. HeS.HanY.WangY.ZhaiH.LiuQ.In vitro selection and identification of sweetpotato (Ipomoea batatas (L.) Lam.) plants tolerant to NaClPlant CellTissue and Organ Culture2009966974
  71. 71. GandonouC. B.ErrabiiT.AbriniJ.IdaomarM.SenhajiN. S.Selection of callus cultures of sugarcane (Saccharum sp.) tolerant to NaCl and their response to salt stressPlant CellTissue and Organ Culture.2006879
  72. 72. OchattS. J.MarconiP. L.RadiceS.ArnozisP. A.CasoO. H.In vitro recurrent selection of potato: production and characterization of salt tolerant cell lines and plantsPlant CellTissue and Organ Culture1999551
  73. 73. BarakatM. N.Abdel-LatifT. H.In vitro selection of wheat callus tolerant to high levels of salt and plant regenerationEuphytica199691127
  74. 74. PurushothamM. G.PatilV.RaddeyP. C.PrasadT. G.VajranabhaiahS. N.Development ofin vitroPEG stress tolerant cell lines in two groundnut (ArachishypogaeaL.) genotypes. Indian Journal of Plant Physiology.1998349
  75. 75. GangopadhyayG.BasuS.GuptaS.In vitroselection and physiological characterization of NaCl- and mannitol-adapted callus lines in Brassica juncea. Plant Cell Tissue and Organ Culture.199750161
  76. 76. Errabii T., Gandonou C.B., Essalmani H., Abrini J., Idomar M., Senhaji N.S. Growth, proline and ion accumulation in sugarcane callus cultures under drought-induced osmotic stress and its subsequent relief.African Journal of Biotechnology.200651488
  77. 77. RoyB.MandalA. B.Towards development of Al-toxicity tolerant lines in indica rice by exploiting somaclonal variationEuphytica2005145221
  78. 78. MariskaI.Peningkatan Ketahanan terhadap Alumunium pada Pertanaan Kedelai melalui Kultur in Vitro. Laporan RUT VIII. Bidang Teknologi Pertanian. Bogor: Balai Penelitian Bioteknologi Pertanian & Kementerian Riset dan Teknologi RI-LIPI.2003
  79. 79. SamantarayS.RoutG. R.DasP.In vitroselection and regeneration of zinc tolerant calli from Setariaitalica L Plant Science.199931201
  80. 80. LaurieS.FeeneyK. A.MathuisF. J.HeardP. J.BrownS. J.LeighR. A. A.rolefor. H. K. T.insodium.uptakeby.wheatroots.PlantJournal.200232139
  81. 81. LiuK.LeiW.XuY.ChenN.MaQ.LiF.ChongK.Overexpressionof.OsC. O. I. N. a.putativecold.induciblezinc.fingerprotein.increasedtolerance.tochilling,salt.droughtenhancedpraline.levelin.ricePlanta20072261007
  82. 82. VanjildorjE.BaeT. W.RiuK. Z.KimS. Y.LeeH. Y.Overexpression of Arabidopsis ABF3 gene enhances tolerance to drought and cold transgenic lettuce (Lactucasativa). Plant Cell, Tissue and Organ Culture.20058341
  83. 83. ZhuY. L.Pilon-SmitsE. A. H.JouaninL.TerryN.Overexpression of Glutathione synthetase in indian mustard enhances cadmium accumulation and tolerancePlant Physiology19991197380
  84. 84. MckersieB. D.ChenY.BeusM. D.BowleyS. R.BowlerC.InzeD..HalluinK. D.BottermanJ.Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (MedicagosativaL.). Plant Physiology.199310311551163
  85. 85. PaytonP.WebbR.KornyeyevD.AllenR.HoladayA. S.Protecting cotton photosynthesis during moderate chilling at high light intensity by increasing chloroplastic antioxidant enzyme activityJournal of Experimental Botany2001522345
  86. 86. BabuV.BansalK. C.Osmotin overexpression in transgenic potato plants provide protection against osmotic stress, 5th International Symposium on Molecular Biology of Potato, Bogensee, Germany, Aug261998
  87. 87. MaqboolS. B.ZhongH.El -MaghrabyY.AhmadA.ChaiB.WangW.SticklenM. B.Competence of oat (AvenasativaL.) shoot apical meristems for integrative transformation, inherited expression and osmotic tolerance of transgenic lines containing HVA1, Theoretical and Applied Genetics.2002105201
  88. 88. KumarS.DhingraA.DaniellH.Plastid-expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots, and leaves confers enhanced salt tolerance.Plant Physiology.20041352843
  89. 89. RuidangQ.MeiS.HuiZ.YanxiuZ.JurenZ.Engineering of enhanced glycine betaine synthesis improves drought tolerance in maizePlant Biotechnology Journal20042477
  90. 90. TangW.PengX.NewtonR. J.Enhanced tolerance to salt stress in transgenic loblolly pine simultaneously expressing two genes encoding mannitol-1-phosphate dehydrogenase and glucitol-6-phosphate dehydrogenase.Plant Physiology and Biochemistry.,2005200543139
  91. 91. DuN.LiuX.LiY.ChenS.ZhangJ.HaD.DengW.SunC.ZhangY.PijutP. M.Genetic transformation ofPopulustomentosato improve salt tolerance. Plant Cell Tissue and Organ Culture2012108181
  92. 92. MolinariH. B. C.MarurC. J.FilhoG. C. B.KobayashiA. K.PileggiM.RuiP. L.JnrLuiz. F. P. P.LuizG. E. V.Osmotic adjustment in transgenic citrus rootstock Carrizo citrange (Citrus sinensisOsb. x Poncirustrifoliata L. Raf.) overproducing proline.Plant Science,20041671375
  93. 93. YamadaM.MorishitaH.UranoK.ShiozakiN.Yamaguchi-ShinozakiK.ShinozakiK.YoshibaY.Effects of free proline accumulation in petunias under drought stressJournal of Experimental Botany2005561975
  94. 94. HonjohK.ShimizuH.NagaishiN.MatsumotoH.SugaK.MiyamotoT.IioM.HatanoS.Improvement of freezing tolerance in transgenic tobacco leaves by expressing the hiC6 gene.BioscienceBiotechnology, and Biochemistry.2001651796
  95. 95. GasicK.KorbanS. S.Expression of Arabidopsis phytochelatin synthase in indian mustard (Brassica juncea) plants enhances tolerance for Cd and ZnPlanta2251277
  96. 96. SavitchL. V.AllardG.SekiM.RobertL. S.TinkerN. A.HunerN. P. A.ShinozakiK.SinghJ.Theeffect.ofoverexpression.oftwo.brassicaC. B. F. D. R. E.B1-liketranscription.factorson.photosyntheticcapacity.freezingtolerance.inBrassica.napusPlant Cell Physiology.2005
  97. 97. FurukawaJ.YamajiN.WangH.MitaniN.MurataY.SatoK.KatsuharaM.MaK. T. J.An aluminum-activated citrate transporter in barleyPlant Cell Physiology.2007481081
  98. 98. IngleR. A.MugfordS. T.ReesJ. D.CampbellM. M.SmithJ. A. C.Constitutively high expression of the histidine biosynthetic pathway contributes to nickel tolerance in hyperaccumulator plantsPlant Cell,20052005172089

Written By

Rosa Ma Pérez-Clemente and Aurelio Gómez-Cadenas

Submitted: November 17th, 2011 Published: October 17th, 2012