IJOEAR-SEP-2017-10

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The utilities of Citrus tissue culture

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-9 September- 2017 Page | 36 The utilities of Citrus tissue culture Firoozeh Chamandoosti Iranian Research Institute of Plant Protection Agricultural Research Education and Extension Organization AREEO Tehran Iran PhD of Cellular and Developmental Biology Assistant Professor of Iranian Research Institute of Plant Protection Department of Plant Diseases Abstract — Citrus is the third most important fruit crop in the world after apple and bananas and the total area cultivated with the various Citrus cultivar covers over 7.2 million hectares yielding total annual production of approximately 100 million metric tons of fruit. The genus Citrus possesses several undesirable characteristic including salt and cold sensitivity they are also susceptible to diseases caused by fungi bacteria and viruses. Despit substantial genetic diversity and interspecific fertility the genus Citrus includes some of the most difficult species to breeding. For example most species are highly heterozygous and produce progeny that segregate widely for many characters when crosses are made. The juvenile periods are often very long self- and cross-incompatibility and pollen and/or ovule sterility are relatively common and the presence of adventitious somatic embryos in the nucellus of developing ovules of the most of Citrus greatly limits hybrid production. Genetic transformation is an alternative to overcome these difficulties. For successful transformation regeneration of whole plants from the transformed cells is a prerequisite. On the other hand production of virus – free plants development of new cultivars and production of seedless fruit production of flavonoid micropropagation germplasm conservation and cryopreservation are the other utilities of in vitro culture of Citrus. Keywords — Citrus Improvement Tissue culture. I. INTRODUCTION Tissue culture is the in vitro aseptic culture of cells tissues organs or whole plant under controlled nutritional and environmental conditions Thorpe 2007 often to produce the clones of plants. It is clear that in vitro culture is an essential component of plant – biotechnology offers innovative approaches in several research areas Annarita and Laura 2012. The importance of plant cell tissue and organ culture is more cleared when it includes in major fruit crop in vitro culture regarding to micropropagation the induction of somatic embryogenesis the improving of methodologies through the analysis of medium components and the use of additives to increase the frequency of regeneration and the production of the in vitro cultures for the important species such as species belong to genus Citrus. The genus Citrus is cultured in more than 100 countries making it one of the most important commercial fruit crops in terms of economic value and human nutrition Barlass and Skene 1986. Citrus is the most important fruit crop in the world and is produced in all five continents and it is often regarded as golden fruit or queen of all fruits Nito 1996. Despite substantial genetic diversity and interspecific fertility the genus Citrus includes some of the most difficult species to breed Gmitter et al. 1992 Perez – Molphe – Balch and Ochoa – Aljeo 1998. This is due to several obstacles for conventional breeding. For example most species are highly heterozygous and produce progeny that segregate widely for many characters when crosses are made. The juvenile periods are often very long self – and cross – incompatibility and pollen and/or ovule sterility are relatively common and the presence of adventitious somatic embryos in the nucellus of developing ovules of the most of Citrus greatly limits hybrid production Perez – Molphe – Balch and Ochoa – Aljeo 1998 Moore et al. 1993. Genetic transformation is an alternative to overcome these difficulties. For successful transformation regeneration of whole plants from the transformed cells is a prerequisite Duan et al. 2007. In this review some of the most important utilities of Citrus tissue culture that are based on result of different experiments in this field were explained. II. THE CITRUS GENIUS NEED TO IMPROVEMENT Citrus belongs to family Rutaceae having 150 genera and 15000 species and it is distributed mostly in tropical and temperate region of the planet Ladania 2008. A number of major genera of family Rutaceae are Citrus Murraya Zanthoxylum and Ruta etc. Perveen and Qaiser 2005. As mentioned earlier Citrus has high dietary value and is a prosperous source of vitamin C in combination with macromolecules such as amino acids organic acids and sugar as well as minerals comparable to magnesium and calcium in sufficient quantity Niaz et al. 2004. The genus Citrus possesses several undesirable characteristics including salt and cold sensitivity Garcia – Agustin and Primo – Millo 1995 Van Le et al. 1999 they are also susceptible to diseases caused by fungi bacteria and viruses such as Citrus exocortis viroid CEV

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-9 September- 2017 Page | 37 Citrus infectious variegation virus CIVV Citrus cachexia viroid CCaV and Citrus tristeza closterovirus CTV Van Le et al. 1999 Greno et al. 1988. These biotic and abiotis stresses cause to low productivity. So Citrus genius has a great need to improvement. III. TISSUE CULTURE IS A BASIC PREREQUISTIC FOR GENE TRANSFER IN CITRUS In recent years there has been a major thrust in Citrus improvement as competition from international Citrus markets disease and pest pressure and other abiotic and biotic stress conditions stimulate worldwide interest Grosser et al. 2000 Dutt and Grosser 2010. Genetic transformation of Citrus is a valuable technique for Citrus improvement due to difficulties of conventional Citrus breeding. Recently Citrus improvement using genetic transformation is being used frequently as increasing competition in international markets and disease pressure have stimulated worldwide interest in Citrus improvement Grosser et al. 2000. In Citrus gene transformation is carried out by three different techniques i.e. particle bombardment Jia – Long et al. 1996. protoplast transformation Fleming et al. 2000 and Agrobacterium Nuria et al. 2012 De Oliveria 2009 Yang and Hu 2011 Cervera et al. 1998. Currently Citrus canker and huanglongbing HLB are the two major diseases threatening the Citrus industry. Canker results in leaf – spotting and blemishing on the surface of the fruit often resulting in defoliation shoot dieback and fruit drop. HLB affects all cultivated Citrus varieties and causes a rapid decline of trees and the production of unmarketable fruit. Both these diseases are caused by non – indigenous bacterial pathogens and cause substantial economic losses. Incorporation of genes via genetic engineering can potentially confer resistance in susceptible cultivars while maintaining the varietal fidelity. Transgenic Citrus plants have been obtained by direct DNA trasfer into protoplasts Vardi et al. 1990 co – cultivation of internode or epicotyl segments with Agrobacterium Moore et al. 1992 Pena et al. 1995a Gutierrez et al. 1997. And particle bombardment of nucellar embryogenic cell suspentions Yao et al. 1996. At persent the most widely used method of gene trasfer in Citrus is the Agrobacterium – mediated transformation of epicotyl segments with 1 cm of length. Using this system transgenic plants of Citrus sinensis Pena et al. 1995a Bond and Roose 1998 Citrus aurantifolia Gutierrez et al. 1997 Citrus aurantium Pena et al. 1997 Citrus paradasi Luth and Moore 1996 Citrus sinensis x Poncirus trifolia Moore et al. 1992 Pena et al. 1995a and Citrus trifoliata Kaneyoshi et al. 1994 have been obtained. However this method is not suitable for the transformation of any seedless cultivar. Also special cultivars in themandarin group remain robust to transform using this method Dutt and Grosser 2010 Dutt et al. 2010 Khawale et al. 2006. Due to the limitations of this method it seems that in order to carry out successful gene transformation studies in Citrus optimized in vitro regeneration protocol is needed. Researchers should optimize efficient regeneration protocol before starting transformation studies. There are also many efficient regeneration protocols published in different Citrus species. In Citrus callus cultures have been established in species such as Citrus grandis L. Osb. Citrus auruntifolia Christm. Swingle. Citrus medica L. Citrus sinensis L. Osb. Citrus madurensis L. Citrus paradisi Macf. Citrus reticulata Blanco and Citrus limon Sabharwal 1963 Murashige and Tucker 1969 Grinblat 1972 Chaturvedi and Mitra 1975 Moore 1985 Duran-Vila et al. 1989 Gill 1992 Gill et. al. 1994. Responses to different culture media are often genotype – specific. So the more optimization of tissue culture techniques is essential for gene transfer in Citrus. IV. TISSUE CULTURE MICROPROPAGATION IS A POWERFUL TOOLS FOR PRODUCTION OF VIRUS – FREE CITRUS SPECIES Micropropagation is an important asexual method that can be used for the production of virus – free rootstock plants Roistacher et al. 1976. As mentioned earlier Citrus due to pedoclimatic conditions often suffers from abiotic stresses such as salinity drought and iron deficiency induced chlorosis nevertheless a major constraint for its cultivation is represented by graft – transmissible agents viruses virus – like viroids prokaryotes that may cause considerable losses in crop yield and quality of plants and plant products. These agents may often remain symptomless thus representing a special risk in their spread over long distances through Citrus infected propagating material. The worldwide movement of graft – transmissible pathogens and relative vectors along with the Citrus material has increased in the last years due to the strong globalization and the lack of adequate quarantine measures. Once they invade a cultivated area the most severe pathogens can be rapidly spread by insect vectors and their control becomes even more difficult Carimi et al. 2013. Virus and viroid‟s have been recognised as serious problem limiting the vigour yield quantity and quality. Severe infections have resulted in the exclusion of some cultivars from commercial usage reported that viral diseases are major threats affecting Citrus industry Vishwanath and Narayan 2015. The diseases are graft – transmissible through grafting infected

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-9 September- 2017 Page | 38 bud sticks Santos et al. 1984. Hence rising of disease – free foundation plants is imperative to provide certified bud sticks to the growers and to encourage the planting of grafts instead of seedlings Mukhopadhyay et al. 1997. The elimination of viruses viroids and phytoplasmas from infecte initial mother propagation material is a prerequisite for the production of healthy vegetatively propagated crop material. Methods used are thermotherapy meristem tissue culture in vitro micrografting in vitro chemotherapy and cryotherapy of shoot tips followed by shoot – tip tissue culture or in vitro micrografting Christina 2015. As all of us know meristem tissue culture in vitro micrografting and cryotherapy of shoot tips followed by shoot – tip tissue culture or in vitro micrografting are the basic tissue culture methods for virus elimination in plants. But we have to keep in mind that all metdods arenot suitable for all plants. For example meristem culture that is used for the elimination of viruses and related pathogens from a large number of vegetatively propagated plants and it is the main method used in plant virus elimination programs for some plants such as Citrus stone fruits and other woody species meristem cultures are not successful George 1993b Navarro 1988. In these cases the meristem tip is grafted onto a virus free rootstock. The micrografting technique was first used for the elimination of viruses and viroids in Citrus by Navarro et al. 1976. Several scientists thereafter adopted this method to produce virus free plant material in Citrus Kapari –Isaia et al. 2002 2007 Mukhopadhyay et al. 1997 Navarro Civerolo Juarez and Garney 1991 Navarro Juarez and Pina 2001 Navarro et al. 1975 Singh 2001. In fact the use of tissue culture methods for Citrus crop species has already had practical benefits. Most notable among these are techniques for obtaining virus – free and mycoplasma – free stocks using in vitro grafting of apical meristems from infected plants onto decapitated seedlings Navaro et al. 1975. The issue that has been considered in recent years is that growing nucellar seedlings was the only method available for producing disease free Citrus cultivars from clones infected with virus or other grafttransmissible pathogens. The primary disadvantage of producing Citrus budlines through nucellar embryony is the phenomenon of juvenility. Young nucellar seedlings exhibit excessive thorniness vigorous and up – right habit of growth slowness to fruit alternate bearing in early years and physical differences in fruit characteristics which are often detrimental in marketing the fruit. These characteristics may persist for many years and over many budded generations. Nucellar budlines usually produce higher yields of fruit than their parental clones over a period of 8 – 10 years or more Cameron et al. 1968 Nauer et al. 1983. The portion of this higher yield that can be attributed to elimination of virus and virus – like pathogens in the parental bud – line has not been determined. Variations among Citrus nucellar budlines and differences other than juvenility from the parental budline have been reported Frost et al. 1957 Nauer et al. 1983 in numbers indicating that genetic variants may occur more often during production of nucellar bud – lines than occur during standard nursery trees production by bud propagation. Therefore a method to recover Citrus plants free of all virus and virus – like diseases and without juvenile characters was needed. The first attempts in this direction were made by shoot –tip culture in vitro a technique widely used to recover healthy herbaceous plants. However attempts to develop Citrus plants from shoot – tips failed Edriss et al. 1984. Constraints of this method the use of alternative methods such as somatic embryogenesis can be useful. Somatic embryogenesis is a developmental process where a plant somatic cell can dedifferentiate to a totipotent embryonic stem cell that has the ability to give rise to an embryo under appropriate conditions. This new embryo can further develop into a whole plant. In woody plants somatic embryogenesis plays a critical role in clonal propagation and is a powerful tool for synthetic seed production germplasm conservation and cryopreservation Yuan et al. 2016. Also somatic embryogenesis can be used to eliminate many virus diseases Bitters et al. 1970 DOnghia et al. 1997 DOnghia et al. 2001 the plant material obtained by somatic embryos regenerated in vitro can be used to establish healthy Citrus stocks. On the other hand while other plant micro – organisms are in many cases controlled by therapeutic treatments directly performed in the field graft – transmissible agents cannot be eliminated by these means because of the peculiarity of their replication cycle. Pro – active strategies which primarily rely on lower disease incidence and restrain virus dissemination can prevent the introduction of these agents into new plantings and new areas this is less hard and expensive than eliminating them once they are already present. Within this context strict phytosanitary regulations and certification programmes for the production of „healthy‟ Citrus nursery plants are amongst the most efficient preventive strategies Carimi et al. 2013. In Citrus the production of embryogenic callus lines was reported from excised nucelli Rangan et al. 1968 abortive ovules Bitters et al.1970 unfertilized ovules Button and Bornman 1971 undeveloped ovules Starrantino and Russo 1980 juice vesicles Nito and Iwamasa 1990 anthers Hidaka et al . 1981 styles and stigmas Carimi et al. 1995 as well as from leaves epicotyls cotyledons and root segments Gill et al. 1995. The embryogenic potential of Citrus varied with genotype and type of explant. in vitro culture of ovules from ovaries and immature fruits was initially used to obtain

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-9 September- 2017 Page | 39 virus – free nucellar plants from polyembryonic Citrus cultivars Bitters et al. 1970 Navarro et al. 1979. Somatic embryos embryogenic callus and cell cultures recovered from in vitro cultured ovules have also been used to develop cryopreservation strategies for germplasm conservation Kobayashi et al. 1990 Marin et al. 1993 Engelmann et al. 1994 Sakai et al. 1990 and 1991 Duran – Vila 1995 and protoplast technologies Vardi and Galun 1989 Grosser and Gmitter 1990 a and b Gimitter et al. 1992. Recent studies have indicated the embryogenic potential of somatic tissues which are neither nucellar nor ovular in origin. Nito and Iwamasa 1990 obtained eight somatic embryos from cultures derived from Satsuma juice vesicles. Carimi et al. 1995 induced formation of embryogenic cultures from styles of different species of Citrus. Gill et al . 1995 obtained somatic embryos from leaf epicotyl cotyledon and root segments of in vitro grown nucellar seedling of C. reticulata Blanco. V. OTHER UTILITIES OF CITRUS TISSUE CULTURE 5.1 Development of new cultivars Citrus propagation by conventional means is restricted to particular season and availability of plant material. It doesn‟t guarantee trueness of cultivars and mass production of certified Citrus plants throughout the year. Plant tissue culture has emerged as a powerful tool for propagation and improvement of many woody plant species including Citrus. Citrus also stands among difficult to root crops and micropropagation offers rapid propagation of such crops in limited space and time under controlled conditions throughout the year Usman 2005. In vitro culture further eliminates diseases Grosser and Chandler 2000 provides scope for the development of new cultivars through somaclonal variation Hammschlag et al. 1995 and somatic hybridization Al – Bahrany 2002 Grosser et al. 1996 Louzada et al. 1996 Mendes – da – Gloria 2000 Ollitrault et al. 1996 Ollitrault et al. 2000 Paudyal and Haq 2000 that have improved Citrus rootstock resistance against nematode infestation and other pests as well Bouquet et al. 2003 Grosser et al. 1998 Guo and Deny 1998 Guo and Deny 2001. Industry the micropropagation of Citrus has always aroused great interest among scientists. There is a growing demand to develop new varieties of plants resistant to pathogens and adverse environmental conditions and characterized by high quality of fruits Yaacob et al. 2014. Traditional techniques for creating new species are not effective in the case of Citrus lemons due to the problems that have already been mentioned such as physiological barriers associated with sexual reproduction such as heterozygosity and polyembriony Tusa et al. 1990 Carimi et al. 1994 Savita et al. 2010 Benabdesselam et al. 2011 Lombardo et al. 2011. Citrus Lemons plantations face a number of problems such as pests slow growth susceptibility to disease sensitivity to low temperatures and substantial losses during storage Mukhtar et al. 2005a b Savita et. al. 2010 Sarma et al. 2011. In vitro culture is a technique that can solve these problems. In addition this technique can also produce crops on a relatively large scale in comparison with traditional plant breeding. Furthermore in vitro cultures eliminate infections and can be faster than traditional plant cultures Savita et al. 2011 Singh and Kaur 2011. Development of new cultivares by tissue culture that mentioned above is due to a phenomenon called somaclonal variation. The term somaclonal variation was coined by Larkin and scowcraft 1981 to define genetic variation present in regenerated plants that is uncovered or induced by a tissue culture process. Somaclonal variation has been reported in a wide range of traits including plant height overall growth habit flower fruit and leaf morphology juvelinity maturity date diseases resistance yield and biochemical charactristics. However most reports generally deal with either solanaceous or cearal crops but little information has been reported in woody prenial fruit crops Grosser et al. 1997. 5.2 Production of seedless Citrus fruit In recent years there has been a shift in the world Citrus market towards seedless Citrus fruits and considerable energy has been devoted towards their production. The seedless trait in Citrus is related to male or female gametophyte sterility self incompatibility or early embryo abortion Reforgiato Recupero et al. 2005 and several methods exist for the production of seedless Citrus of which mutation breeding somaclonal variation and triploid breeding are the most important. In Citrus triploid seedless cultivars are obtained by breeding between elite monoembryonic diploid cultivars as female parent with tetraploid cultivars as pollen parent Esen and Soost 1973. Sterility in such fruits is caused due to the odd number of chromosomes that are unable to undergo successful meiotic pairing to produce chromosomally balanced gametes Reforgiato Recupero et al. 2005. Colchicine is an alkaloid obtained from the meadow saffron Colchicum autumnale L.. This alkaloid inhibits mitosis by hampering the development of the nuclear spindle Blakeslee and Avery 1937 and is most commonly used to obtain tetraploid plants artificially Notsuka et al. 2000. In Citrus tetraploidy has been induced by treatment of axillary buds with

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-9 September- 2017 Page | 40 colchicine as was done with the cultivars Ellendale and Clementine. The treated buds upon grafting on rootstock produced several tetraploid plants Oiyama 1992. However a disadvantage of using axillary buds in colchicine experiments is that most of the recovered plants end up being unstable chimeras and do not have applications in a breeding program Barrett 1974 Jaskani et al. 1996. This is due to the use of multicellular tissue as a source of explants for colchicine treatment. Using such tissues usually result in production of a large proportion of chimeric tetraploids Kadota and Niimi 2002. Non – c-himeric autotetraploid Citrus plants have been obtained from in vitro colchicine experiments via embryogenesis of underdeveloped ovules from immature Citrus fruits Gmitter and Ling 1991 Gmitter et al. 1991. 5.3 Production of flavonoid Citrus and Citrus peels contain common flavonoids such as hesperidin naringin neohesperidin narirutin eriocitrin didymin and rutin among others Benavente – Garcia et al. 2007 Tripoli et al. 2007 Gattuso et al. 2007. A number of studies have demonstrated the biological properties of these Citrus flavonoids including anti – carcinogenic anti-oxidant and anti-inflammatory properties that promote and benefit human health Tripoli et al. 2007 Gattuso et al. 2007 Lopez – Lazaro et al. 2002 Wang et al. 2014 . In addition to Citrus flavonoids Citrus peels are also the sole and rich source of polymethoxylated flavonoids which were found to exert many biological properties particularly anti-cancer and anti – inflammatory activity Li et al. 2009 Gosslau et al. 2014 Li et al. 2014 Li et al. 2014. Recent studies have also demonstrated potent anticarcinogenic and anti – inflammatory efficacy of 5-demethylated polymethoxyflavones in single molecules Li et al. 2014 Ma et al . 2014 Lai et al. 2007 or in multiple 5-demethylated polymethoxyflavones Lai et al. 2011. The natural content of 5 –demethylated polymethoxy flavones in Citrus peels is low in percentage but it has been confirmed that they have more potent biological activity than their non-demethylated counterparts such as anticancer activity Lai et al. 2014 Lai et al. 2007 Lai et al. 2011. In essence there are three subclasses of Citrus flavonoids exiting abundantly in Citrus peels namely polyhydroxy flavonoids polymethoxy flavonoids and mixed substituted flavonoids with both hydroxyl and methoxyl groups particularly 5 – demethylated polymethoxyflavnoids. These flavonoids have demonstrated effective anti – cancer property both in vitro and in vivo either in a form of individual compounds or in a mixture of Citrus flavonoids. The anti – cancer study of these flavonoids has progressed well in recent years owing albeit in the initial steps to the modern chemical analysis and isolation and the biological activity testing. However with the exception of nobiletin the relationships between each individual flavonoid in Citrus peels and its bioactivity such as anti – carcinogenesis remain untouched to some extent. Relationships among the naturally proportioned flavonoidsin Citrus peels and their biological activities are even more complex and unexplored. Biotechnology uses techniques and processes that involve living organisms to obtain specific products and/or modifications that increase the production of chemical substances of interest in less time and less capital investment Davies and Deroles 2014. Secondary metabolites such as Citrus flavonoids that are found in plants are generally produced in low concentrations compared with primary metabolites. Therefore different strategies including in vitro culture systems have been extensively studied to increase the production of secondary metabolites in plants Smetanska 2008 Muranaka and Saito 2010 Gill et al. 2013. In vitro cell cultures represent an interesting alternative because secondary metabolites of interest are obtained in a controlled environment that is not influenced by changes in climate or soil conditions Goncalves and Romano 2013 Collin 2001. Plants that are grown in their natural habitat generally have varying concentrations of compounds of interest depending on the particular crop season Salmore and Hunter 2001 Puricelli et al. 2002 Ralphs and Gardner 2001. Moreover their exploitation in their natural environment can cause gradual genetic erosion Sidhu and Bel 1996. Also callus cultures and cells in suspension have been used to study the biosynthesis of economically important secondary metabolites enabling the propagation of cell lineages that contain alterations in biosynthetic capabilities. he production of different compounds in plants is generally mediated by environmental factors that vary according to physiological conditions and seasonal variations Gill et al. 2013. Thus cell cultures ensure controlled conditions that circumvent environmental changes. 5.4 Micropropagation Germplasm conservation and Cryopreservation Citrus trees are propagated both by seed and by vegetative means. There is huge demand of planting material. Non availability of scientifically propagated planting material from elite clones for plantation are the main constraints in Citrus cultivation. In recent years tissue culture techniques micropropagation are increasingly used for rapid clonal propagation of several economic plants restoration of vigour and yield due to infection and preservation of germplasm. Hence

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International Journal of Environmental Agriculture Research IJOEAR ISSN:2454-1850 Vol-3 Issue-9 September- 2017 Page | 41 micropropagation is a very useful tool for a production of large number of planting materials. Besides this technique is also useful for saving the Citrus species which are facing extinction singh 2002. Conservation of Citrus germplasm in the field requires great space labour and costs the risk of damage by natural calamities and pathogen infection that may be always incurred. Therefore in vitro conservation can easily overcome these difficulties and ensuring the maintenance of healthy Citrus germplasm. Moreover for several Citrus species in vitro culture may play a major role as a conservation strategy or even be the only option available. For an efficient in vitro conservation of healthy germplasm we need suitable protocols of plant regeneration. Fortunately there exist many reports on organogenesis from different types of explants of Citrus and Citrus rootstocks. The morphogenic responses of Citrus cultured in vitro are influenced by the genotype the explant type and the culture medium. Explants include shoot tips Barlass and Skene 1986 stem sections Grinblat 1972 Chaturvedi and Mitra 1974 Raj Bhansali and Arya 1979 Barlass and Skene 1982 root sections Sauton et al. 1982 Burger and Hackett 1986 Sim et al. 1989 Bhat et al. 1992 leaf sections Chaturvedi and Mitra 1974 Hu and Kong 1987 stem internodes Duran – Vila and Navarro 1989 epicotyl segments Edriss and Burger 1984 and transverse thin cell layer tTCL explants excised from stem options Van Le et al. 1999. The regeneration of adventitious shoots has been obtained either directly from the explant or from an intermediate callus phase. Also as noted above traditionally Citrus germplasm is preserved in clonal orchards where it is susceptible to pests diseases and climatic catastrophes Duran – Vila 1995. Cryopreservation of embryogenic calli at ultra – low temperatures –196 °C in liquid nitrogen LN2 is an excellent mean to overcome the challenges inherent to maintaining embryogenic materials and to provide long – term conservation of valuable embryogenic lines Gonzales – Arnao et al. 2008. There are many studies on Citrus cryopreservation using very different materials. Efficient vitrification and dehydrationbased cooling procedures have been reported for various Citrus organs and tissues including shoot tips Wang and Deng 2004 seeds Kaya et al. 2016 embryonic axes Cho et al. 2002 somatic embryos Marin and Duran – Vila 1988 ovules Gonzales – Arnao et al. 2003 embryogenic calli Perez et al. 1997 Olivares – Fuster et al. 2000 and nucellar cells Sakai et al. 1990. And ultimately as we all know the success of any of these methods requires the establishment of appropriate tissue culture systems. VI. CONCLUSION Like the vast majority of genera and species of plants especially economically important genera and species different species of Citrus genius need to improvement. Citrus improvement by conventional methods due to difficulties such as has limitations that all of them solve by modern methods of biotechnology or in other words transformation. Low plant regeneration frequencies especially for many of the economically important Citrus species is the most important difficult within this almost new and advanced method. So the progression of tissue culture methods leads to solving these problems. REFERENCES 1 Al – Bahrany A.M. 2002. Effect of phytohormones on in vitro shoot multiplication and rooting of lime Citrus aurantifolia Swing.. Scientia Horticulturae. 95: 285 – 295 2 Annarita L. and Laura M.R.R. 2012. 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