Proliferation of Citrus aurantifolia by In Vitro Epicotyl Segment Culture
Abstract
It is clear that, beneficial species of Citrus need to improvement especially by new methods due to limitations of conventional methods. New methods like biotechnology and gene transfer need to establishment of regeneration plants by tissue culture. Shoot proliferation in Citrus is easy but rooting of proliferated micro shoots has been discussed in various articles. The goal of this study is presentation of a proper method for rooting of micro shoots in Citrus by manipulation of media content. Hypocotyl segments of Citrus aurantifolia from 45 day old seedlings and 0.5 – 0.7 cm in length were cultured on MSmedia supplemented with different kinds and concentrations of plant hormones suitable for shooting and rooting such as BA, IBA and NAA alone or together. 1 mg/l BAand 1.5 mg/l NAA on MSmedia was the best treatment for shooting and rooting respectively. In this study we can overcome one of the most important problems of establishing regeneration system in Citrus and opening the way for biotechnology and gene transfer for this important and economic plant.
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Introduction
Citrus fruits duo to extremely pleasant aroma and taste also effect on human health for its useful substances, which is one of the most important fruits in Iran like allover the world. There are many biotic and abiotic stresses that have negative and harmful effects on cultivation of these valuable horticultural products. For example fungal diseases that cause significant problems in the production of Citrus fruits, such as postbloom fruit drop, caused by Colletotrichum acutatum; Alternaria brown spot, caused by Alternaria alternata; scab diseases, caused by Elsinoe fawsettii and E. australis; melanose, caused by Diaporthe citri; and greasy spot caused by Mycosphaerella citri1[1] and bacterial diseases such as Citrus canker, Citrus variegated chlorosis, and Huanglongbing (HLB) that caused by members of the bacterial species Xanthomonas citri, Xylella fastidiosa and ‘Candidatus Liberibacter’ respectively[2] and Virus and Virus – Like diseases as Citrus Tristeza Virus (CTV), Citrus Psorosis (CPV), Citrus Exocortis Viroid (CEVd), and also some of the agents pathogen such as Candidatus Phytoplasma aurantifolia that causes WBDL (Witches Broom Disease) [3 and 4]. Some of these diseases are very important in Iran. So it is very clear that Citrus industry mainly need to a strong program for improvement for supply of new cultivars with desirable characteristics like resistance against diseases.
Conventional breeding methods have demonstrated limitations with respect to Citrus improvement due to some of the biological characteristics of woody plants such as nucellar polyembryony, high heterozygosity, long juvenile period, and auto incompatibility [5 and 6].Genetic engineering of Citrus presents the possibility to produce Citrus plants with resistance to different diseases. Genetic engineering needs to viable shoots which can be regenerated via organogenesis firstly. Regeneration of plants from single cells and complex explants is therefore the key process in every genetic manipulation work; unless this can be achieved consistently and efficiently, no genetic improvement by somatic methods is possible. To accomplish this, a source of genetically homogeneous cells and tissues is necessary. Micropropagation cultures are an ideal source of homogeneous cells and tissues and this system is useful for the propagation of plants emanating from experiments of genetic variation [7]. So we need to a proper regeneration system via regeneration of shoots and roots and whole regenerated plants finally for gene transfer protocols.
There are several reports indicative problems of rooting of regenerated shoots in Citrus [8; 9 and 10]. It is clear that, this seemingly insignificant problem complicates the transformation process. Therefore optimizing as much as possible of regeneration systems is necessary. The goal of this study is presentation of simple and repeatable proliferation system for one of the most important species of Citrus (Citrus aurantifolia).
Conclusion
Biotechnology programs and gene transfer studies need to establishment of a proper, repeatable and easy plant regeneration method by tissue culture systems. Proliferation of shoots in Citrus species is feasible but rooting of proliferated shoots and whole plantlets regeneration need to optimizing of method as it mentioned in discussion sections. In this study we present a simple and repeatable method for rooting of regenerated micro shoots in Citrus so it was prepared an introduction of gene transfer program in this very important species in order to attaining new cultivars.
FIGURE 2-8: Plantlet regeneration of Citrus aurantifolia on MSmedia with different kinds and concentrations of PGRs. 2. Shoot proliferation on MSmedia supplemented by 1 mg/l BA. 3. Shoot proliferation on MSmedia supplemented by 2 mg/l BA. 4 and 5. Growth and foliation of micro shoots on MSmedia supplemented by 1 mg/l BA. 6. Rooting of micros hoots on MSmedia supplemented by 1.5 mg/l NAA. 7. Weak rooting of micro shoots on MSmedia with 1 mg/l IBA and 0.3 mg/l BA(the arrows show only 2 roots). Whole regenerated plantlet of Citrus aurantifolia).
