ESTABLISHMENT OF TISSUE CULTURE TECHNIQUES IN CITRUS SPECIES (original) (raw)

Regeneration of Citrus via Shoot Apecies

HortScience

Viral damage is a major problem in citrus. As most citrus are asexually propagated, it is necessary to have an alternative way of regenerating virus-free plants from infected plants. Shoot apicies are the most suitable explant material for this purpose because that part of the plant is virus-free. Fifty sour orange shoot tips and 22 Swingle shoot tips, 1 mm - 1.5 mm long, were excised from in vitro germinated seedlings and cultured on semisolid Murashige and Skoog medium, without growth regulators, containing 0.2 % Gelrite. After 8-10 weeks, shoots and leaves developed in 68'% of the sour orange explants, and in 77% of the Swingle explants. Some explants produced roots, after 11-12 weeks, and could be removed from culture and established in soil medium.

Proliferation of Citrus aurantifolia by In Vitro Epicotyl Segment Culture

Agriculture Journal IJOEAR, 2020

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 MS media 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 BA and 1.5 mg/l NAA on MS media 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.

Effect of various factors on shoot regeneration from citrus epicotyl explants

Journal of Applied Horticulture

The effect of various treatments on shoot organogenesis from seedling epicotyl explants from various scion and rootstock polyembryonic citrus types was determined. Treatments included water source, gelling agent, explant insertion, seed size, light intensity, malachite green, nonionic surfactants, and sodium sulphate. Tap water, with the highest levels of SO 4 2-, Ca 2+ , K + , Mg 2+ , and Na + , resulted in the most shoots compared to the other 5 sources, suggesting a mineral nutrient effect. Carrageenan produced fewer shoots than agar and gellan gum. Explants inserted into the medium produced more shoots than those cultured on the surface, presumably because of better exposure to water and nutrients. Seed size, light intensity, malachite green, and sodium sulphate had no effect on the number of shoots

Citrus TISSUE CULTURE WITH TWO DIFFERENT APPROACHES

International Journal of Biosciences, 2020

Citrus is an important genius for economy and human health but a susceptible genius against biotic and abiotic stresses, so it needs to improved programs. Different basal media (MS, 1⁄2MA, 1⁄3MS and DKW) and different kind and concentrations of plant growth regulators i.e. BA, KIN, 2ip, ZE and TDZ (0 – 2 mg/l) and NAA, IAA and IBA (0 – 2 mg/l) added with 30 g/l sucrose, 3 g/l active charcoal and 7.5 g/l bacteriological agar] were used for organogenesis include shooting and rooting, also callusing from nodal explant of Citrus latifolia. MS medium supplemented with 1 mg/l BA, and 0.01 mg/l NAA is the best media for multiple shoot induction on nodal explants and elongation of them. Other cytokinins had not significant effects on shoot induction and multiplication. Using of 0.01 mg/l IBA instead of 0.01 mg/l NAA on medium with 1 mg/l BA, led to multiple shoot induction on nodal explant indirectly. Rooting was induced on DKW medium plus 1.5 mg/l NAA in the best way compared another media...

In vitro regeneration of sour orange (Citrus aurantium L.) via direct organogenesis

2013

Low cell competency for regeneration and transformation is the main cause of so-called recalcitrance to transform a species or a genotype. A research was conducted to determine the optimum conditions for in vitro plant regeneration involving organogenesis in Citrus aurantium, which is an important rootstock worldwide. Seeds with peeled teguments were germinated in vitro, either kept in dark for 6 weeks or maintained in the absolute dark for 4 weeks followed by 10 days in 16-h photoperiod (56 µmol m-2 s-1) at 27 ± 2°C. Epicotyl-originated explants were cultured in MS medium supplemented with 6-benzylaminopurine (BAP) (0, 1, 2 and 3 mg L-1) and Naphthaleneacetic acid (NAA) (0, 0.05, 0.1 and 0.2 mg L-1) to induce organogenesis. Effects of Pre-culture in liquid MS medium (0, 1 and 2 days) on the number of responsive explants (RE) have been also evaluated. In the next step, explants having buds were transferred to MS medium containing Gibberellic Acid (GA 3) (0, 0.5 and 1 mg L-1) and the size and number of shoots, which have been produced by RE are then measured. The highest percentage of responsive explants (90%) obtained by using 2.5 mg L-1 BAP in combination with the 0.05 mg L-1 NAA which had 2 days pre-culture period of epicotyls for allowing to grow in the absolute darkness for 4 weeks, followed by 10 days in 16-h photoperiod (56 µmol m-2 s-1). The highest number of well-developed shoots was 4.2 shoots per explant and obtained with medium containing 0.5 mg L-1 GA 3. These protocols are suitable in association with Citrus aurantium Agrobacterium-mediated genetic transformation.

In vitro Propagation of Citrus Species through Callus Induction and Regeneration: A Review

International Journal of Current Microbiology and Applied Sciences

Citrus, one of the most important group of fruit crops around the world, are propagated at large scale with many difficulties. Propagation through seeds is challenging because of Phytophthora foot rot together with recalcitrance of citrus seeds. Vegetative propagation of Citrus species is mainly performed now-a-days by budding on seedling rootstocks. As heavy losses are experienced among the susceptible seedlings due to Phytophthora and Citrus tristeza virus (CTV), the interest in resistant rootstocks has greatly increased. The potential of conventional methods of citrus plant breeding of rootstocks are limited by physiological factors such as heterozygosity, inbreeding depression, nucellar polyembryony and juvenility. Under such conditions advanced tissue culture techniques provide best possible alternative for producing large number of resistant progenies from elite citrus genotypes. Plant tissue culture provides reliable and economical method of maintaining pathogen free plants that allows rapid multiplication and international exchange of germplasm. Generally, when in vitro propagation protocols are developed for any specific plant species, specialized conditions for individual genotypes, elite species and even various developmental stages of the explants plants are selected via error-andtrial experiments. Because large diversity is observed in Citrus plant family, it takes many months to develop protocols for most suitable culture medium, best concentrations and combinations of plant growth regulators and other supplements for better development of explant cultures. Therefore, in this review, we tried to put together results from difficultto-find literatures and listed all the identified findings, in which callus induction or somatic organogenesis was used to develop citrus plants. Successful protocols of surface sterilization method, culture establishment, shoot regeneration, in vitro rooting and acclimatization are presented systematically.

In vitro organogenesis optimization and plantlet regeneration in Citrus sinensis and C. limonia

Scientia Agricola, 2002

Exogenous genes can be introduced in plants by genetic transformation techniques. However, an efficient tissue culture system with high rates of plant recovery is necessary for gene introduction. This work aimed to define organogenesis and plant regeneration protocols for sweet orange varieties Natal, Valencia and Hamlin (Citrus sinensis L. Osbeck) and Rangpur lime (Citrus limonia L. Osbeck) which can be used in plant transformation experiments. Seeds of which teguments were removed, were germinated in vitro and maintained in the dark for three weeks, followed by one week at 16-h photoperiod (40 µmol m -2 s -1 ) and 27 ± 2°C. Organogenesis induction was done by introducing epicotyl segments in MT medium with 25 g L -1 sucrose and different BAP concentrations. After adventitious bud growth, the shoots were transferred to MT medium with either NAA or IBA (1 mg L -1 ), or absence of auxin, for rooting. The best results were obtained with 1 mg L -1 BAP for bud induction and 1 mg L -1 IBA for rooting for all three sweet orange cultivars. The use of 0.5-2.5 mg L -1 BAP, followed by 1 mg L -1 IBA were the best growth regulator combinations for bud induction and rooting, respectively, for 'Rangpur' lime. The protocols presented in this work are suitable for associations with genetic transformation experiments for these cultivars.