LIBRO-CONGRESO-CITRUS

efficient for elimination of all citrus graft-transmissible pathogens from local or imported varieties. It has
allowed worldwide the recovery of hundreds of healthy cultivars and the planting of hundreds of millions of
healthy certified trees. Only in Spain about 135 million certified nursery plants propagated from micrografted
plants have been planted. STG is also a very useful technique for regeneration of elite genotypes in several
areas of research. In vitro grafting for these purposes may be done using larger shoots (up to 1 cm). STG is
being routinely used for the following purposes: (i) Regeneration of somatic hybrids from embryos difficult to
germinate; (ii) Regeneration of plants from irradiated shoots to produce seedless varieties; (iii) Regeneration
of plants from haploid embryos that are very difficult to germinate. STG was used to regenerate the
‘Clemenules’ haploid plant that has been used by the International Citrus Genome Consortium to sequence
the whole citrus genome; (iv) Production of stable tetraploid plants of monoembryonic genotypes, which are
very useful for triploid breeding; (v) Regeneration of transgenic plants from shoots that are very difficult to
root in vitro. STG has become a routine application in citrus genetic transformation.
S05P25
Microshoot tip grafting in vitro-a technique for establishment of disease free scion bank of Citrus
reticulata var. ‘Khasi’ mandarin
Sanabam R.S., Huidrom S.D., and Handique P.J.
Institute of Bioresources and Sustainable Development (IBSD), Medicinal Plants and Horticultural Resources Division, India.
sbnm_rakesh@yahoo.com
This study has been carried out to produce viral disease free scion bank of ‘Khasi’ mandarin (Citrus reticulata)
using four citrus cultivars namely ‘Kachai’ lime, ‘Champra maounthabi’, ‘Solom’ and ‘Phouheiree’ through
micrografting techniques as viral disease affects the longevity of citrus orchards and quality of fruits. Success
of micrografting depends on the type of cut for scion insertion, age of seedlings of rootstock, conditions of
light or darkness, concentration of sucrose used and also on cultivar used for micrografting. Frequency of
success improved considerably from 36.24% success at 3% sucrose to 40.05 % success at 6% sucrose with 0.5
mg/L benzyl amino purine (BAP) in Murashige and Tucker (MT) liquid media. Response of micrografting also
significantly improved when inverted T type of cut was used for scion insertion giving 37.07% compared to
28.13% of success in that of wedge type of cut. Highest rate of success was obtained in ‘Champra maounthabi’
with success rate of 40.74% followed by ‘Phouheiree’, ‘Solom’ and ‘Kachai’ lime of 34.26%, 32.24% and 27.53%
success respectively. Analysis of 8-10 months old micrografted plants for the presence of graft-transmissible
Citrus tristeza virus (CTV) following double antibody sandwich-enzyme linked immunosorbent assay (DAS-
ELISA) technique was performed giving negative results for the virus.
S05P26
Elimination of Spiroplasma citri by somatic embryogenesis from citrus stigma and style culture:
preliminary results
Frasheri D. 1 , Moujahed R. 1 , Djelouah K. 1 , Carra A. 2 , Carimi F. 2 , Valentini F. 1 , and D’Onghia A.M. 1
1 CIHEAM-Mediterranean Agronomic Institute of Bari (CIHEAM-MAIB), Integrated Pest Management, Italy; and 2 Consiglio Nazionale
delle Ricerche, UOS Palermo (CNR-UOS Palermo), Istituto di Genetica Vegetale, Italy. frasheri@iamb.it
Spiroplasma citri, the causal agent of Citrus Stubborn disease, is spread by infected budwood and, in nature,
by phloem-feeding homoptera insects, predominantly leafhoppers. This pathogen can be usually eliminated
by shoot-tip grafting. In this work, somatic embryogenesis from stigma and style culture, which proved to be
effective in the elimination of several infectious agents of citrus, has been firstly applied in the elimination of
this pathogen. Flowers were collected from an infected Egyptian source of sweet orange ‘Washington’ navel.
Pistils were tested by DAS ELISA using monoclonal antibodies (SEDIAG-INRA, France) and PCR using specific
primers targeting the spiralin; 5 out of 70 pistils were found infected by S. citri. About 75 flowers were surface
sterilized in ethanol and vertically placed in Petri dishes in contact with Murashige and Skoog (MS) medium
supplemented with 6- benzylaminopurine. Explants were in vitro cultured and subculturing was carried out
every 3-4 weeks. Thirty-nine explants produced callus about 20-30 days after culture initiation, twenty-eight
of which regenerated embryos 4 months later. Embryos were individually cultured in test tube and in vivo
acclimatized after 6 months from embryo formation by minigrafting regenerated plants onto 6 month-old
XII INTERNATIONAL CITRUS CONGRESS 2012 - 89
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