Production of triploids by interploidy hybridization (2n x 4n) and embryo rescue

1. PRODUCTION OF POLYPLOID CITRUS HYBRIDS BY
INTERPLOID HYBRIDIZATION AND EMBRYO RESCUE
BY: NOORANI GUL NABI KHAN
ADVISOR: DR. CHITOSE HONSHO
DATE: 16-04-2015
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2. Introduction:
• Seedless citrus cultivars are highly desired by consumers.
• Breeding of triploid consists of conventional and modern techniques
• Interploidy hybridization is one of the promising technique
Interploidy hybridization
◦ Diploid x Diploid
◦ Tetraploid x Diploid
◦ Diploid x tetraploid
◦ Each type of above crosses have merits and demerits
Research aim: Production of triploids with 2x x 4x hybridization and its
confirmations by Karyotyping studies.
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3. Diploid x Tetraploid
Diploid x Tetraploid sexual hybridization is widely used by breeder for seedless citrus production.
(Williams and Roose. (2000); Cameron and Soost 1969; Esen and Soost, 1971a, b; Oiyama and Okudai,
1988; Oiyama et al., 1982; Tachikawa et al., 1961; Oiyama and Kobayashi, 1990; Aleza et al., 2012).
So on
Limitations:
• Low no. of triploid with unexpected diploid, tetraploid and aneuploidy production.
• triploid embryo abortion
• Empty seeds (without embryos)
Solution:
• In vitro culture of immature embryos to avoid embryos abortion.
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4. Materials and Methods
Plant materials:
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5. Pollination, Fruit set and Measurement
• A control hand pollination will be preformed according to the procedure
(Aleza et al., 2010; 2012; Guardiola et al., 1984 and Honsho et al., 2009).
• In addition, 2x Natsudaidai pollen will be collected for Pollen Size measurement experiment.
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6. Embryo Rescue and Plant Recovery
Immature embryos will be rescued after 10-15 weeks of pollination (Jaskani et
al., 2005; Usman et al., 2002; Raza et al., 2003; Fatima et al., 2002; Asim, M. et al., 2011; Aleza et al., 2010;
Oiyama and Kobayashi., 1990 and Satarrantino and Resupero., 1981).
 Three immature fruits will be harvested in each cross every week (10-15)
In addition, two immature fruits will be harvested in control treatments.
Ten immature embryos will be rescued in vitro each week.
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7. Potting and acclimatization
 After two months of growth on MT media supplemented with hormone and hormone less media.
Seedlings will transfer to artificial stem sterilized soil
a) 40% black peat, 29% coconut fibre, 24% washed sand and 7% perlite (Aleza et al., 2010; 2012)
b) Coco peat (Chabukswar and Deodhar et al., 2005)
c) soil, sand and leaf mold 1:1:1 (Jaskani et al 2005; Usman.M et al, 2002; Fatima et al., 2002)
d) garden soil, sand and vermiculite 1:1:2 (Siwach et al., 2012)
e) vermiculite+ peat moss+ sand 1:1:1 v/v/v (Sherif et al., 2009)
f) Vermiculite ( Toolapong, P. et al. (1996); Yahata et al., 2006)
 Pots will be covered with Plastic film and tighten with rubber bands to prevent desiccation and to avoid rapid
changes in environment (Aleza et al., 2010; Sherif et al., 2009).
 Low concentration of Fungicide will be used during acclimatization (Fatima et al., 2002; Usman.M et al, 2002)
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8. CHARACTERIZATION OF PLOIDY
Molecular characterization: The hybridity of offspring obtained by 2x x 4x hybridization
during 2014-2016 years and their parents will be analyzed by Microsatellites or SSRs genetic
markers.
Ploidy level analysis: Ploidy level and genome size will be determined by flow cytometry
(Cell Lab Quanta SC MPL) according to the methodology described by Honsho et al., 2012.
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9. Continue…
Chromosome Observation: Prometaphase chromosome samples will be prepared by using
young leaves according to the procedure of Oiyama, (1981); Befu, M., et al. (2000, 2001, 2002);
Kitajima et al.(2001; 2007; 2011) ; Ma, Y. et al., 1996 etc.
Morphological and Histological study:
cell size, leaf morphology, growth of the plants, size and density of stomata, relative water
content and water loss from leaf.
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10. Thank You for your kind attention!
Questions, comments and recommendations are most welcome
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