This is my research plan presentation for postgraduate studies in Miyazaki University. Recently, I am carrying out this research for the development of citrus industry.

1. Production of polyploid Citrus hybrids by
interploid hybridization and embryo rescue
Presenter :NOORANI GUL NABI KHAN
M1 student of Pomology lab at UOM
Advisor: Dr. Prof. Chitose HONSHO
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2. Contents
• Key words of research title
• Introduction
• Problem statement
• Purpose of research
• Plant materials and methods
• Characteristics of parents plants
• Experiments and its objective
1. Exp 1: Pollination and observation of Embryo Development
2. Exp 2: Embryo rescue and plant recovery
3. Exp 3: Characterization of ploidy plants.
• Expected results
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3. Title’s key words
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Production of Polyploid Citrus hybrids by
interploid hybridization and embryo rescue
• Polyploid
• Hybrids
• Interploid hybridization
• Embryo rescue

4. Introduction
• Citrus economically important…
• Seedlessness
• Market demand
• Engage or remain in competition.
• Various breeding approaches
 conventional techniques
 Modern techniques
, protoplast fusion, cybirds,
irradiation, interspecific or generic crosses,
flowcytometry and citrus transformation.
• More productive and less time consuming
Pict 1 shows seedless citrus fruits
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5. Problem statement
• Large quantity importation
• Market dominated
• Lack of Triploid (seedless) citrus varieties.
• Low skill of citrus breeding.
• Lack of advanced propagation and breeding technologies.
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Why did I select this topic for my research

6. Purpose of study
Specific:
Seedless hybrid citrus plant production
Contribution:
1. Above mentioned deficiencies.
2. vital role, development of fruit industry.
3. Economic prosperity
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7. Plant materials and methods
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Following genotype, 2X x 4X hybridization
Table 1 shows parent plants

8. Characteristics of Parents
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Table 2 shows characteristics of parent plants

9. Experiments & Objectives
Objective:
• Hand or control pollination of
parents to avoid contamination.
• To find specific time of embryo
development after pollination for
plant recovery.
1. Collection of pollen, balloon
2. Incubation of excised anthers
3. Emasculation
4. Bagging
5. Harvest 12-20 WAP
6. 5 fruits each time, 2 embryo
observation, 3 embryo rescue
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Exp 1: Pollination and Observation
of Embryo Development
Pict2 shows balloon stage of flower Pict3 shows excised anthers
Pict 4shows removal of stamen
from female bisexual flower.
Pict 5 shows bagging of flower

10. • Embryo abortion and competition
• Rescue immature embryos
• Interploidy hybridization is more efficient…
• Success development stage and nutrients
• 3 harvested fruit – ethanol 70%
• Immature embryos excision using clean bench.
• Immediate culture on nutrient media
• Using test tube
• 28C and light exposure.
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Exp2: Embryo Rescue and Plant recovery
A: Embryo Rescue

11. Continues …
• 2-3 weeks days after culture, transfer to another test tube.
• Same media
• Particular stage of growth
• Artificial soil (vermiculite)
• Keep in green house for ploidy level analysis.
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B: Potting and Acclimatization

12. Exp 3 : Characterization of Polyploidy plants
• What is ploidy level or genome size?
• Determine by flow cytometry.
• Advantages
• Leaf samples pomology lab protocol
• Buffer to preserve sample in life like state
• PI (( Propidium Iodide)
• Cell lab Quanta SC-MPL Instructions
guide.
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A: Ploidy level or genome size analysis
Pict 6 show cell lab Quanta SC-MPL flow cytometry

13. B: Chromosomes Observation
• Confirmation of triploidy, No of chromosome and morphology
• Many techniques but EMA method
• Root tips and aerial parts
• Pomology lab protocol
Steps:
1. Samples preparation
2. Pre –fixation
3. Treatment of Carnoy’s solution
4. Enzymatic maceration .
5. Slides preparation and staining with 2% Giemsa soln
6. Observation
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Preparation of small chromosome samples by EMA.
Pict 7 shows chromosome shapes under
microscopic observation

14. C: Morphological and Histological study
• Morphological and histological study will help us to estimate ploidy
of plants for breeding purposes.
• Several characteristics of polyploid plants will be analyzed and
compared while conducting this experiment.
• Cell size measurement, leaf morphology, growth of the plants, size
and density of stomata, relative water content and water loss from
leaf.
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15. Expected Result
• Seedless (triploid) citrus hybrid cultivars
• Fundamental techniques of breeding.
• Research methodology
• Thus, it will help us to develop fruit industry within my
beloved country Afghanistan.
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Thank you for your kind attention!
I welcome your questions,
recommendations, suggestions and
comments.
NOORANI
Thank you all for your support.