In vitro induction of Haploid Plants from Anther Culture in Citrus
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Transcript of In vitro induction of Haploid Plants from Anther Culture in Citrus
In vitro induction of haploid plants from Anther culture in Citrus
Presenter: GUL NABI KHANAdvisor: Dr. Chitose HONSHO
Laboratory of Fruit Science, Dept. of Agriculture and Environmental Sciences, Uni. Of Miyazaki , Japan
05/01/2023
Presentation Outline • Learning Objectives • Introduction • 1st Report: Plantlet Formation by Anther Culture of Citrus aurantium L. • 2nd Report: The Effect of Light Quality on Anther Culture of Citrus
clementina Hort. ex Tan. • Questions and Answers session
Learning Objectives • Haploids and Double haploids (DHs)• Importance of Haploids in fruit breeding and genetics • Spontaneous Haploids in fruit trees • Scientific Methods to produce haploids in fruit trees • Gametophytic Embrogenesis (Androgenesis and Gynogenesis) • Factors affecting induction of haploids in fruit trees
Introduction • Shorten juvenile or non-fruiting period • Increasing yield • To pre-long ripening season• Regular fruit bearing • Induction of seedlessness • Improvement of external and internal quality of the fruits • Tolerance to biotic and abiotic stresses
How to induce these properties in new varieties of fruit trees?
Through Conventional and Biotechnological techniques 1. Spontaneously2. Conventional methods like
• Hybridization • Selfing • Selection • Mutation
3. Biotechnological techniques • In vitro tissue culture• Regeneration from protoplasts• Somatic hybridization• In vitro mutant selection• Genetic transformation• Haploid and Double haploids (DHs) production
What is haploids and Double haploids (DHs)?
Haploids and Double haploids (DHs)Haploid: An individuals with the gametic chromosome number (n) in its somatic cells. A Doubled Haploid: is a genotype formed when haploid cells (n), i.e. egg or sperm cell undergo chromosome doubling (2n).
The resulting individual is completely homozygous
How to induce haploidy in fruit trees for breeding purposes?
1. Spontaneous HaploidsParthenogenesis: The production of an embryo from egg cell without the participation of male gamete.
Apogamy: The production of an embryo from gametophytic cell other than ovum.
2. Conventional Breeding Approaches • Long reproductive cycle of fruit trees • Large size • High level of heterozygosity • Parthenocarpy • Self-incompatibility • Male sterility
In each generation hetrozygosity reduces by 50%
3. In vitro (Biotechnological) Production of Haploids
Haploids from male gametes (Androgenesis) a) Anther culture b) Pollen/Microspore culture
Haploids from female gametes or Megaspores (Gynogenesis) pollinated by irradiated or triploid pollens
a) Immature ovary culture b) Immature Ovule culture
also called in vivo modified pollination method
Direct and Indirect Embryogenesis
Factors influencing Androgenesis
• Genotype of donor plant • Stage of pollen development • Physiological condition of the donor plant • Pre-treatment • Composition of culture media • Organic supplement or Carbon source • Plant growth regulators • Physical factors during tissue culture (light, temperature)• Mode of inoculation
1st Report:Plantlet Formation by Anther Culture of Citrus aurantium L. Hidaka et al., 1982
Research objective: • Therefore, the present paper described the formation of plantlets
from anthers of Citrus aurantium L. and comparison with that of Poncirus (Hidaka et al., 1979).
Materials and Methods • ‘Sour Orange’ (Citrus aurantium L.), ‘Kabusu (Shu-to)’, ‘ Choshu-to’
and ‘Bouquet’
Surface sterilization, 70% ETOL
Aceto carmine squash
MS, 50g/l Sucrose, pH 5.8, 8g/l agar, IAA, Kinetin28+1°C Dark until embryoid induction, 16 h light,
Sampling for observation of pollen development
‘Sure orange’3~21 days (6 days interval)
21~93 days (9 days interval)
Fixed in FAA Soln
Dehydrated in ethyl and n-butyl alcohol series
Placed in paraffin, cut 8-12 µm thickness, stained with Safranin O and Fast green FCF.
Results and Discussion Effect of developmental stages of the pollen grains, additions to the media, and varieties on embryoid differentiation
Fig 2: An embryoid formed from an anther of sour orange after 16 weeks of culture at late uninucleate stage.
Effect of Culture media on embryoid formation
Varietal comparisons of embryoid formation
Histological Observations
Induction of Roots
Discussion Poncirus (Hidaka et al., 1979)
• Embryoid appeared after 3W of inoculation.• Embryoid differentiated from pollen grains ranging
tetrad~ uninucleate stages.• Calli were obtained from the somatic tissue of
anthers at all stages. • Poncirus have higher ratio of embryoid induction. • Diploid, hyperhaploid, and mixoploid besides
haploid
‘Sour orange’ (Hidaka et al., 1982)
• C. aurantium required longer time than Poncirus.• Late uninucleate stage pollen produced embryoids. • Calli formation is like to Poncirus • Developmental phases from pollen to embryoid were
not yet resolved b/c of low ratio of embryoid induction.
• Only Diploids • No haploid was confirmed in this study.
2nd Report:The effect of light quality on anther culture of Citrus clementina Hort. ex Tan. (Antonietta et al., 2005)
Research Objective: • Therefore, in the present study was carried out to evaluate the effect of the light
quality on the anther culture of C. clementina Hort. ex Tan., cultivar Nules.
Materials and Methods
DAPI (4,6 diamidino 2-
phenylindol dichloride) Staining
3.5~4.5mm in length, Microspores at vacuolated stage
Dark (D)4°C, 14 days
Flow cabinet, petals were removed with forceps
10 cm3 induction medium (Germana and Chiancone 2003),
27+1 °C, One month under Dark conditions
Light Conditions Blue (B), 24+1 °C, 600 Red (R), 24+1 °C White (W), 24+1 °C Far Red (FR), 24+1 °C
Experimental Design and Analysis • Not developed, swollen, and anther with callus were recorded. • The effect of light were tested by analysis of variance at P≤0.05. • Difference among means by fisher’s (LSD) multiple comparison test.
Results and Discussion
Effect of light on Callus quality
Light quality affected the type of callus, R and FR produce higher % of green, compact and not morphogenic callus.
Morphogenic callus: Callus that shows same morphological characteristics with donor plant.
Importance of Dark induction and W light • In Citrus, dark and light seems to be a key factor in embryogenic induction of
microspore.
Conclusion • W and R together, as well as the control D conditions, were the most
suitable light qualities and regimes to promote callus formation. • B light significantly decreased the induction and growth of callus.
References 1. HIDAKA, T., YAMADA, Y. and SHICHIJO, T. (1982). Plantlet Formation by Anther
Culture of Citrus aurantium L. Japan. J. Breed., 32(3), pp.247-252.2. Antonietta, G., Benedetta, C., Calogero, I. and Rosario, M. (2005). The effect of
light quality on anther culture of Citrus clementina Hort. ex Tan. Acta Physiologiae Plantarum, 27(4), pp.717-721.
THANK YOU FOR YOUR KIND ATTENSION Questions and suggestions are welcome