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