Production of Autotetraploid Citrus Plant using active seeds and cell suspension culture techniques

Presented by: NOORANI, M1

Advisor: Dr. Chitose HONSHO

• Autotetraploid citrus plant • Colchicine treatment •Citrus paradise (plant material)•Flow cytometry •Interploidy crosses•Cell suspension •Citrus reticulata (plant material) • chimeric plant

At the end of the session participants will be able to,

1. Describe the difference between autotetraploid and allotetraploid?

2. Explain the importance of tetraploid citrus plants in breeding?

3. Identify at least two methods of autotetraploid citrus plant production?

4. Have knowledge about colchicine and its treatment?

5. Discuss unstable chimeric plants?

6. An idea about Embryogenesis callus production?

• Seedless trait in citrus

• Various breeding approaches

• Scarcity of tetraploid

• Several breeding techniques to Produce (allotetraploid and autotetraploid)

1. Screening of seedlings

2. In vitro colchicine

3. Somatic hybridization

4. Protoplast fusion

4n x 2n

Monoembryonic seed parent, efficient, yield max triploid, normal sexual fertilization (18n +9)

2n x 4n

polyembryonic seed parent, zygotic embryo abortion, immature embryo rescue and yield max population

Efficient Interploidy hybridization

Autotetraploid : An organism, individual or cell with four set of chromosome derived from same species. Or An individual or strain whose chromosome complement consists of four copies of a single genome due to doubling of an inherited chromosome complement.It occurs with three known mechanisms1. Fusion of unreduced gamete 2. Cross fertilization of an unreduced

gamete and diploid gamete(TB).3. Artificially chemical induction using

colchicine The pink arrow indicated unreduced or diploid gametes whereas the blue refers to haploid gamete.

1

2

Dorone, Y. (2012), Diploidization of meiosis in autotetraploids

• Definition: A hybrid cell or individual having four sets of chromosomes derived from two different parental species.

Schematic diagram of allopolyploidy.

Kellogg E A PNAS 2003;100:4369-4371

• Induction of Autotetraploids in Pummelo (Citrus grandisL. Osbeck) through Colchicine Treatment of Meristematically Active Seeds In Vitro:

(Kainth et al. 2010)

• Knowledge gape:

• What is the objective of this research paper?

• Why this research has been carried out?

Because auxiliary buds, shoots tips and somatic embryogeic callus has been

used to produce monoembryonic autotetraploid through colchicine treatment

but up to date no report has been published using germinating seed in citrus.

• Seed extracted from three red fleshed pummelo selections 1. 5-1-99-2

2. C2-5-12

3. UKP-1

12-14 d, until hypocotyls emerge 5-8mm

Dried, peeled, sterilized , rinsed

NaCLO, 8 min

High meristematic activity (SAM+RAM)

Ready for colchicine treatment

Let us talk about what is colchicine?

• Colchicine is a medication that treats gout(disease). It is a toxic natural product and originally extracted from plants of meadow saffron (Colchicum autumnale).

• It is alkaloid in nature and inhibit mitosis by delaying the development of nuclear spindles due to its toxicity nature.

• It is high toxic poison must be handle carefully.

• Dissolve in few drops of DMSO (dimethyel sulfoxide)

• Final concentration 1g/L, 2g/L, 3g/L

• Experimental design two-way factorial consisting 3 concentrations (1g/L,2g/L, 3g/L) and 2 exposure time(12 h and 24h)

• Three replication of each treatment.

• Per treatment 15 germinated seeds used.

Falcon tube having 10ml of germinating media. Different concentration of Colchicine

Rotary shaker 30rmp, dark

chamber 25+2C

Solid germination media, dark condition

Rooting media with NAA (16h light/ 8h dark

Flow cytomerty, micro grafted

• Generally colchicine induced tetraploid in all accession.

• Some cytogenic chimeric plants too

• Higher concentration and long exposure time delay the growth of seedling, cause hyperploidy, browning, necrosis and death of seedling.

• Survival and death rate

• Effective conc+time

• Inversely proportional to the concentration and time.

• 0-20% drop at 0.3%

TIE= % Seedling survival X % tetraploid seedlings

Definition:

• An organism, organ, or part consisting of two or more tissues of different genetic composition, produced as a result of organ transplant, grafting, or genetic engineering.

• Unstable

• Not using in breeding purposes.

• Later on tetraploid chimeric plant change to diploid.

In vitro production of autotetraploid Ponkan mandarin (Citrus reticulata Blanco) using cell suspension cultures:

(Dutt et al. 2009)

Knowledge gape:• Axillary buds, disadvantage, recovered plant unstable chimeras plant

• The use of cell suspension cultures, non chimeric tetraploid plants

• Low frequency

• This research paper describe a protocol for the production of stable

autotetraploid with high frequency.

Immature Ponkan mandarine(150d), sterilization, rinse

Unfertilized ovule

1st culture: DOG medium (MT salts+ vitamins+ sucrose + Malt extract + agar+ Kinetine)

Production of suspension cells:

Plant materials Ponkan mandarin

2nd Culture: ovule on monthly base until yellow callus emerge

Cell suspension culture:

Monthly transferred to EME( Callus-maintenance media) free of growth regulator.

3rd suspension culture: 5 g callus incubated in 25ml of H+H Cell proliferation medium

After 7 days cell were treated with colchicine

Final concentration of 1g/L colchicine

1 g callus in H+H medium4-8d incubation on platform shaker at 30rpm

• After incubation with colchicine 4 and 8 d.

• Suspension cells washed and culture on solid EME supplemented with 50g/L maltose for rapid reproduction. (28c, 16 h light/ 8h dark)

• Two treatment

1. Culture just on EME

2. Culture on EME overlaid with 2ml of 1:2 (0.6M BH3: 0.15M EME+maltose liquid medium)

• Regenerated embryoids cultured over 0.22mm cellulose acetate membrane to normalize and enlarge.

• Plantlet observed to be tetraploid ( flow cytometery+ chromosome staining) transfer to RMAN rooting medium and then potted into metro mix

commercial potting media.

Grosser and Gmitter 2011

• It has been found that suspension cells that treated with colchicine after 7d of 3rd suspension culture. Majority cell are in S-phase of mitotic cycle on 6 and 7 day of their subculture.

• This stage is ideal for colchicine application.

• H+H considered best medium for rapid reproduction of suspension cells.

• Liquid medium has advantage b/c of nutrients.

Effect of colchicine on development of somatic embryos:

• Proliferation time depends on colchicine treatment time.

• 2 weeks later, 8day treated cells turned dark brown.• 8 days treatment failed to advance from globular stage into torpedo.• Higher concentration and long time reduce the survival of seedlings.

• The majority tetraploid obtained from 4d treatment with liquid medium.

• No chimeric plant has been identified in treatments.

• B/c the origin of somatic embryogenesis is single cell (ovule) . It is therefore it reduced the possibility of chimeric plants.

• In vitro colchicine treatment can fulfill the deficiency of tetraploid citrus plants for breeding purposes.

• According to results of research papers , seed treatment technique produced tetraploid as well as mixploid plants but cell suspension technique produced only non-chimeric tetraploid.

• It has been proved by both researches that higher concentration and long exposure time reduce the germination and survival of seedlings.

At the end of the session participants will be able to,

1. Describe the difference between autotetraploid and allotetraploid?

2. Explain the importance of tetraploid citrus plants in breeding?

3. Identify at least two methods of autotetraploid citrus plant production?

4. Have knowledge about colchicine and its treatment?

5. Discuss unstable chimeric plants?

6. An idea about Embryogenesis callus production?

• Kainth, D and Jude W. (2010) Induction of Autotetraploid in Pummelo (Citrus grandis L. Osbeck) through Colchicine Treatment of Meristematically active seed in vitro. Proc. Fla. State Hort. Vol. 123, pp. 44 -48.

• M.Dutt et al. (2009) In vitro production of autotetraploid Ponkan mandarin (Citrus reticulate Balanco) using cell suspension cultures. Euphytica. Vol.173, pp. 235- 242.

Thank you for your kind attention!

I welcome your questions, recommendations, suggestions and

Comments b/c it will open a new window of study.

NOORANI