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In vitro culture for production of quality banana and

plantain planting material

GUEYE Badara RTB in vitro propagation and conservation specialist (In vitro Genebank)

International Institute of Tropical Agriculture (IITA)Headquarters: PMB 5320, Oyo Road, Ibadan, Nigeria

Web: http://www.iita.org/genetic-resources-center

2nd International Workshop of the Learning Alliance on Banana Bunchy Top Disease Control in Africa.

9 to 14 March 2015. IITA Ibadan, Oyo Road, Ibadan, Nigeria

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PTC gives opportunity to grow plant material in artificial conditions with advantages such as:

-Possibility for sanitation (culture in sterile conditions, pathogen cleaning)

-Rescue and conservation (continuous availability, space saving, cost-effectiveness, cryo)

-Micro-grafting and micro-propagation (mass multiplication: Bioreactor, cell suspension, polyshoot…)

-Germplasm exchange

-Door opened for more research (genetic transformation, somatic embryogenesis, haploid production, embryo rescue, molecular and pathological/virological investigations…)

INTRODUCTION

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Musa spp. GERMPLASM SANITATION

Vegetatively propagated crop: accumulation of pathogens over the reproductive cycles

3 major viruses: BBTV, BSV and CMV

Disease control: Production of clean planting material through virus eradication

Cleaning techniques through in vitro culture: Meristem regeneration, Thermo-treatment and Chemo-treatment

Factors: (a) the virus characteristics, (b) the type of tissues treated (c) the plant species(d) the genotype

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In vitro VIRUS ELIMINATION TECHNIQUES FOR Musa spp.

In vitro Virus elimination techniques

Meristem regeneration Thermo-treatment Chemo-treatment

(+)

- Most efficient on phloem-associated viruses ( BBTV)

- Association with other techniques

- « the smaller, the better »

- Mainly heat-treatment, cryo-treatment less popular

- Widely used in vivo/in vitro and associated with meristem culture

- A wide range of potentially antiviral molecules tested

- More efficient on CMV

(-) - Less efficient for virus in meristematic dome (CMV)

- Depends on the virus type

- mode of action not very known

- Time consuming and harmful

- Phytotoxicity and negative effect on regeneration

- Mode of action (inhibition, elimination, « silencing ») ???

Schematic representation of virus eradication processes for banana. The green , blue, and red arrows represent the process of chemotherapy, meristem culture, and

thermotherapy, respectively (Lassois et al., 2012. In Maurizio Lambardi et al. (eds.), In press)

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MICROPROPAGATION

Musa spp. are very responsive to in vitro PTC

Meristem regrowth in 4 to 6 weeks, followed by shoot development

Proliferation: subculture of fully developed plantlet

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MICROPROPAGATION

Musa spp. multiplication using semi-solid culture medium

Plantain/banana proliferation medium

Average multiplication rate onto semi-solid culture medium

4:1

Components Qty / LMS basal medium 4.43 g Myo-inositol 100 mg Sugar 30 g IAA (Indol-Acetic Acid) 0.18 mg BAP (Benzyl Amino Purine)

4.5 mg

Ascorbic acid 10 mgAgar 7 g

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Large scale multiplication

MICROPROPAGATION

DeRoi, SA

National genebank, SA

Vitropic, France

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MICROPROPAGATION

Musa spp. multiplication using Temporary Immersion System (TIS) Bioreactor

Rocket kit shaker & shouthern sun bioreactor

Air-lift Method

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MICROPROPAGATION

Musa spp. multiplication using Temporary Immersion System (TIS) Bioreactor

NACGRAB, Nigeria

Setis Method

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MICROPROPAGATION

Musa spp. multiplication using Temporary Immersion System (TIS) Bioreactor

Average multiplication rate using TIS

16:1

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HARDERNING

Acclimatization processMore than 95 % success

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HARDERNING

Nursery, a vital step (DeRoi, SA)

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Virus cleaning by meristem culture+ Most popular and more efficient (on phloem-associated viruses like BBTV)

+ « the smaller, the better »

+ Association with other techniques

e.g.: On Musa acuminata (AAA, cv Williams)

- BBTV eliminated at 99 % elimination

- BSV at 76 % and 41 % for in vivo and in vitro respectively

-Balance with regeneration capacity: « the bigger the better »

- Less efficient on CMV, virus in meristematic dome (1 à 7 % clean)

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Virus cleaning by Thermo-treatment

+ Mainly heat-treatment, cryo-treatment less popular

+ Widely used in vivo/vitro and associated with meristem culture

- Depends a lot on the virus type

- mode of action not very known

-Time consuming and harmful

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Virus cleaning by Chemo-treatment

+ A wide range of potentially antiviral molecules tested

+ More efficient on CMV

+ Example: Virazole ® (Ribavirin, 1-B-D-ribofuranosyl-1-2-4-triazole-3 carboxamide),

synthetic analogue of guanosine, replication inhibitor)

-Phytotoxicity and negative effect on regeneration

- Mode of action (inhibition, elimination, « silencing »)

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Recommandation: combination of techniques

- Meristem culture + thermo-treatment:

e.g.: On Musa acuminata (AAA, cv Williams)

- CMV eliminated at 38-70 % elimination

- BSV at 79 % and 67 % for in vivo and in vitro respectively

Banana and plantain virus cleaning

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Banana and plantain virus cleaning

CHALLENGES

- Clean or not expressed (« opportunism » of the virus)???

-What about B genomes???

- Integration of the virus in the genome (BSV)

- Inhibition of the expression (BSV)

- Policy issues (germplasm exchanges)…

Recommandation: combination of techniques

- Meristem culture + thermo-treatment:

e.g.: On Musa acuminata (AAA, cv Williams)

- CMV eliminated at 38-70 % elimination

- BSV at 79 % and 67 % for in vivo and in vitro respectively

Banana and plantain virus cleaning

Banana and plantain virus cleaning

CHALLENGES

- Clean or not expressed (« opportunism » of the virus)???

-What about B genomes???

- Integration of the virus in the genome (BSV)

- Inhibition of the expression (BSV)

- Policy issues (germplasm exchanges)…