micropropagation/ tissue culture ... -...

Micropropagation/ Tissue Culture

Technology Updates

Taiwan Banana Research Institute

Sin-Wan Lee

Nov. 2014

Taiwan Banana Research InstitutePingtung, Taiwan, R.O.C.

Banana Micropropagation in Taiwan

Established in 1983 to supply clean planting material to check the spread of Fusarium wilt

Adopted the meristem culture technique of

Ma and Shii (1972, 1974)

Rapid propagation of superior commercial cultivars ‘Pei-Chiao’ and Foc TR4 resistant / tolerant cultivars ‘Formosana’ , ‘Tai Chiao No. 5’, and ‘Tai Chiao No. 7’,

Up to 2014 (Oct), produced over 70 million Fusarium-free (BBTV, CMV) indexed plantlets

Commercial Micropropagation

TBRI has a network of over 15 nurseries operated by the Fruit Cooperative located in the banana growing areas.

Superior quality of plantlets :

* free from Fusarium wilt (soil-less potting mix)

* virus indexed (BBTV, CMV)

* acceptable percentage of off-types ( 3 - 5%)

* technical services after sale of plantlets

Banana plants infected with

Foc TR 4

(A) BBTV

(B) CMV

Virus Indexing (ELISA) for BBTV and

CMV of suckers for micropropagation

Smaller size of TC

plantlets saves labor cost

well developed roots

increase survival rate

T C

Sucker

Number of plantlets propagated

(1995 – 2013)

Nu

mb

er of p

lan

ts in m

illion

s

(1) Select suckers

(2) Culture initiation

(3) Induction of buds

(4) Multiplication of

shoots

(6)Acclimatization in nursery

(7) Planting in field

(5) Regeneration

of plantlets

Micropropagation Protocol

Stage 0: Preparative stage

Stage 1: Culture initiation

Stage 2: Multiplication of buds

Stage 3: Regeneration of plantlets

Stage 4: Acclimatization in nursery

Stage 0: Preparative stage Select suckers from true-to-type mother plant

Stage 0 : Preparative stage

Cleaning and trimming of sucker

Stage 0 : Preparative stage

Surface clean with cotton (75% alcohol)

inside laminar flow work bench

Stage 1: Culture Initiation

Remove leaf base of sucker to

expose meristematic region

Isolate meristematic

block from sucker

Stage 1. Culture initiationInduction of buds from meristematic block

Stage 1: Culture initiation

Subdivide meristematic block

Stage 2 : Multiplication of buds

(Subculture – 1)

Stage 2 : Multiplication of buds(Subculture – 2)

Stage 2 : Multiplication of buds(subculture – 3)

Stage 2 : Elongation of buds before

plantlet regeneration

(Subculture - 6 )

Stage 3: Regeneration of plantletsDouble layer technique : adding liquid regeneration

mediumto established shoot clusters in agar

Elongation and rooting of shoot in screened

house

Double layer technique :elongation of plantlets

Deflasking after rooting of shoots

Stage 3 : Regeneration of plantlets

Sorting of plantlets according to size

(Large, Medium, Small)

L M S

Stage 4: Transplanting in screened nursery

Off-types commonly

found at nursery stage

Commercial Banana Micropropagation

Tissue culture plantlets

established in field

TBRI demonstration farm

established with TC plantlets

Banana from tissue

cultured plantlet

Application of Tissue Culture Technology

Micropropagation

1. TP multiplication medium

Thidiazuron (TDZ) and

Paclobutrazol (PP333)

2. Temporary Immersion System (TIS)

TP medium

(TDZ + PP333)

Effect of TP medium

on multiplication of buds in ‘Pei Chiao’

TDZ + PP333 medium increased the

multiplication rate of shoots in three

commercial Cavendish cultivars

Mu

ltiplica

tion

rate

Aventages of TP (TDZ+PP333) medium in

commercial micropropagation

TP medium gives a 3 to 5 fold increase in the

cumulative multiplication rate of shoots

compared with BA

Sequence of application of TP medium is crucial

in the subculturing schedule

Multiplication rate in TP medium increased

with time of incubation ranging from 30 to

90 days with the optimum at 45 to 60 days

2. Temporary Immersion System (TIS)

Periodic medium immersion achieves

uniform diffusion of nutrients

Gaseous exchange improves the

development of plants

Programmed interval and duration

of liquid immersion can

synchronize the rate of growth

Digital

timer

Air compressor

Comparison of BA and TP (TDZ + PP333)

multiplication medium

Comparison of multiplication rate of shoots

in TIS and agar medium containing

TDZ and PP333

Mu

ltiplic

atio

n ra

te

Regeneration of plantlets

after adding liquid medium

Acclimatization of plantlets in nursery

Application of Tissue Culture Technology

Musa improvement

3. Multiple Bud Clumps (MBCs)

(a) MBCs for in vitro selection

(b) MBCs for induction of callus

4. Callus (induced from MBCs)

for in vitro selection

3. Induction of Multiple Bud Clumps (MBCs):(A) meristem of sucker in BA medium. (B), (C), (D) small

buds from (A) subcultured in TDZ+ Paclobutrazol medium

A

C D

B

Multiplication of Multiple Bud Clumps

(MBCs) in TP medium

Multiple bud clumps ready for in vitro selection

4 week culture of MBC

Explant ( 0.5 x 0.2 cm)

In vitro selection of MBCs‘ Pei-Chiao’ in Foc TR4 culture filtrate

0%

check

10%

Culture

filtrate

15%

Culture

filtrate

20%

Culture

filtrate

MBCs of ‘Pei Chiao’ and ‘Tai Chiao no. 2’ after 3 successive selections in Fusaric Acid

(A) 0.175→0.175→0.22mM (B) 0.2→0.2→0.22mM

(A) 0.175→0.175→0.22mM (B) 0.2→0.2→0.22mM

Pei

Chiao

Tai

Chiao

no.2

Selection - 1

FA 0.2 mM x 1

(22/167=13%)

Selection – 2

FA 0.2 mM x 2

(31/33 = 94%)

Selection – 3

FA 0.2 mM x 3

(65/83 = 78%)

in vitro selection of ‘Pei Chiao’ Survial rate of MBCs after

3 successive selections in Fusaric acid 0.2 mM

in vitro selection of ‘Latundan’ Survival rate of

MBCs after 5 successive selection cycles

Foc 10% x 2 →Foc 12% x 2 → Foc12%

( 83.5%)

Foc 10% x 2 →Foc12% x 2 → FA 0.2mM

(45.0%)

Net house screening of plantlets

resistant to Foc TR4 pathogen in potting mix

Evaluation of resistance of in vitro selected

MBCs to Foc TR4 in net house

Evaluation of disease resistance of selected

somaclones in Foc race 4 infested orchard

In vitro selected somalcones in field trial

In vitro Selected somalcones in field trial

‘Tai Chiao no.6- 59’ ‘Pei Chiao -72’

Results of in vitro selection of MBCs

Preliminary field trials of 2,132 clones obtained from different in vitro selection cycles and different cultivars, 83 clones (3.9%) did not show disease symptoms 6 to 8 months after planting in the Foc infested orchard.

Tissue culture plantlets were propagated from the suckers of these selected clones for further evaluation of stability in disease resistance, horticultural traits and post harvest fruit quality.

Conclusion

In the in vitro selection of MBCs tolerant to Fusaric Acid, a tremendous number of compact tiny meristems could be screened with much reduced time, space and man-power.

in vitro selection of MBCs, in combination with net house and field evaluation, will accelerate the progress of the screening program for Fusarium wilt resistant cultivars at TBRI.

4. Induction of callus from MBCs

for in vitro selection

Multiple Bud Clump Callus

Comparison of callus induction media

Cultivar(genotype)Callus induction formulation

Da Dg Pa Pg P3 DP

Pei Chiao (AAA) ＋＋ ＋＋＋ － ＋ ＋ ＋＋

Tai Chiao #2(AAA) ＋＋ ＋＋ ＋ ＋ ＋＋＋ ＋＋

Tai Chiao #5(AAA) ＋＋ ＋＋＋ ＋＋＋ ＋ ＋＋＋ ＋＋＋

Morado (AAA) ＋＋ ＋＋ － － ＋＋ ＋＋

Cultivar Rose(AA) ＋＋ ＋ － － － ＋＋

K K (AA) － － － － － ＋

Latundan (AAB) ＋＋＋ ＋＋＋ ＋ ＋＋ ＋＋ ＋＋＋

Namwa (ABB) ＋＋＋ ＋＋ ＋ － ＋ ＋＋

(16＋) (16＋) (6＋) (5＋) (12＋) (17＋)

Induction of callus in Pei Chiao : Picloram 1.5 mg/L (C-2)

Differentiation in ½ MS TDZ 2 mg/L

Plantlets regenerated from differentiated

from calli in Pei Chiao

Callus induction in Pei Chiao

2,4-D + P Picloram 4 mg/L 2,4- D 4mg/L

Induction of callus in Picloram (4 mg/L)

‘Pei Chiao’ (AAA) ‘Tai Chiao no. 2’(AAA)

Induction of callus in Picloram (4 mg/L)

KK (AA) Namwa (ABB)

Field trial of ‘Pei Chiao’ plantlets

regenerated from callus

Field trial of plants

regenerated from callus

1,106 plantlets showed

normal growth during the

nursery stage.

After field planting,

off-type plants were

a. 1/158 = 0.6％b. 1/198 = 0.5％

Appication of callus in in vitro selection

Two steps Method

1. Callus induced from MBCs in Picloram (4 mg/L)

2. Buds differrentiated in ½ MS TDZ (2 mg/L)

+ Fusaric Acid ( 0.1 mM)

One step Method

Callus induction and differentiation in

one medium (P4T) : Picloram (4 mg/L)

+ TDZ (2 mg/L)

+ Fusaric Acid (0.1 mM )

2 steps method of callus in in vitro selection Step 1 : Callus induction in Picloram (1.5 mg/L ),

P1.5 → ½ T2 (ck)

7/7 = 100%

P1.5 → ½ T2 + 0.1mM FA

2/7 = 28.6%

Pei Chiao Pei Chiao

2 steps method of callus in in vitro selection

Step 2 : Differentiation

1/2MS T2 + Fusaric acid (0.1 mM) (C-23)

P1.5 → ½ MS T2 (ck) P1.5 → ½MS T2 +

0.1mM FA

Evaluation of plants derived from calli induced from

MBCs for resistance to Foc in net house

Two steps method ( P4, 1/2MS T2) C-20

Cultivar No. of calli

Differentiated

To form shoots

(%differentiation)

No. of plantlets

Regenerated

For net house

selection

No. of plants

with disease

symptoms

No. of plants

without disease

symptoms

Pei Chiao 7 (15%) 608 608 0

Pei Chiao

941

5 (13%) 144 143 1

Tai Chiao

No. 2

14 (31%) 725 720 5

Tai Chiao

No. 5

8 (15%) 431 426 5

Tai Chaio

No. 6

7 (15%) 418 417 1

KK 3 (8%) 54 54 0

Efficiency of selection = 0.5%

One step method (P4T) for induction and

differentiation of calli in Pei Chiao

P4T (check) Regeneration of bud

P4T + Fusaric Acid 0.1 mM Regeneration of bud

One step method (P4T) for induction and

differentiation of calli in Tai Chiao #5

P4T check Regenration of bub

P4T + Fusaric Acid 0.1 mM Regeneration of bud

One step method (P4T) for induction and

differentiation of calli in Cultivar Rose

P4T check Regeneration of bub

P4T + Fusaric Acid 0.1 mM Regeneration of bud

One step method (P4T) for induction and

differentiation of calli in Latundan

P4T check Regenration of bub

P4T + Fusaric Acid 0.1 mM Regeneration of bud

Evaluation of plants derived from calli by

One step method for resistance to Foc in net house

One step method (P4T) :

(3T-P4T-BA-BA-BA-BA) C-21

(without adding Fusaric acid)

No. of plants tested = 1,650

No. of plants did not show

disease symptom = 23

Efficiency of selection = 1.4%

Innoculum of pathogen

（4.2×103 spores/g soil)

Conclusion

1. Micropropagation

Commercial propagation of Fusarium wilt

free tissue culture planting materials in

Taiwan was established in 1983.

In the past three decades, over 70 million

plantlets have been supplied, including

Foc TR4 resistant/tolerant cultivars to

sustain the banana industry .

Conclusion

2. Tissue Culture Technology

TP medium

(Thidiazuron + Paclobutrazol )

Multiple Bud Clumps (MBCs)

Induction and multiplication

in vitro selection

Callus

Induction of calli and differentiation

in vitro selection

Thank you