Micropropagation/ Tissue Culture ... -...
Transcript of 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