FNCA Mutation Breeding
Sub-Project Meeting on Disease Resistance in Banana
●Ⅲ.Summary of Progress Reports ●IV.Midterm Report ●V.Work Plan
Attachment III

SUMMARY OF PROGRESS REPORT

INDONESIA
  • Results observation in hot spot area showed that one plant can be infected by two or three disease such as sigatoka and bunchy top virus or Fusarium and S igatoka or Fusarium, bunchy top virus and S igatoka
  • Putative mutant indicated tolerance to Fusarium was attacked by another disease such as S igatoka and bunchy top virus
  • Around 446 plants were attacked by S igatoka and bunchy top virus
  • Banana mutant which provided fruit, their male bud was taken for cloning and propagation
  • During observation of banana grown in hot spot area showed that 38 plants indicated disease tolerant, but three months later most of plants were infected by S igatoka and Fusarium
MALAYSIA

   One of the strategies for banana improvement is through mutation induction that can alter one or more characteristics of generally acceptable cultivars while retaining the original genotype. In-vitro mutation induction is particularly important for vegetatively propagated plants such as banana. This approach may be important for sterile Musa species, where there is no sexual reproduction that could generate variation. Any stable genetic change can be fixed and multiplied for evaluation and utilization. Therefore, the efficiency of classical breeding may be improved by incorporating in-vitro mutagenesis to generate genetic variability. Research activities will be focused mainly on screening and selection for mutants tolerance for resistance for Fusarium wilt disease using popular local banana cultivars.

PHILIPPINES
  • Thirty two selected G1 mutant lines were evaluated in the field under high disease incidence. Five resistant mutant lines (PR 25-28, PR 23-30, PR 2-45, PR I3-30, and PR 22-28) were selected.
  • Nine mutant lines were planted in farmer's field and evaluated for BBTV disease reaction under natural field infection. Three mutant lines (PR 1-28, PR 13-30, PR 23-30) were selected.
  • Four mutant lines were evaluated for nematode ( Radopholus. similis) resistance. Based on the results on root damage and nematode reproduction, the three mutant lines PR 2-45, PR 4-45, and PR 10-45 were resistant under greenhouse condition.
VIETNAM
  • In-vitro shoot tips 2-3 leaf primordial. ABB genome (Musa sp. ABB)
  • Shoot tips culture on MS medium supplemented with BAP at 4mg/l and 0.5mg/l NAA rooted on half strength MS medium
  • Optimum gamma radiation is 10 or 20 doses
  • Artificial pathogenous infection (wounded, infection injection, dipping root, cultured hydroponics
  • Field experiments in a Fusarium wilt infected ‘hot spot'
  • Leaf symptoms on susceptible plants were observed within 55-65 days of cultivation
  • Infected rates: mutant lines 6% control 32%.
Attachment IV

Midterm Report for Disease Resistance in Banana


1. The selection of Starting breeding Materials.
- Whether it was appropriate or not.

<Fusarium wilt>
Indonesia : Cv Berangan (AAA), Race 4
Malaysia : Cv Berangan (AAA), Race 4
Vietnam : Cv Tay (ABB), Race 1

<Banana Bunchy top (Nematodes, Sigatoka) >
Philippines : Cv Lakatan (AA/AAA)
2. The determination of culture methodologies
- Tissue culture of clones, and Isolation and multiplication of selected mutant clones. 		- Explant and medium
Indonesia : shoot tips (meristem) from male buds/ MS +3 mg/l BAP +0.5 mg/l IAA
Malaysia : shoot tips (meristem)
Cell suspension MS + 5mg/l BAP
Vietnam : shoot tips/ MS + 4mg/l BAP + 0.5 mg/l NAA
Philippines : proliferating shoot cultures/ MS + 5 mg/l BAP
3. Irradiation to Starting Materials.
- The determination of the methodologies, e.g. optimum dose, radiation sensitivity, etc. 		a) Radiosensitivity test / no. of plants/dose
Indonesia : 0 - 80 Gy, 10 shoot tips/dose
Malaysia : 0 - 100Gy, 100 meristems, 5 replicates
Vietnam : 0, 10, 20, 30, 40, 50Gy; 10 shoot tips/dose
Philippines : 0, 5, 10, 20, 25, 30, 40, 60, 80, 100Gy; 50 clumps/dose

b) Radiation dose
Indonesia : LD 50 - 40 Gy, LD 100 - 80 Gy
Optimum dose; 10 - 20Gy
Malaysia : LD 50 - 50 Gy, LD 100 - 80 Gy
Optimum dose; 20, 30, 40 Gy
Vietnam : LD 50 - 30 Gy, LD 100 - 60 Gy
Optimum dose; 20 Gy
Philippines : LD 50 - 20 - 25 Gy, LD 100 - 80 Gy
Optimum dose; 20 and 25 Gy
4. Selection of Mutants Clones with Disease Resistance.
- Establishment of screening technique.
5. Testing and Evaluation of Selected Mutants in the field.
- The data of field trials and estimate of their ability. 		[ Indonesia ]
greenhouse screening: Soil from ‘hot spot' are placed in plastic pots and 10 day-old tissue culture plants are planted (1 plant per pot), evaluate for 3 months.

Field screening: Those that survived in the greenhouse are planted in the ‘hot spot's. No. of plants; 489

[ Malaysia ]
Screening:
a) Dipping method- 1-2 hrs soaking in spore suspension (10 6 spores/ml)
b) Double tray method; hardened plants for 4 weeks are planted in sterile sand media in tray and Fusarium spores are poured in the first tray.
c) Plants are hardened for 4-8 weeks in individual polybags and transfer in tray with coir dust containing Fusarium for 2 weeks. Evaluation is done 4-6 weeks after planting.

Field screening: Those that survived from the above screening method are transferred to hot spot. Those resistant plants are multiplied and transferred back to hot spot for three generations.

[ Vietnam ]
Greenhouse screening:
a) Hydrophonic; 1 week
b) Dipping method: 24 hrs soaking plants in spore suspension (10 5 spores/ml)
No. of plants; 500 soaked in container at the same time

Field screening: hot spot
a) Condition of ‘hot spot': more than 50 % infection of the susceptible check
b) Maximum number of plants to be tested for in vitro: 5,000 plants
c) Parameters for evaluation: 1) yellowing of the leaves/ browning of the pseudostem
d) ELISA test: Indexing virus using ELISA test;
for BBTV compulsory
for CMV and BBrMV optional

[ Philippines ]
Greenhouse screening: Tissue-cultured plants are planted in plastic pots and inoculated with viruliferous aphids (10 aphids/ plant). Evaluation is done 1 to 9 months after inoculation. ELISA test is done on plants without BBTV symptoms. No. of lines; more than 6,000 plants were screened and 114 were selected without BBTV symptoms.

Field screening: Those plants with negative ELISA are multiplied by tissue culture and planted in the field under high disease pressure for further evaluation. Evaluation will be done for 3 generations either in the field or farmers' field. No. of lines; 32 (Generation 1) and 10 (Generation 2). No. of plants per line; 30. All the mutant lines are maintained in tissue culture.
6. Publication of the results (Reports etc.)


Internal report, seminar and conferences, publication in journals, public patent
Indonesia :
1. Ishak and Ita Dwimahyani (2005) Evaluation of banana mutant lines growth and tolerance to Fusarium f.sp cubense (FOC), Stigma Vol. XIII (1) pp:26-29
2. Ishak, Sasanti, W. and Yulidar (2004) Cloning and propagation of banana mutant line for selection purposes (Technical report 2004, CAIRT)
3. Ishak, Sasanti, W. Yulidar (2005) Cloning and propagation of banana mutant lines for selection purposes (Technical report 2005, CAIRT)
7. Other result and ripple effects of the project, if there are. Tissue culture using meristem-six months
Suspension cell cultures- 12 months
8. Opinion about the project (problems, ideas, remarks, proposal, etc.) Difficulty in finding and building hot spot
Limited budget ( Indonesia , Malaysia , Philippines , Vietnam )
National program of banana is ending ( Malaysia )
9. Collaboration with other institution
Malaysia ; University of Malaya , private company
Indonesia : Andalas University , private company
Vietnam ; Vegetable and Fruit Research Institute, private company
Philippines ; PNRI, IAEA, PCARRD, banana farmers
10. End Users
Malaysia ; Farmers (1 ha) cooperative and private plantation for banana (more than 40 ha)
Indonesia ; Traditional farmers (0.5 - 2 ha)
Vietnam ; Small farmers (less than 1 ha)
Philippines ; Small farmers (less than 5 ha)
Attachment V

3 year-plan for FNCA Mutation Breeding Project

As of July 28, 2006
FY2005 FY2006 FY2007 FY2008
Sub-Project 3
Disease Resistance in Banana"		 Induction of embryogenic callus from male inflorescence or other explants etc.
Irradiation of materials etc.		 <BBTV>
Field evaluation of material (Generation 1&2)

<Fusarium>
Field Screening in hot spot (Q4-2006)

*Mid-term Review		 <BBTV(other diseases)>
Greenhouse and field screening (Generation 3) (Q4-2007)

<Fusarium>
Greenhouse and Field screening in hot spot (Q3-2007)		 <BBTV>
Multi location trials and performance evaluation

<Fusarium>
Performance evaluation

*Final Repor
●III.Summary of Progress Reports ●IV.Midterm Report ●V.Work Plan