Field Screening of Gamma-Irradiated Cavendish Bananas

Abstract

In our search for Cavendish bananas to withstand Fusarium oxysporum f. sp. cubense (Foc TR4) and other diseases, field screening of tissue-cultured Grand Nain banana seedlings derived from gamma-irradiated shoot tips was explored. Six months after irradiation and multiplication in the laboratory, the plantlets (M₁V₆) were individually grown in seedling bags under screen house conditions for 8 weeks, side-by-side with non-irradiated plantlets of the same clone. Once acclimatized, the banana plants were grown in an area confirmed positive of Foc TR4 (based on previous farm records stating that more than 50% of the plant population succumbed to the disease). Seedlings from each treatment (dose of radiation) were divided into four replicates, regardless of the number of plants. Each plant was given a unique identification code for traceability during disease monitoring, bunch and fruit quality evaluation.

Incidences of Foc TR4, Moko disease (Ralstonia solanacearum) and virus diseases were monitored weekly. Plants found positive of any disease were eradicated immediately. The plant population for the succeeding generation was managed by removing the unwanted suckers, 12 weeks from planting using a spade gouge and keeping only one sucker per plant for the next generation. Agronomic characters of each plant were taken at the flowering stage. These included age to flower, height, pseudostem circumference, number of leaves and height of the sucker. The bunch was harvested 12 weeks from flowering. The number of hands in a bunch, the number of fingers and weight of a hand were recorded. The same agronomic characters of the plant were taken for the succeeding generations.

Plants left standing in the field without any disease symptoms 3 years after planting were considered as putative mutants and were selected as candidate lines for multiplication and second-generation field screening. Only healthy suckers (free from viruses) were further multiplied via tissue culture technique to reach M₁V₆. Clean suckers from each line free of soil debris or dirt were sent to the laboratory for multiplication. At least 1000 plantlets were produced from each line for the second-generation field screening. These were grown in two locations – with and without records of Foc TR4. Field monitoring activities including plant population management, disease incidence assessment and fruit quality evaluation were carried out following the same protocols used in the establishment of the first-generation plants. Lines with population showing ≤10% Foc TR4 after the first harvest, with good vigor, fruit quality and productivity were considered as candidates for further multiplication, farmers distribution and field planting under semi-commercial scale.

1 Introduction

The choice of planting Cavendish bananas in the Philippines particularly Grand Nain resides in its high-yielding capacity, fruit quality and export market demands. After decades of cultivation, the fungal disease, Fusarium oxysporum f. sp. cubense (Foc TR4) debilitated the plant population, and its productivity per unit area. In some locations, the disease affects more than 50% of the population in a short period of time.

To date, there is no single effective treatment against the disease. The variety GCTCV 218 introduced from Taiwan Banana Research Institute (TBRI) through the Bureau of Plant Industry has helped to improve some devastated farms. Yet it has been found that this variety shows susceptibility to Foc TR4 at varying degrees, depending on the location. The availability of a resistant clone therefore, with qualities at par or better than Grand Nain is a welcome development to help manage, if not totally address the complexities of banana pest and disease problems. This will help growers and farmers especially in regions that are vulnerable and are positive of the disease.

Bhagwat and Duncan (1998) reported banana mutation breeding for tolerance to Foc TR4 using gamma irradiation. In 2006, Damasco et al. published a paper “Banana Bunchy Top Virus (BBTV) Resistance in Cultivar Lakatan Developed via Gamma Irradiation of Shoot Tips”. This prompted Dole Research to seriously engage in experiments using Grand Nain clone of Cavendish. We have coordinated with the Philippine Nuclear Research Institute (PNRI) equipped with facilities to cover the gamma irradiation treatments. The protocol of Novak et al. (1990) served as an important guide in this project. Radiation treatment is at M₁V₁ stage of the shoot tips while inside the culture bottles and further multiplied to reach M₁V₆. Some aspects of Novak et al.’s (1990) protocol have been modified to better understand the characteristics of the plants in the field in addition to its susceptibility to diseases.

The field screening project started in year 2013 in Malandag, Malungon, Sarangani Province, and after 4 years, it was extended to two other regions namely, Maragusan and, Compostela Valley. Both locations are situated in the islands of Mindanao and Philippines.

This protocol describes the field screening tests of plants exposed to gamma irradiation treatments, their multiplication and further field screening tests of those that escaped from Foc TR4 and other diseases (putative mutants) after completing at least three harvest cycles. Disease incidence, plant vigor, growth and development patterns, productivity, and fruit quality were recorded. These parameters describe the tolerance of a putative mutant to a certain disease and ensure that fruit quality and productivity are within farmers’ and consumers’ level of acceptability.

2 Materials

2.1 Planting Material

• 1. Hardened banana seedlings (Fig. 7.1 and see Note 1).

Fig. 7.1

Establishment of healthy seedlings in the screen house for 8 weeks (a) and a healthy seedling during field planting (b)

2.2 Land Preparation and Planting

• 1. Farm record showing the historical Foc TR4 cases of the area, ≥50% of the population.

• 2. Heavy equipment for land ripping and harrowing.

• 3. Soil penetrometer.

• 4. Transect line.

• 5. Wooden stakes.

• 6. Digging bags.

• 7. Shovels.

• 8. Fertilizer.

• 9. Calibrated scoops for fertilizer application.

• 10. Cutting knife.

• 11. Prepared plant labels (with treatment number, plant number).

2.3 Disease Survey and Eradication

• 1. Template pictures of disease symptoms.

• 2. Eradication solution: systemic herbicide (20%) in 1% ammonium sulfate solution.

• 3. Eradication injection equipment.

2.4 Bunch Care

• 1. Fruit bags.

• 2. Markers.

• 3. Data sheets.

• 4. Pens.

• 5. Clip boards.

2.5 Harvest and Transport

• 1. Ladder.

• 2. Harvesting knife.

• 3. Weighing scale.

• 4. Table.

• 5. Measuring tape.

• 6. Data sheets.

• 7. Pens.

• 8. Clip boards.

• 9. Paper boxes or wooden crates.

2.6 Fruit Quality Assessment

• 1. Digital weighing scale.

• 2. Oven.

• 3. Refractometer.

• 4. Ripening chamber.

• 5. Reefer container.

• 6. Color meter.

2.7 Selection and Transport of Putative Mutant Suckers from Field to Laboratory

• 1. Gouging bars.

• 2. Knife.

• 3. Bolo.

• 4. Labels and tags.

• 5. Pens.

• 6. Fine mesh to cover the suckers during transport.

• 7. Plastic sacks to contain the suckers.

• 8. Fine mesh to cover the suckers.

• 9. Refrigerated container or air-conditioned vehicle to transport the suckers from the field to the laboratory.

3 Methods

3.1 Selection of Land for Planting

• 1. Select an area which is confirmed to have Foc TR4 (see Note 2).

3.2 Land Preparation and Planting

• 1. Deep plough and rip at 50–60 cm depth to reach soil compaction to level < 2 Kg/cm² (see Note 3).

• 2. Plant in a single line pattern at population of 2222 per hectare at 3-m distance between lines and at 1.5-m distance between plants, orientated East to West (see Note 4).

• 3. Prepare planting holes of the size 20-cm wide and 30-cm depth (see Note 5).

• 4. Depending on the number of treatments (radiation doses) and the number of seedlings produced, the layout planting design is obtained by dividing the seedlings from each treatment to cover at least four replicates in the field.

• 5. Give a permanent and unique identification code to each plant after planting.

• 6. Prepare a field map to indicate full details of plant identification and location.

• 7. Record the date and week of the year of planting.

3.3 Disease Monitoring

• 1. The entire population is surveyed at least once a week.

• 2. Individual plant is inspected if showing any disease symptoms.

• 3. Diseases to be recorded include Foc TR4, Moko, virus diseases (BBTV, BBrMV, CMV) and other abnormalities.

• 4. Plants with disease are eradicated within 24 h from planting.

3.4 Disease Eradication

• 1. Surveyed plants with disease will be further confirmed by the eradicator (see Note 6).

• 2. If confirmed positive of the disease, the plant will be injected with 50–100 ml of the eradication solution.

• 3. If sucker/s are present, injection will also be performed using ~50 ml eradication solution.

• 4. Gather and contain the debris of eradicated plants within the same planting spot.

3.5 Plant Care and Population Management

• 1. At 6 weeks from planting, dried or burnt leaves due to sigatoka or natural senescence are trimmed.

• 2. Leaf trimming is done once every 2 weeks.

• 3. After 12 weeks from planting, each plant is surveyed for presence and density of suckers.

• 4. Count and record the number of suckers in a plant.

• 5. Select one sucker nearest to the mother plant to serve as the second-generation plant.

• 6. Remove the other suckers (unwanted suckers) using a spade gouge.

• 7. Repeat removal of unwanted suckers once every month until the plant starts to flower.

• 8. At flowering stage, select the third-generation sucker, or until available.

• 9. The monthly schedule of sucker removal is followed for the succeeding generations.

3.6 Bunch Care

• 1. At flowering stage, the following parameters are collected:

  • (a) Number of leaves

  • (b) Plant height

  • (c) Height of the selected sucker (next generation)

  • (d) Pseudostem circumference (1-m from the base)

• 2. When all the hands in a bunch are exposed, the bunch is cleaned by removing the dried flowers, bracts and the male flower.

• 3. Tag the bunch to indicate date of flowering and to track harvest dates.

• 4. Place plastic inserts in between fingers (if necessary) to avoid fruit bruises.

• 5. Cover the cleaned bunch with a bag (paper, fabric or polyethylene) if, and when available.

• 6. Protect the fruit-bearing plant by installing bamboo poles (and/or its equivalent) as propping materials.

• 7. Harvest the bunch 12 weeks from flowering (see Note 7).

3.7 Harvest

• 1. Using a special harvest knife, harvest the bunch by cutting one hand at a time starting from the last hand (see Note 8).

• 2. The following information should be recorded:

  • (a) Number of hands

  • (b) Number of fingers in a hand

  • (c) Weight of each hand

• 3. Use the second and last hand of each bunch for fruit quality reading.

• 4. Pack and label fruits before dispatching them to the laboratory in a refrigerated condition (13.5 °C).

• 5. Leave the pseudostem of the harvested plant intact with the youngest two leaves (see Note 9).

3.8 Fruit Quality Reading

• 1. For each bunch, divide the second and last hand specimens into two.

• 2. The first half will be read for pulp moisture content at color green.

• 3. The second half will be subjected to standard ripening procedure.

• 4. Fruit will be read for peel thickness, pulp moisture content, total soluble solids and/or brix at color yellow, 4 days after gassing.

3.9 Overall Plant Population and Farm Status

• 1. After harvest of the first generation, the remaining plants free from Foc TR4 symptoms and other diseases are inventoried (Table 7.1).

• 2. Follow above methods Sects. 3.3 to 3.8 for the succeeding generations until two to three harvest cycles are completed.

• 3. When population left in the area is ≤20% from the initial population, allow two or more suckers to grow from each plant.

• 4. Assess the number of individual plants that survived from each treatment and of the entire experimental lot.

• 5. Each of this survived plant from the gamma irradiated meriplants are considered putative mutant lines.

• 6. Archive data on plant growth, vigor and fruit quality of each putative mutant to decide if characteristics are qualified for further field evaluation (see Note 10).

Table 7.1 An illustration of survived plants in the field after 3 years of screening seedlings from shoot tips subjected to gamma irradiation

3.10 Handling of Putative Mutant Plants from an Infected Field for Laboratory Multiplication and Second-Generation Field Screening

• 1. Once the putative mutant plants are selected, the available suckers are tagged.

• 2. Obtain tissue samples to test and verify for fungal or virus diseases (if any) via PCR in the laboratory (Company Standards).

• 3. Only suckers free from any disease will be dug and cleared from soil or dirt and sent to the laboratory.

• 4. Maintain a quarantine condition during transport of suckers by containing individually in a sack and enclosing with a fine net.

• 5. Transport suckers in a refrigerated van (if possible) or inside an air-conditioned vehicle.

• 6. Process the selected suckers immediately upon arrival in the laboratory.

• 7. Follow the standard tissue culture procedure to reach M₁V₆.

• 8. If untreated plants are not available in the experimental field, source the untreated plants of the same clone (Grand Nain) and include them in the batch of tissue culture intended for second generation field screening.

• 9. Produce at least 1000 plantlets from each line (see Note 11).

• 10. At M₁V₆ stage, submit sample plantlets from each line to a reputable laboratory, side-by-side with the untreated control (original clone) to identify presence or absence of mutations.

• 11. In the screen house, nurture the plantlets into healthy seedlings for 8 weeks, following the standard commercial practices.

• 12. Plant the 8-week-old seedlings in two locations – (1) confirmed positive of Foc TR4 where ≥50% of the population succumbed to the disease; and (2) confirmed negative of Foc TR4 (see Note 12, Table 7.2).

• 13. Gather the same data and information collected in the first-generation planting.

• 14. Select at least 30 bunches from each line when the population reaches 25, 50 and 75% harvests for quality readings in the laboratory following Sect. 3.8 above. Figure 7.2 illustrates plants in a population of putative mutant lines under second generation field screening. Both plants are clones of Grand Nain but received different doses of gamma irradiation treatment and survived during the first-generation planting.

• 15. Summarize cases of diseases, plant growth and productivity data after completing at least 85% of the first harvest (see Note 13, Table 7.3 and Fig. 7.3).

• 16. Decide which line to further multiply for farmer distribution and commercial-scale field planting after completion of at least three harvest cycles.

• 17. Apply for a patent any line deemed important.

• 18. Register to IAEA database (Mutant Variety Database) as product of induced mutation breeding via gamma irradiation.

• 19. Submit candidate Foc TR4 tolerant line/s to International Transit Centre (https://www.bioversityinternational.org/banana-genebank/) for preservation and as backup specimens for further research investigations.

• 20. Perform molecular characterization of identified mutant lines (see Note 14).

• 21. Direct field screening of banana seedlings from gamma irradiated shoot tips is time consuming, laborious and expensive. From the point of view of a banana grower, the measure of significance of an introduced variety covers both its tolerance to the disease of interest and the resulting agronomic character of the novel variety. Our project was launched in 2013 and is estimated to take five to 10 years to achieve successful results, with great possibility that mutant line/s will be produced (see Table 7.4). In order to meet this target, the screening process for disease tolerance need to be planned and implemented correctly.

Table 7.2 Seedlings multiplied from the putative mutant plants for second generation field screening

Fig. 7.2

Sample fruit under second generation field screening of putative mutant lines originally from one Cavendish clone (Grand Nain) at different doses of gamma radiation – (a) 8 Gy and (b) 10 Gy

Table 7.3 Sample field screening data from two different locations showing incidence of diseases, after completing the first-generation harvest

Fig. 7.3

A graphical presentation of the weekly cumulative flowering of putative mutant lines (a–e) in an area negative of Foc TR4, compared with the untreated control

Table 7.4 Program of activities and estimated time frame in the field screening of gamma irradiated Cavendish bananas for disease tolerance, plant vigor and fruit quality

4 Notes

• 1. Seedlings from Grand Nain shoot tips were used as planting materials. They were subjected to different doses of gamma radiation, while the untreated control seedlings were grown in the screen house, provided with proper irrigation and nutrition for 8 weeks. Healthy seedlings are advantageous for quick recovery from stress during transport from the screen house and during field planting.

• 2. The area must be confirmed positive of Foc TR4 to challenge the response of the plants under natural, field conditions. Farm records must show that more than 50% of the host plants in the previous cropping succumbed to the disease in 1 year or less from planting.

• 3. Proper land preparation is required according to soil compaction data. It is believed that the process facilitates mixture of fungal inocula in the soil.

• 4. Planting pattern follows the commercial practice and population per hectare of the clone.

• 5. No other soil amendments are required, while fertilization follows the recommended protocol for Cavendish (Robinson and Sauco, 2010).

• 6. Both the disease surveyor and the disease eradicator must be knowledgeable of any disease symptoms or abnormalities of bananas. The confirmation by the eradicator will avoid mis-eradication of the plant and wrong record entry. Whenever a new disease symptom is observed, both the surveyor and eradicator report to the scientist/s for further confirmation.

• 7. If a plant with a hanging bunch is positive of any disease (Foc TR4, Moko or any virus disease), it will be eradicated similar to schedule and procedures described in Sect. 3.4 above.

• 8. If the plant is tall, a sturdy ladder will be required during harvest.

• 9. The natural decomposition of the pseudostem of the mother plant will facilitate growth of the succeeding generation.

• 10. It is the prerogative of the scientist to decide what other features, aside from disease tolerance are necessary to proceed with multiplication of a putative mutant line. In general, productivity (weight of the bunch and age from planting to harvest), moisture content and brix are important parameters to consider.

• 11. A ‘line’ is defined in this article as the resulting tissue culture plants obtained from a putative mutant.

• 12. Planting of putative mutant lines in an area confirmed free of Foc TR4 will help understand and establish the productivity potential of a new line.

• 13. Plant growth and vigor in an area free of Foc TR4 can be illustrated by the flowering pattern vis-à-vis control (Fig. 7.3). These can also be supported by productivity data such as bunch weights, number of hands in a bunch and fingers in a hand. Under commercial scale, a plant population of a line, variety or clone with ≤10% Foc TR4 infection in the first cropping (from planting to completion of first harvest cycle) is a better alternative than the susceptible Grand Nain.

• 14. Samples of putative mutant lines have been submitted to Plant Breeding and Genetics Laboratory, Joint FAO/IAEA Centre on Nuclear Techniques in Food and Agriculture, Department of Nuclear Applications, International Atomic Energy Agency for molecular analysis together with the untreated control.