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NATIONAL UNIVERSITY OF RWANDA NATIONAL UNIVERSITY OF RWANDA FACULTY OF AGRICULTURE FACULTY OF AGRICULTURE DEPARTMENT OF CROP OF CROP PRODUCTION AND DEPARTMENT PRODUCTION AND HORTICULTURE HORTICULTURE RESEARCH PROPOSAL

IN VITRO PROPAGATION OF TAMARILLO IN RWANDA
IN VITRO PROPAGATION OF TAMARILLO IN RWANDA

BY BY ISHIMWE RODRIGUE ISHIMWE RODRIGUE

SUPERVISORS: Prof PETER YAO KANZE SALLAH SUPERVISORS: PROF PETER YAO KANZE SALLAH Dr JANE KAHIA DR JANE KAHIA

2011

2011

ii TABLE OF CONTENTS
TABLE OF CONTENTS..................................................................................................ii LIST OF ABBREVIATIONS AND ACRONYMS.................................................................iii CHAPTER 1: GENERAL INTRODUCTION.......................................................................1 CHAPTER 2: LITERATURE REVIEW...............................................................................7 2.1. Tissue culture - Review.................................................................................7 Justification.......................................................................................................... 8 CHAPTER 3: GENERAL METHODS AND MATERIALS...................................................10 3.1. Plant materials............................................................................................10 3.2. Plant growth regulators...............................................................................10 3.3. Media preparation.......................................................................................10 3.4. Surface sterilisation of explants..................................................................11 3.5. Aseptic techniques......................................................................................11 3.6. Dissecting tools..........................................................................................11 3.7. Incubation conditions..................................................................................11 3.8. Cleaning of glassware.................................................................................12 3.10. Photography..............................................................................................12 3.12. Data to be collected..................................................................................12 WORK PLAN..............................................................................................................13 REFERENCES............................................................................................................14

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LIST OF ABBREVIATIONS AND ACRONYMS 2iP: 2-isopentyl adenine 2, 4-D: Dichlorophenoxy – acetic acid ADAR: Rwanda Agribusiness Development Assistance ANOVA: Analysis of varieties BA: 6-benzyladenine Cm: Centimeter GA3: Gibberellic Acid HCL: Hydrochloride acid IAA: Indole-3-acetic acid IBA: Indole-3-butyric acid ICRAF: World Agroforestry Center ISAR: Institut des Sciences Agronomique du Rwanda Kg: Kilogram l: Litre mg: milligram ml: Milliliter MS: MURASHIGE and SKOOG (culture media) NAA: 1-naphtylacetic acid

iv NaOH: Sodium hydroxide
O

C: Celsius degrees

TDZ: Thidiazuron v/v: Volume by volume %: Percent

χ: Chi-square

LIST OF PLATES Plate 1.1: Tamarillo roots Plate 1.2: Tamarillo leaves Plate 1.3: Tamarillo flowers Plate 1.4: Tamarillo fruits Plate 1.5: Tamarillo seeds inside the fruit

LIST OF TABLES

Table 1: Solvents used to solubilise plant growth substances. Table 2: Composition of Murashige and Skoog’s Medium

1

CHAPTER 1: GENERAL INTRODUCTION
1.1.

Classification of Tamarillo (CAB Abstracts, 1998):

Domain: Eukaryota Kingdom: Viridiplantae Phylum: Spermatophyta Subphylum: Angiospermae Class: Dicotyledonae Order: Solanales Family: Solanaceae Genus:Cyphomandra Species: Cyphomandra betacea
1.2.

General description of Tamarillo

Cyphomandra betacea (Cav.) Sendtn. is a woody plant of the Solanaceae family commonly known as tamarillo or tree tomato (Correia et al., 2009). It is the best-known of about 30 species of Cyphomandra (family Solanaceae) (Morton, 1987). pix (Guatemala); tomate de palo (Honduras); arbre à arbol (Colombia); tomate dulce (Ecuador); tomate Among its various regional names tomates (France); arvore do tomate, cimarron (Costa Rica); ikinyomoro are: tomate, tomate extranjero, tomate de arbol, tomate granadilla, granadilla, pix, and caxlan tomate de arvore (Brazil);lima tomate, tomate de monte, sima (Bolivia); pepino de (Rwanda); Mgogwe (Kenya); Munyanya (Uganda) and tomate francés (Venezuela, Brazil). In 1970, or shortly before, the construed name "tamarillo" was adopted in New Zealand and has become the standard commercial designation for the fruit (Morton, 1987). 1.3. Uses

Tamarillos are grown mainly for their edible fruits and, to a lesser extent, as an outdoor ornamental (Guimaraes et al., 1996). The fruits, when ripe, have the same application as the common tomato, and are a good substitute during the winter months when tomatoes are difficult

2 to obtain. They have several culinary uses and can be eaten raw in salads or as dessert but preferentially cooked (Guimaraes et al., 1996). In addition, they can be used as a condiment with grilled steak or ham, for making preserves, pies, sauces pickles, chutneys, and also in the canning industry. In all cases, the seeds and especially the skin should be removed, since the latter is fairly tough and quite disagreeable. In Jamaica and the West Indies, tamarillo fruits are considered to have beneficial effect in relieving disorders of the liver, and for that reason they were given the name vegetable mercury (Guimaraes et al., 1996). Tamarillo is widely grown throughout Rwanda (ADAR, 2002). The fruit are larger and better tasting than those from Zimbabwe, which exports a small amount as fresh fruit, and may be of interest (albeit on a small scale) to European fresh produce importers. Consumption of tamarillo fruit is traditionally recommended by Rwandans for people suffering from stomach ailments. Tamarillos are mainly used in Rwanda for making jams and juices but unfortunately in low quantities because of its low area coverage which can mainly be due to some viral diseases. 1.4. Ecology

Although C. betacea can be grown in a variety of soils and climates, there are some limitations to its cultivations imposed mainly by temperature, wind and soil conditions. Tamarillos do not withstand very low temperatures (frost may kill the leaves and tender growths), or the very high tropical heat. They do best in subtropical conditions or in tropical regions at altitudes between 700 and 2000 m. Wind may cause damage by breaking leaves and branches, or uprooting the plant. They do not tolerate waterlogged conditions, requiring light, and well – drained soils. 1.5. Structure

Cyphomandra betacea is a semi-woody shrub or small tree 2-3 m high, rarely 5 m. It is unarmed, pubescent, with a short trunk and stout lateral branches (ICRAF, 2009). The bark is grey. 1.6. Roots

Tamarillo has shallow root system (Plate 1), it has little ability to withstand drought and is easily blown over (ICRAF, 2009). The shallow roots and large, soft leaves of C. betacea make it particularly susceptible to wind damage. They are apparently intolerant to constant high temperatures and often their fruits fail to mature in lowland tropical climates owing to excessive heat.

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Plate 1.1: Tamarillo roots 1.7. Leaves

Leaves alternate, simple, entire, usually grouped at the branch tips, with a robust petiole, 4-8 cm long. The limb is large, 15-30 x 10-20 cm, ovate, shortly acuminate, with a cordate base (Plate 1.2). Young leaves covered on both surfaces with a soft pubescence; with age, the upper surface becomes glabrous. Midrib and principal veins are prominent on both surfaces.

Plate 1.2 Tamarillo Leaves 1.8. Flowers

Flowers of tamarillo are fleshy pink (Plate 1.3), in groups of 3-10 in axillary cymes or racemes, near the ends of the branches. They are hermaphroditic, pentamerous, fragrant, pedicellate, 13-15 mm diameter. The calyx is campanulate with broadly ovate, subacute lobes, which are thick and crescent in fruit. The corolla rotate-campanulate, 12 mm long, with 5 long, narrow, lanceolate segments; reflexed at the apex and the stamens 5, yellow, inserted at the throat of the corolla.

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Plate 1.3 Tamarillo Flowers 1.9. Fruits

Fruits are ovoid berry, measuring 4-6 (max. 10) cm long and 3-5 cm wide (ICRAF, 2009). They are suspended at the end of a long stalk (Plate 1.4), and surrounded at the base by the persistent green calyx. Skin is thin, smooth, and reddish-brown to violet changing to orange-red at maturity. Some varieties become deep purple at maturity. The pulp contains numerous small seeds that are circular, flat, thin and hard (Plate 1.5).

Plate 1.4: Tamarillo fruits 1.10. Pollination

Plate 1.5: Tamarillo seeds inside the fruit

Tree tomato flowers are normally self-pollinating (Morton, 1987). If wind is completely cut off so as not to stir the branches, this may adversely affect pollination unless there are bees to transfer the pollen. Unpollinated flowers will drop prematurely. 1.11. Germination

Seeds should be selected from true to type plants. Chilling is claimed to result in virtually 100% germination within 4-6 days (Morton, 1987). Seedbeds should be prepared with manure or compost and lightly shaded.

5 1.12. Chemical composition

The pulp, which varies in colour from yellow to orange – red, is relatively acidic (pH 3.7 – 3.8; Cacioppo 1984) and has an agreeable aromatic flavour. Tamarillos are an interesting crop from the nutritional viewpoint. They are comparatively high in protein (1.5 – 2 g /100 g of food), vitamin C (30 – 45 mg/100 g) and E (1.85 mg/100 g), provitamin A, mineral elements (K, P), and low in carbohydrates (4.7 g/100 g) and in calories (about 28 Kcal/100 g) (Guimaraes et al., 1996).

1.13.

Propagation

Tamarillos may be raised from seeds, cuttings or by grafting onto wild tobacco tree (Solanum mauritianum). Seeds germinate easily, but a pretreatment with fungicide may be recommendable to prevent “damping off”. Mature cuttings (1 – 2 – year – old wood), 30 to 40 cm in length and 1 to 2.5 cm diameter, are indicated for rooting. Tamarillo scions have been successfully grafted onto Solanum mauritianum in New Zealand and in Australia with the main goal of improving the tolerance to wet soil conditions by reducing susceptibility to root rot. Propagation from cuttings is an alternative, but it is difficult to ensure freedom from virus infections. Container growing can cut planting-out losses. Cuttings of 1- to 2-year-old wood 10-30 cm thick and 45-100 cm long can be defoliated and planted directly in the field. In Trees on the stock are slightly dwarfed, but they bear prolifically and need to be staked. Fruit will keep up to 12 weeks at 3.54.5 deg. C with a 7-day shelf life afterwards (ICRAF, 2009). 1.14. Major diseases

Tamarillo is susceptible to several viral infections like the tamarillo mosaic virus (TaMV; (Guimaraes et al., 1996)) which is also a problem which is affecting tamarillo tree in Rwanda, especially in areas where the crop has been grown for some time (ADAR, 2002). It makes the plants to become stunted, with puckered and mottled leaves. Also others can be seen in various regions in the World: cucumber mosaic virus, and potato virus Y, which not only affect the vigor and health of the plant, nut also cause distortions in the leaves and blemishes on the skin, its commercial value.

6 1.15. Major pests

Tamarillos is attacked by aphids, white flies, caterpillars, and the green vegetable bug as the main pests, and powdery mildew, sclerotina, bacterial blast, leaf spot, and root rot (in waterlogged conditions) as the principal diseases (Guimaraes et al., 1996).

7 CHAPTER 2: LITERATURE REVIEW 2.1. Tissue culture - Review Tissue culture is an in-vitro method for the development of new plants in artificial medium under aseptic conditions from plant tissues. Different plant parts: leaves, nodes, roots, internodes, stems of plants can be used during tissue culture. The requirement to rapidly multiply important cultivars of tamarillo can be achieved through tissue culture which has great potential for facilitating rapid propagation of elite materials. Obando et al.,1992 regenerated Cyphomandra betacea axillary buds plantlets using MS basal medium and phytohormones NAA, BA, and GA3 all in concentration 0.2 mg/l. Omitting GA3, lowering NAA to 0.02, and adding Zeatin 2.5 mg/l instead of BA, resulted in 70% of explants with multiple shoots but no roots. Shoots formed easily from leaf explants when only TDZ was used as cytokinin. Leaf sections also showed morphogenic responses, forming abundant shoots. The incorporation of TDZ (5 and 10 mg/l) promoted this response in the absence or presence of IAA (1 mg/l). Barghchi, 1998, established a protocol for the efficient micropropagation and plant improvement of Cyphomandra betacea. Explants from axillary and flower buds of 2 mature tamarillo plants were cultured on MS medium supplemented with a combination of different growth regulators (BA 0.1-4.0 mg/l, kinetin 0.25-4.0 mg/l and NAA 0.25-4.0 mg/l). Regeneration of plantlets was studied using 3 types of in vitro regeneration methods: axillary shoot culture, terminal bud culture and adventitious shoot regeneration. Obando and Jordan, 2001, examined the potential of in vitro regeneration in Cyphomandra betacea (Cav.) Sendt. Morphogenic responses were observed in a series of organs, mainly through somatic embryogenesis, adventitious shoots from leaf explants and root formation in axillary buds leading to plantlets. Somatic embryos were induced from ovaries, petioles and cotyledons. In axillary buds and leaf explants it was found that, at the same time related with initiation of morphogenic events, a substantial increase in soluble protein synthesis occurred, while browning and necrosis decreased. Tamarillo propagation through tissue culture has been successfully done by the India’s Tissue Culture Lab Department’s located in the Government fruit Garden in Shillong in 2007(Indian Agriculture Information, 2009). During the trial, yellow colour tamarillo has been found to be very promising. They first initiated the seeds in kinetin media after sterilizing them in mercury

8 chloride for 7 minutes. In three to four weeks, the seeds started to germinate with one plumule. This plumule was then used as a base for various micro propagation processes in the lab. The regenerated plantlets were rooted in a basal media for about 5-6 weeks. Once hardened, the plantlets were then hardened in coco peat in a poly house. Correia et al., 2009, attempted to induce somatic embryogenesis from adult plants of Cyphomandra betacea with the objective of cloning selected genotypes. To achieve this goal assays with the pith stem and floral tissues were performed. The ability of different tamarillo genotypes to undergo somatic embryogenesis was also tested, with preliminary results indicating that some genotypes are more suitable than others for somatic embryo formation.
2.2.

Statement of the problem

The cultivation of Tamarillo in Rwanda is facing challenges caused mainly by viral diseases like mosaic virus (TaMV) (ADAR, 2002) caused by pathogens which are difficult to control and are transferred by vegetative propagation often resulting in loss of plant production and a poor quality product. The lack of adequate, healthy planting materials is hampering tamarillo production in Rwanda. Justification Tamarillo seed propagation systems faces limitations such as undesirable variability, diseased seedlings, inadequate and seasonal supply, and the tissue culture of tamarillo can bring an answer to this problem as it offers large scale production of disease free planting materials. Micropropagated tamarillo plants give higher yield and shorter gestation period compared to traditional methods (Indian Agriculture Information, 2009). Besides, the other advantages offered by tamarillo tissue culture methods is the large scale availability of its planting materials at any time of the year without depending on the season of production. Although in vitro propagation of tamarillo is more expensive than naturally propagated seedlings, they offer to the horticulturist the advantages of uniform fruits characteristics which are important in international market. Multiplication by tissue culture techniques could provide a viable alternative to these traditional methods of tamarillo propagation. Tissue culture methods permit the production of relatively uniform plants on a massive scale in a shorter period than under conventional methods. Tamarillo plants produced by tissue culture flowers earlier than the plants produced through conventional methods.

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2.3. General objective

To develop an efficient tissue culture protocol for propagating Tamarillo (Cyhomandra betacea) plantlets.
2.4.

Specific objectives

To determine the optimum sterilization technique for Cyphomandra betacea

To compare effects of Cyphomandra betacea

different cytokinins in regenerating plantlets of

2.5.

Hypothesis

Tissue culture through use of nodes for regenerating plantlets provides a feasible method for mass crop propagation of Cyphomandra betacea.

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CHAPTER 3: GENERAL METHODS AND MATERIALS 3.1. Plant materials The nodal explants will be obtained from tamarillo growing in the green house at ISAR Rubona. 3.2. Plant growth regulators Plant growth regulators will be weighted and stocks prepared using appropriate solvents (Table 1). The stocks will be clearly labelled and stored in the refrigerator at 8oC. Table 1: Solvents used to solubilise plant growth substances. PLANT GROWTH SUBSTANCES BA Kinetin IAA IBA NAA 2, 4-D TDZ 2iP 3.3. Media preparation The MS (1962) media will be used for all the experiments (Appendix 1). Media will be prepared by dissolving the organic and inorganic components in distilled water. The solutions will be stirred until they dissolved and made up to final volume. The media pH will be adjusted between 5.7 and 5.8 by using either 1N HCL or 1N NaOH before the gelling agent is added. Media will then be heated on a hot plate with continuous stirring using a magnetic stirrer until agar is dissolved and media dispensed in the culture vessels. The culture vessels will be capped with lids and placed in trays and autoclaved. Autoclave will be set at a temperature of 121oC and a pressure of 1.1kg/cm2 for 20 minutes. All media will be autoclaved within 12 hours of preparation and when possible freshly autoclaved media will be used. However, when it will not be possible to use the media immediately it will be stored in a refrigerator at 4 oC for no longer than two weeks before use. SOLVENT 0.1N NaoH & heat if required 0.1N NaoH Absolute alcohol Absolute alcohol 90% alcohol Absolute alcohol NaOH Acidified water

11 3.4. Surface sterilisation of explants Healthy looking leaf explants will be selected from healthy plants. They will be put into a beaker containing tap water and taken to the laboratory for cleaning. The explants will then be transferred to the lamina flow cabinet, immersed in 70% (v/v) ethanol for 30 seconds and rinsed twice with sterile distilled water. This will be followed by surface sterilization using different JIK concentrations and time intervals. They will then be rinsed twice 4 times in sterile distilled water. 3.5. Aseptic techniques The process of sterilization and dissection of plant materials will be carried out under sterile conditions in lamina flow cabinet. The cabinet will be switched on and swabbed down with 70% ethanol using cotton wool or sterile towel and kept running for about 15 minutes before the work in the cabinet starts. All the plant materials will be dissected on the sterile pieces of paper. The lamina flow cabinet will be frequently swabbed down with 70% alcohol and sterile pieces of paper will be used between each transfer in order to ensure that sterile surfaces are used for each culture. Hands will be sprayed with 70% ethanol at suitable intervals while working for protracted periods in front of the cabinets. Following completion of transfer work, Lamina flow cabinets will be swabbed down with 70% ethanol. Personal hygienic precautions will be observed by wearing a clean lab coat and gloves while carrying out experiments in the lamina flow cabinet. 3.6. Dissecting tools All tools will be placed in an aluminium foil and sterilised in an autoclave. During their use in the cabinet, tools will be dipped in 70% ethanol followed by heat sterilization in steribead steriliser maintained at 250 o C. In between operations, the tools will be frequently sterilized by dipping them in ethanol and in steribead sterilizer. 3.7. Incubation conditions For regenerating tamarillo nodes, the cultures will be incubated in growth rooms maintained at 25oC and 16 hours photoperiod.

12 3.8. Cleaning of glassware All glassware and vessels will be washed in hot water to which few drops of liquid detergent will have been added. The glassware will then be rinsed in cold water three times followed by a final rinse in distilled water with a few drops of commercial bleach (JIK). All this will be carried out in a clean dust free washing room. The glassware will then be dried in the oven at 600C in a clean dust free place. 3.10. Photography Photographs of the experimental materials will be taken using a Sony Digital Camera. 3.11. Experimental design and statistical analyses All experiments will be laid out in Completely Randomised Design. The level of replication which will be used per treatment combination may vary depending upon the availability of experimental materials. For the micropropagation experiments 10 replicates per treatment will be used at the outset of experiments. Chi-square tests will be used in the case of percentages (frequency data); were chi-square values will be presented, the figure in the brackets following the derived value should be the one obtained from statistical tables. A number of hypotheses should be used for χ2 test. For the majority of the data analysed statistically, a standard Analysis of Variance (ANOVA) will be used on either transformed or untransformed data values. The level of significance used for treatment comparison will be either p≤0.01 or p≤0.05. Analysis of variance and data transformation will be performed using Genstat Statistical Software. The Genstat Statistical Software will be used in cases of unequal numbers of replications were present in some batches due to accidental contamination or in those cases where it will be relevant, e.g. number of roots per rooted microshoots. Standard errors of means (SE) will be indicated in tables where a statistical analysis would be conducted. 3.12. Data to be collected The following data will be collected:
 Percent clean explants  Number of microshoot per node

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 Length of microshoot

WORK PLAN Task February March April May June July August September

Project proposal writing Setting up the experiment Data collection Data analysis Data interpretation report writing

14 REFERENCES 1. ADAR (Rwanda Agribusiness Development Assistance) . (2002). Developing a Horticultural Export Industry in Rwanda Progress Report: May - June 2002. Kigali: USAID. 2. Barghchi, M. (1998). In vitro regeneration, plant improvement and virus elimination of tamarillo (Cyphomandra betaceae (Cav.) Sendt.). In Davey M. R., Alderson P. G., Lowe K. C., & Power J. B., Tree biotechnology: towards the millennium (pp. 173185). Nottingham, UK: Nottingham University Press. 3. Correia S. I., Lopes M.L. and Canhoto J.M.. (2009). Somatic embryogenesis in tamarillo (cyphomandra betacea): recent advances. In F. D. M.-V. Hanke, I International Symposium on Biotechnology of Fruit Species: Biotechfruit2008. Dresden, Germany: ISHS Acta Horticulturae. 4. Guimaraes M.L., Tomé M.C., and Cruz G.S. (1996)., Cyphomandra Betacea (Cav.) Sendtn. (Tamarillo). In Y. P. Bajaj, Biotechnology in Agriculture and Forestry 35 (pp. 120 - 135). New Delhi: Springer. 5. ICRAF (World Agroforestry Center). (2009). Cyphomandra betacea. Retrieved March 21, 2011, from Agroforestry Tree Database: http://www.worldagroforestrycentre.org/sea/Products/AFDbases/AF/asp/SpeciesInf o.asp?SpID=639 6. Morton, J. F. (1987). Tree Tomato. In J. F. Morton, Fruits of warm climates (pp. 437–440). Miami, FL. 7. Obando M. and Jordan M. (2001). Regenerative responses of cyphomandra betacea (Cav.) Sendt. (Tamarillo) cultivated in vitro. In S. K. S. Sorvari, IV International Symposium on In Vitro Culture and Horticultural Breeding. Tampere, Finland: ISHS Acta Horticulturae. 8. Obando M., Goreux A. and Jordan M. (1992). In vitro regeneration of Cyphomandra betacea (Tamarillo) and Andean fruit species. Ciencia e Investigacion Agraria , p. 125-130 9. The Indian Agriculture Information Wing. (2009). Micropropagation of Tamarillo. Agricultural Newsletter , p. 8. Agricultural Information Offset Press, Fruit Garden, Meghalaya, Shillong, India.

15 APPENDIX Table 2: Composition of Murashige and Skoog’s Medium:
Stock solution A B C Constituents Concentration in stock solution g/l 82.5 95.0 1.24 34 0.166 0.05 0.005 88.0 74.0 4.46 1.72 0.005 7.45 5.57 0.02 0.1 0.1 0.4 5 5 5 5 Volume of stock solution in final medium ml/l 20 20 5 Final concentration in medium mg/l 1650 1900 6.2 170 0.83 0.25 0.025 440.0 370.0 22.3 8.6 0.025 37.35 27.85 0.1 0.5 0.5 2.0

NH4NO3 KNO3 H3BO3 KH2PO4 KI Na2MoO4.2H2O COcl2.6H2O

D E

Cacl2.2H2O MgSO4.7 H2O MnSO4.4 H2O ZnSO4.7 H2O CuSO4.5 H2O

F

Na2.EDTA FeSO4.7 H2O

G

Thiamine HCL Nicotinic Acid Pyridoxine HCL Glycine

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