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Uploaded bySudesh Jayashantha
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Seminar II

Banana hybridization was studied through natural breeding behavior observations and artificial crosses. Natural breeding of banana cv. 'Seeni Kesel' showed maximum stigma receptivity from 0800-1000h, correlating with peak nectar production in the morning. Artificial crosses of cv. 'Seeni Kesel' with M. balbisiana resulted in hybrid seed set but abnormal embryo development led to poor germination. M. balbisiana pollen germinated and grew tubes down the cv. 'Seeni Kesel' pistil but attempts to culture hybrid embryos did not produce plantlets. The study provides insights into banana breeding but overcoming barriers to hybrid embryo development requires further research.

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Sexual Hybridization in Musa P. G. S. Jayashantha S10369 Plant Biotechnology Department of Plant Sciences University of Colombo 1
Banana Breeding Through Sexual Hybridization • Knowledge on the breeding system in banana is far from complete • However, It is essential to produce new cultivars to overcome various stress conditions • Objective of this study was to, • Examine natural breeding behavior of banana • Attempt artificial hybridization in banana 2
Natural breeding behavior of banana cv ‘Seeni Kesel’ • Study location - Ethbatuwa, Angunakolapelessa. • 3 female stage plants and 5 male stage plants were used • Studies on,  Floral phenology  Stigma receptivity  Nectar production 3
Inflorescences 4 • Inflorescence differentiated to produce 2 types of flowers in  Two development stages – Female flowers - Female stage – Male flowers - Male stage • Flowers covered with bracts • Bract opening allows exposure of flowers to visitors Male stage Female stage
Floral phenology • Female flower are larger than male flowers • Male flower length 7.4 cm (SD = ± 0.8 cm) • Female flower length 13.9 cm (SD = ± 0.5 cm) • Female flower ovary  More or less four-rowed ovule arrangement 5
• Time of bract opening • Period of flowering • Persistence of flowers and flower whorls • Observations made, • from 0400 h to next day 0200 h • At hourly intervals 6 Flowering duration
Flowering duration (cont.) 7 • Female stage inflorescence • First and second bracts did not curl after opening • All other bracts → Same pattern in floral behavior • Day 1 • The 3rd bract has the first cluster of flowers • Bract opened between 0400 – 0500 h • Bract curling started during 1400 – 1500 h Female stage
Flowering duration (cont.) • Female stage inflorescences.. • Day 2 • Bract lifted almost completely at 0700 h • Day 3 • Bract fell off during 0800 – 0900 h • Flower petals and sepals fell off 0800 to 1100 h • Most of the styles fell off by 1800 h 8
Flowering duration (cont.) • Male stage inflorescence Day 1 • Bract opening started between 1600 – 1700 h • The bract was lifted and opened at 1700 - 1900 h • 90 𝑜 angle with the peduncle at 2200 h • Day 2 • Falling of flowers started between 0600 – 1000 h • Bract falling happened between 1000 – 1200 h • Some flowers remained after bract falling 9
Stigma receptivity of female flowers • Stigma receptivity is a crucial stage in maturation • Stigma maturity Peroxidase activity • There is a correlation between stigma receptivity and peroxidase activity (McInnis et al., 2006) • 5 flowers from each 3 plants → a drop of 3% H2O2 placed on the stigma • Time of the first appearance of bubbles & intensity was observed 10
Stigma receptivity (cont.) • 0800 to 0900 h stigma receptivity • Peak time of receptivity - 0900 h • After 1000 h • Stigma color also changed with time • After 1500 h, stigma browning & senescence started 11 Not receptive Receptive
Nectar production • Inflorescences were enclosed in polythene bags to exclude flower visitors • Female stage • Initial nectar collection was carried out at 0720 h • Periodic collection after 6 hr/12 hr durations for female stage plants • Male stage • One time collection in the evening just after the opening of the flower (1700 h – 1800 h) 12
Nectar production 13 Nectarvolume(µl) Time from 1st collection Initial nectar production Female flower = 59 μl Total nectar = 117 μl (24 hrs.) Nectar production of female flowers vs Time Initial nectar production Male flower = 46 μl
Natural breeding behavior conclusion • Cv ‘Seeni Kesel’ shows adaptations to cross pollination • Maximum stigma receptivity observed between 0800 h – 1000 h • Nectar production also correlates with the stigma receptivity • Almost 75% of nectar produced in the morning • Therefore the best time for artificial pollination could be 0800 h to 1000 h 14
Pollination of cv ‘Seeni Kesel’ with M. balbisiana • Cv ‘Seeni Kesel’  Male sterile  Posses residual female fertility • Possible cross Cv ‘Seeni Kesel’ × M. balbisiana 15 M. Balbisiana anther Cv ‘Seeni Kesel’ anther Cv ‘Seeni Kesel’ do not produce pollen
M. balbisiana pollen • Pollen size was determined using microscope graticule and stage micrometer • Histochemical test for • Starch – KI3 test • Lipid – Sudan dye test • Protein – Coomassie brilliant blue test 16
M. balbisiana pollen cont. • Diameter 122 μm (± SD = 7.66 μm) • Production - Mature anthers of male flowers • Small pollenkitt around the pollen • Contain starch lipids and proteins • Since pollen contains food reserves, – Able to germinate & reach the ovule – May serve as a reward for foraging pollinators 17
Pollination of cv ‘Seeni kesel’ with M. balbisiana pollen • Natural pollination was observed in the University premises. • Seed set observed in cv ‘Seeni Kesel’ fruits • Artificial pollination carried out in the field (Angunakolapelessa) using pollen collected from Atwelthota 18
Growth of M. balbisiana pollen in cv ‘Seeni Kesel’ pistils • Anthers (pollen) - collected from male stage inflorescence of M. balbisiana • Cv ‘Seeni Kesel’ stigmas pollinated – M. balbisiana pollen • Pistils collected –1, 2, 3, 4, 6 and 8 hours after pollination • Fixed immediately in 1:3 acetic alcohol • Pistils softened overnight in 2 M NaOH • Stained with Aniline blue (in 108 mM K3PO4) • Observed under BV light for florescence (OLYMPUS-BH2) 19 In the Field In the Lab
M. balbisiana pollen tube growth on cv ‘Seeni Kesel’ pistils • Pollen started to germinate after 1 hr. • Germinated pollen observed on the stigma for 2 – 4 hrs. 20 Pollen started germination after 1 hr Pollen germinated after 2 hr Pollen growth after 4 hr (× 100)
Pollen tube growth (cont.) 21 • Pollen tube growing down the style after 6 hrs. Pollen growth after 6 hrs. (× 100) Pollen tube growth towards the ovary (× 100) • After 24 hours pollen tube passes the style
Pollen tube growth (cont.) • In the style pollen tube produces callose plugs 22 Pollen tube plugs (x 100) (arrows) Xylem (x 100)
Pollen tube growth (cont.) • M. balbisiana pollen germinated successfully after artificial pollination on cv ‘Seeni Kesel’ pistil • Artificial pollination • Open pollination 23 No pre-fertilization barriers to crossing
Seed set and seed germination • Measurements on seed size weight and germination were recorded from, – M. balbisiana seeds – Hybrid seeds obtained from natural pollination of cv ‘Seeni Kesel’ with M. balbisiana pollen 24
Seed set and seed germination (cont.) 25 Seed type Weight of 100 seeds (g) Seed diameter (mm) Seed thickness (mm) Seed germination After 21 d (%) Hybrid seeds 7.801 (± SD = 0.245) 6.51 (± SD = 0.68) 4.46 (± SD = 0.43) 1.33% M. balbisiana seeds (natural pollination) 4.599 (± SD = 0.050) 4.94 (SD = ± 0.57) 4.00 (± SD = 0.49) 71.33% 2 mm Hybrid seeds were heavier & larger But have a poor germination capacity
Structure and embryo arrangement of hybrid seed • A of sample 100 hybrid seeds collected & Soaked overnight • Split into two halves & observed under stereo microscope 26 A. Longitudinal split of a hybrid seed B. Longitudinal split of a M. balbisiana Seed B
Embryo arrangement of hybrid seeds 27 1. M. balbisiana seed 2-8. Abnormalities found in hybrid Seeds 1
Embryo culture Surface sterilization of seeds - 2 methods used Method I Washed with tap water 30 min → Kept 10 min in AgNO3 solution → washed 3 times → sterile 5% NaCl solution 30 sec → AgCl containing suspension removed → washed 3 times (Bakry, 2008) Method II Washed with tap water → 30 min 20% Clorox solution for 20 min → stirred in 20 ml of 70% ethanol for 5 minutes → washed 3 times • Surface sterilization results after 3 days • Method I high rate of contamination (> 77 %) • Method II contamination was very low (<15%) 28
Embryo extraction and culture • Under aseptic conditions, • Embryo extracted • Inoculated onto germination medium • Kept at 25 °C culture room in dark conditions • Germinated embryos (1.5–2.0 cm) transferred onto growth medium • Kept under light conditions • Only M. balbisiana embryos were germinated up to two weeks 29 Mushroom shaped embryo
10 days after embryo culture, • Germinating embryos of M. balbisiana on the germinating medium after 10 days. A. totally brown embryo, B. initiation of epicotyl. C. immerged epicotyl. D. browning parts of the embryo. E. root-like structures of the embryo. F. cauliflower-like cell masses on the embryo (special structural differences shown by arrows) 30
12 days after embryo culture, 31 Twelve days old embryos on germinating medium; after 10 days dark and 2 days continuous light. (Shoots are indicated in S and roots in R) X is an embryo after 3 days of inoculation onto the germination medium But, Hybrid seeds did not develop plantlets even after 2 weeks
Conclusion • Basic studies on ecology, morphology and biomolecules involved in reproduction can lead to better understanding of the mechanisms involved in seed set in Musa (L.) • Both artificial pollination and natural pollination of cv ‘seeni Kesel’ with M. balbisiana produce hybrid seeds • Difficult to obtain plants from hybrid seeds resulting from crossing M. balbisiana and cv ‘Seeni Kesel’ due to abnormalities in the seeds 32
References • Ortiz, R., & Vuylsteke, D. (1995). Factors influencing seed set in triploid Musa spp. L. and production of euploid hybrids. Annals of Botany, 75(2), 151-155. • Simpson, B. B. & Neff, J. L. (1983) Evolution and diversity of floral rewards. In: Jones CE, Little RJ, eds. Handbook of experimental pollination biology. New York: Van Nostrand Reinhold Company Inc., 142 – 159. • Ortiz, R., & Crouch, J. H. (1997). The Efficiency of Natural and Artificial Pollinators in Plantain (Musa spp. AAB group) Hybridization and Seed Production. Annals of Botany, 80(5), 693 – 695. • McInnis, S. M., Emery, D. C., Porter, R., Desikan, R., Hancock, J. T., & Hiscock, S. J. (2006). The role of stigma peroxidases in flowering plants: insights from further characterization of a stigma- specific peroxidase (SSP) from Senecio squalidus (Asteraceae). Journal of experimental botany, 57(8), 1835-1846. • Bakry, F. (2008). Zygotic embryo rescue in bananas. Fruits, 63(02), 111-115. • Dafni, A. (1992). Pollination ecology: a practical approach. IRL Press Ltd, London, UK. • Faegri, K., & Van der Pijl, L. (2013). Principles of pollination ecology. Elsevier. • McGahan, M. W. (1961). Studies on the Seed of Banana. I. Anatomy of the Seed and Embryo of Musa balbisiana. American Journal of Botany, 48, 230 – 238 33
Acknowledgement • I would like to express my special thanks and my heartiest gratitude to my supervisor Prof. T. D. Silva for her inspiring guidance • I would like to thank Dr. R. M. C. S. Ratnayake, Department of Botany, Faculty of Science, University of Kelaniya and other people who shared their knowledge to help me to complete this research • I would like to thank Staff of the department of Plant Sciences and all my colleagues for their continuous support 34
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