INEA HYBRIDISATION PROTOCOLS 2011 Anton Ivancic Hybridisation of taro (Colocasia esculenta) Floral characteristics of taro Colocasia esculenta is an allogamous, protogynous species, for which the main pollinators are insects. Its inflorescence structure is shown on the drawing below (Fig. 1). Fig. 1. Spadix of Colocasia esculenta: a peduncle, b female part, c sterile region between female and male parts, d fertile male part, e sterile appendix). Each inflorescence consists of a spadix covered by a spathe. The spadix is divided into a female part (lower part), a sterile region, a male part, and a sterile appendix. Female (pistillate) flowers are sessile and green, with well developed ovaries and stigmas. They are usually mixed with pistoids (sterile female flowers), which are distinguished by their light colour. The male part consists of sessile staminate (male) flowers. The spathe consists of two parts. The lower part is usually green or red, and forms the floral chamber in which female flowers are located. The upper part is predominantly yellow but sometimes can be red, purple, or blotched. Flowering Some varieties rarely (or never) produce inflorescences. In many cases, flowering can be successively induced by spraying plants with gibberellic acid (GA 3 ). The treatment should take place 3-5 weeks after replanting (depending on climatic conditions and growth vigour). The most efficient concentrations are between 200 and 300 mgl -1. As taro leaf blades are
waxy, GA 3 is usually mixed with a detergent. The first inflorescences usually appear 3-5 weeks after the treatment. The first visible indication of the approaching flowering is the appearance of the flag leaf (a modified membranous leaf). Once the flag leaf is exposed, the first inflorescences appear within 1-3 weeks. The development of a taro inflorescence and flowering is shown in Fig. 2. The beginning of flowering is usually associated with the emission of strong odour, mainly from the spathe. Its main purpose is to attract insects-pollinators. An odorous inflorescence is usually erect and its spathe is loose (Fig. 2-6), enabling small pollinators (coming from a day older, pollinating inflorescences) to enter the floral chamber and distribute pollen on stigmas of the female flowers. Most of these insects will remain inside the inflorescence until the next morning, when the spathe will be fully open and pollen will be released. Fig. 2. Crucial stages of development of a taro (Colocasia esculenta) inflorescence: 1, 2, 3, 4 stages before flowering, 5, 6 female phase, 7 male phase, 8, 9 early stages of fruit development. The most appropriate time for emasculation is between the developmental stage 4 and 5 (24-48 hours before the inflorescence reaches the temperature peak). Wind pollination can be significant only for openly flowering genotypes (with a fully exposed male portion of the spadix) in a dense population characterised by synchronised flowering. Rain can cause self-fertilisation by washing pollen grains from the male part of the spadix to the pistillate region. Self-fertilisation is possible, because the incompatibility system of selfincompatible genotypes, at the end of flowering, becomes less efficient, and because there is an overlap between stigma receptivity and pollen release. Taro is a thermogenic species. Our studies indicated that significant thermogenic activity of taro inflorescences took place during two successive nights: during the night when an inflorescence became odorous (the female phase) (Fig. 2-6), and a night later, when microsporogenesis approached its final phase (the male phase) (Fig. 2-7). During the female phase heat was generated by the fertile male part and the sterile appendix. The highest average difference between mean temperatures of the ambient air and inflorescences was documented during the female phase, at 05:00 hours (it was 6.8 C above the temperature of the ambient air). Thermogenic activity was synchronised with the protogynous nature of this 2
species and insect pollination in the early morning hours. During the male phase, heat was generated only by the fertile male part. Emasculation The most suitable time for emasculation is early morning, usually between 06:00 and 10:00, when the selected inflorescence gradually approaches the female phase (Fig. 2 developmental stage between 4 and 5; Fig. 3 inflorescences 4, 5 and 6; Fig. 4-1). Such an inflorescence is more or less erect and the upper part of the spathe is gradually changing its colour. The odour has not yet been released. 1 3 5 7 2 4 6 8 Fig. 3. Taro inflorescences in various developmental stages. The inflorescences suitable for emasculation are 4, 5 and 6. The emasculation procedure is relatively simple and is demonstrated in Fig. 4. At first, we have to cut the inflorescence in the middle of the sterile zone, between female and fertile male portions. The cut should not be too low because it may damage the female portion. Then we carefully remove the lower part of the spathe (green part) which protects the floral chamber. The lower part of the spathe should not be removed completely (i.e., the spathe should not be cut too low). The best way is to leave about 5-8 mm high ring of the spathe which protects the base of the inflorescence. When the spathe is cut too low, the base of the inflorescence can be damaged and in this case the inflorescence will not produce any seed. Pollination The emasculation is usually followed by immediate pollination (due to protogyny). For pollination, we use inflorescences which are two days older (Fig. 2-7). One vigorous male inflorescence can pollinate several females, depending on quantity of pollen available. When 3
there are several genetically different plants flowering in the same area, it is recommended to protect (to isolate) the male components. If not, the male portions of the inflorescences will contain pollen grains from different sources (brought by insects and/or wind). Male inflorescences can be protected in different ways (e.g., by keeping them in a cup with some water in an isolated place or by keeping the upper part of the spathe closed Fig. 5). There are several possible ways to distribute pollen on stigmas: by brush, fingers or directly by the male portion of the spadix (Fig. 4). Most of the breeders pollinate directly by the male portion of the spadix. 1 2 3 4 Fig. 4. Taro crossing procedure: 1- removal of the upper part of the inflorescence; 2 removal of the lower part of the spathe which protects the female part of the spadix, and pollination by using the upper (male) part of the spadix of the male component; 3 - protection of the pollinated inflorescence with the lower part of the spathe; 4 a labelled cross. Crosses have to be labeled. The best labels for taros are made of paper which can resist heavy rains (Fig.4-4). Information recorded on a label must include three essential data: the parents, the date of pollination and the name or the initials of a person who pollinated the inflorescence. The data should be written clearly and with a pencil. An example can be the cross VU 373 PH 16. VU 373 is the female ( ) and PH 14 is the male parent ( ). For self-pollination we use the symbol (e.g., VU 373-1 ). 4
Self-pollination Self-pollination is frequently used in studies of genetic inheritance and for induction of genetic segregation (taro plants are predominantly heterozygous). It may also occur naturally, when there is only one flowering plant in the field or when the flowers remain closed during flowering. Hybridisation within a clone can also be considered as genetically equivalent to self-pollination. Artificial self-pollination is very simple. One needs only to protect inflorescences from contamination with pollen originating from other plants. The most appropriate are small elongated bags made of materials which enable aeration. The bags are placed on the selected inflorescences early in the morning, a day before they become intensely odorous (20-24 hours before reaching the temperature peak) (Fig. 2 developmental stage between 4 and 5; Fig. 3 inflorescences 4, 5). If the constriction of the spathe in the sterile region of the spadix is too narrow, the spathe has to be removed. 1 2 3 Fig. 5. Protection of the male component from pollinating agents: 1 inflorescences are kept in a glass of water in an isolated place; 2, 3 upper part of spathe is kept closed. Harvesting and preparation of seeds for germination The success of a cross (or self-pollination) becomes visible within a week. Green, fast developing fruit clusters, 10 and 20 days after pollination, are shown on Fig. 6. The fruit is ready for harvest when the berries become soft and their colour becomes lighter green or sometimes yellowish or orange (in the tropical lowlands, it usually happens 4-5 five weeks after pollination). At the same time the peduncle becomes soft. If the pollination was not successful, the peduncle becomes soft much earlier. The fruit of each cross has to be harvested together with the label. Seeds have to be extracted from the fruit by washing in water. Most of the breeders use special, very fine screens so that the seeds cannot be washed through. Clean seeds have to be dried and put into labeled plastic or paper bags. For a longer storage, seeds have to be sealed in small plastic bags and kept in a desiccator inside a refrigerator. In such conditions, seeds remain viable for at least two years. 5
B A Fig. 6. Successful crosses: A - 10 days after pollination, 20 days after pollination (photo was taken in January 2011, at the Vanuatu Agricultural Research and Technical Centre, VARTC, on Espiritu Santo). Seed germination For a successful and fast germination, seeds require light sterile soil, high humidity and temperature above 24 C. The easiest way to obtain such conditions is to place seeds in pots which are kept in special water pools inside cages protected by transparent plastic sheet (Fig. 7). Seeds are distributed close to the soil surface. In a screen house, pots can be covered directly by transparent plastic sheet. Once a day the sheet has to be removed and the pots sprayed with clean rain or sterile water, using a hand sprayer. Seeds start germinating about one week after sowing. After the seed germination, the transparent plastic sheet has to be removed or placed a little higher above pots. Fig. 7. A special water pool constructed for germination of taro seed. 6
Two or three weeks after germination, seedlings are ready for transplanting. One to five plants are transplanted to each pot, depending on pot size. When plants are strong enough, they are transplanted outside, under shade cloth and finally to the field. It takes from 3.5 to 5.5 months from germination to field. Literature Ivancic, A, Lebot, V. 2000. The genetics and breeding of taro. Séries Repères; CIRAD, Montpellier, France. Ivancic, A., Lebot, V., Roupsard, O., Quero-García, J., and Okpul, T. 2004. Thermogenic flowering of taro (Colocasia esculenta, Araceae). Can. J. Bot. 82: 1557-1565. Ivancic, A., Roupsard, O., Quero-García, J., Melteras, M., Molisale, T., Tara, S., and Lebot, V. 2008. Thermogenesis and flowering biology of Colocasia gigantea (Araceae). J. Plant Res. 121: 73-82. 7