As early as 1926, Dahlgren reported fertilization and seed development in Codonopsis ovata fol- lowing in vitro pollination of the stump after cutting off the style at its base. That was perhaps the beginning of what almost 40 years later developed into the technique of IVP (Kanta et al.
1962).
IVP of excised pistils (stigmatic pollination) resulting in the production of viable seeds has been achieved following self (Antirrhinum majus) as well as cross-pollination (Nicotiana rustica, Petunia violacea). The presence of intact calyx lobes and pedicel was found to be essential for viable seed formation following in vitro stigmatic pollination in Trifolium
S. S. Bhojwani and P. K. Dantu, Plant Tissue Culture: An Introductory Text, DOI: 10.1007/978-81-322-1026-9_13,Ó Springer India 2013
species. Considerable success has been achieved with IVP and IVF in maize (Bhojwani and Raste 1996; Kranz and Kumlehn 1999; Okamoto and Kranz 2005; Kranz and Scholten 2008). Dhaliwal and King (1978) and Raman et al. (1980) reported seed-set following both stigmatic and ovular IVP in maize.
13.2.1 Terminology
Kanta et al. (1962) and, following them, several other authors (Zenkteler 1965, 1967, 1970; Balatkova and Tupy 1968; Rangaswamy and Shivanna 1969) have described the technique of seed development through IVP of ovules as ‘‘Test Tube Fertilization’’. Seed development following stigmatic pollination of cultured pis- tils has been referred to as ‘‘In Vitro Pollina- tion’’. However, in either case, fertilization of egg occurs inside the ovule by sperms delivered by the pollen tube almost in a natural fashion. Strictly speaking, the term ‘‘Test Tube Fertil- ization’’ or ‘‘In Vitro Fertilization’’ should, as in animals, refer to plant regeneration through in vitro fusion of isolated single egg and sperm cells as reported, first time, by Kranz et al. (1991; see also Kranz and Lörz 1993). There- fore, in this chapter, in vitro application of pol- len to excised ovules is referred to as ‘‘In Vitro Ovular Pollination’’, to the ovules attached to a piece of placenta as ‘‘In Vitro Placental Polli- nation’’ and to the stigma of intact pistil as ‘‘In Vitro Stigmatic Pollination’’ under the general term of in vitro pollination (Fig.13.1).
13.2.2 Technique
The success with IVP depends, to a great extent, on a thorough knowledge of the floral biology of the experimental materials, such as time of anthesis, the time of anther dehiscence, viability of pollen grains, viability of the female gametophyte, receptivity of stigma to pollen grains, pollen ger- mination and pollen tube growth, fertilization and embryo and endosperm development. If the
flowering seasons of the desired parents are different or they are growing in geographically different locations, suitable methods for pollen storage and transport need to be adopted. The choice of nutrient media to support optimal pollen germination and pollen tube growth as well as development of in vitro fertilized ovules to form viable seeds is most critical. In some instances, full development of seeds and embryos may require a transfer of ovules, 7–10 days after IVP, to fresh medium of a different formulation (Marubashi and Nakajima 1985; Bino et al. 1992).
13.2.3 Preparation of Explant
In order to prevent chance pollination, flower buds of the female parent are emasculated and bagged. The buds are excised 1–4 days after anthesis and prepared for aseptic culture. Both, sepals and petals are removed, and the pistil with pedicel rinsed quickly in 70 % ethanol, surface sterilized with suitable sterilizing agent and, finally, washed thoroughly with sterile distilled water before planting them on the culture med- ium under aseptic conditions. For in vitro
Fig. 13.1 Diagrams depicting various types of in vitro pollination. a, b Intra-ovarian pollination; pollen suspen- sion is injected into the ovary through a hole or a slit. cStigmatic pollination; pollen are applied to the stigma of a cultured pistil. d Placental pollination; pollen are dusted on the ovules cultured along with a piece of placental tissue. e Ovular pollination; individual ovules are cultured and pollinated
stigmatic pollination, the excised pistils are sterilized without wetting the stigma and wiped dry with a sterile filter paper before implanting them on the culture medium.
For placental and ovular pollination, the stigma and style are removed and the ovary wall is peeled off to expose the ovules. The whole placenta, bearing the ovules on a short pedicel, is generally used for placental pollination. The placenta may also be divided into two or more pieces, each carrying a certain number of ovules and cultured individually. In species such as Trifolium, where removing the ovary wall or cutting a slit along the length of the ovary wall causes browning of the ovules, the best method to achieve IVP is to introduce pollen grains into the ovary through a pore in the ovary wall (in vitro intra-ovarian pollination).
For collecting pollen, undehisced anthers are aseptically removed from surface-sterilized flower buds and kept in sterile Petri plates until dehiscence. When anthers are taken from already opened flowers, they need to be surface sterilized and kept on a filter paper inside a sterilized Petri plate. The pollen grains are aseptically collected and deposited on the ovules or the stigma as the case may be. Pollen deposited on the ovules or placenta have per- formed better than those placed on the medium around the ovules.
The procedure described above may require modification according to the type of experi- mental material. In maize, where ovaries are well protected by several layers of husks, surface sterilization is not necessary. Ears are bagged prior to the emergence of silks (styles) to prevent uncontrolled pollination. Such bagged ears are collected 2–6 days after the emergence of silks. The protruding silks are excised and all but the innermost husks are removed. The removal of the innermost husk and all subsequent operations are performed aseptically. Pieces of cob, each car- rying a total of 4–10 ovaries in two rows are placed on the medium so that their silks hang out of the Petri plate after replacing the lid. It is also possible to keep this plate inside a larger Petri plate so that the silks are contained within it. The
silks are pollinated, not necessarily aseptically. Twenty-four hours after pollination, the silks are clipped off and the Petri plate sealed with Para- film. Several reports have described single-pol- len pollination in maize using this technique.
For in vitro pollination of ovules, pollen are deposited directly on the ovules either attached to the placenta or detached from it. However, mostly the ovules are left attached to the pla- centa as the pollination of detached ovules has not been successful except in Brassica oleracea (Kameya et al. 1966; Kameya and Hinata 1970). In vitro self-pollination has been attempted in 47 species belonging to 34 genera, and viable seeds have been obtained in 28 species (Bhojwani and Raste 1996). Interspecific, intergeneric and interfamily in vitro cross-pollination of ovules has been attempted in 37 crosses, but success has been achieved in only 16 combinations.
13.2.4 Factors Affecting Seed-set
Following IVP
The number of viable seeds produced is the measure of success in IVP. Some of the factors that affect the success of the technique are the nature of the explant, culture medium and the culture conditions.
In Petunia axillaris, excised ovules or pieces of placenta each bearing a group of ovules did not set viable seeds following IVP. However, pollination of the ovules on intact placenta resulted in normal germination of pollen and development of viable seeds. In P. hybrida, even the removal of stigma and style proved delete- rious. In maize, the number of kernels formed on the pieces of cob after IVP was directly related to the number of ovaries attached to the explant and the amount of accompanying cob tissue (Gengenbach 1977). The ovular and stigmatic tissues in the explant should be dry. Traces of water at the site of pollination lead to pollen bursting. The ovules for IVP should be excised 1–2 days after anthesis instead of on the day of anthesis; for maize, the optimal stage of spike for IVP is 3–4 days after silking. The
physiological state of pistil is affected by polli- nation and pollen tube growth. In many cases, unfertilized ovules excised from pollinated pis- tils responded better than those from unpolli- nated pistils.
The efficacy of IVP, to a large extent, depends on the composition of the culture medium, which should support pollen germina- tion and pollen tube growth to cause fertilization and, secondly, development of the fertilized ovules into mature seeds with a viable embryo. At times, it may be necessary to transfer the fertilized ovules, 7–10 days after IVP, to a dif- ferent medium to support normal seed develop- ment. It is, therefore, imperative to optimize, before proceeding with IVP, the nutritional and hormonal requirements of fertilized ovules (containing zygote or 2-celled proembryo) of the plant to be used as the female parent.
The genotype of the parents also influences the response. The mineral salts commonly used in the medium for IVP are those prescribed by Nitsch (1951) for ovary culture. To this, sucrose and vitamins are added as recommended by White (1943). The composition of the modified Nitsch’s medium widely employed for IVP is given in Table13.1. The addition of growth regulators and supplements to this medium needs to be optimized for every system.
The in vitro pollinated ovule cultures are generally kept in dark at normal culture room temperature (25°C).