Let us make an in-depth study of the meaning, categories, principles and protocols of embryo culture.
What is Embryo Culture?
The embryo of different developmental stages, formed within the female gametophyte through sexual process, can be isolated aseptically from the bulk of maternal tissues of ovule, seed or capsule and cultured in vitro under aseptic and controlled physical conditions in glass vials containing nutrient solid or liquid medium to grow directly into plantlet.
Different Categories of Embryo-Culture and their Objectives:
Culture of embryo (Fig 10.1) may be divided into the following categories:
1. Culture of Mature and Intact Seed Embryo:
The aim of this study is to analyse the various parameters of embryonic growth and the metabolic and biochemical aspects of dormancy and germination.
2. Culture of Surgically Disected Embryo:
The mature seed embryo can be dissected surgically into a number of segments. Such embryo segments are cultured to analyse the relationship of different parts of the embryo to its final form in culture.
3. Culture of Immature Embryos or Pro-embryo’s:
The term pro embryo means the early developmental stages of the embryo that precede cotyledon initiation. Globular and heart-shaped stages of embryo are appropriately called as pro embryos. The objective of such culture is to understand the control of differentiation and the nutritional requirements of such progressively developing embryos.
4. Culture of Intact Seed Containing Undifferentiated Embryo:
Each fruit of an orchid plant develops several thousand tiny seeds which contain morphologically undifferentiated embryos. These embryos are the spherical mass of tissue lacking both radicle and plumule. There is even no storage tissue in the seeds and the seed coat is reduced to a membranous structure. For this reason the entire seed of orchid containing undifferentiated embryo is cultured and treated as embryo culture. In nature, these seeds germinate only in association with a proper fungus or else they perish. As a result, numerous seeds are lost. In vitro culture of orchid seeds is routinely employed for orchid propagation.
5. Culture of Adventives Embryos from Polyembryonic Seeds:
Besides the zygotic embryo produced from egg cell, some additional embryos are produced from nuclear tissue in polyembryonic seed like lemons and oranges. Such additional abortive embryos can be exploited in culture for clonal propagation.
6. Culture of Inviable or Abortive Embryos:
In many inter-specific or inter-generic breeding experiments, sometimes inviable or abortive embryos may develop due to unsuccessful crosses. As a result, the non-viable seeds do not germinate normally. But it is now possible to raise a hybrid plant by culturing the inviable embryos in vitro.
Principles of Embryo Culture:
The underlying principle of the method is the aseptic excision of the embryo and its transfer to a suitable nutrient medium for development under optimum culture conditions. In general, it is relatively easy to obtain pathogen-free embryos, since the embryo is lodged in the sterile environment of the ovule or seed or capsule or fruit. So, surface sterilization of the embryos as such is not necessary. Thus the entire seeds or fruits containing the ovule are surface sterilized and the embryos are then aseptically separated from the surrounding tissues.
Seeds with hard seed coats are generally surface sterilized and then soaked in sterile water for a few days aseptically so that seed can be cut easily to free the embryo. Although seeds are surface sterilized before soaking they may need to go through a second sterilization before embryo excision. Splitting open the seeds and transferring embryos to the nutrient medium directly is the simplest technique that can be done with seeds.
In the isolation of comparatively smaller embryos, it is important that they are removed intact carefully from the ovule without any injury. This can be best achieved by carrying out the operation under a specially designed dissecting microscope. In case of orchid seeds, the entire seed or ovule is cultured because the seed contain morphologically undifferentiated spherical embryo having no functional storage tissues like endosperm and the seed coat is reduced to a membranous structure.
Although the entire ovule containing undifferentiated embryos is cultured, but it is referred to as embryo culture. Each fruit of an orchid contains thousands and thousands of tiny seeds, so a large quantity of sample can be cultivated easily just after excision of surface sterilized fruits. It is also important that the freed seeds containing the embryos not become desiccated during above operation.
The most important aspect of embryo culture work is the crucial selection of the medium necessary to sustain continued growth of the embryos. The formulation of nutrient media may vary depending upon the species used for study and many of them have not been rigorously determined.
Nevertheless it is possible to make certain generalization i.e. the younger the embryo the more complex is its nutrient requirements. Thus while the mature embryo can be grown in an inorganic salt medium supplemented with a carbon energy source such as sucrose, relatively young embryos require in addition, different combinations of vitamins, amino acids, growth hormones and in some cases natural endosperm extracts such as coconut milk. Since pro embryos are often submerged in the ovular sap under considerable osmotic pressure, culture of such embryo in presence of an osmoticum such as mannitol is often suggested.
The changing nutritional requirement for successful embryo culture has often meant transferring the embryo from one medium to another for optimum growth. Monnier devised a culture method (Fig 10.2) which allowed for the uninterrupted growth of globular embryo to maturity. By this method, embryo can be grown in both solid and liquid medium at the same time.
The composition of both media is different. Monnier also stressed the importance of obtaining uninjured embryos with their suspensor for successful culture. Suspensor is important for the growth of immature embryos. But embryo selected at later stages do not require the attached suspensor.
In culture, the embryos are not induced to form callus tissue but they are allowed to form a plantlet. After the embryos have grown into plantlets in vitro, they are generally transferred to sterile soil or vermiculite and grown to maturity in the green house.
Protocol for Embryo Culture:
The following protocol for embryo culture (Fig 10.3) is based on the method used for Capsella bursapastoris. With modification, this basic protocol should be applicable to embryo culture in general.
The steps are given below:
1. Capsules in the desired stages of development are surface sterilized for 5-10 minutes in 0.1% HgCl2, either in a closed small room previously illuminated by UV lamps or in a Laminar air flow.
2. Wash repeatedly in sterile water.
3. Further operations are carried out under a specially designed dissecting microscope at a magnification of about 90X. The capsules are kept in a depression slide containing few drops of liquid medium.
4. The outer wall of capsule is removed by a cut in the region of the placenta; the halves are pushed apart with forceps to expose the ovules.
5. A small incision in the ovule followed by slight pressure with a blunt needle is enough to free the embryos.
6. The excised embryos are transferred by micro-pipettes or small spoon headed spatula to standard 10 cm petridishes containing 25 ml of solidified standard medium. Usually 6-8 embryos are cultured in a petridish.
7. The petridishes are sealed with cello-tape to prevent desiccation of the culture.
8. The cultures are kept in a culture room at 25 ± 1oC and given 16 hrs. illumination by cool white fluorescent tube.
9. Subcultures into fresh medium are made at approximately four weeks intervals.
In case of fresh seeds or dry and imbibed seeds, the schedule is slightly changed. Seeds are cleaned by 5% Teepol (a liquid detergent) for 10 minutes and dipped in 70% ethyl alcohol for 60 seconds. Surface sterilization in 0.1% HgCl2 is followed by washing in sterile water, then the seeds are decotylated using a sharp scalpel and embryos are transferred to solid nutrient medium. In case of orchid seeds, after following the scheduled surface sterilization procedure, the fruits are excised and dusty seeds are gently spread over the medium.
Precocious Germination Embryo in Culture:
A lot of cellular, physiological and biochemical changes take place during embryo development from the zygote to the fully formed embryo stage. After its full-term development, the embryo becomes dehydrated and enters a phase of metabolic quiescence and developmental arrest (dormancy) which may last from a few days to several months or even years.
During this stage, embryo is normally incapable of germination. Embryos of mangroves and some viviparous varieties of cultivated plants (e.g. Sechium edule) germinate while still attached to the parent plant. They are able to bypass the stage of dormancy.
Similar phenomenon has been observed when excised immature plant embryos are grown in vitro. In culture, excised immature embryos do not proceed further then the embryo genic development for its maturation and start to germinate.
The immature embryos develop into weak seedling showing only those structures which are already present at the time of embryo excision. This phenomenon of seedling formation without completing normal embryo genic development is known as precocious germination. LaRue and Avery (1938) have demonstrated in vitro growth of Zizania aquaiica embryos excised from ovules at different stages of development (Fig10.4).
Embryos 0.05 mm long show very little in vitro growth. Older, immature embryos (3.5 mm long) germinates precociously and form seedlings which are not as well developed as those formed by mature embryos in the same culture period. The main objective of culturing immature embryos is to stimulate normal embryological development in order to understand the factor(s) that regulate the orderly development of embryos in nature.