In this article we will discuss about the procedure of embryo culture.
Physiologically uniform embryos of the same size and same growth phase should be used for culture, which can be achieved if the plants are raised and maintained under controlled condition.
For supply of materials regularly the selected plants should bear the flowers at regular intervals; to obtain the embryos of a specific age, artificial pollination of freshly opened flowers is necessary and it may be desirable to prepare a chart showing the different stages of embryo development with days after pollination.
Where the embryos become abortive after development of seeds, the embryos should be excised before the onset of abortion.
As the embryo grows within the seed which is within a totally sterile environment, so surface sterilization of the embryo is not needed unless there is an injury on seed-coat, or any kind of systemic infection is present.
Otherwise, mature seeds, entire ovules or fruits are surface sterilized and the embryos are removed aseptically from the surrounding tissue. In case of orchid seeds the entire seed contains the morphologically undifferentiated spherical embryo. As there is no nutritive tissue and the seed coat is very thin, the whole seed is cultured in place of embryo.
Excision of Embryo:
The embryo-excision operation is performed aseptically in a laminar air flow cabinet with the help of a stereomicroscope, and using the dissecting tools like forceps, needles, scalpels, razor blades, etc.
If the seed coat is hard then seeds are soaked in water and cotyledons are splitted open and embryos are excised. But in case of immature embryos where these are embedded in endosperm, the incision is made at the micropylar end and pressure is applied to isolate the young embryo.
Nutritional Requirements in Culture Medium:
There are mainly two phases of embryo development, heterotrophic phase—where the embryo is dependent on the nutritional tissue or maternal tissue, another is the autotrophic phase—where the embryo is metabolically capable of synthesizing substances required for growth, i.e., becoming independent for nutrition.
The media composition for embryo culture differs for young or immature embryos from those of mature embryos. When immature or heterotrophic embryos are cultured, the medium requirement becomes progressively complex to permit the expression of the total developmental potential of an embryo.
This is dependent upon the progressive activation of critical enzyme systems or biochemical pathways concerned with the synthesis of substances necessary for the growth of embryos at a specific age. But the embryos of mature stage are completely autotrophic and grow on a simple medium comprising of mineral salts with carbohydrate energy source.
Mineral salts are used according to MS, B5 or White’s media with some modifications. Among the carbohydrates, sucrose is the main source, addition of maltose, lactose, raffinose or mannitol may be required for some species of embryo culture. The carbohydrate concentration may also vary according to the maturity of embryo.
Presence of ammonium salt has been found essential for proper growth and differentiation of embryos. Various amino acids or their amides are sometimes essential for embryo growth. Casein hydrolysate (CH), an amino acid complex has been widely used as an additive to embryo culture media.
Coconut milk helps the young excised embryos to increase in length of various species like sugarcane, barley, tomato, carrot, or some interspecific hybrids. Among the growth regulators, GA is used at low levels which effectively stimulate excised heart shaped embryos.
The incubation temperature for embryonic growth and germination may vary with the genotype and species. Initial dark incubation for 4 days of embryo culture is essential, following which these can be grown to maturity under light condition according to requirement.
The protocol of embryo culture has been depicted in Fig. 19.1.
Morphogenesis in Partially Differentiated Embryo:
Embryo development is a process from zygote formation to embryo germination, the stages can be classified into five stages:
I. Cleavage and differentiation:
Cell division with little growth; differentiation of all major tissues.
Rapid cell expansion and division.
Little or no cell division or expansion, synthesis and storage of reserve materials.
Renewed cell expansion and division; embryo growth.
Detailed cultural studies have revealed the role of suspensor in embryo development. Suspensor is an ephemeral structure which is found at the radicular end of the pro-embryo that attains its maximum development by the time the embryo reaches the globular stage. In some cases the presence of suspensor with the immature embryo is essential for maturity. The requirement of the suspensor may be substituted by the addition of GA or ABA to the culture medium.
After full development of the embryo it enters into a phase of dormancy characterised by metabolic quiescence and development arrest of cells. Dormancy may last for different time periods for different kinds of crop species.
Excised immature plant embryos on a nutrient medium tend to bypass the stage of dormancy and cease to undergo the linear embryogenic mode of development. The phenomenon of seedling formation without completing normal embryogenic development is called precocious germination. Casein hydrolysate, high sucrose level, extension, elevated temperature; high light intensity and also ABA like substances prevent precocious germination of embryo.
In many plant species fully developed seeds contain embryos that lack differentiation into radicle, plumule and cotyledons. In these cases the globular stages are attained and the embryonal end proximal to the micropyle regarded as the ‘radicular pole’ and distal to the micropyle is regarded as the ‘plumular pole’. In case of orchid, the plumular pole enlarges to form a spherical structure which turns green and after attainment of certain size differentiates into roots and shoots.