It has been observed that even after gametic union has taken place something may arrest the growth of the embryo so that the resulting seeds are non-viable. In some cases the embryos also may not develop fully. In such cases, it is possible to excise the young embryo from the ovules and grow them in artificial media. This technique is termed embryo culture.
The first attempt to grow the embryos of Angiosperms invitro under aseptic conditions, was made by Hannig in 1904. It represents a cornerstone in the study of embryo culture. Using completely aseptic techniques, Hannig cultured relatively mature embryos of Raphanus sativus, R. landra, R. candatus and Cochlearia danica of different ages in a mineral salt medium supplemented with sugar, and obtained transplantable seedlings. Brown (1966) studied the relative efficiency of various nitrogen com¬pounds on the growth of barley embryos cultured in a medium containing mineral salts and sucrose. Knudson (1922) succeeded in germinating orchid embryos into plant-
lets in the absence of the symbiotic fungus by growi th«ifl on nutrient agar medium containing sugar.
Dietrich (1925) recorded that on semi-solid medium containing minerals and 2.5 - 5% sucrose mature embryos showed normal growth but the embryos excised IK mi immature seeds failed to achieve the organisation of | mature embryo. Instead, they grew directly into seedling! skipping the stages of normal embryogenesis. Dietrich described this phenomenon of precocious germination nl embryo as "Kunstliche Fruhgeburt".
Laibach (1925, 1928) demonstrated the practical appli cation of this technique. In certain interspecific crosses of the genus Linum, Laibach observed that the seeds were greatly shrivelled, very light and incapable of germination. By excising embryos from such seeds and growing them on moist filter papers or on cotton wadding containing sucrose or glucose, he was able to raise the hybrid plants. In recent times this technique has been used by plant breeders for raising many more such hybrids which were not possible due to the abortion of the embryos.
The nutrition of the embryo in vivo and in vitro is well known and the nutritional requirements of differentiated embryos are simple. They can be grown successfully in a medium containing^ few mineral salts and sucrose only. At this stage which is also called the autotrophic phase of embryo development, cultured embryos of most plants show almost similar requirements. On the other hand, younger embryos represent the heterotrophic phase of embryo development and are dependent for most of their growth requirements. In nature these requirements are met by the endosperm or some other maternal tissue. For growing such embryos in vitro, an elaborate nutrient medium is required. Globular embryos and undifferentiated post-globular embryos can be reared to maturity by adding nutritionally rich substances (coconut milk, caesin hydrolysate, yeast extract) and/or growth substances to the basal medium. The requirements of cultured young embryos may vary with the plant.
Preglobular embryos when grown in cultures grow in an ■norganised fashion. This suggests that the maternal tissue ■orming immediate environment of the embryos not only provide nutrition but also imposes a formative influence on ■he young embryos to follow the pre-determined course of Embryogenesis. Norstog (1965) observed that on an appro¬priate medium, the in vitro growth of barley embryos larger than 0.15mm was closely parallel to their in vivo develop¬ment. However, proembryos 0.1mm or smallar in length failed to undergo the normal mode of development. Instead, they grew into an undifferentiated mass of tissue which later formed many plantlets. Similarly, globular embryos of Dendrophthoe (Johri and Baiai, 1962) and young embryos of Cuscuta (Maheswari and Baldev 1962) did not pass through the normal stages of embryogenesis in cultures but formed a callus which subsequently differentiated many embryoids. Osmotic value of the nutrient medium seems to be an important factor for the proper growth of the embryo. A common experience is that younger embryos grow better on a medium with high osmotic value, and it decreases with the age of the embryo. This has been observed by Ryczkowski (1960-1972). He has shown that in monocotyledons as well as in dicotyledons, the values of osmotic pressure, viscosity, specific gravity and concentration of sugar and aminoacids of ovular sap decrease with the age of the ovule.
Microsurgical experiments with embryos in culture: Such experiments are aimed to study two morphogenetic problems; (a) Regeneration potentialities of various parts of the embryo, and (b) interaction between different parts of the embryo during germination.
Lee (1955) cultured various segments of lupine embryos and observed that only those segments that include the plumule formed full seedlings. Hypocotyledonary segments and radicular segments gave rise to same type of organi¬sation as is characteristic of the region. Segments taken from just below the plumule formed callus only. Bajaj (1966) made similar observations with Dendrophthoe falcata.
The cotyledons have been shown to play an important role in seedling development, especially in the growth of plumule. In Avena, Hordeum, Pennisetum and Zea th< cultures of embryonal axis in the absence of scutellum did not show the development of plumule or radicle. Hotta (lit.1 >7 i reported that in Vigna sesquipedalis the growth of root, hypocotyl and shoot depends on the amount of cotyledons severed from the embryo before culturing; larger portion of cotyledons severed greater is the suppression of seedling development. Rangaswamy and Rangan (1971) have made similar observations on Cassytha filiformis, a stem parasi t.c In recent years, intensive methods on the artificial culture of embryos have been developed. In several crosses which were formerly unsuccessful, the hybrid embryos have been* successfully reared to maturity. In mentioning some specific crosses where this technique has been employed with success, we may refer stone fruits - Prunus avium (Sweet berry), P. domestica (plum) and P. persica (peach) -similar instances of the artificial cultures of hybrid embryos have been recorded in various other plants. To obtain hybrids between Solanum nigrum x S. lutteum and Lycopersicon lycopersicum x L. peruvianum, this method has been successful.
