Studies on the culture of ovules removed before fertilization, or within several hours or days after fertilization, have been undertaken by a group of workers in the Department of Botany of Delhi University in the period since 1958. N. Maheshwari (461) first succeeded in rearing ovules of Papaver somniferum which contained a zygote (or 2-celled proembryo) and a few endosperm nuclei, into viable seeds; this was done by the use of Nitsch's medium (542) supplemented with 0.4 ppm kinetin. More recently, Kanta, Ranga Swamy, and P. Maheshwari (368) have cultured, in the same vial, ripe pollen and ovules of this species and have observed all the stages from germination of pollen to double fertilization to development of mature seeds (Fig. 30).
Sachar and Kapoor (637) cultured ovules of an unidentified species of Zephryanthes using as a basal medium that of Nitsch supplemented with Β vitamins and calcium pantothenate. Ovules removed before pollination, and cultured in this medium containing gibberellic acid, developed into seeds with normal testas, but they lacked embryos and endosperm and were about half the normal size. Ovules excised 2 days after pollination (zygote stage with undivided primary endosperm nu
cleus) when cultured in the above medium grew up to the formation of a late globular embryo, but the endosperm development was in
hibited. Further development was not achieved by additions of kinetin, auxins, or gibberellic acid. However, ovaries excised at this stage and
FIG. 3 0 . Cultures of ripe pollen and ovules of Papaver somniferum, leading to normal double fertilization in culture and the development of mature seeds. (A) Whole mount of ovule from a 3-day old culture showing germinating pollen grains.
(B) 7-Day-old culture—the white bodies are developing seeds. (C) Globular embryo dissected from developing seeds in a 9-day-old culture. ( D ) Fully formed embryo of a seed in a 22-day-old culture. From Kanta, Ranga Swamy, and Maheshwari ( 3 6 8 ) .
cultured in the basal medium gave seeds with fully differentiated em
bryos, thus indicating that the ovary wall contributes certain substances which are essential for the normal differentiation of the embryo. If excision of the ovules was delayed until 4 - 5 days after pollination (when they contain a young globular proembryo and a free nuclear endosperm) normal differentiation of the embryo occurred in culture and the seeds germinated in situ. Differentiation of the embryo required simultaneous endosperm development. In a later paper, Kapoor (369) showed that normal development of viable seed could be achieved from ovules excised 2 days after pollination if the culture medium was supplemented by coconut milk and casamino acids. The casamino acids could be replaced by either histidine or arginine at appropriate concen
tration. The importance of endosperm for embryo differentiation is also indicated by the studies of Chopra and Sabharwal (156) with Gynan-dropsis gynandra. Ovules of this plant could only be raised to maturity if they had, at the time of excision, a globular proembryo, and a few endosperm nuclei. When cultured at this stage and in a medium sup
plemented by IAA and casamino acids (but not coconut milk), it was found that the rate and extent of development and size of the embryo were all increased by leaving the ovules attached to placental tissue.
Mature seeds raised in vitro did not become dormant (as occurs in vivo) but could germinate immediately.
The Delhi School have also studied the behavior in culture of excised Citrus ovules in which embryos can arise adventitiously from cells of the nucellus (nucellar embryos). In some species, such as Citrus mitts (C.
microcarpa), polyembryony occurs naturally in this way. In 1961, and using this species, Ranga Swamy (598a) split the ovules open and
obtained a callus of nucellar origin in continuous culture. The callus gave rise, in its growing upper part, to embryolike structures which were termed "pseudobulbils," and from these, plantlets arose (Fig. 3 1 ) . Sab
harwal (633) cultured ovules, nucelli, and embryos of a second species, C. reticulata, in which nucellar embryos also arise. Callus arose from proembryos in the ovule and from the nucellar tissue and both types of callus gave rise to pseudobulbils and to weakly seedlings. Such callus cultures therefore represent tissue "banks" capable of initiating clones of adventive embryos, a phenomenon of both practical value and theoretical interest.
Ovoid embryos (0.5-1 mm) in Cuscuta reflexa, cultured in White's medium supplemented with IAA and casamino acids, were found by P. Maheshwari and Baldev (463) to give rise to adventive embryos.
From older embryos of this species a continuously growing callus cul
ture was obtained which, on each subculture, gave rise to a fresh crop
FIG. 31. (A) A callus culture derived from the nucellus of Citrus mitis forming pseudobulbils and plantlets; ( B ) Stages in differentiation of pseudobulbils into embryos. ( C ) Longitudinal section of a fully formed embryo from the nucellar callus. From Ranga Swamy (598a).
of adventive embryos. Similarly a callus obtained from globular pro-embryos of Dendrophthoe falcata (358) gave rise to numerous adventive embryos. These studies, together with the induction of embryos from cultured phloem cells of carrot (710) and more prolifically from dis
persed carrot embryo cells (709) clearly demonstrate that the capacity to develop into a new plant does not reside in the zygote alone (cf.
Chapter 8 ) . Although embryonic development can be initiated from somatic cells, it seems that the physiology of cells in growing tissue aggregates recently derived from the embryo or nucellus is particularly favorable for the expression of this totipotency of the cells of higher plants.