Embryo germination

Occasionally, hips from a desirable cross will not reach full maturity (turn red or orange) and thus only a few seeds may germinate or no germination may occur at all.  Why germination is inhibited is unknown at this time but may be related to the biochemical status of the seed/embryo required for germination or dormancy effect or a combination of these factors.  When this occurs, the embryo culture technique can be used to rescue the embryo.

Seed sterilization

Pierek (1987) defines embryo culture as "the sterile isolation and growth of an immature or mature embryo in vitro with the goal of obtaining a viable plant".  This discussion relates to embryos which are mature or nearly mature.  Seeds which are sterilized in a 49 to 51 (v/v) sodium hypochlorite to sterile water solution for 20 minutes and swirled with a magnetic stirrer.  They are then rinsed in three successive solutions of sterile distilled water.  Removal of the embryo is done under asceptic conditions in a transfer chamber.

Embryo excision and medium

Excision is done under a stereoscope which is placed within a transfer chamber.  The seed coat, held with a pair of tweezers whose ridges have been filed sharp to better hold the seed, is pared away at the end farthest from the radical.  After seed coat removal, the embryo is still enveloped in a seed skin (Krussmann, 1981) which must also be removed for germination to occur.  The recovered embryo is then placed on a solid agar medium (pH 5.5 – 6.0) containing salts of Murashige and Skoog (1962) and supplemented with 4% sucrose and 0.6 % agar.  Reducing the salt concentration in the media to ½ strength has proved to be beneficial.  Growth regulators need not be added.

Embryo growth

A period of dark for 10 days or more may be required for germination.  Cultures are then transferred to a cool white fluorescent lighting of about 4000 lux or less for a period of a 16-hr day.  If nutrient deficiencies become evident in the seedlings light reduction netting is placed between the cultures and the fluorescent lights.  When 2 to 3 true leaves have developed the seedlings are transplanted to vermiculite or a comparable substrate and shaded for two to three weeks under mist conditions.  During this time a very weak solution of soluble fertilizer is applied – a small pinch in a gallon (4 liters) of water every 2 to 3 days.  After this period the seedlings are moved out of the mist to a screen shaded part of the greenhouse or growth chamber or growth room.  The screen is slowly withdrawn over a period of two weeks until the seedlings have sufficiently hardened and then placed in full sunlight.

More and other details on embryo rescue can be obtained from books on this subject and in scientific publications e.g. (Lauzer, D. and Laberge, C., 1996) and from various sites on the web.  It should be noted that the above information works well for Northern Hybrid Roses but changes are made as the occasion arises.