EXPERIMENTAL STUDIES ON THE CULTIVATION OF EXCISED ANTHERS IN NUTRIENT SOLUTION

GENOTYPIC CONTROL of the size of mitotic chromosomes, chiasma frequency and position and meiotic failure of various kinds have been described in many plants (Darlington 1937). Beadle (1930) showed that in Zea mays meiosis may have a genetic basis similar in its behavior to any ordinary genetic factor. Exceptional failure of pairing not due to hybridity has been described in Nicotiana, Zea mays, Ranuncutlus acris, Datura, Crepis, Triticum and many other plants (Darlington 1937). The question of the origin of this genotypic control of chromosomes, i.e. whether it proceeds locally from the chromosomes involved in a given mitosis or remotely from the same genes acting elsewhere in the organism to produce mitosis controlling materials, becomes of prime importance in a consideration of the localization of meiosis. It has been shown that the initiation of the special structures in which meiosis occurs in flowering plants is due to a special diffusible substance (Hamner and Bonner 1938) (for review see Loehwing 1938). Furthermore, it has been strongly suggested that this substance is non-species specific as well as non-photo-period specific (see review in Loehwing 1938). The problem of the genetic control of meiosis has been approached both by cytological and genetic analyses. The question of the localization of this control has, however, evaded researches of this kind. One obvious method which has not been specifically applied to this problem lies in the separation of the meiotic cells from the parent plant and the culturing of these cells in vitro away from the influences of the plant. The cultivation of small excised parts of plants has been practiced with varying degrees of success since the problem was first formulated by Haberlandt in 1902 (White 1936). During the years following a great deal was learned about the nutrient requirements of excised plant parts, but it was not until 1934 that potentially unlimited growth of excised meristems was accomplished (White 1934). At this time White had developed a nutrient solution for excised tomato roots which contained the usual nutrient salts, sucrose, accessory salts, and an unknown amount of organic accessory materials in the form of an extract of dried "Brewers" yeast. Since this time worliers have turned their attention to the substances in the yeast extract necessary for the growth of excised roots. Bonner (1937), culturing pea roots, Robbins and Bartley (1937), growing tomato roots, and somewhat later White (1937a), also working with tomato roots, showed that the yeast extract was partially replaceable by vitamin B1. White (1 937b) showed that a combination of several amino acids and vitamin B1 were capable of replacing the yeast extract for tomato roots. Addicott and 1 Received for publication May 23, 1940. Bonner (1938) and Addicott and Devirian (1939) have shown that nicotinic acid is an essential factor for continued growth of excised pea roots. Finally, Robbins and Schmidt (1939) have suggested vitamin B6 as an additional factor in the growth of excised tomato roots. Efforts to grow the meristematic parts of flowers have not met with the same success as the work with root tips. (It should be mentioned here that the remarkable regeneration experiments from flower parts, etc., of Drosera carried out by Behre (1929) hardly constitute successful organ or tissue culture.) According to Barber (1939) divisions cease within a week to ten days even though, according to La Rue (1938) the tissues may remain alive for as long as twelve months. Gregory (1939a) has shown however, that anthers of Lilium longiflorum excised early in their development make good growth in nutrient solution, some individuals far exceeding the normal size for anthers of this species; but that in such anthers the sporogenous cells, after a mitotic period of about two weeks, differentiate into thinwalled parenchvma. Later the same author (Gregory 1939b) showed that anthers of Datura and Lycopersicum excised at similar stages of development made no growth in nutrient solution. Addicott (1940) writes that anthers of Tropaeolum and Convallaria react somewhat like those of Lilium in nutrient solution (see Gregory 1939a and 1939b). In liquid paraffin mitosis will proceed for at least twelve hours and a large proportion of prophases go through to telophase. Mitosis ceases completely in preparations twenty-four hours old even though active protoplasmic streaming may continue for fourteen days or longer. Mitosis also stops after a comparable length of time in sugar solutions (Barber 1939). Shimakura (1934) was able to get Tradescantia pollen mother-cells to proceed from M I through both divisions in saccharose solution by carefully adjusting the solution to 7.93 per cent saccharose and to a pH of 7.2 to 7.3. In order to determine what causes may be associated with the initiation of meiosis and to ascertain whether this initiation proceeds from the sporogenous tissue involved or is brought about by substances originating elsewhere in the plant, it becomes necessary to remove the sporogenous cells from the influence of the plant and place them under physiologically controlled conditions. The object of the present study has been to isolate the sporogenous cells at different stages of growth of the anthers of several flowering plants and grow them in nutrient solution in order to see if growth proceeded and if meiosis continued normally and if not what changes took place in the sporogenous tissue.