A method has been developed that extracts DNA from stem tissue of flowering tobacco plants, Nicotiana By in vitro cultivation of excised stem segments, flowering tobacco plants have been shown to have a pronounced apex to base gradient in the capacity to form floral buds (1, 2), which truly reflects a difference in the capacity to flower and not merely a decrease in the over-all capacity to form buds (32). Appropriate exogenous supplies of IAA effected a transition from floral to vegetative bud formation in such segments (31).

Abstract. The formation of flowers has been studied in stem tissue excised from flowering plants of Nicotiana tabacum variety Wisoonsin No. 38, and cultured in vitro on Murashige and Skoog nutrient medium. A procedure for quantitative evaluation of factors influencing floral expression has been developed and effects of the growth substances, indole-3-acetic acid (IAA), kinetin and gibberellic acid (GA,), on the process are reported.Although a low (1 juM) level of IAA was required for the development of normal flowers on stem segments, higher concentrations tended to inhibit flowering. The decrease in floral buds was rapid in the 3 to 15 AM range. IAA concentrations up to 75 aM increased vegetative bud formation so as to effect a transition from floral to vegetative buds rather than merely an inhibition of bud formation. Higher IAA concentrations inhibited both vegetative and floral bud formation.Kinetin in high concentrations greatly increased the number of vegetative buds but had no significant effect on the number of floral buds per segment. High kinetin concentrations also permitted branching of floral shoots so that flower clusters were formed.GA3 applied in the medium from the start, strongly inhibited bud formation on the stem segments, but when applied to young floral buds after they had formed, it promoted their further development ("bolting").Flower formation occurred in complete darkness, but light of moderate intensity was required for the development of normal flowers.Experimentation on the physiology of flowering has been limited mainly to whole plants, but a new approach to regulation of flowering has been opened wvith the dcemonstration that under certain conditions tobacco stem segments cultured in vitro form floral instead of vegetative buds (12, 13). Materials and MethodsIn preliminary experiments it was found that the Murashige and Skoog "revised medium" (11) minus CoCl, and with 1.4 poI indole-3-acetic acid (IAA) and 7.5 X 10-2 gm kinetin would serve as a standard medium for culturing stem segments. Stock solutions of ingredients were kept separately refrigerated for use no longer than 6 weeks. Thiamine-HCl and IAA solutions were prepared fresh for each experi-

Abstract. The RNA base analogues, 2-thiouracil, 6-azauracil and 8-azaguanine incorporated singly into the medium, increased the number of floral buds in excised stem segments of Nicotiana tabacum variety Wisconsin No. 38 cultured in vitro. Combined treatments with 2 and 3 base analogues were even more effective. The effects were prevented by the corresponding natural counterparts, uracil, uridine, and guanosine respectively. These nucleic acid constituents added to cultures without base analogues did not affect the number of floral buds formed. In stem segments from the lower internodes treatments with the analogues effected a transition from vegetative to floral bud formation, thus in a sense removing the floral gradient as defined by Chouard and Aghion.The inhibition of flower bud formation ordinarily achieved even with low concentrations of indole-3-acetic acid (IAA) was counteracted by low concentrations of 2-thiouracil, 6-azauracil and 8-azaguanine. Reversal to vegetative bud formation was not achieved even with high IAA concentrations in the presence of the base analogues.Stem segments excised from young, vegetative tobacco plants never formed floral buds in vitro. Thus, the inhibiting effect of IAA and the promoting effect of base analogues seem to be on the expression and not on the induction of flowering. The regulatory aotion of the IAA and base analogues in flower bud formation is tentatively ascribed to their differential effeots on RNA synthesis.Chouard and Aghion (3) found that flower buds formed de nozo on stem segments derived from Nicotiana tabacumn variety Wisconsin No. 38 plants which were already flowering. The closer the segments were cut from the top of the plant, the greater the ratio of floral to vegetative buds. They suggested that this apex to base floral gradient may be a result of endogenous gradients of growth substances in the stem. Aghion-Prat (1) demonstrated that the floral gradient also occurred in vivo. Inducing tumor formation along the stem of a flowering tobacco plant by injection with Agrobacterium tumnefasciens, she showed that tumors which were near the top of the plant formed flower buds, whereas those which were located further down the sLcm Materials and MethodsTobacco stem segments (Nicotiana tabacum variety Wiscon in No. 38) were cultured, and growth substances were added to the medium as described (13). The base analogues [2-thiouracil (2-TU), 6-azauracil (6-AU), 8-azaguanine (8-AG)] and their respective counterparts [uracil (U), uridine (UR) and guanosine (G)] as well as gibberellic acid (GA3) when used were always prepared fresh, filter sterilized and pipetted into each separate culture flask after the medium had been autoclaved. The flasks were then cooled rapidly in cold water.In all experiments except those with high IAA concentrations ca. 90 % of the segments formed buds.UJnless specified the calculations of the average numbers of floral and vegetative buds per segment are based on differentiated segments only.

The transition of a flowering plant from vegetative to reproductive growth is preceded presumably by biochemical changes within the shoot apex that render the apex capable of forming flowers instead of leaves. Any apex which has attained a flowering capability is considered to be in a floral state. Recently, several investigators (3,8,9,14,18,19,22,23,25,26,31) utilizing segments cut from floral branches or cut from along the stem axes in the inflorescences of flowering plants have shown that these segments, like floral-induced apices, initiate flowers instead of leaves. Thus, the in vivo inductive attainment of a floral state by plant tissues can be extended below the apex into the stem.The floral state in stems has been studied most thoroughly for cv. Wis. 38 tobacco plants. In this cv., Aghion-Prat (1, 2) found that in the internodes along the stem axis of a flowering plant that was an inhibitor gradient in flower bud formation (the floral gradient), and Wardell and Skoog (32) found that the inhibitor gradient in flower bud formation occurred simultaneously with a promotive gradient in vegetative bud formation. The floral gradient, therefore, was a gradient change in bud expression rather than a gradient decrease in the over-all capacity to form buds. Wardell and Skoog (31) showed that segments cut from the top of a flowering plant (intemodes 1-4) and treated with The extent to which the floral gradient may be due to an inverse gradient in endogenous IAA has been investigated (25, 32) by completely counteracting with RNA base analogs the inhibitory action of IAA on floral expression. As expected, below-gradient segments treated with base analogs increased (8-fold) in their floral expression to the extent that they became identical in bud expression to top segments not treated with base analogs. However, the floral expression of top segments treated with base analogs also increased (3-fold). And the floral expression of base analog-treated segments cut from along the region of the stem corresponding to the floral gradient all increased so that the floral gradient remained. On these bases, Wardell and Skoog have suggested that some factor, besides IAA, with a gradient distribution, also is involved in the regulation of floral expression.Another gradient factor correlated with floral expression of tobacco stem segments has been reported (33). Using methodology developed specifically for extracting DNA from tobacco stems already in a floral state, it has been found that a gradient in DNA roughly paralleled the floral gradient, and that the low concentrations of IAA effective in inhibiting floral expression also inhibited thymidine incorporation into DNA. In striking contrast, low concentrations of IAA promoted thymidine incorporation into DNA in stem sections cut from young vegetative plants. Leaves

The short term effect of 11.4 AM indoleacetic acid on the incorporation of (methyl-'H)