Cleland detected the florigenic activity in honeydew produced by aphids infecting to flowering Xanthium strumarium. The aphid inserts its styllus into phloem, sucks phloem sap, and digests sugars in it. The components other than sugars in the sucked phloem sap are discharged as honeydew. Lemna gibba cultured on the medium containing the honeydew was induced to flower. The active component was identified as salicylic acid. Salicylic acid and related compounds induce flowering in several species of the Lemnaceae. However, photoperiod-related changes in the endogenous level of salicylic acid have not been detected.

　　Phloem sap of Perilla frutescens and that of Pharbitis nil have also been assayed. Photoinduced leaves were cut off, and the cut ends placed in water to allow phloem sap to diffuse into water. Thus collected phloem sap was added to medium on which shoot meristem was cultured. Flowering was reported to be induced, but the details are unknown.

　　Many attempts have been made to isolate the florigenic activity by the extraction with organic solvents. Takimoto and his group extracted fronds of Lemna paucicostata with water, and found flower-inducing activity. The active principle was identified as α-ketol-linolenic acid. This compound has not been proven to act as an endogenous flowering regulator.
　　Lemna spp. are also induced to flower by lysine, L-pipecolic acid and some other chemicals, but their endogenous levels have been reported to be similar in flowering and vegetative plants.

　　Known plant hormones have been assayed for their activities to induce flowering. Their endogenous levels have been compared in flowering and vegetative plants.

　　The flower-inducing activity of exogenously applied gibberellins is clear in rosette-forming long-day plants such as carrot. From this fact, gibberellins have been considered as florigen. This conclusion was not accepted because gibberellins do not induce flowering in short-day plants. However, there is no reason to assume that florigen must be the same in different plant species. Whether gibberellins are florigen or not should be examined from the view point of whether endogenous gibberellins were actually involved in the mechanism regulating flowering in one fixed plant species.
　　Endogenous levels of gibberellins have been compared between flowering and vegetative plants of spinach, a long-day plant. The gibberellin level increased under long-day conditions. However, inhibitors of gibberellin synthesis did not inhibit flower formation although they inhibited stem elongation. It was concluded that gibberellin was not involved in the flowering of spinach.
　　The role of gibberellins in flowering of short-day plants has been well studied in Pharbitis nil. Endogenous gibberellin contents increase under short-day conditions, inhibitors of gibberellin biosynthesis inhibit flowering and this inhibition is nullified by gibberellin. However, application of gibberellins did not induce flowering under long-day conditions. The process promoted by gibberellin was found to be flower evocation. Therefore, gibberellin is a factor promoting the action of florigen.

　　Abscisic acid has a dual function in flowering of Pharbitis nil. It inhibits flowering when given before or early in the dark period, and promotes it when given after or later in the dark period. Together with the estimation of endogenous levels and the effect of inhibitors of biosynthesis, abscisic acid has been concluded to inhibit the time-measuring process and promote florigen transport and flower evocation.

　　Ethylene induces flowering of pineapple and plant species belonging to the Ananaceaceae and the bulbious plants. Ethylene is commercially used as a flower-inducing agent in pineapple cultivation. On the other hand, ethylene inhibits flowering in many other plant species. It is possible that ethylene is a florigen of the Ananaceaceae. However, it is not clear whether ethylene synthesis is induced under inductive photoperiods, or whether ethylene is actually involved in the regulation of flowering.

　　The endogenous level of polyamines, especially putrecine is known to increase in photoinduced leaves of Sinapis alba. Difluoromethylornitine, an inhibitor of putrescine biosynthesis inhibits flowering, and this inhibiting effect was nullified if putrescine is applied simultaneously. However, polyamines alone do not influence flowering.
　　Benzoic acid, nicotinic acid, nicotinamide and L-pipecolic acid have been isolated and identified as flower-inducing substances from the acetone extract of fronds of Lemna gibba. Differences in their levels between flowering and vegetative plants could not be detected, and thier role as an endogenous regulator of flowering has not been proven.
　　Asparagus grows vegetatively for 2 to 3 years after the seed germination prior to flowering. The treatment of seeds with s-triazole compounds or carbamate compounds induced flowering in about 1 month after seed germination. These compounds are artificially-synthesized chemicals and therefore may not be involved in the natural regulation of flowering.

　　Attempts to detect florigen have not been successful, mainly because of the lack of a suitable bioassay system. To establish a bioassay system for detecting florigen we need samples with apparent florigen activity, but again we cannot obtain such active samples because a suitable bioassay system is not available. This is a vicious circle.
　　Generally, the sample has to be incorporated efficiently by the assay plant. The sample is applied to the surface of the assay plant in many bioassay systems. The plant tissue surface has a function to prevent the intrusion of invading substances. Therefore, macromolecules such as proteins and nucleic acids may not be incorporated into the plant tissue by this application method, although known plant hormones and small active compounds can be incorporated. The chemical nature of florigen is unknown, and the possibility that it is a macromolecule can not be excluded. 　 Furthermore, florigen is believed to move only in living tissue. Therefore, it is said that the sample may not be incorporated from the cut-end of a shoot even in an in vitro assay using a shoot apex. Considering these problems, the so-called perfusion system may be a theoretically better assay method. In this system, the aqueous sample solution is forced directly into the apoplast of the assay plant from the cut-end of stem using compressed air.
　　Another problem is that the failure to detect flowering activity by the bioassay does not always mean that the assayed substance does not have the activity. The assay plants would not detect any activity, if the assay sample was deactivated by the assay plants, or when the assay plants did not have the sensitivity to the substance given. Many problems remain to be solved before the bioassay system for detecting florigen can be established.

　　One can search for the substances generated specifically in the photoinduced plants by physicochemical methods. The known substances such as plant hormones can be easily detected using already-established methods. However, the methods to detect florigen can not be determined logically because its physicochemical nature is totally unknown.
　　Pharbitis nil can be induced to flower even under long-day conditions when exposed to stress such as poor nutrition, low temperature or high fluence light. The endogenous level of phenylpropanoids has been found to correlate with the flowering response. Analysis for phenylpropanoids revealed a few specific peaks in the high-performance liquid chromatography profiles of the extract from flowering P. nil cotyledons. These peaks were identified as chlorogenic acid and some other phenylpropanoids. A positive correlation has been found between the flowering response and the endogenous level of chlorogenic acid. The role of chlorogenic acid in stress-induced flowering and that in photoperiodic flowering are still unknown.

　　The attempt to clone cDNAs of the genes which are specifically expressed under a flower-inducing condition is quite natural. The genes which may be involved in the production of florigen could be found among those cloned genes. However, the number of papers reporting the isolation of such genes is surprisingly small. It is not clear either whether the products of those reported genes are actually involved in the flowering.
　　Molecular genetics of flowering is being actively studied in Arabidopsis thaliana. Many mutants related to flowering have been isolated, and the responsive genes isolated. Analyses of these genes have revealed the interactions between genes in the cascade resulting in flowering. However, those papers reporting the flowering genes do not refer to florigen. The molecular genetical approaches in A. thaliana do not seem to be contributing to the study on florigen.