Chemicals in Plant Breeding

Too often, when beginners think of chemical treatments to use in plant breeding, they immediately mention colchicine and various chemical mutagens. These substances have their places, but there are others that are also of value. First, some information on colchicine.

According to the IPCS Inchem entry on Colchicum autumnale:

All parts of the plant contain toxins. The greatest concentration of toxins is found in the seeds and the bulb (corm) (Cooper & Johnson, 1984; Frohne & Pfänder, 1983).

Colchicine is present in the flowers (0.1 to 0.8% in fresh flowers; up to 1.8% in dried flowers), in the seeds (0.2 to 0.8%) in the bulb (corm) (0.4 to 0.6%). The leaves contain very low amounts of colchicine (Gessner & Orzechowski, 1972).

And in Toxicity of Houseplants by David G Spoerke Jr and Susan C Smolinske we learn that, " little as 20 grams of G. superba tubers provide 60 milligrams." That's Gloriosa superba, the beautiful Gloriosa lily.

Most blue and purple flowers achieve their colors by combining red pigments (mostly anthocyanins) with co-pigments that usually both intensify the color and shift it towards the blue end of the spectrum. These co-pigments are often colorless or pale yellow — perphaps so pale that they are not easily seen. However, they can be exposed simply by "fuming" the flowers with ammonia. That is, the flowers are suspended in a container over a small quantity of ammonia. The vapors are enough to change the color of anthocyanins and flavones in definite ways that help us identify which anthocyanins are present, and whether a white flower contains flavones.

W. J. C. Lawrence, J. Genetics 21: (1929)
(c) Reactions of anthocyanins and flavones with ammonia and SO2
As indicated on p. 128 the change of colour occurring when white, ivory or yellow petals are fumed with ammonia is as follows:

Petal colour Changes to
White No change
Ivory Lemon yellow
Yellow Intense orange

The ammonia test is a well-known method used for detecting the presence of anthocyanin in plant tissues. If anthocyanin is present a green or bluish colour usually develops upon fuming, though possibly other substances in the cell sap may modify this green or bluish appearance. In addition to the many fumings made with ammonia in the course of these experiments, petals have been bleached with SO2, and the reactions of different flower colours with these two reagents noted.

The observations may be briefly summarised as follows:

SO2. SO2 does not affect the flavones. It half bleaches the deeper coloured petals and almost entirely bleaches tinged varieties. Penetration is increased if the petal is first fumed in ammonia and then bleached—complete removal of the anthocyanin pigments resulting, thus revealing the flavone ground.

Ammonia (a) Magenta and purple flowers give green and bluish-green reactions. (b) Orange and scarlet give an intense reddish-brown coloration. (c) Intermediate forms give intermediate reactions.

The intense reddish-brown which develops when orange or scarlet petals are fumed is of some interest, since this is not typical reaction of anthocyanin with ammonia.

All the other species I have grown conform, after their kind, to the above results.

W. J. C. Lawrence, Genetics & Cytology of Dahlia Variabilis, J Genet 24:257-301 (1931)

In mosaic forms the distribution of the anthocyanin is discontinuous in both stems and ray florets. It occurs in flecks and streaks and sometimes in larger areas (Plate IX, figs. 12 and 13). The flavone ground of the rays is never mosaic. The size of the coloured area may vary from one cell to the whole of the capitulum. Both the pale and deep pigments may be mosaic, but the pale anthocyanin is never mosaic unless mosaic deep anthocyanin is present. This is demonstrated particularly in families segregating forms with pale anthocyanin only, mosaic forms with deep anthocyanin only, and mosaic forms with both pale and deep. In this last class both pigments are indiscriminately mosaic; in the mosaic individuals with deep anthocyanin only, absence of the deep pigment reveals the clear yellow or ivory ground colour; but all the pale individuals are self-coloured, and crossed between themselves and with normal-coloured forms never give mosaics.


Petals are first macerated in a dilute aqueous solution of HCl. In this acid solution pure pelargonin gives an orange-red, a pure cyanin a cherry-red color. On the addition of a few cc. of saturated aqueous solution of sodium carbonate the following reactions are found: pure pelargonin gives a red-violet, pure cyanin a a clear green-blue. In Dahlia pelargonin with yellow flavone gives a red-brown, and pelargonin plus cyanin gives shades of red-brown-violet to violet-blue according to the proportion.

Onslow: The Anthocyanin Pigments of Plants p. 282 (2014)
Gawalowsky, A., 'Künstliche Blatt- und Blütenfärbungen,' Wiener landw. Zeit., 1911, LXI, p. 616.
Author finds that treatment of soil with sodium orthophosphate gives a deep red or blue-violet colour to petals of Rosa centifolia; also to Malva tinctoria a deep brown-red. Potassium carbonate causes development of anthocyanin in Lactuca sativa.

Alcohol is another useful tool where it is necessary to identify traces of lycopene masked by deep yellow carotenes.

Dr. Peter Werckmeister. Iris colors and pigments. Bull. Amer. Iris Soc. no. 158: 25-33 (1960)

Now I have found that it is easy to extract the yellow pigment with alcohol, but lycopin is insoluble in alcohol and is not removed. If then orange-colored beards are immersed in alcohol the yellow color is removed and they turn pink if they contain lycopin; if no lycopin is present they become white. Also, plants heterozygous for the tangerine gene, which appear among F2 progenies are of T1t3 genetic constitution, can be detected by this test. Thus such tests are important for the breeder or geneticist because in some irises with bright orange beards the color is due entirely to orange colored carotenoids.

Cells possess a mechanism that detects levels of DNA. If the mechanism finds too much DNA, it can induce the cell to divide without duplicating the chromosomes. This can occur following colchicine treatment, leading to the production of "sports" that have the original chromosome number but are not identical to the original plant. E.g., suppose we have a diploid plant with semi-double pink flowers, raised from a cross between a double white and a single red. Colchicine treatment doubles the chromosomes, leading to a tetraploid with semi-double pink flowers. As a cell doubles its chromosomes in preparation for mitosis, the DNA-detecting mechanism may kick in and force an extra cell division. This results in four diploid daughter cells instead of the expected two tetraploids. The diploid cell-lines may express various recombinations: single white, semi-double red, double pink, semi-double white, etc.

The DNA-detecting mechanism apparently cannot distinguish caffeine, uracil or sodium-nucleate from DNA. Thus, cells cultured in a nutrient solution containing, or a plant injected with, a solution containing one of these substances may be induced to produce cell lines with half the normal number of chromosomes.

Induced meiosis might be useful for transferring traits between diploid species with incompatible chromosomes. For example, some of the dwarf bearded Irises have a patch on the falls known as the Pumila spot. This trait is lacking in the tall bearded species. And when DBs and TBs are crossed, the Pumila spot tends to be recessive. DBs and TBs have different chromosome numbers, so a direct transfer of the spot in diploid crosses is unlikely to occur.

However, among several hundred seedlings of a tetraploid intermediate bearded Irises (with 8 pairs of DB chromosomes and 12 pairs of TB), a spotted tetraploid may be found. Presumably, a gene or chromosome segment associated with the Pumila spot has been transferred to a TB chromosome. If we could induce somatic meiosis in such a plant, it might be possible to isolate a diploid TB displaying the Pumila spot.

Furthermore, it has long been known that crossover frequency in a pair of chromosomes declines as the cell-size increases. More importantly, as the crossover frequency changes, so do the most likely positions of the crossovers. Thus, some linkage groups that are almost never broken at the diploid level, may be disrupted when the chromosomes find themselves in the larger cell of a tetraploid.

E.g., Sinningia concinna and S. pusila are tiny diploid species that can cross breed. Continued breeding from the hybrids could blend the characteristics of the species in a variety of ways, but not all recombinations are equally probable.

Both species have been crossed with the larger Sinningia eumorpha, and the weakly fertile diploid hybrids have yielded fully fertile tetraploids. If we cross the two tetraploids, we will still have concinna chromosomes pairing with pusila chromosomes. However, we can expect somewhat different recombinations. If we then induce somatic meiosis, some shoots that emerge may revert (more or less) to concinna-pusila diploids that could be crossed with the other diploid line derived from concinna-pusila hybrids.

Peña, et al. Bimeiosis induced by caffeine. Chromosoma 83(2): 241-248. (1981)
A 0.1% caffeine solution has been injected into plants of rye at various stages of spike development. Cytokinesis was inhibited in the germ line, and the resulting binucleate cells underwent bimeiosis. Nuclear fusions occurred during cell divisions of the germ line, giving rise to mononucleate tetraploid PMCs which showed 14 bivalents instead of the expected up to 7 quadrivalents. A decrease in chiasma frequency was also noted.

Amino Shin-ichi; Nagata Toshiyuki: J. Plant Res. 109: 219-222. (1996)
Caffeine induced a mitosis-like state in cultured tobacco (Nicotiana tabacum L) BY-2 cells after DNA synthesis had been arrested by aphidicolin. Cells were synchronized upon removal of aphidicolin. When aphidicolin was readded, the cell cycle was again interrupted and caffeine, when added with aphidicolin, induced the mitosis-like state in 5-10% of cells.

Schlegel R, Pardee AB. Caffeine-induced uncoupling of mitosis from the completion of DNA replication in mammalian cells. Science. 1986 Jun 6;232(4755):1264-6.
Caffeine was shown to induce mitotic events in mammalian cells before DNA replication (S phase) was completed. Synchronized BHK cells that were arrested in early S phase underwent premature chromosome condensation, nuclear envelope breakdown, morphological "rounding up," and mitosis-specific phosphoprotein synthesis when they were exposed to caffeine. These mitotic responses occurred only after the cells had entered S phase and only while DNA synthesis was inhibited by more than 70 percent. Inhibitors of protein synthesis blocked these caffeine-induced events, while inhibitors of RNA synthesis had little effect. These results suggest that caffeine induces the translation or stabilizes the protein product (or products) of mitosis-related RNA that accumulates in S-phase cells when DNA replication is suppressed. The ability to chemically manipulate the onset of mitosis should be useful for studying the regulation of this event in mammalian cells.

Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 452 (1) (2000) pp. 67-72
Inducing somatic meiosis-like reduction at high frequency by caffeine in root-tip cells of Vicia faba
Yihua Chen, Lihua Zhang, Yihua Zhou, Yuxuan Geng and Zhenghua Chen

Abstract: Germinated seeds of Vicia faba were treated in caffeine solutions of different concentration for different durations to establish the inducing system of somatic meiosis-like reduction. The highest frequency of somatic meiosis-like reduction could reach up to 54.0% by treating the root tips in 70 mmol/l caffeine solution for 2 h and restoring for 24 h. Two types of somatic meiosis-like reduction were observed. One was reductional grouping, in which the chromosomes in a cell usually separated into two groups, and the role of spindle fibers did not show. The other type was somatic meiosis, which was analogous to meiosis presenting in gametogenesis, and chromosome paring and chiasmata were visualized.

Acta Hort. (ISHS) 300:377-380
Cytological characterization of cell suspension cultures of fruit trees
Blando, F., Giorgetti, L., Tonelli, M.G. and Nuti Ronchi, V. 1992.

Abstract: Using a new suitable method to initiate a cell suspension culture in apple (Malus x domestica Borkh.) and quince (Cydonia oblonga Mill.), it has been possible to detect some cytological mechanisms of chromosome reduction, firstly reported in carrot cell cultures (Nuti Ronchi, 1990; Nuti Ronchi et al., 1990). These segregational events, namely somatic-meiosis and prophase-reduction, are present in all analysed cultures, haploid metaphases (n=17) being 3.2% in apple lines. The importance and role of these phenomena in cell cultures of different non-embryogenic species are discussed in comparison with the carrot cell culture model.

J. Hered. 39: 311-325 (1948)
Segregation and Reduction in Somatic Tissues
C. Leonard Huskins

By growing bulbs of Allium cepa in an aqueous solution of 1-4% sodium nucleate, chromosome segregation and/or reduction of the chromosome number has been induced in root-tip cells. In treatments of freshly harvested bulbs made November, 1947, with the Schwarz sample S.N. 4509 both pairing and segregation of metaphase-like chromosomes was obtained. A second division which separates sister chromatids and resembles that of a normal gonocyte meiosis occurred in a number of cells. More commonly the two divisions overlap and separation of chromatids occurs during the equivalent of gonocyte meiotic anaphase I.

Sugars and mineral salts have been found to induce mutants.

MacDougal, et al. Mutations, variations, and relationships among the Oenotheras (1907)
Some decisive results were also obtained from Raimannia odorata, a member of a separate genus of the evening-primrose family from Patagonia. During the first season (1905) injections of the ovaries were made with several substances, with the result that an atypic form identical in all cases was found in seeds from ovules that had been treated in various ways. Two such mutants were secured from seeds of an ovary that had been treated with a 10 per cent sugar solution, 10 from one that had been injected with a solution of calcium nitrate 1 part to 1000 of distilled water, and one was also found in the progeny from seeds taken from a capsule which had been exposed to the action of a radium pencil.

Individuals grown from seeds taken from one capsule. The large rosette is of the parental type, the smaller are derivatives, two of which have come into bloom at the same age and are scarcely so high as the normal rosette.

Singleton: Vegetable oils as mutagens (1962)
The amazing discovery was that the mutation rate induced in T. aestivum by peanut oil was higher than that produced by X rays, fast neutrons, P32 and S35, nitrogen mustard, and the other vegetable oils used. The mutation frequency was 156% per plant progeny. Some of the mutant characters induced are shown in Fig. 17-17. Castor oil produced 61% and mustard oil 4% per plant progeny.