Wide Hybridization in Plants pp.39-47 (1960; English Trans. 1962)

D. D. Brezhnev
First Vice-President of the All-Union Academy of
Agricultural Sciences im. V. I. Lenin

Academician N. V. Tsitsin has dealt exhaustively with the theoretical basis and history of wide hybridization in his report, and this eases our task considerably. We would, nevertheless, like to mention some work having a general theoretical importance, which also touches upon the history of the problem.

Wide hybridization dates from the second half of the eighteenth century, when Koelreuter obtained a hybrid between Nicotiana rustica and Nicotiana paniculata for the first time.

Academicians Pallas, Gaertner, and Naudin could be cited among the first workers who obtained data entirely in accordance with Koelreuter's.

*The source cited in the Russian original in Darwin, Proiskhozhdenie vidov, pp. 433-434.

Darwin, too, paid considerable attention in his investigations to problems of wide hybridization. He was the first to draw attention to causes of incompatibility, and in his famous work "The Origin of Species" he already stressed both morphological and physiological causes. On causes of noncrossability between species, Darwin wrote: "In the case of first crosses, the greater or lesser difficulty in effecting a union and in obtaining offspring apparently depends on several distinct causes. There must sometimes be a physical impossibility of the male element reaching the ovule, as would be the case with a plant having a pistil too long for the pollen-tubes to reach the ovarium. It has also been observed that when the pollen of one species is placed on the stigma of a distantly allied species, though the pollen-tubes protrude, they do not penetrate the stigmatic surface. Again, the male element may reach the female element but be incapable of causing an embryo to be developed .... Lastly an embryo may be developed, and then perish at an early period". Darwin, Ch. The Origin of Species*.

The interest in a practical utilization of interspecific and even intergeneric hybrids has grown with the development of science and the accumulation of new data. Interest in this problem was largely provoked by the discovery in nature of wild plant species possessing numerous valuable distinguishing characters (disease resistance, cold-hardiness, a high dry-matter content, etc).

At the end of the nineteenth and the beginning of the twentieth century, Luther Burbank began applying interspecific hybridization to fruit and other cultures.

During this period the scope of investigations in wide hybridization was greatly expanded, thanks, in particular, to the work of our compatriot, I. V. Michurin. He not only demonstrated the possibility of obtaining interspecific and intergeneric hybrids, but developed a whole series of new methods for overcoming incompatibility, and ultimately formulated a sound theory of wide hybridization in plants.

Already in his early articles he noted in wide hybridization "an infinite vista of possibilities for obtaining entirely new fruit species, the form and properties of which are yet unknown" (Michurin, I. V., Sochineniya (Works), 1:422. 1948).

Michurin severely criticized those botanists who maintained that it is impossible to obtain interspecific and intergeneric hybrids, he proposed concrete methods to ensure success in wide hybridization.

The most significant achievements of Michurin and a number of other investigators in wide hybridization were attained in fruit breeding. This advance was mainly because fruit cultures are generally propagated vegetatively, so that the modifications obtained are more readily preserved in the descendants. In other cultures, and particularly in annual grass and vegetable crops, there are numerous difficulties in the production of interspecific hybrids. But in this field also, scientists and practical growers have considered methods of combining certain traits inherent in particular species and genera.

Indeed, the desirable characters of different species and genera, not otherwise occurring together within a single species, can be combined only by wide hybridization. Consider the sensation a tomato-Physalis cross would cause! The fruit being enclosed within a bladder-like calyx, as in the ground cherry, is the one character which would solve the problems of tomato storage and transport. Again, fruits with a high dry-matter and vitamin content might be obtained by bringing together certain characters of peppers and tomatoes. Further, the cold-hardiness of some species might be successfully combined with the superior taste qualities of others, by crossing melon with squash.

In leguminous plants, a combination of the stem characters of beans and peas would facilitate mechanized harvesting of these crops. In addition to being economically valuable, such forms would be scientifically interesting. I. V. Michurin himself said that wide hybridization opens a wide field of activity to original breeding work, as hybrid forms with extremely varied characters are highly adaptable to the external environment of a new locality. The prospect of creating such interesting and useful forms attracts scientifically-minded vegetable breeders, and much attention has been paid to this problem for a considerable time.

For convenience, we will briefly survey wide hybridization achievements, according to groups of crops, omitting the older material.

Interspecific and intergeneric tomato hybrids have been thoroughly investigated in Russia and elsewhere.

The studies of the following workers are noteworthy: O. I. Asfarova-Ryabova (1938), V.A. Borkovskaya (1938), N. V. Tsitsin and M. Z. Nazarova (1953), E. V. Ivanovskaya (1954), K. V. Ivanova (1954, 1957), F. D. Kryzhanovskii (1955), N. A. Solov'eva (1956) and T. B. Batygina (1957).

The following investigations by foreign scientists are noteworthy: G. W. Skirm (1942), P. G. Smith (1944), H. B. Walker (1945), W. A. Frazier (1947), R. E. Lincoln and G. B. Cummins (1949), M. Lesly and J. W. Lesly (1952), McGuire and K. M. Rick (1954), and other workers, who studied wide hybridization in the genus Lycopersicon Tourn.

The production of hybrids with economically valuable properties by crossing L. esculentum with such tomato species as L. peruvianum or L. hirsutum is an important problem. The species peruvianum and hirsutum possess very valuable properties. Both species are resistant to a number of widespread diseases: tobacco mosaic, leaf mold (Cladosporium fulvum), septoria leaf spot, and others. Also, L. hirsutum is valuable for its frost hardiness (it can withstand frosts to -2°C). Its fruit contains three to four times as much carotene as cultivated tomato fruit. Hence it is valuable breeding material. However it is not easy to obtain hybrids between these species, as they intercross with the greatest difficulty. The production of varieties that possess all the characters listed is particularly difficult.

*State Breeding Station in the Kamennaya Steppe.

In 1933, O. I. Asfarova-Ryabova crossed L. esculentum and L. peruvianum at the Kamennostepnaya Station*, both by using a pollen mixture which was applied repeatedly to the same flower, and also by crossing with polyploid forms. She used these methods to overcome incompatibility, and only succeeded in obtaining one hybrid plant, which flowered abundantly but was almost completely sterile.

Lesly, in 1953, observed that only parthenocarpic fruits with empty nongerminating seed are obtained by crossing the cultivated tomato with L. peruvianum.

We are still studying this question with K. V. Ivanova and T. B. Batygina at the All-Union Institute of Plant Cultivation.

In our study, we first had to work out methods of overcoming the incompatibility between L. esculentum, L. peruvianum and L. hirsutum. We used several known methods, such as single, double and triple pollination, pollination with small anther columns, and pollination by mixed pollen.

After three years we concluded that backcrossing and pollination by mixed pollen are the most effective.

The interagent method (used by K. V. Ivanova) is another possibility. The maternal plant of L. esculentum was grafted, in its cotyledon stage, onto more developed stocks of L. peruvianum (stage of five to six real leaves); later, L. peruvianum pollen was placed on the stigmas of emasculated L. esculentum flowers for three to four days.

*Presumably L. esculentum

Using all these methods, we obtained sound seed which yielded good seedlings when sown. The seedlings of hybrid fruit were already distinguished by a large variability in shape, size and leaf characters. Moreover, the germination of the seeds was uneven. Later, this same seed was sown near Leningrad and in the Maikop Experimental Station (Krasnodar Territory). In the first and second generations a considerable segregation of characters was observed. Plants of an intermediate type as well as a series of completely new forms were obtained in addition to the two parent types. On the whole, plants of the seed-parent type* dominated. Thus, in 1953, of 606 hybrid plants sown near Leningrad 453 were of the seed-parent type, 148 of the pollen-parent type, and only five were intermediates.

Hybrid plants of the paternal type proved to be sterile in crosses with L. peruvianum. A high resistance to Macrosporium and Phytophthora was observed in hybrid plants of the seed-parent. The most interesting and valuable feature of these crosses is the appearance of plants of an intermediate type. Among such plants a variable range of morphological characters is observed. It appears that hybrid seed is more easily obtained from L. esculentum X L. hirsutum — with L. esculentum as maternal form — than from L. esculentum x L. peruvianum. From a breeding point of view, L. peruvianum is the more interesting partner.

These investigations show that the segregation of F1 and F2 hybrids depends on the conditions under which the F1 plants germinate. This is very important, as it indicates methods of directing this process.

Similar work has been done by S. S. Voskanyan of the All-Union Institute of Plant Cultivation. She obtained a form of practical value, resistant to Septoria and Macrosporium. This form was selected from hybrids between the cultivated variety Shtambovyi karlik [Dwarf-stemmed] and L. hirsutum.

Workers in other countries have also obtained interesting hybrids in recent years. W. S. Porte and H. B. Wacker (U.S.A., 1945) obtained hybrids between L. esculentum and L. peruvianum by backcrossing.

Like many of their predecessors, these authors failed to obtain seed in a whole series of combinations. Viable seed was eventually obtained by crossing with the Prince Borghese variety. There were forms resistant to Cladosporium and root eelworm among the F2 plants. In 1953 variety Waltham Mold-proof Forcing was obtained at the Massachusetts Experimental Station.

A number of workers used L. peruvianum for obtaining eelworm-resistant forms: F. A. Romsac (1942), D. E. Ellis (1949), V. M. Watts (U.S.A., 1947); W. A. Frazier and R. K. Dennet (U.S.A., 1949). V. M. Watts obtained resistant forms which were fertile and had small orange-red fruit.

A. F. Jaeger and H. J. Purinton (1946) at the Hampshire Experimental Station also obtained fertile forms with a high ascorbic acid content from crosses between L. esculentum and L. peruvianum. The largest fruits were selected, and their seed sown. The plants obtained were backcrossed to L. esculentum.

The fruit of the F3 hybrid has a 46 to 67 mg% ascorbic acid content (against 19 mg% in the standard). The result of these crosses was the New Hampshire Victor variety, with an ascorbic acid content of 39 mg%.

Cases of crossing of L. esculentum with L. hirsutum are also known from the literature.

R. E. Lincoln and G. B. Cummins (U.S.A., 1949) crossed L. esculentum with L. hirsutum to obtain Septoria-resistant varieties. They obtained six hybrid generations, but resistance to septoria leaf spot in the obtained forms was less than that of L. hirsutum.

J. W. Lesly (U.S.A., 1948) obtained hybrids that (like L. hirsutum) for several years were hardly ever infected with mosaic.

The practical results of wide hybridization work in tomatoes must be stressed. In 1950, six hybrid varieties were obtained at the experimental station of the University of Honolulu in Hawaii by including L. peruvianum in the crosses. These varieties (Hawaii, Kawai, Lanai, Mani, Molonai, Niihau) were resistant to fusarium and a number of other diseases.

In 1954, the variety Valabond V-508, resistant to nine races of Cladosporium, was obtained at the experimental station of Ontario (Canada) from crosses with L. hirsutum. A number of other disease-resistant varieties which were obtained by wide hybridization are also known.

Inviable seed is often obtained on crossing species of the genus Lycopersicon Tourn. There are many careful studies of this phenomenon.

The work of G. Smith (1944) is noteworthy. He observed that the fruits setting from L. esculentum - L. peruvianum crosses had small elongated embryos. From a study of the embryo development he established that the endosperm begins dying off on the 30th to 40th day after pollination, and that later, the embryo itself dies off. Smith found a method of excising the embryo from the seed. These embryos were put into Petri dishes with a nutrient solution and placed in a thermostat at a temperature of 25° C. After two to three days, they were exposed to diffuse light — and, a week later, normal plants began developing from the embryos. Analogous investigations were carried out by G. W. Skirn (1942) and B. Choudhury (1955).

Much research has been done on the crossing of cultivated tomato with lines of the currant tomato (L. pimpinellifolium), which we consider a subspecies. Lines of this subspecies are distinguished by a high dry-matter content (as much as 8 to 10%), a high sugar content and a fair resistance to soil drought and certain diseases.

The utilization of these lines in breeding often gives good results. Daskalov (Bulgaria, 1957) succeeded in obtaining valuable forms from crosses with a line of the currant tomato (cited by him as L. racemigerum). The varieties which he obtained are superior in yield and in additional valuable qualities to other varieties grown in Bulgaria.

Many workers overcome incompatibility in intergeneric and interspecific vegetable crosses by bringing the partners close to each other vegetatively (graft hybrids).

N. V. Tsitsin, F. D. Kryzhanovskii, E. V. Ivanovskaya, and M. Z. Nazarova obtained a tomato - Cyphomandra hybrid. It had been found impossible to cross these genera previously. It was decided to produce hybrids vegetatively. In this vegetative hybridization, the scion was systematically deprived of its leaves and, as a result, a series of morphological and physiological modifications appeared. All these modifications were fully transmitted to the progeny. In the third generation derived from Cyphomandra stock grafted with the tomato variety Bizon, one plant of an intermediate type was obtained. Its seed was fully sound. From 123 seedlings produced, only one plant resembled Cyphomandra. Thus the first intergeneric hybrid was obtained.

It was also I. V. Michurin who began producing new, more highly resistant melon varieties by wide hybridization. Michurin obtained the new, early-maturing melon variety Kommunarka by crossing selected local and Far-Eastern early-maturing, resistant varieties with varieties of European origin. He also carried out crosses between cucumbers, melons and squashes with the preliminary use of graft hybrids, and also crosses with a wild squash — Thladianta sp. As a result, hybrids with fruits of varying shape, size and chemical composition were obtained.

F. Belik (1956) showed that certain melon varieties intercross with the utmost facility, but hybrids obtained from crosses between cultivated melons and wild ones must be further selected and trained. Table water melons cross well with fodder varieties, but are not easily crossed with wild species. These wild species are particularly valuable because of their immunity characters. The various squash species intercross with the utmost difficulty.

*Vavilov, N. I. Trudy po prikladnoi botanike i selektsii (Work, in Applied Botany and Breeding). — 14 (2), Leningrad. 1925.

N. I. Vavilov made crosses between melons, water melons, and squashes. He did not obtain positive results. He wrote: "The species of the Cucurbitaceae have drifted so far apart in their evolution that insurmountable obstacles prevent fertile hybrids between them..." (Works in Applied Botany and Breeding)*.

It is only possible, as N. I. Vavilov assumed, to obtain hybrids between Cucumis melo and Cucumis trigonus Roxb. (the cultivated and the wild melon species). This is not surprising, as a series of intermediate forms exists between the two.

N. I. Vavilov quotes several unsuccessful attempts to obtain wide hybrids in the Cucurbitaceae. In Becker's experiments with squash-cucumber crosses, one seedless fruit was obtained; in Naudin's crosses between various squash species no hybrid seed was obtained, and the extensive attempts of Locy to cross different species of squash led him to the conclusion that these species are mutually incompatible. But one must not conclude from these unsuccessful attempts that it is impossible to obtain such hybrids. It seems that new methods are required for hybridization here.

A. F. Makarovskii and M. 1. Podmogaev (1952), at the Biryuchekutsk Experimental Station obtained a series of hybrids with normal seed. The success in obtaining hybrids can be explained by the fact that, in all the combinations, morphologically balanced hybrids took part, (i.e., forms with a shattered heredity), and that pollination was done by pollen mixtures.

In recent years, extensive research on wide hybridization in the Cucurbitaceae has been clone by N. A. Khokhlacheva in the Krasnodar Vegetable and Potato Experimental Station. Crosses between Cucurbita maxima and Cucurbita moschata were effected. Khokhlacheva did not succeed in obtaining hybrids in the normal way for a long time. To overcome incompatibility, the preliminary use of graft hybrids was resorted to. The seed-parent form was used as the stock. After bringing partners close to each other vegetatively in this manner, sexual hybrids could be produced. However, the fertility of these hybrids was low in the F1 and F2 generations. Maximum segregation was observed only in the F3 generation, when economically valuable hybrid forms, high-yielding and with a high sugar content, were obtained.

N.A. Khokhlacheva also grafted cucumbers on squashes. In the south, where frequent dry winds reduce cucumber yields, varieties which are resistant to this are required. To obtain such varieties, grafting was resorted to. On grafting, the cucumber undergoes a series of modifications (particularly if grafted a second time): the inflorescence becomes racemose, flowering starts seven to ten days earlier, and yields increase.

It is desirable to extend melon cultivation northward.

S. P. Lebedeva began her studies in the grafting of melon on squash in 1925. She examined the various grafting methods carefully and selected the best stocks and scions. The most suitable scions appeared to be late and midseason melons from Uzbekistan. The Espeel' variety (S. P. L.) or Podmoskovnaya Lebedevoi, which succeeds well in the Moscow area, is a result of her work.

The success of interspecific and intergeneric crosses can be aided greatly by grafting.

O. V. Yurina obtained the relatively cold-resistant melon varieties Gruntovaya gribovskaya and Gribovskaya rassadnaya 13 by employing vegetative and sexual hybridization in conjunction with each other.

Further studies in wide hybridization between squashes and melons are: V. G. Smirnov and O. A. Smirnova (1939), O. Gashkova (1944). Foreign scientists also give due consideration to these questions in their investigations. Noteworthy among many others are the studies of G. P. Eseltine (1936); J. R. Wall (1954) in U.S.A.; and T. W. Whitaker (1951), who carried out crosses in the genus Cucurbita in an attempt to clarify the origin of C. maxima; J. Jamane (1953), Fakashima (1953) and H. Hayase (1954) in Japan; Batra (1953) in India; Grebenscicov (1955) in the German Democratic Republic; R. Schagen (1956) and F. Weiling (1956) in the Federal Republic of Germany.

Wide hybridization plays a very important part in the genus Allium. As early as 1935, work was begun on crossing the common onion (Allium cepa) with Welsh onion (Allium fistulosum) and Altai Onion (Allium altaicum).

In 1935, S. L. Emsweller and H. A. Jones (U.S.A.) crossed Allium cepa with Allium fistulosum. Seven seeds were obtained by hand pollination, and one plant was grown. Morphologically this plant was of an intermediate type; it developed vigorously, was almost completely sterile and resembled A. fistulosum in being perennial.

*Bulb formation and frost-hardiness are obviously meant.

In the U.S.S.R., A. A. Krivenko and G. V. Fedorov worked on the same crosses. Allium cepa is nonresistant to frost; A. fistulosum is frost-hardy but does not form bulbs. A combination of these two properties* would be of great practical importance. In 1934, G. V. Fedorov obtained 12 viable A. cepa X A. fistulosum hybrids. In 1935 he crossed A. cepa with A. altaicum. F1 hybrids between the common onion and the Welsh onion or the Altai onion are marked by heterosis. They have very delicate green tops and, as they can be propagated vegetatively, the author recommends the utilization of these F1 hybrids for onion tops.

A. A. Krivenko's work in wide hybridization of onions merits note. In crosses with common onion he used Welsh onion, Altai onion, chives, and leeks. He carried out reciprocal crosses between the common onion and these latter Allium species. He found that A. cepa is best used as seed-parent, especially the older varieties with balanced characters where there is less influence of the wild forms. Economically valuable forms can be obtained more easily using the older varieties. Accordingly, Krivenko stresses the importance of suppressing certain wild traits, and recommends crossing the common onion with wild species in their first year of flowering, and to backcross the F1 hybrids to the common onion. Stable forms can be selected from the ensuing progeny.

The F1 Allium cepa X fistulosum hybrid Troitskii mnogognezdnyi [Multilobate], produced by A. A. Krivenko, has a number of desirable characters: it is perennial, frost-resistant, high-yielding and of pleasant taste. It was approved for market cultivation in 1940.

The achievements of wide hybridization in legumes are also notable. Nonlodging pea forms suitable for mechanized harvesting, immune varieties, extra early-maturing ones, etc, can be produced. K. Tjebes (Sweden, 1927), M. M. Sasonkina (1935), Sousa Bourdouil (1938), V. V. Novikov (1939), M. Shel'khorn (1940), Lamprecht (Sweden, 1941, 1952), P.I. Shul'ga (1952), S. Honma (U.S.A., 1955) and Buishand (Netherlands, 1956) have worked on this subject.

Wide hybridization in legumes has been studied in detail from 1936 to 1940 by V. V. Novikov. He pollinated Vicia faba with a mixture of pollen of other Vicia species when direct crosses of peas with the broad bean did not succeed. In 1937, crosses between broad bean, winter vetch (Vicia villosa) and common vetch (V. sativa) led to the production of intermediate forms, which were further utilized for crossing with peas (with pure lines or with the hybrid Alaska X Capital). From these crosses three valuable forms were obtained: 1) a nonlodging, high-yielding pea-bean; 2) an immune form of pea-bean; 3) an extra early-ripening form of pea-bean, which completes its vegetative growth within 54 days.

M. M. Sasonkina made use of preliminary grafting for producing hybrids between Lupinus polyphyllus and the garden pea (Pisum sativum), Lupinus polyphyllus and the field pea (P. arvense), Lupinus angustifolium and the garden pea, and many others. Field pea X Lupinus polyphyllus and field pea X L. angustifolium hybrids were obtained from crosses made after double grafting. Nonlodging pea forms and alkaloid-free lupine forms might have resulted from further investigations in this direction.

The work of A. M. Drozd (at the Crimean Experimental Breeding Station) in wide hybridization involving kidney bean is also of great interest.

A. P. Lorts (U.S.A., 1952) studied hybrids between lima bean and the wild species Phaseolus polystachyus. Crosses were made with the object of introducing into the lima bean such valuable traits as underground germination and resistance to various diseases. After numerous attempts, seven hybrid plants were obtained, in all of which the cotyledons remained underground during germination.

Much work has been done on intercrossing species and genera of the Cruciferae in the U. S. S. R., as in other countries. G. D. Karpechenko (1937 to 1938) intercrossed Brassica oleracea, Br. chinensis and Br. carinata, and obtained fertile hybrids.

A detailed study of F1 hybrids between cabbage and radish is being made in Japan by Fukushima; the first Brassica oleracea X Raphanus hybrids were obtained in the U.S.S.R. by G. D. Karpechenko.

In Hungary, A. Kiss (1956) experimented with hybridization between rape (Brassica napus) and turnip (Brassica rapa).

An intermediate (rape x cabbage) hybrid fodder plant called "rapko" was obtained in Germany in 1936.

The work of R. A. Calder (New Zealand, 1937), on pollination in the genus Brassica, should be noted. He showed that rape (Brassica napus L.), turnips (Brassica rapa L.), and swedes (Br. napobrassica Mill.) can intercross under natural conditions, but that none of these species intercrosses naturally with Brassica oleracea. This must be taken into account in work with these particular species, as under natural open cross-pollination the seed is apt to become contaminated. Plants for seed-growing have to be rigorously isolated. Work on interspecific Brassica crosses is being done in China (Tsai I-sin', 1955). All these studies are accompanied by extensive cytological investigations.

*Institut maslichnykh i efiromaslichnykh kultur.

G. S. Voskresenskaya has sent a communication about mustard, rape, and common winter cress (Barbarea vulgaris) cabbage hybrids, obtained in the Institute of Oil and Essential Oil Crops*.

Note should be made of work being done on the interspecific hybridization of red peppers by Swami Rao R., M. H. Narasimaha Rao, and T. V. Subramanian (India, 1942), and on interspecific crosses involving eggplants by T. Tatebe (Japan, 1941). R. C. Thompson (U.S.A., 1943) worked on interspecific hybridization in the genus Lactuca with the purpose of elucidating genetic relationships between species.

This brief and incomplete survey shows that work on wide hybridization in vegetable cultures is being done by many scientists in the U. S. S. H., and in other countries. One can now list dozens of vegetable crop varieties obtained in this way. It is true that work on the wide hybridization of vegetable cultures is accompanied by considerable difficulties. In fruit, the modifications obtained are easily preserved in the clonal, vegetatively propagated descendants; vegetables, on the other hand, are almost invariably propagated by seed. Yet these difficulties are not insurmountable.

In work on wide hybridization, obstinacy and, above all, patience are important, This was stressed by I. V. Michurin. All work should be carried through to the end and not be left half-finished, as has often happened.

Our investigations on wide hybridization are one-sided. The crosses are made, but the resulting hybrids are rarely trained. The dominance of characters can be modified and guided in the desired direction by clever training. Almost no cytological and embryological studies of interspecific and intergeneric hybrids have been made in recent years, although these most essential investigations could be of great help in discovering reasons for incompatibility, sterility, and so on.

Success of crosses often depends on the conditions under which the parental forms were grown, but there are almost no investigations of this.

The method of raising embryo cultures of interspecific and intergeneric hybrids on an artificial nutrient medium is not utilized at all. In many cases hybrids between wide forms could be obtained much more easily by this method. More consideration should be given to the possible use of radiation of both hybrid seed and parental forms, to increase the viability of intergeneric and interspecific hybrids.

Future studies on vegetable cultures should take note of all these shortcomings. More publicity should be given to current investigations on wide hybridization. Research projects should be developed on a larger scale, and be carried out more competently, in conjunction with cytologists, embryologists, biochemists and physiologists, and with full consideration for the achievements of modern science.