Interspecific hybridization in cucumis

Interspecific Hybridization in C u c u m i s 1

J O H N R. DEAKIN, G. W. BOHN AND THOMAS W. W H I T A K E R

Introduction

Among the genera of the Cucurbitaceae (Gourd Family) used for human food, the genus Cucumis contains two species of great economic importance. Cucumis melo L., the muskmelons, and C. sativus L., the cucumbers, are widely grown for their edible fruits or seeds in nearly all of the tropical, semi- tropical, and temperate regions of the world. A few other species such as C. anguria (the gherkins) and C. metuliferus (the African Horned Cucumber) are grown on a smaller scale for food or ornament (38).

Cucumis melo is extremely variable. Some forms produce round fruits ranging in size from 1 inch in diameter in feral forms to several inches in diameter and weighing up to 20 pounds in cultivated varieties (cantaloup, Honeydew, Crenshaw, Persian, and Golden Beauty Casaba). Other forms are moderately to extremely elongated, reaching lengths of 2 to 3 feet in some cultivars such as Banana melon and Chinese cucumber. The species is equally variable in other vegetative and fruit characters so that a wide range of variability is available for exploitation by plant breeders. Valuable genes within the species include those con- ferring resistance to several diseases such as powdery mildew (4), downy mildew (3), Alternaria blight (3), gummy stem blight (32), watermelon mosaic virus 1 (35, 37), and cucumber mosaic virus (4, 35) and resistance to at least one insect (12, 13).

Despite great variability in Cucumis melo and diligent search among its multitude of cultivated, semi-feral, and feral forms from all parts of the world, no plants have been found with sharply defined, potent resistance to crown blight, watermelon mosaic virus 2, Verticillium wilt, several nematodes, and several insects. Screening studies for nematode resistance by Winstead & Sasser (39), Fassuliotis & Rau (10), and Fassulio-

1 Geneticists, Plant Science Research Division, Agricultural Research Service, Charleston, South Carolina, and La Jolla, California. Submitted for publication February 24, 1971.

tis (9), and for resistance to several diseases by Leppik (18, 19, 20), Corley (7), Sowell et al. (32), and Leppik et al. (21) suggest that resistance to still other destructive pests may be found within the genus in species uther than C. melo.

An exchange of genes between the cultivated and feral or semi-feral species of Cucumis would open a vast potential re- source of variability for exploitation by breeders attempting to improve muskmelons and cucumbers. The exploratory studies reported here failed to achieve that objec- tive, but some successful species crosses were achieved. These studies extend our knowledge of this large but little-known genus and will aid the work of other inves- tigators in achieving crosses between C. melo or C. sativus with the noncultivated species.

Literature

A major deterrent to investigations of species crosses in Cucumis has been the absence of an accurate monographie treatment of the entire genus. The only com- prehensive monograph was published by Cogniaux (6) and was based entirely on herbarium specimens. Cogniaux described many species that were separated from one another by minute differences poorly defined in keys. Systematic studies by taxonomists have reduced many of Cogniaux' species to synonymy and have removed a few species to other genera.

Since our own studies were initiated, parts of the genus have been studied critically by Meeuse (23) and Jeffrey (14). Meeuse collected and studied plants in their natural habitats in southern Africa and in experi- mental plantings used for Enslin & Rehm's (8) work on bitter principles. Meeuse (loc. cit.) gives good descriptions of the species, their habitats, and ranges. Also there is a good key to 17 species of Cucumis found in southern Africa. Jeffrey (loc. cit.), an au- thority on the family Cueurbitaeeae, collected and studied plants in their natural habitats. His treatise of the Cueurbitaeeae

195

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TABLE I CUCUMIS SPECIES USED IN HYBRIDIZATION STUDIES

Chromo- some

Entry Species No. (2n) Source Origin

la C. sativus L. 14 lb C. hardwickii Royle 14 2 C. melo L. 24 3 C. humi#uctus Stent. 4a C. dinteri Cogn. 24 4b C. sagittatus Peyr. 24 5 C. metuli[erus Naud. 24 6a C. anguria L. 24 6]3 C. anguria var. longipes

( Hook. f. ) Meeuse 24 7 C. dipsaceus Spach. 24

8 C. a#icanus L.f. 24 9a C. leptodermis Schweik. 24 9b C. myriocarpus Naud. 24

10a C. prophetarum L. 24 10b C. zeyheri Sond. 24 l l a C. fici[olius A. Rich. 48 l lb C. heptadactylus Naud. 48

Breeding lines P. I. 183,967 Breeding lines ( Meeuse ) P. I. 282,444 ( Rehm ) P. I. 282,441 (Rehm) P. I. 202,681 (Rehm) P. I. 196,477 (Rehm) P. I. 282,442

United States Himalayan foothills, India United States South Africa Southwest Africa Southwest Africa Northern Transvaal Brazil Southern Rhodesia

P. I. 193,498 Ethiopia P. I. 282,440 (Rehm) Northern Cape Province,

Witfontein, Transvaal P. I. 282,447 (Rehm) Orange Free State P. I. 282,449 Pretoria P. I. 292,396 Israel P. I. 282,450 (Rehna) South Africa P. I. 196,844 Ethiopia P. I. 282, 446 (Rehm) Northern Cape Province

(in Flora of Tropical East Africa) contains a critical treatment and key to 13 species of Cucumis native to tropical eastern Africa. Rosette Fernandes & A. Fernandes" treatise (11) has keys and descriptions in Portuguese of seven species of Cucumis collected in Angola. These studies in combination give an excellent picture of the genus in areas where species of Cucumis are relatively abundant.

The first recorded attempt to cross CItcIt- mis melo with other species appears to have been made by Naudin (24). He was unsuccessful in obtaining a cross between C. melo and C. myriocarpus. Koslov (16), Vavilov (33), and Pangalo (25) apparently reported artificial and natural hybrids within C. melo. Whitaker & Davis (38) failed to secure hybrids from crosses among the three cultivated species, C. anguria, C. melo, and C. sativus. Batra (2) and Smith & Venkat Ram (30) were unable to cross C. melo with C. sativus at the tetraploid as well as the diploid level. In unreported studies performed at that time we also failed to secure species hybrids from diploid and

tetraploid clones of C. melo and C. sativus cross-pollinated in all combinations. Shan- mugasundaram et al. (28) reported a successful cross of C. me/o with C. pubescens. This cross appears to be an intraspecific rather than an interspecific cross.

Andrus & Fassuliotis (1) obtained 13 interspecific crosses including eight with Cucumis anguria, but pollinations with C. melo or C. sativus as a parent yielded no viable progeny. The present report confirms and extends that of Andrus & Fassuliotis (loc. cir.). Studies on morphology, physiology, and compatibility are combined to aid a phylogenetic interpretation of available species of Cucumis.

Methods

Standard pollination techniques for Cucu- m/s (38) were used for crosses in greenhouse and field.

In addition to the standard techniques, some special techniques were used in attempts to secure crosses of Cueumi8 melo with other species: (1) fruit-setting hor-

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Fro. 1. Cucumis sativus, C. hardwickii, their F~ hybrid, and the backcross to C. hardwickii have sparsely stiff-spined fruits. Size, shape, and color vary in different cultivars of C. satiw,s, and the spines may not persist.

mones were applied to pistillate flower buds at pollination; (2) chemical senescence in- hibitors were applied to pistillate flowers at pollination; (3) mixtures of pollen from several species were applied to stigmas of single flowers; (4) pistillate flowers were pollinated at anthesis and at 1 and 2 days before anthesis; (5) F1, F o, and three-way interspecific hybrids were used as parents in reciprocal crosses with C. melo; (6) tetraploid lines of C. melo and some other species were used in reciprocal crosses; and (7) embryo culture was attempted with a few minute embryos from several interspecific crosses. Several of the wild species were used as parents in those crosses, but in all cases, C. melo was used as one of the parents.

Putative hybrids were compared with

their parents and used as parents in controlled pollinations. Controlled pollinations were made to secure selfs, backcrosses to both parents, and outcrosses to other species. Pollen fertility was determined by effective- hess in pollinations and by counts of stained pollen in aeeto-carmine or laeto-phenol/ fuehsin smears.

Materials

Several species of Cucumis exist as parts of the native flora in Africa, India, and neighboring lands. Until recently seeds of most of these Old World species were not available to Western plant scientists. Seeds of some species are still unavailable to us.

We obtained seeds of 16 species of Cucu- m/s (Table I) through the United States Department of Agriculture, Agricultural Re-

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FIG. 2. Mature fruits of C. melo vary greatly in size, shape, color, aroma, and ornamentation. Soft hairs on the young ovaries may persist, or the mature fruits may be smooth, ribbed, or ornamented with corky net. Cultivars usually weigh in the range of 2 to 20 lbs., but wild types may weigh as little as 10 grams, as in (b), from India, and its F1 hybrid with cultivar Georgia 47 (a).

search Service, New Crops Research Branch, as Plant Introductions (P. I. numbers), and through the courtesy of A. D. J. Meeuse, formerly with the Division of Botany, Pre- toria, and S. Rehm, formerly at the Horti- cultural Research Station, Pretoria, Trans- vaal, South Africa.

Fruits of the 16 species are illustrated in 11 figures. Fernandes & Fernandes (11), Meeuse (23), Jeffrey (14), and others have given good technical descriptions of them.

1. Cultivars of the predominantly monoecious annual cucumber, Cucumis sativus, bear young fruits with scattered short, sharp, stiff spines that may persist. The mature fruits vary in size, shape, color, and surface markings. Many are large and elongate (Fig. 1 and Table I entry la) .

Fruits of the wild, monoecious, annual, bitter cucumber of India, C. hardwickii, also bear scattered, short, sharp, stiff, often eva- nescent spines. The small, ellipsoidal (5 X 4 cm) mature fruits are green with ivory or

Fic. 3. The white, subterranean fruits of C. humifructus are ornamented with net-like ridges. The oblate fruits average 35 mm long X 40 mm across.

yellow stripes (Fig. 1). Both species have seven pairs of chromosomes (Table I).

2. Cultivars of the annual muskmelon, C. melo, bear young fruits with abundant, long, soft, fragile hairs that may persist. The mature fruits vary greatly in size, shape, color, surface ornamentation, and other characters. Cultivars include cantaloup, honeydew, casaba, Chinese cucumber, and other types. Many are large and ellipsoidal (Fig. 2), but fruits of the Indian cultivar Kakri may measure 3 feet long X 3 inches wide. Most American cultivars are andromonoecious, but those of other lands usually are monoecious, and other sex forms occur.

Young fruits of monoecious, annual, wild C. melo from Africa and elsewhere also are ornamented with hairs. The small, usually ellipsoidal, mature fruits are often green with darker mottle or stripes, but bright yellow-orange and other colors occur. Original collections of wild and cultivated C. melo have 12 pairs of chromosomes (Table I), but 48-chromosome breeding stocks have been derived from natural and colchicine- induced mutants of muskmelons as well as squashes (2, 27 fig. 3).

3. Other wild species of Cucumis with hairy rather than spiny young fruits include the monoecious annual C. humifructus and the monoecious perennials C. dinteri and C. sagittatus. These species also have 12 pairs of chromosomes (Table I).

The pedicels of pistillate flowers of C. humifructus elongate after fertilization and push the small, pointed pistil into the ground

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FIG. 4. Ovaries of wild, African C. dinteri ( a ) and C. sagittatus ( b ), like those of C. melo, bear hairs that may persist on the brownish green or yellow mature fruits. The short, ellipsoidal fruits average 45 X 35 mm and 50 X 40 ram.

before the carpels start to enlarge. The ivory-white, oblate fruit, 5 to 9 cm in diameter, has a rough, check-marked, waxy surface (Fig. 3). Meeuse (22) has shown that this unique species is partly dependent for dissemination on the aardvark, which digs up and eats the geocarpic fruits.

4. The nearly globose (3.5 cm), green, stripe-mottled fruits of C. dinteri (Fig. 4a) (see Meeuse, 23) turn yellow when fully mature. The flowers in our cultures were pleasantly aromatic. The slightly larger (4 to 5 cm) fruits of C. sagittatus (in Fernandes & Fernandes, 11, = C. angolensis Hook. f. ex Cogn. in Meeuse, 23) tuna pale yellow more readily (Fig. 4b). The flowers in our cultures, like those of most Cucumis species, were not aromatic. Both species have 12 pairs of chromosomes.

5. The African "Horned Cucumber," C. metuliferus, is eaten in Africa but grown as an ornamental in the United States. Wild,

inedible, bitter forms occur (Meeuse, 23). The 12 X 7 em fruit is sparsely covered with large, very stout, stiff spines (Table I entry 5 and Fig. 5). The red-orange fruits have thin white flesh, green jelly-like pulp, and seeds covered with fine hairs. The monoecious, annual plants have 12 pairs of chromosomes.

6. Fruits of the sparsely short-spined, edible gherkin of the West Indies, C. anguria var. anguria (Fig. 6a), and the abundantly long-spined, bitter C. anguria var. longipes of Africa (Fig. 6b and see Meeuse, 23) are usually 5 to 6 cm long and 3 to 5 cm in diameter. Both monoecious annuals have 12 pairs of chromosomes.

7. Fruits of the "dipsaceus gourd," C. dipsaceus, measuring about 7 • 5 cm, are densely covered with long, thin, soft spines, with each spine ending in a hyaline bristle (Fig. 7). The pale green fruits turn uniform yellow at maturity. This species is native

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FIc. 5. The brownish ring-mottled, orange fruits of C. metuliferus, the African 'Horned Cucu her,' have unique, thick, fleshy spines. The long ellipsoidal fruits average 115 • 70 ram.

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to NE tropical Africa (Jeffrey, 14) but is cultivated for ornament and is sometimes adventive elsewhere. The monoccious, annual plants have 12 pairs of chromosomes.

8. The nearly cylindrical (8 X 4 cm), brownish, ivory flecked and striped fruits of C. africanus are well covered with stout, blunt, conical spines (Fig. 8). Edible forms, like those shown, and smaller, poisonous forms occur in southern Africa (Meeuse, 23). The monoecious, annual plants have 24 pairs of chromosomes.

9. The small, nearly spherical (20 X 19 mm), pale greenish yellow, faintly striped fruits of C. leptodermis are sparsely ornamented with short, soft spines (Fig. 9a). The sharply ivory and brownish green striped, ellipsoidal (25 X 22 mm) fruits of C. myriocarpus are densely spined on the

ribs (Fig. 9b). Both species are monoecious annuals with 24 chromosomes.

10. All surfaces of the 44 • 35 mm, uniformly pale yellowish green fruits of C. prophetarum are moderately well covered with short, curved, soft spines (Fig. 10a). Collections differ in number and length of spines, and some are faintly striped. The 52 • 37 mm, pale and dark green striped fruits of C. zeyheri are similarly spined on all surfaces (Fig. 10b). Both species are monoecious perennials with 24 chromosomes.

11. All surfaces of the 50 X 35 mm, indistinctly pale ,and dark green striped fruits of C. ficifolius are sparsely ornamented with very short, soft spines (Fig. l l a ) . The plants are monoecious. The thick-spined, brownish green, oval (53 X 45 mm), striped fruits of C. heptadactylus resemble those of

<.- FIG. 6. Fruits of the pale yellow, cultivated West Indian Gherkin, C. anguria var. anguria, and

the pale green, wild African C. anguria var. longipes differ greatly in spine ornamentation. The short ellipsoidal fruits average 60 • 45 mm and 45 K 38 nun.

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FIG. 7. The densely soft-spined, apple green fruits of C. dipsaceus, from NE tropical Africa, make it useful as an ornamental plant. The ellipsoidal fruits average 70 • 45 ram.

C. africanus and C. myriocarpus (Fig. 11b); the linear leaflets are unique and the plants are dioecious. Both species are perennial with 48 chromosomes.

Results

A. Primary crosses, selfs, and backcrosses

1. Cucumis sativus and C. hardwickii

Cucumis sativus and C. hardwickii, indig- enous to India, are diploids with seven pairs of chromosomes. They set fruits with full complements of plump seeds from reciprocal cross-pollinations. The hybrids were mostly fertile but with slight sterility similar to that found in wide crosses within C. melo. Those results support Rehm's statement 2 that morphological characters and cucurbitacin con- tent fail to differentiate the two entities as distinct species. Perhaps C. hardwickii bears a relationship to C. sativus similar to that of

" In personal correspondence to T. W. Whit- aker 3.9.68.

C. anguria var. longipes to C. anguria var. anguria.

Cucumis hardwickii and C. sativus failed to set fruits, or set fruits with seeds lacking embryos from cross-pollinations with the 24- and 48-chromosome species (Table II) .

2. Cucumis melo

All collections of wild C. melo and all cultivars of C. melo cross readily with one another. More cross-pollinations were attempted with C. melo than with other species. Fruits were set from several crosses with and without the aid of fruit-setting hormones. Interspecific pollinations, in all combinations, between diploid and tetraploid lines of Cucumis melo and C. sativus cultivars set some fruits, especially on C. sativus, without hormone aids. Such fruits contained ovules that were sometimes enlarged, but they did not contain embryos visible to the naked eye. The cross C. melo • C. hardwickii gave similar results.

Pollinations on Cucumis melo with pollen from C. dinteri, C. sagittatus (=C. ango-

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Fro. 8. The blunt-spined, nearly cylindrical, brownish green-mottled and ivory-striped fruits of C. a#icanus average 80 • 40 ram.

lensis), or C. humifructus initiated fruit development, but the fruits either failed to mature or they matured with partly developed seeds. Visible embryos were absent in the seeds, or they were small and failed to emerge from soil or sand plantings. Re- ciprocal crosses gave similar results.

Cross-pollinations between Cucumis melo and the remaining 10 species usually failed to set fruits without the aid of fruit-setting hormones. The fruits set with such aid contained nonenlarged ovules. There was

little indication that C. melo could be crossed directly in either direction with any of those species.

3. Cucumis humifructus

The unique subterranean fruits of Cucu- mis humi#uctus resembled those of C. melo more closely than did those of other species. The plants, also, were similar to those of C. melo. However, the pistillate flowers of C. humifructus are uniquely specialized to force the pistil into the soil after pollination. C. humi[ructus set fruits that failed to de- velop from cross-pollinations with pollen from C. melo, C. dinteri, and C. sagittatus (Table I I ) . It failed to set fruits from cross- pollinations with 12 other species.

4. Cucumis dinteri and C. sagittatus

Cucumis dinteri and C. sagittatus yielded fertile hybrids from reciprocal cross-pollinations with each other (Table II). This agrees with S. Rehm's work as reported by Meeuse (23, p. 65) and supports Fernandes & Fer- nandes' (11) opinion that the two entities are members of a single species.

Cucumis dinteri, used as a female, set fi'uits with seeds lacking embryos or with minute embryos that failed to emerge from sand plantings from crosses with C. melo and C. humifructus. It set fruits with nonenlarged ovules or failed to set fruits from cross-pollinations with 12 other species. Cucumis sagittatus set a single fruit that contained enlarged ovules lacking embryos from a flower pollinated by C. zeyheri. It set fruits with ovules lacking embryos from

Fie. 9. (a) The small, nearly spherical (21 • 20 ram), pale greenish yellow, striped fruits of C. leptodermis are sparsely ornamented with short, soft spines on the darker stripes. (b) The small, short-ellipsoidal, brownish green-ribbed, ivory-striped, and densely spined fruits of C. myriocarpus average 25 • 22 mm.

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Fro. 10. (a) The ellipsoidal (44 • 35 mm), pale greenish yellow fruits of C. prophetarum may be concolorous or indistinctly striped. All surfaces bear curved, soft spines that vary in length and abundance in different collections. ( b ) The ellipsoidal ( 52 • 37 mm ), dark and pale green-striped fruits of C. zeyheri turn yellow at full maturity. All surfaces bear curved, short, soft spines.

crosses with C. melo and C. prophetarum. Cross-pollinations with seven species failed to set fruits (Table I I ) .

5. Cucumis metuliferus

The uniquely thick-spined, monoecious, mesophytic, diploid cultivar Cucumis metuliferus, "Horned Cucumber" failed to set

fruits from reciprocal cross-pollinations with 15 other species and C. anguria var. longipes (Table I I ) .

6. Cucumis anguria and its variety longipes

The cultivated variety anguria of Cucumis anguria crossed readily with its wild variety longipes. The hybrids were vigorous and

DEAKIN ET AL.: INTERSPECIFIC HYBRIDIZATION 2 0 5

Fie. 11. (a) The ellipsoidal (50 X 35 ram), striped green fruits of C. ficifolius are sparsely ornamented with very short, soft spines. (b) The ellipsoidal (53 • 45 ram), striped, brownish green fruits of C. heptadactylus bear short, thick spines intermediate between those of C. a[ricanus and C. myriocarpus.

fruitful (Table II) . They produced flowers with 97% stainable pollen and set fruits with abundant seeds from selfed flowers. Our results agree with those of Meeuse (22) and support his view that the two entities are varieties of a single species.

Cucumis anguria varieties could be crossed with C. dipsaceus, C. africanus, C. leptodermis, C. myriocarpus, C. fici[olius, C. prophetarum, and C. zeyheri, and they yielded partly fertile F 1 hybrids. Stainable pollen ranged from 14% in the F1 C. prophetarum • C. anguria to 24% in the F1 C. zeyheri • C. anguria. Backcrosses to the pttrental species were either unsuccessful or yielded only a few plump seeds. C. anguria vats. anguria and longipes failed to yield viable seeds from cross-pollinations with C. heptadactylus, C. dinteri, C. melo, C. metuli[erus, C. hardwickii, and C. sativus.

7. Cucumis dipsaceus

In addition to the crosses with Cucumis anguria mentioned above, C. dipsaceus was successfully crossed with C. a[ricanus, C. [icifolius, and C. zeyheri. It set fruits lacking plump seeds from crosses with C. Ieptoder- mis, C. myr~ocarpus, C. melo, and C. prophetarum. C. dipsaceus failed to set fruits

from cross-pollinations with C. heptadactylus, C. dinteri, C. metuliferus, C. hardwickii, and C. sativus.

The successful crosses yielded F1 hybrids that ranged from partly fertile ones with Cucumis africanus and C. anguria to sterile or nearly sterile ones with C. ficifolius and C. zeyheri. Most male flowers on plants from the latter two crosses aborted, but stainable pollen from FI flowers that did reach anthesis averaged 9% in F1 C. dipsaceus • C. zeyheri and 4% in F1 C. [ici- [olius X C. dipsaceus. The greater fertility of the hybrid C. dipsaceus x C. zeyheri was indicated, also, in fruits set from backcrossed flowers. Such fruits averaged 39 and 38 plump seeds, respectively, from pollinatio~as with C. dipsaceus and with C. zeyheri. Fruits set on the F 1 C. ficifolius • C. dipsaceus averaged only one partly plump seed from backcrosses to either parental species.

Fertility was partly restored in plants from the backcross (Cucumis dipsaceus • C. zeyheri) x C. zeyheri. Stainable pollen in four plants ranged from 20% to 76%.

8. Cucumis a[ricanus

Cucumis africanus crossed readily with C. anguria vars. anguria and longipes; also with

206 E C O N O M I C B O T A N Y

T A B L E I I

S E L F - AND CROSS-COMPATIBILITIES a AMONG CUCUMIS SPECEES. SPECIF~ PERFORMANCE AS F E M A L E

PARENTS ARE INDICATEJ) IN HORIZONTAL ROWS; THEIR PERFORMANCES AS M A L E

PARENTS ARE INDICATED IN VERTICAL COLUMNS.

Species

C. sativus a in 14 5 4 1 0 1 1 0 0 0 0 0 0 0 0 0 0 C. hardwickii a m/a 14 4 5 0 0 0 0 0 0 0 0 0 0 0 C. melo a m/a 24 0 0 5 1 1 1 0 0 0 0 0 0 0 0 0 0 0 C. humifructus a In 0 0 1 5 1 1 0 0 0 0 0 0 0 0 0 0 C. dinteri p in 24 0 0 1 1 5 4 1 1 1 0 0 0 1 0 0 0 C. sagittatus p m 24 1 4 5 0 0 0 0 0 0 1 1 0 C. metuliferus a m 24 0 0 0 0 0 0 5 0 0 0 0 0 0 0 0 0 C. anguria a In 24 0 1 5 5 3 3 3 3 0 2 a

var. longipes a m 24 0 0 0 0 0 5 5 3 2 3 0 C. dipsaceus a m 24 0 0 1 0 0 3 0 5 3 1 0 2 0 0 C. africanus a In 24 0 0 0 0 0 0 0 3 3 3 5 3 3 0 0 C. leptodermis a rn 24 0 5 5 C. myriocarpus a m 24 0 0 1 5 5 1 C. prophetarum p na 24 0 1 1 3 1 3 3 5 2 C. zeyheri p m 24 1 3 2 1 3 2 3 5 2 d C. [ici[olius p m 48 0 0 0 2 2 2 d 5 C. heptadactylus p d 48 0 0 0 0 0 0 0 0 0 0 1 5

a Classes of cross-compat ibi l i ty are indica ted numer ica l ly : 5, fully ferti le progeny; 4, p rogeny modera te ly self-fertile; 3, p rogeny spar ingly self-fertile; 2, p rogeny self-steri le bu t spar ingly fertile in crosses; 1, f ru i t set bu t no progeny; 0, no frui ts set. Blanks, poll inations too f ew to cert i fy compat ib i l i ty status.

b a, annual ; p, perennial . e a, andromonoecious ; d, dioecious; m, monoecious. d Tr ip lo id offspr ing were observed.

C. dipsaceus, C. leptodermis, C. myriocarpus, C. prophetarum, C. zeyheri, and C. ficifolius but not with the remaining eight species (Table II) . Some of the crosses could be made more easily in one direction. For example, C. africanus crossed easily and yielded vigorous, normal green, moderately prolific F1 hybrids from crosses with C. anguria used as a female parent. The reciprocal cross yielded fewer viable seeds, and the few resulting seedlings were initially weak and light green in color. Plant color improved in the field, however, and the mature plants were comparable in vigor and prolificacy with those of the reciprocal. Some crosses were successful in only one direction.

Male flowers aborted on F1 hybrids from some of the successful crosses. However, some mature flowers with nearly normal anthers occurred on most of the hybrids.

Stainable pollen ranged from 17% to 34% in seven species hybrids. The backcross to C. anguria using the F 1 as a male parent demonstrated viable pollen in the C. anguria • C. africanus hybrid. Seeded fruits from naturally pollinated flowers indicated that most or all of the F 1 hybrids produced func- tional pollen.

Most of the F 1 hybrids backcrossed to their respective parents yielded 30 to 50 plump seeds in a fruit. Curiously, the hybrid from the wild Cucumis anguria var. longipes • C. africanus yielded only about 10% to 15% as many seeds in a fruit as did the hybrid from the cultivated C. anguria var. anguria • C. africanus.

Most of the backcross plants were robust and fruitful, but they varied greatly in size, appearance, and percentages of stainable pollen. Occasional dwarf-like B 1 plants occurred. Percentages of stainable pollen

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ranged from 29% to 90% in individual plants, and averages from five representative B1 populations ranged from 49% to 68%.

9. Cucumis leptodermis and C. myriocarpus

Cucumis leptodermis crossed readily with C. myriocarpus and yielded vigorous, fully fertile F1 hybrids. Our results agree with those of Meeuse (23), who stated: "C. leptodermis hybridizes quite easily with C. myriocarpus, apparently not only after artificial cross-pollination, but also in nature." The two entities yielded similar results in attempted crosses, yielding weakly fertile hybrids with C. africanus and C. anguria but none with eight other species (Table II) . C. myriocarpus yielded weakly fertile hybrids also from crosses with C. prophetarum and C. zeyheri. Our results support Meeuse's suggestion that " . . . perhaps C. leptodermis is not more than a variety or a subspecies of C. myriocarpus" ( loc. cit.).

10. Cucumis prophetarum and C. zeyheri

Cucumis prophetarum X C. zeyheri yielded vigorous F1 hybrids that were weakly fertile (Table II) . Male flowers yielded an average of 37% stainable pollen. One or both species yielded weakly or very weakly fertile hybrids from crosses that were attempted with C. anguria, C. dipsaceus, C. africanus, and C. myriocarpus. Both species failed to set fruit or set fruits lacking viable seeds from attempted crosses with the first seven species in Table II.

The breeding results failed to furnish strong evidence to support Jeffrey's statement (14) that C. zeyheri is a subspecies of C. prophetarum. We believe that for the present C. zeyheri should be retained as a distinct species separate from C. prophetarum because they yielded weakly fertile hybrids from crosses with one another. In addition, their distributions differ; C. prophetarum is a native of eastern tropical Africa, but C. zeyheri is a native of southern temperate Africa.

11. Cucumis ficifolius

According to Shimotsuma (29), the monoecious, perennial Cucumis ficifolius is tetraploid. In addition to several of our accessions labeled C. ficifolius, two lines labeled C. membranifolius and C. pustulatus respectively were studied. Both were similar

in morphological characteristics to C. ficifolius, and the tetraploid accession, labeled C. membranifolius, was finally identified as C. fici[olius. The other accession, labeled C. pustulatus, was a diploid with some characters in common with both C. zeyheri and C. ficifolius. These findings indicate that it may be either a diploid race of C. ficifolius or that it represents a link between C. fici[olius and C. zeyheri.

Cucumis fici[oIius yielded self-sterile hybrids from crosses with C. africanus, C. anguria, C. zeyheri, and C. dipsaceus (Table II) . The last three were sparingly fertile in crosses. Chromosome counts indicated that the first three species hybrids were triploid. Cross-pollinations between C. fici[olius and the first five species in Table II failed to set fruits.

12. Cucumis heptadactylus

The dioecious, xerophytic, tetraploid Cu- cumis heptadactylus has uniquely linear leaflets. It set fruits lacking viable seeds from cross-pollinations with C. myriocarpus. It failed to set fruits from reciprocal cross- pollinations with 10 other species (Table II).

B. Outcrosses to a third species

Work by Kehr & Smith (15) in Nicotiana, and by Wall & York (34) and Whitaker & Bemis (36) in Cucurbita demonstrated that interspecific incompatibilities can be over- come in some plant genera by using hybrids from compatible species crosses as one or both parents. Thus the germplasm from two species that refuse to cross can be combined in a single individual by using a third species compatible with both of the others as a "bridging" species.

Repeated attempts were made to break the compatibility barrier to the Cucumis melo • C. anguria cross. No species was found that was cross-compatible with C. melo, trot several were cross-compatible with C. anguria. Accordingly, numerous cross- pollinations were made between C. nwlo and the several F1 hybrids described above. Most of the cross-pollinated flowers failed to set; the few that did set produced fruits with nonenlarged ovules, or with partly enlarged ovules lacking embryos.

To broaden the germplasm base within the cross-compatible group, pollinations were made between F 1 species hybrids and a

208 E C O N O M I C BOTANY

third species partly cross-fertile with both parents of the F~ hybrid. The close relationship between C. anguria and the other seven species in the anguria group was borne out in hybrids from such three-way crosses. The Fi hybrid from the cross C. dipsaceus • C. anguria was outcrossed to C. africanus, C. myriocarpus, and C. zeyheri, yielding hybrids with a full genome of one species and partial genomes of the other two. Small (eight-plant) populations varied in stainable pollen percentages from 1% to 35%. Fur- thermore, nearly all of the plants set fruits with some seeds from natural pollinations, and some of them were surprisingly prolific and fertile.

The Fi hybrid from the cross Cucumis anguria • C. africanus was outcrossed to C. myriocarpus, yielding two three-way hybrids with 36% and 76% stainable pollen. Both plants produced numerous seeded fruits from naturally pollinated flowers.

A promising indication that cross-incompatibilities can eventually be broken in Cu- cumis was secured in a successful three-way cross from the Fi (C. anguria X C. africanus) • C. heptadactylus. That cross yielded robust, prolific plants with 47% to 81% stainable pollen. The fruits resembled those of C. heptadactylus in shape, color pattern, and spine size; in other characters the three-way hybrids resembled C. anguria more than the other parents. The fertility of these triple hybrids, which was much greater than either that of the F 1 hybrid or that of C. heptadactylus itself, is impressive for several reasons: (1) C. heptadactylus failed to cross directly with either of the other two species; (2) C. heptadactylus is a perennial, the other two species and their F 1 hybrid are annuals (Meeuse, 23) ; (3) C. heptadactylus is dioecious, the others monoecious; (4) C. heptadactylus is a tetraploid with 48 chromosomes, the others are diploids with 24 somatic chromosomes (Shimotsuma, 28).

High fertility in each of the trihybrids may indicate that they have resulted from fertilization of unreduced megagametes. Unreduced microgametes are frequently observed in pollen mounts from the F1 hybrids. If that hypothesis is correct, the trihybrids would contain a full genome from each of the three parents. They furnish an excellent opportunity for combined genetic,

cytogenetic, phylogenetic, and physiological study. They may also have greater potential than the parents in crosses with species in the Cucumis melo group at the diploid or tetraploid level.

The successful cross of Cucumis heptadactylus with the F i hybrid from the cross C. anguria • C. africanus adds that species to the group related to C. anguria.

The Fi Cucumis dipsaceus • C. zeyheri was outcrossed to C. africanus and to C. anguria. The first three-way cross yielded seven plants with 8% to 40% stainable pollen. The second yielded five plants with 3% to 18% stainable pollen. All of the plants produced seed.

The results indicate that an admixture of genes from a third species within the anguria group did not destroy the ability of the plants to produce stainable pollen and viable seeds. In fact, the greater variability in all characters in the three-way hybrids and their surprisingly high, partial fertility suggest that they may be useful in securing hybrids with species outside this large group of partly cross-fertile species.

Discussion

The interbreeding results indicate that several spiny-fruited wild African species of Cucumh" are related to the West Indian Gherkin, Cucumis anguria var. anguria. Cu- cumis anguria is economically the most im- portant member of this diverse group of naturally or artificially interbreeding species. These species can be termed, collectively, the anguria group for convenience.

The anguria group varies greatly in morphology, ecology, sex differentiation, and chromosome number. It includes (1) the cultivated C. anguria var. anguria and the three mesophytic, bushland-inhabit ing, climbing or trailing, monoecious, diploid (2n = 24), slightly cross-fertile, annual herbs C. africanus, C. anguria var. longipes, and C. dipsaceus (Figs. 6, 7, and 8); (2) the two xerophytic, trailing, monoecious, diploid (2n = 24), interfertile, annual herbs C. leptodermis and C. myriocarpus (Fig. 9) ; (3) the two xerophytic, prostrate, monoecious, diploid (2n = 24), moderately cross-fertile, perennial herbs C. prophetarum and C. zeyheri (Fig. 10) ; (4) the xerophytic, trailing, monoecious, tetraploid (2n = 48) perennial

DEAKIN E T A L . : I N T E B S P E C I F I C H Y B R I D I Z A T I O N 209

herb C. [icifolius (Fig. 11); and (5) the xerophytic, prostrate, dioecious, tetraploid (2n = 48) perennial herb C. heptadactylus (Fig. 11).

No associations as close as those among members of the anguria group were found in the other species studied. Morphological characters of Cucumis metuliferus (Fig. 5), especially its "homed" fruits and hairy seeds, suggest that it is unique. The breeding results confirmed this view; no fruits were set from cross-pollinations with C. metuliferus as either the male or female parent. Cucumis metuliferus is therefore set apart with no close relatives among the other species?

Similarly, the chromosome complement of Cucumis sativus and C. hardwickii (seven pairs compared with 12 and 24 pairs for other species), their harsh foliage and stiff- spined fruits (Fig. 1), and the natural dis- tribution of the wild C. hardwickii in India, indicate that they are not closely related to the cultivated C. melo or C. anguria and their wild relatives from Africa. Breeding results confirm the distant relationship. All crosses of C. hardwickii and of C. sativus with other species either failed to set fruits or set fruits with ovules lacking visible con- tents.

All of the species discussed above have spined ovaries. The remaining entities, C. melo, C. humi#uctus, C. dinteri, and C. sagittatus (Figs. 2, 3, and 4), lack spines on the ovaries. Instead, the ovaries are ornamented with fragile hairs that often dis- appear by the time the fruits mature. The perennial C. dinteri and C. sagittatus resemble one another and are interfertile. They bear little resemblance to either C. humi[ructus or C. melo, and crosses have failed to yield viable progeny. The closeness of their relationship to those species remains in question.

The plants, seeds, and fruits of Cucumis humi#uctus resemble those of C. melo more closely than do those of any other species studied. However, the curious structure and

a Since this was written, J. D. Norton reported a successful cross between C. metuli[erus and feral C. meh~ (P. I. 140471) in the 1969 Bien- nial Report of Vegetable Breeding in the South- ern United States, Hawaii, and Puerto Rico, p. 53.

behavior of the pistillate flower with its tiny ovary, which delays its development after fertilization, and its unique pedicel that resumes growth after fertilization and forces the ovary into the soil, present obstacles to cross-fertilization not found in any of the other species. C. humifructus, therefore, also remains unique with undetermined affinities in the genus.

The studies reported here have demonstrated that several wild species of Cucumis from Africa can be crossed with C. anguria, the West Indian Gherkin. Such crosses com- bining germplasm from two, three, or perhaps several species furnish potential parents for use in crosses with C. melo. Those crosses are desirable in order to transfer resistance to nematodes and other parasites from C. anguria into C. melo. Attempts to make such crosses using C. melo with C. anguria and its hybrids have failed to yield seeds with visible embryos.

In the search for a wild species that can be crossed with Cucumis melo to break the incompatibility barrier, C. sagittatus, its close relative C. dinteri, and C. humi[ructus failed to yield viable hybrids from crosses with C. melo. They also failed to cross with each other or with other Cucumt~ species. It seems that the greatest likelihood of success in achieving the ultimate goal can be ex- pected from work with little-known but presently unavailable relatives of C. melo, such as C. sacleuxii, 4 or with wild forms of C. melo, reported by Meeuse (23) to occur in Africa.

Conclusions

A. The genus Cucumis and its close relatives in the family Cucurbitaceae have long supported a disorganized tangle of misinfor- mation resulting from incorrect identification of plant cultures. Several disease resistance surveys and other studies on exotic species of cucurbits have reported information on unverified and often misnamed cultures, but during the past decade many identity errors in Cueumis have been corrected by critical inquiry.

B. The data from cross-fertility tests are compatible with data from studies on mor-

4 Mentioned in personal correspondence by C. Jeffrey.

210 ECONOSIIC BOTANY

phology, eytogenetics, phytogeography, and physiology (cueurbitaeins). They indicate:

1. C. anguria var. longipes is closely related to C. anguria var. anguria and may be its progenitor.

2. C. leptodermis and C. myriocarpus are related as varieties or subspecies rather than as distinct species.

3. C. zeyheri is no closer than a subspecies to C. prophetarum, and it may be a distinct species.

4. C. dinteri and C. sagittatus are related as varieties or subspecies rather than as distinct species.

5. C. hardwickii and C. sativus are related as varieties rather than as distinct species.

C. The cross-fertility studies indicate that Cucumis species can be tentatively orga- nized into four cross-sterile groups:

1. Eight spiny-fruited, African species or varieties of Cucumis are closely related to C. anguria var. anguria, yielding part ly fertile hybrids with it.

2. C. metuliferus is not closely related to any other species used in these studies.

3. C. sativus, including many cultivated forms and the spiny-fruited, seven-chromosome C. hardwickii from India, is not closely related to any of the spiny-fruited, 12-chromosome species from Africa or their cultivated relative, C. anguria var. anguria.

4. The three African species with young fruits hairy and lacking operculate spines ( C. humifructus, C. melo and its cultivated forms, and C. sagittatus including C. din~eri) are more closely related to one another than they are to any of the other species studied.

D. Crosses of C. melo with C. sagittatus yield part ly developed seeds. Work with additional collections of those species and their close relatives may yield a "bridging species" or form that will permit transfer of genes from other species into C. melo. Embryo culture techniques may be useful in culturing the minute embryos observed in these crosses.

E. The abundance and specific structure of soft spines on fruits in the anguria group appear to be unrelated to cross-fertility bar-

riers. Spine differentiation occurs within as well as between interfertile entities.

L i t e r a t u r e C i t e d

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Interspecific hybridization in cucumis