J. A m e r . S o c . H o r t . S c i . 111(4):627-630. 1986.

Inter generic Hybridization in the J u g la n d a cea e:

P tero ca rya sp. X J u g la n s regia
G .H . M cGranahan1, W. Tulecke2, S. Arulsekar3, and J.J. Hansen4
Agricultural Research Service, U.S. Department o f Agriculture, and Department o f Pomology,
University o f California, Davis, CA 95616
A d d itio n a l index w ords, w in gn u t, w aln u t, rootstock , b reed in g, som atic e m b ry o g en esis, iso z y m e s
Abstract. Intergeneric hybrids between wingnut (Pterocarya sp.) and walnut (Juglans regia) were developed by
regenerating plants from somatic embryos produced on immature cotyledons of seed from control-pollinations. Hy-
bridization was confirmed by isozyme analysis using starch gel electrophoresis. To the best of our knowledge, this is
the first report of hybrids between wingnut, which has a high level of resistance to Phytophthora spp. and nematodes,
and walnut. Wingnut may now be used as a source of germplasm for improving walnut rootstocks.

Rootstocks for Persian walnut (Juglans regia L.) cultivars
include seedling J. hindsii Jeps. ex R.E. Smith, ‘Paradox’ (J.
hindsii X J. regia), J. regia, and Pterocarya stenoptera C.D.C.
The relative merits of each type of rootstock, as well as their
response to pests and diseases recently have been reviewed (13).
Foremost among the root-related problems in walnut are root
lesion nematodes (Pratylenchus vulnus Allen and Jensen) (8)
and the Phytophthora root and crown rots caused by several
species of Phytophthora (14). Recently, wingnut (P. stenoptera)
has been shown to have substantially higher levels of resistance
to these pests than other potential rootstock species tested (9,
11, 14). It has also been shown to have higher tolerance to
water logging (3).
Wingnut is used as a walnut rootstock in the northern prov-
inces of China (4). Graft compatibility with J. regia was first
reported in the United States in 1948 (18). However, subsequent
reports (15) indicated that it was not fully compatible with all
walnut cultivars, and growers have been discouraged from using
P. stenoptera even in compatible combinations because it suck-
ers profusely and roots tend to be close to the soil surface. These
disadvantages suggested that P. stenoptera might be most useful
as a source of germplasm for breeding pest- and disease-resistant
rootstocks rather than as a rootstock itself. No literature is avail-
able on crossability between these 2 genera.
The purpose of the following study was to determine the
feasibility of producing walnut X wingnut hybrids for use in
rootstock improvement. This paper reports the successful pro-
duction of 2 hybrid clones using controlled pollination followed
by ovule culture and somatic embryogenesis. Hybridization was
confirmed by isozyme analysis.
Materials and Methods
The parent trees were grown in collections at the Univ. of
California, Davis. The wingnut parents had been identified in
the collections as P. stenoptera, but examination of the taxo-
Received for publication 3 Oct. 1985. This material is based on work partially
supported by the Walnut Marketing Board and ARS/USDA Cooperative Agree-
ment NO. 58-9AH Z-1-626. The use of product names does not imply endorse-
ment by U S D A . W e w ould like to thank E. Sutter and C. L eslie for critical
review o f the manuscript. The cost of publishing this paper was defrayed in
part by the payment o f page charges. Under postal regulations, this paper there-
fore must be hereby marked advertisement solely to indicate this fact,
horticulturist ARS/USDA.
2Professor, Dept, o f Biology, Antioch College, Ohio.
•Postgraduate research geneticist.
4Agriculture research technician, ARS/USDA.
nomic features of the fruits indicated that they were probably
hybrids between P. stenoptera and P . fraxinifolia (Lam.) Spach.
This origin was confirmed by W.E. Manning (personal com-
munication) after examination of the fruits.
Controlled pollinations (12) between J. hindsii, J. regia, and
Pterocarya sp. were performed in the field in 1983 and 1984.
In 1983, pollen germination on the stigma in all possible com-
binations (including reciprocals) was observed using methods
described by Martin (10). Twelve flowering shoots (10-15 cm
long) from one individual per species were collected several
days prior to anthesis and forced in tap water in the laboratory.
Male and female parents were selected solely on their devel-
opment stage. When flowers were at peak receptivity (5), they
were placed in 4 physically isolated groups. Each group con-
tained 3 shoots from each species. The flowers were then hand-
pollinated with one of the 3 sources of pollen. One group served
as an unpollinated control. Twenty-four hours after pollination
flowers were excised for storage and later microscopic exami-
nation.
Because results in 1983 suggested that a crossing barrier be-
tween Pterocarya and Juglans might occur after fertilization,
but before seed maturity, immature seeds derived from the con-
trolled pollination (Pterocarya sp. X J. regia ‘Gustine’) were
collected about 2 months after pollination in 1984. A total of
22 ovules was removed from surface-sterilized fruits and placed
on the basal medium used for walnut culture (16). A subset of
7 ovules was placed on the basal medium with one-half the
normal concentration of dipotassium sulfate (K2S 0 4). No in-
duction medium (16) was used to stimulate somatic embryo-
genesis. The culture methods and techniques for regenerating
plants were the same as those used for walnut (16).
Plants were obtained by selecting normal, vigorous somatic
embryos from among the many abnormal and fused embryos
produced in culture. Five embryos with cotyledons and radicle
were given a cold treatment of 2°-6°C for 8 weeks to break
dormancy.
After cold treatment, embryos were transferred to fresh basal
medium and grown under lights (87 |xmoFs_1*m_2, 16-hr pho-
toperiod) at 24°C for 4-6 weeks. Germinated embryos were then
moved to peat plugs (Castle and Cook, Techniculture, Salinas,
Calif.) for further root and shoot development.
After 2-4 weeks, growing plantlets were transferred to ster-
ilized soil in 23.7-ml (8-oz) foam cups for one month and then
to 20.3-cm (8-inch) pots covered with polyethylene. Acclima-
tization occurred as the polyethylene was perforated over a 2—
4 week period.
Starch gel electrophoresis was used to confirm parentage of

J. Amer. Soc. Hort. Sci. 111(4):627-630. 1986. 627

the embryogenic cultures and resulting plants. Results were based bryos on their cotyledons. One of these, the first to exhibit
on differences in the isozymes of 2 enzyme systems, phospho- somatic embryogenesis (8 weeks after initiation in culture) had
glucomutase (PGM) and leucine amino peptidase (LAP). The been grown on the low K2S 0 4 culture medium, but this medium
enzyme extraction procedures, and gel and electrode buffer sys- did not appear to be essential for somatic somatic embryogen-
tems have been described (1, 2). esis. Somatic embryos removed from the cotyledons of hybrid
The gels were stained in the following reaction mixture and embryos continued to produce additional somatic embryos through
incubated in the dark at 37°C for 45 min. repetitive embryogenesis (Fig. 2). The 2 resulting full-sib em-
PGM: glucose-1-phosphate (85 mg); NADP (10 mg); MTT bryogenic lines have been maintained in the dark at ambient
(15 mg); phenazine methosulfate (5 mg); distilled H20 (93 ml); room temperature for over a year with monthly transfers.
1 m Tris-HCl pH 8.0 (2 ml); 0.1 M MgCl2 (5 ml); glucose-6- After one year, 10 plants are in pots and acclimatized and 6
phosphate dehydrogenase (40 units). to 10 others are in various stages of development beyond ger-
LAP: Leucyl-naphthyl amide, HC1 (20 mg); black K salt (20 mination. Each plant is derived from a somatic embryo from
mg); distilled H20 (30 ml); 2% NaOH solution (20 ml); 0.2 m one of the 2 embryogenic lines.
Tris-maleic buffer (50 ml). Confirmation of hybridization was obtained through isozyme
analysis. The parent clones of J . regia and P terocarya sp. ex-
Results hibited distinct banding patterns for PGM and LAP. The hybrids
have the isozymes of both parental species.
Pollen germination and growth (Fig. 1) were observed in all
There are 2 loci that control the PGM isozymes in walnuts,
crosses, indicating that there was no barrier to germination at
Pgm-1 and Pgm-2 (2). The J. R egia cultivar ‘Gustine’, which
the stigmatic surface. No pollen was present on stigmas of the
was the male parent of the hybrids, was homozygous at both
unpollinated controls. Quantitative measurements were not made,
loci, exhibiting a single band at Pgm-1 and Pgm-2. The P ter-
but it appeared that the most vigorous and directed pollen tube
ocarya sp. parent had a slow-moving isozyme in the Pgm-1
growth occurred when wingnut was pollinated by walnut. Thus,
region and 2 bands at the Pgm-2 region. The hybrids possessed
in 1984, wingnut alone was used as the female parent.
the fast-moving Juglans isozyme and the slow-moving P tero-
The results from 1983 field crosses were inconclusive. No
carya isozyme at the Pgm-1 locus. At Pgm-2, one of the P ter-
seeds were produced from the 46 J. hindsii flowers pollinated
ocarya isozymes (slow) appeared in combination with the fast-
with wingnut pollen. Twelve seeds resulted from the 10 J. regia
moving band of Juglans (Fig. 3). Embryogenic tissue and leaf
‘Franquette’ flowers pollinated by wingnut, but only one ger- tissue of the hybrids showed identical patterns.
minated and it was determined to be J. regia. Viable seeds (3-
9 per raceme) were produced in all crosses in which wingnut
was used as the female parent, including unpollinated checks.
The resulting seedlings appeared to be wingnuts and isozyme
analysis confirmed that either apomixis or stray pollen contam-
ination had occurred.
In 1984, seeds again were produced in all crosses where wingnut
was used as a female parent, including unpollinated controls.
Number of seeds set per raceme ranged from 7 (unpollinated
control) to 28 (Pterocarya sp. X P terocarya sp.). Intergeneric
hybrid crosses resulted in an average of 15 seeds set per raceme.
The majority of P terocarya sp. X P terocarya sp. seed germi-
nated, but no seeds from unpollinated controls or intergeneric
hybrid crosses germinated. Dissection of the seed showed that
these did not contain viable embryos.
Of the 22 hybrid ovules cultured, 2 produced somatic em-

Fig. 1. Germination of J. regia pollen on stigma of Pterocarya sp. Fig. 2. Repetitively embryogenic culture of Pterocarya sp. X J . regia
(30 x). culture shows embryos in various stages of development.

628 J. Amer. Soc. Hort. Sci. 111(4):627-630. 1986.

Fig. 3. Zymogram patterns of PGM isozymes. Lanes 1, 2, 3, and 4
represent walnut leaves, wingnut X walnut hybrid embryos, wingnut
x walnut hybrid leaves, and wingnut leaves, respectively.
' iii«
l— — 1 ....... .. } l a p - i
cr= 3 t= i L ' ^ LAP -2
2 3 4 contain traits that will be beneficial in walnut rootstock breed-
Fig. 4. Zymogram patterns of LAP isozymes. Lanes 1, 2, 3, and 4
represent walnut leaves, wingnut X walnut hybrid embryos, wingnut
x walnut hybrid leaves, and wingnut leaves, respectively.
A similar situation was present in the LAP isozymes (Fig.
4). The hybrids exhibited the fast-moving LAP-1 isozyme of J.
regia and the slow-moving LAP-1 isozyme of Pterocarya. In
the LAP-2 region the hybrids had the fast-moving Pterocarya
band along with the slow-moving J. regia band. These results
confirmed that intergeneric hybrids had been produced.
Phenotypically, the hybrid plants (Fig. 5) appear to be inter-
mediate between the parents. Both lines perform similarly. The
leaf rachis is slightly winged. Leaflets number between 2 and
7. Further studies will be needed to determine the characteristics
of more mature plants.
Discussion
To the best of our knowledge, this is the first report of elec-
trophoretically confirmed intergeneric hybrids within the Jug-
landaceae. There have been reports in the literature from the
USSR (7) on putative hybrids between Juglans and Cary a, but
these have not been confirmed. Isoenzymes have been success-
fully used to identify interspecific and intergeneric hybrids in
other genera. Gleba and Hoffman (6) used isozymes to identify
hybrids between Arabidopsis thaliana and Brassica campestris.
J. Amer. Soc. Hort. Sci. 111(4):627-630. 1986.
Fig. 5. Pterocarya sp. X J . regia hybrid.
Wetter (17) used isozymes to identify intergeneric hybrids be-
tween Nicotiana glauca and Glycine max.
Somatic embryogenesis was useful in obtaining plants from
intergeneric crosses in the present study. Additional work is
needed to determine whether conventional ovule culture meth-
ods (i.e., without somatic embryogenesis) can be applied suc-
cessfully to wingnut x walnut hybrids and whether there is an
advantage to conventional culture. Currently, it appears that
removal of intact embryos from seeds after cotyledons have
developed is a difficult process due to the bony pericarp. Thus,
very small embryos, decotylated embryos, or somatic embryo-
genesis from cotyledons are the most promising target tissues
for rescue of wide crosses in the Juglandaeae.
It appear that Pterocarya can be used as a source of germ-
plasm for the genetic improvement of walnut rootstocks. It re-
mains to be seen whether its hybrid progeny will be fertile or
ing.
Literature Cited
1. Arulsekar, S., D.E Parfitt, and G.H. McGranahan. 1985. Iso-
zyme gene markers in Juglans species. Inheritance of GPI and
ATT in J. regia and J. hindsii. J. Her. 76:103-106.
2. Arulsekar, S., G.H. McGranahan, and D.E. Parfitt. 1986. In-
heritance of phosphoglucomutase and esterase isozymes in Per-
sian walnut (J. regia L.). J. Her. (In press)
3. Catlin, P.B., G.C. Martin, and E.A. Olsson. 1977. Differential
sensitivity of Juglans hindsii, J. regia, Paradox hybrid, and Pter-
ocarya stenoptera to waterlogging. J. Amer. Soc. Hort. Sci.
102:101-104.
4. China Trees Comm. 1978. China Main Forest Planting Tech-
nology. Agr. Publ. Co., Beijing, People’s Republic of China.
5. Forde, H.I. and W.H. Griggs. 1975. Pollination and blooming
habits of walnuts. Univ. of California Lflt. 2753.
6. Gleba, Y. Y. and F. Hoffman. 1978. Hybrid cell lines Arabidop-
sis thaliana X Brassica campestris: no evidence for specific chro-
mosome elimination. Mol. Gen. Genet. 165:257-264.
7. Komanich, I.G. 1980. Biology, cultivation and breedng of wal-
nut. A. A. Chebotar (ed.). Shtiintsa Publishers, (The Botanic Gar-
dens of the Academy of Sciences of the Moldavian SSR), Kishiner,
USSR.
8. Lownsbery, B.B. 1956. Pratylenchus vulnus, primary cause of
the root-lesion disease of walnuts. Phytopathology 46:376-379.
9. Lownsbery, B.F., G.C. Martin, H.I. Forde, and E.H. Moody.
1974. Comparative tolerance of walnut species, walnut hybrids
629
 and wingnut to the root-lesion nematode, Pratylenchus vulnus.
Plant Dis. Rptr. 58:630-633.
10. Martin, F.W. 1958. Staining and observing pollen tubes in the
style by means of fluorescence. Stain Technol. 34:125-128.
11. Matheron, M.E. and S.M. Mircetich. 1985. Relative resistance
of different rootstocks of English walnut to six Phytophthora spp.
that cause root and crown rot in orchard trees. Plant Dis. 69:1039-
1041.
12. McGranahan, G.H. and H.I. Forde. 1985. Genetic improvement,
pp. 8-12. In D.E. Ramos (ed.). Walnut orchard management.
Univ. of California Coop. Ext. Publ. 21410.
13. McGranahan, G.H. and P.B. Catlin. 1986. Juglans. In: R.C.
Rom and R.C. Carlson (eds.). Rootstocks. Wiley, New York.
(In press)
14. Mircetich, S.M. and M.E. Matheron. 1983. Phytophthora root
and crown rot of walnut trees. Phytophatology 73:1481-1488.
15. Serr, E.F. and A.D. Rizzi. 1964. Walnut rootstocks. Univ. Calif.
Agr. Ext. Serv. Publ. AXT-120.
16. Tulecke W. and G.H. McGranahan. 1985. Somatic embryogen-
esis and plant regeneration from cotyledons of walnut, Juglans
regia L. Plant Sci. 40:57-63.
17. Wetter, R.L. 1977. Isoenzyme patterns in soybean—Nicotiana
somatic hybrid cell lines. Mol. Gen. Genet. 150:231-235.
18. Whitehouse, W.E. and L.E. Joley. 1948. Notes on growth of
Persian walnut propagated on rootstocks on the Chinese wingnut
Pterocarya stenoptera. Proc. Amer. Soc. Hort. Sci. 52:103-106.

J. A m e r . S o c . Ho r t . Sc i . lll(4):630-634. 1986.

Bermudagrass Germplasm Adaptation to Natural

Pest Infestation and Suboptimal Nitrogen
Fertilization
Philip Busey
Fort Lauderdale Research and Education Center, IF AS, University o f Florida, 3205 College
Avenue, Fort Lauderdale, FL 33314
Additional index words, breeding, clonal variation, plant introduction, Cynodon spp., Scapteriscus spp.
Abstract. Bermudagrass (Cynodon spp.) turf in subtropical Florida normally requires higher levels of N than other
grasses and frequently requires pesticide applications. Three sequential 2-year cycles of clonal selection were per-
formed in replicated field plots to recognize bermudagrass germplasm adapted to suboptimal fertilization and natural
pest infestation. Low fertility, 19 to 25 g N*m~2, was applied yearly, including the establishment phase. No nematicides,
fungicides, or insecticides were applied. Severely damaging mole cricket {Scapteriscus sp.) populations were left
uncontrolled. Among 95 clones, 4 experimentals (FB-109, PI-291586, T-72-54, and FL-2400) survived repeated cycles
with relatively high turfgrass coverage and quality. Among cultivars, only ‘Tifgreen-II’ and ‘Ormond’ performed
well. African introductions and artificially-induced mutants of hybrid cultivars were the best sources of adapted
germplasm. Although the mechanism of this adaptation is unknown, field tests were an effective prescreening method
for clonal selection.

Bermudagrass was introduced to the United States from Af-
rica by 1751 (9). Its value as a turfgrass was recognized in the
United States by 1917 (19), and it was planted on golf courses
(4) and lawns (6) in Florida at least by the 1920s. Subsequently,
bermudagrass cultivars, including the vegetatively propagated
hybrids ‘Tifway’ and ‘Tifgreen’, were intentionally developed
through breeding (2). Considerable germplasm was evaluated
in the southern United States from 1955 through 1962 (10).
Bermudagrass turf requires high maintenance, particularly in
subtropical Florida, where severe pest populations frequently
require control. Pest problems encountered are mole crickets
(15), tropical sod webworm (12), bermudagrass stunt mite (14),
nematodes (11), and weeds. Nitrogen rates that provide maxi-
mum quality of hybrid bermudagrass in warm humid climates
range from 116 to 180 g N*m-2 *year-1 (1, 16). Bermudagrass
turf grown on sandy soil requires additional N to compensate
for losses due to leaching (17). In a one-growing season study
in north Florida (5), the highest fertilization rate used (45 g
Received for publication 4 Apr. 1985. University of Florida Agricultural Ex-
periment Station Journal Series 6295. Special thanks are given to Dr. W .W .
Huffine, who collected most of the plant materials, and to Ms. B.J. Center,
who provided expert technical assistance throughout these investigations. The
cost o f publishing this paper was defrayed in part by the payment of page
charges. Under postal regulations, this paper therefore must be hereby marked
advertisement solely to indicate this fact.
N-m_2*year-1) provided the highest turf quality on ‘Ormond’
bermudagrass, and 25 g N*m_2*year_1 provided the lowest turf
quality.
Because of its fine-leaved texture, high density, and traffic
tolerance, bermudagrass is appropriate for sports turfs and lawns.
With the limited use of bermudagrass in Florida home lawns
and other low-maintenance areas (8), a search among bermu-
dagrass introductions was undertaken to discover germplasm
that would establish and persist as a turf under natural pest
infestation and suboptimal N fertilization.
Materials and Methods
Cycle 1, 1976-1979. Stolons of 69 introduced clones of ber-
mudagrass were obtained from W.R. Langford (ARS/USDA,
Reg. Plant Intro. Sta., Experiment, GA 30212). Most had been
collected by W.W. Huffine in Zimbabwe and the Republic of
South Africa. During initial expansion, 26 clones were dis-
carded due to duplications of vegetative traits, caterpillar dam-
age, and ungainly stature, leaving 43 clones in Cycle 1. Other
clones selected for comparison included 5 (FB-prefixes) that had
persisted for several years without fertilization in plots at Fort
Lauderdale, and cultivars ‘Tifgreen’ and ‘Tifway’. Sprigs of
these 7 clones were obtained directly from field plots (and not
prepropagated), which may have affected their early perform-
ance.

630 J. Amer. Soc. Hort. Sci. 111(4):630-634. 1986.