HORTSCIENCE 25(5):569-571. 1990. and generating plants from all available seed.

In this regard, Wang et al. (1984) reported

Plant Regeneration from Excised somatic embryogenesis and plant regenera-
tion from immature strawberry embryos.
Embryogenic structures were evident 5 weeks
Cotyledons of Mature Strawberry after culturing, but only 18% of the explants
developed into complete plantlets. Further,
Achenes it was not possible to subculture the embry-
ogenic tissue. The objective of the present
A. Raymond Miller study was to develop a protocol for over-
Department of Horticulture, The Ohio State University, Ohio Agricultural coming germination failures, thus rescuing
more gene combinations and generating nu-
Research and Development Center, Wooster, OH 44691 merous plantlets from a single embryo,
through continuous shoot proliferation.
Craig K. Chandler Strawberry achenes for this study were from
Agricultural Research & Education Center, IFAS, University of Florida, the hybrid cross of ‘Chandler’ × ‘Totem’,
13138 Lewis-Gallagher Road, Dover, FL 33527 and from the hybridization of other selected
clones. Achenes were harvested, dried to 10%
Additional index words. Fragaria × ananassa, tissue culture, organogenesis.
to 14% water content, then stored at 4C for
Abstract. A protocol was developed for excising and culturing cotyledon explants from at least 4 months before dissection and cul-
mature achenes of strawberry (Fragaria × ananassa Duch.). Cotyledon explants formed ture. For culturing, achenes were surface-
callus with multiple shoot buds on agar-solidified Murashige and Skoog media con- sterilized with 10% (v/v) Clorox (5.25%
taining several combinations of hormones (1 µ M 2,4-D; 10 µ M 2,4-D; 1 µ M BA + 1 NaOCl) for 10 rein, followed by four rinses
µ M 2,4-D; 1 µ M BA + 10 µ M 2,4-D; 5 µ M BA; 5 µM BA + 1 µ M 2,4-D; 5 µ M B A in sterile double-distilled water. The achenes
+ 10 µ M 2,4-D; 5 µ M BA + 5 µM NAA; 5 µ M BA + 15 µ M NAA). After three remained in the final rinse for ≈ 12 hr. Co-
subcultures, only tissues maintained on the medium containing 5 µM BA + 5 µM NAA tyledon explants were prepared by making a
continued to form shoots. Tissues transferred to other media eventually died (1 µ M transverse cut across the embryo axis of ach-
2,4-D; 1 µ M BA + 10 µ M 2,4-D; 5 µ M BA; 5 µ M BA + 1 µ M 2,4-D), became enes (Fig. 1) with a scalpel. Explants were
unorganized (1 µ M BA + 1 µ M 2,4-D; 5 µ M BA + 10 µ M 2,4-D; 5 µ M BA + 15 µ M placed on MS (Murashige and Skoog, 1962)
NAA), or formed roots (10 µM 2,4-D). Whole plantlets were produced by transferring medium solidified with 1% (w/v) purified
callus with buds to medium lacking hormones. The rapid regeneration of clonal plant- agar, and supplemented with 3% (w/v) su-
lets from cotyledon explants may be useful for reducing variability in future develop- crose and the following hormone combina-
mental studies. Chemical names used: N-(phenylmethyl) -1H-purin-6-amine (BA); (2,4- tions: no hormones; 1 µM 2,4-D; 10 µM
dichlorophenoxy) acetic acid (2,4-D); and 1-naphthaleneacetic acid (NAA). 2,4-D; 1 µM BA + 1 µM 2,4-D; 1 µM BA
+ 10 µM 2,4-D; 5 µM BA; 5 µM BA + 1
Strawberry cultivars and selections are on a given genotype. µ M 2,4-D; 5 µM BA + 10 µ M 2,4-D; 5
highly heterozygous. Therefore, each seed- Strawberry meristem culture has been used µ M BA + 5 µ M NAA; 5 µM BA + 15 µM
ling within a population is genetically unique to clonally propagate plants for commercial NAA. Five or six explants were placed on
(Scott and Lawrence, 1975). Strawberry purposes (Boxus et al., 1977; Damiano, 1980) 30 ml of medium in Parafilm-sealed plastic
breeders are dependent on this inherent seed- and studies on phenotypic and genotypic petri dishes (100 x 15 mm), with at least
ling-to-seedling variability, for it is among variation (Swartz et al., 1981; Shoemaker et three dishes per hormone treatment. Shoot
segregating populations that new cultivars are al., 1985). Plants can also be regenerated formation was scored after 5 weeks, and
selected. This same variation, however, is from leaf disks (Liu and Sanford, 1988) or shoots were placed on MS medium lacking
also a source of experimental error in studies petiole-derived callus (Jones et al., 1988). hormones for root regeneration. The remain-
where individual seedlings are the experi- However, all of these techniques use mature ing tissue was transferred at 5-week intervals
mental units. Additionally, seed germination tissue as the explant source, i.e., only plants to fresh MS medium containing the original
between strawberry cultivars, selections, and from germinating seeds are represented in combination of hormones. All cultures were
crosses is highly variable (Wilson et al., 1973; the population. Further, these techniques in- maintained at 27 ± 1C and a 12-hr photo-
Scott and Draper, 1967; Guttridge and Bright, crease the possibility of somaclonal varia- period was supplied by General Electric
1978), which results in the loss of geneti- tion, since cultures are maintained for long F40WW warm-white fluorescent lamps (flu-
cally unique seedlings. The ability to gen- periods. A better protocol would generate ence of 7 to 9 W·m-2 PAR). Each experi-
erate plants from the nonviable seeds would clonal plants from a primary embryo ex- ment was conducted at least twice.
allow the rescue of potentially valuable plant, thereby minimizing the time in culture Following sterilization and imbibition,
genotypes. Further, the development of clonal
populations would increase the precision of Table 1. Effect of hormones on in vitro shoot formation by cotyledon explants from mature strawberry
studies on seedling ontogeny and juvenility, achenes of a ‘Chandler’ × ‘Totem’ cross.
including studies on responses to biotic and
abiotic stresses, and environmental effects

Received for publication 19 hr. 1989. Salaries

and research support provided in part by state and
federal funds appropriated to the Ohio Agricul-
tural Research and Development Center and a Seed
Grant to A.R.M. from The Ohio State Univ. Jour-
nal Article no. 80–89. We thank Joseph C.
Scheerens for suggestions on the manuscript, Greg
Brenneman, Patricia Erb, and Heike Bruer for their
excellent technical assistance, and Bonnie Beck
for typing the manuscript. The cost of publishing
z
this paper was defrayed in part by the payment of Mean ± SD (n = four replicate plates, each with six explants).
y
page charges. Under postal regulations, this paper Tissue died during the second subculture.
x
therefore must be hereby marked advertisement Tissue formed a proliferating, unorganized callus after the first subculture.
w
solely to indicate this fact. Tissue died during the third subculture.

HORT SCIENCE , VOL . 25(5), MAY 1990 569

 All of the initial cotyledon-containing ex-
plants cultured on medium supplemented with
1 µM 2,4-D, 1 µM BA + 1 µM 2,4-D, 5
µ M 13A, 5 µM BA + 1 µM 2,4-D, 5 µM
BA + 5 µM NAA, or 5 µM BA + 15 µM
NAA formed shoots (Table 1). Initial ex-
plants cultured on media containing the other
hormone combinations also formed shoots,
but at much lower frequencies. The number
of shoots formed per explant ranged from 2
to 11, but did not appear related to the hor-
mone combination (data not shown). Differ-
ences did exist, however, in the amount of
callus formed; initial explants cultured in the
presence of an auxin (2,4-D or NAA) pro-
duced more callus than those cultured in the
presence of BA alone, and 10 µM 2,4-D re-
Fig. 1. Diagrammatic representation of a longitudinal view through a strawberry achene. The dotted sulted in more callus than 1 µ M 2,4-D, re-
line indicates the position of a scalpel cut used to separate the embryo into cotyledon- and shoot gardless of the presence or absence of BA.
apex/radicle-containing portions. The major difference between hormone
combinations was their effect on the organ-
ogenic potential of callus formed by the in-
itial explant. This difference became evident
following successive transfers of callus to
media containing the original hormone com-
binations (Table 1). Only those explants cul-
tured on medium containing 5 µM BA + 5
µM NAA produced callus that retained its
organogenic potential through three trans-
fers. This hormone combination has been
previously reported to be optimal for the de-
velopment of organogenic callus from straw-
berry leaf disks (Yurgalevitch et al., 1985).
By contrast, callus maintained on 5 µM BA
+ 15 µM NAA lost its organogenic potential
after two transfers, and callus maintained on
5 µM BA or 5 µM BA + 1 µ M 2,4-D lost
its organogenic potential after the first trans-
fer (Table 1). The remainder of the hormone
combinations (Table 1) did not support the
development of organogenic callus, although
shoots were formed on the initial explant.
Shoots may have differentiated directly from
cotyledonary cells cultured on these media,
but we have no direct evidence for this hy-
pothesis. Wang et al. (1984), however, have
shown that somatic embryogenesis from im-
mature strawberry embryos is possible.
Fig. 2. (A) Enlarged strawberry cotyledon after emergence from a cut achcne 5 days after culture
(72 ×). (B) Callus formation on an enlarged cotyledon 2 weeks after culture (38.5 ×). (C) Formation The protocol described here for the gen-
of multiple shoots on cotyledonary callus 5 weeks after culture (41.5 ×). (D) Complete plantlet eration of strawberry plantlets from mature
derived from callus of an excised mature cotyledon after transfer of the shoot to hormone-free medium cotyledonary tissue appears to be effective
(36 ×). for producing numerous “copies” of seed-
ling genotypes. Nearly 100% of the cotyle-
strawberry achenes could be easily cut into larged slightly, then died. The shoot apex/ don explants tested to date produced shoots,
cotyledon- and shoot apex/radical-containing radicle-containing portion, by contrast, de- which is higher than that observed by normal
sections (Fig. 1). Initially, the respective veloped into a normal seedling (not shown). seed germination (ranging from 40% to 97%,
embryo tissues were excised from the en- Cotyledon tissue placed on any of the hor- depending on genotype and achene treat-
docarp/seed coat using a dissecting micro- mone combinations tested, however, en- ment; Wilson et al., 1973; Scott and Draper,
scope, but this method proved labor-intensive larged after 5 days in culture and formed 1967; Guttridge and Bright, 1978), or so-
and damaged many of the explants. Alter- callus after 2 weeks. Buds were distinguish- matic embryogenesis from immature zygotic
natively, we found that the intact cotyledon- able on this callus after 3 weeks, and rec- embryos (Wang et al., 1984). In addition,
and shoot apex/radicle-containing sections ognizable shoots were present after 4 weeks the production of shoots can be continuous,
within the endocarp/seed coat, could be placed (Fig. 2 A through C). Transfer of the shoots since the organogenic callus maintains its
directly on culture media without altering the to medium lacking hormones resulted in shoot potential through successive subcultures.
developmental pattern. This technique al- elongation and root formation after 4 weeks Tissue from immature strawberry embryos
lowed the culture of more explants in the (Fig. 2D). Following acclimation to green- loses its morphogenic/embryogenic potential
same period of time than the dissection house conditions, plantlets developed into upon subculturing (Wang et al., 1984). The
method. normal-appearing plants. A similar pattern phenotypic stability of plants derived from
When achene sections from the ‘Chandler’ of development was observed for five other successive subcultures of cotyledon sections
× ‘Totem’ cross were placed on medium hybrid crosses between advanced breeding will be assessed in future greenhouse and
lacking hormones, cotyledon tissues en- lines (not shown). field studies.

570 HORT SCIENCE , VOL . 25(5), MAY 1990

 Literature Cited
Boxus, P. H., M. Ouoirin, and J.M. Laine. 1977.
Large scale propagation of strawberry plants from
tissue culture. p. 130-143. In: J. Reinert and
Y.P.S. Bajaj (eds.). Applied and fundamental
aspects of plant cell, tissue and organ culture.
Springer-Verlag, New York.
Damiano, C. 1980. Strawberry micropropagation.
Proc. Conf. on Nursery Production of Fruit
Plants–Through Tissue Culture Applications
and Feasibility. USDA-SEA, Agr. Res. Results
ARR-NE-11. p. 11-22.
Guttridge, C.G. and S. Bright. 1978. Accelerating
and synchronizing germination of strawberry
seeds by osmotic pre-treatments. Euphytica
27:843-848.
Jones, O. P., B.J. Wailer, and M.G. Beech. 1988.
The production of strawberry plants from callus
cultures. Plant Cell Tissue Org. Cult. 12:235-
241.
Liu, Z.R. and J.C. Sanford. 1988. Plant regen-
eration by organogenesis from strawberry leaf
and runner tissue. HortScience 23:1056-1059.
Murashige, T. and F. Skoog. 1962. A revised me-
dium for rapid growth and bioassay with to-
bacco tissue cultures. Physiol. Plant. 15:473-
497.
Scott, D.H. and A.D. Draper. 1967. Light in re-
lation to seed germination of blueberries, straw-
berries and Rubus. HortScience 2:107-108.
Scott, D.H. and F.J. Lawrence. 1975. Strawber-
ries. p. 71-97. In: J. Janick and J.N. Moore
(eds.). Advances in fruit breeding. Purdue Univ.
Press, West Lafayette, Ind.
Shoemaker, N. P., H.J. Swartz, and G.J. Galletta.
1985. Cultivar dependent variation in pathogen
resistance due to tissue culture-propagation of
strawberries. HortScience 20:253-254.
Swartz, H. J., G.J. Galletta, and R.H. Zimmer-
man. 1981. Field performance and phenotypic
stability of tissue culture propagated strawber-
ries. J. Amer. Soc. Hort. Sci. 106:667–673.
Wang, D., W.P. Wergin, and R.H. Zimmerman.
1984. Somatic embryogenesis and plant regen-
eration from immature embryos of strawberry.
HortScience 19:71-72.
Wilson, D., A. Goodall, and J. Reeves. 1973. An
improved technique for the germination of
strawberry seeds. Euphytica 12:362–366.
Yurgalevitch, C. M., H.W. Janes, and C. Chin.
1985. Somaclonal variation in Fragaria.
HortScience 20:450. (Abstr.).

HORTSCIENCE 25(5):571-572. 1990. root-derived embryogenic callus (Chang and

Hsing, 1980a; Choi, 1988; Furuya et al.,
Callus Induction and Plant 1986), and in vitro flowering of somatic em-
bryos from root-derived callus has been re-
Regeneration of American Ginseng ported (Chang and Hsing, 1980b). Callus
cultures of Korean ginseng have been trans-
Andrew S. Wang
l formed with Agrobacterium rhizogenes to
produce many hairy roots that synthesize the
Sungene Technologies Cop, 2050 Concourse Drive, San Jose, same saponins and ginsenoides as did un-
CA 95131 transformed roots (Yoshikawa and Furuya,
Additional index words. Panax quinquefolium, tissue culture 1987).
Although callus cultures have been estab-
Abstract. Friable embryogenic callus of American ginseng (Panax quinquefolium L.) lished using leaves, roots, and stems of
was induced from root pith on Murashige and Skoog medium supplemented with 2 mg American ginseng (Butenko et al., 1968;
2,4-D and 1 mg KIN/liter. Optimal callus growth occurred on medium containing 1.5 Jhang et al., 1974), plant regeneration from
mg dicamba/liter. Plants were regenerated on MS medium supplemented with various these cultures has not been reported. This
concentrations of plant growth regulators (PGRs); the best PGR combination was 0.5 paper reports studies on embryogenic callus
mg IBA and 0.1 mg NAA/liter. Chemical names used: (2,4 -dichlorophenoxy) acetic acid induction, callus growth, and plant regen-
(2,4-D); 3,6-dichloro-o-anisenic acid (dicamba); 6-benzylaminopurine (BA); gibberellic eration of American ginseng.
acid (GA); indole-3-butyric acid (IBA); kinetin (KIN); and naphthaleneacetic acid (NAA). A 4-year-old fresh field-grown ginseng mot
was washed in 70% alcohol for 40 see, then
The root of ginseng (Panax spp.), culti- requires 3 to 4 years to flower and 4 to 5 blotted and dried with paper towels. The
vated in northern China, Korea. the United years to harvest. Tissue-culture technology cleaned ginseng root was then sterilized in
States, Canada, and the- Soviet- Union, has might be able to accelerate the rate of im- 2.5% sodium hypochlorite for 20 min under
been extensively used as a cure-all drug or provement. For Korean ginseng (Panax gin- vacuum with occasional agitation, rinsed with
tonic in the Orient (Choi, 1988). American seng C.A. Meyer), callus cultures have been sterile, distilled water three times, and sliced
ginseng, grown in North America, is Panox successfully established (Butenko et al., into 2-mm sections. Pith tissue of each sec-
quinquefolium L. (Carlson, 1986; Proctor and 1968), plantlets have been regenerated from tion was removed with a cork borer 1 cm in
Bailey, 1987). In 1987 and 1988, the United
States exported 578 and 448 t, respectively,
of American ginseng root (valued at nearly
$100 million) to other countries, mainly in
Asia (US Bureau of Census). The majority
of exported ginseng is produced in Wiscon-
sin (Wis. Dept. of Agr.). Wisconsin har-
vested 592 and 472 t of the ginseng root in
1987 and 1988, respectively.
Improvement of ginseng root production
through conventional breeding is slow and
difficult, since the plant is a perennial and

Received for publication 1 May 1989. I thank Ross

Eccleshall and Alexi Miller for reviewing the
manuscript, and Faye Wang for technical assis-
tance. The cost of publishing this paper was de-
frayed in part by the payment of page charges.
Under postal regulations, this paper therefore must
be hereby marked advertisement solely to indicate Fig. 1. Net callus growth of American ginseng cultured in MS medium containing 2 mg dicamba ( ❏ )
this fact. or 2 mg 2,4-D ( ■ )/liter. Each point represents the average callus growth from at least four plates.
1
Present address: Northrup King Co. Research Each plate initially contained 50 ml of the maintenance medium and 1 g of callus. The vertical bars
Center, Highway 19, Stanton, MN 55081. represent the SE of the mean.

HORT SCIENCE , VOL . 25(5), MAY 1990 571