In Vitro Cell. Dev. Biol.

Plant 36:287292, JulyAugust 2000

q 2000 Society for In Vitro Biology
1054-5476/00 $10.0010.00

ALGINATE ENCAPSULATION OF AXILLARY BUDS OF OCIMUM AMERICANUM L. (HOARY BASIL),

O. BASILICUM L. (SWEET BASIL), O. GRATISSIMUM L. (SHRUBBY BASIL),
AND O. SANCTUM L. (SACRED BASIL)

JAYDIP MANDAL, SITAKANTA PATTNAIK, and PRADEEP K. CHAND*

Plant Tissue and Cell Culture Facility, Department of Botany, Utkal University, Bhubaneswar 751004, India

(Received 1 June 1999; accepted 2 February 2000; editor S. Merkle)

Summary
Propagation and conservation of four pharmaceutically important herbs, Ocimum americanum L. syn. O. canum Sims.
(hoary basil); O. basilicum L. (sweet basil); O. gratissimum L. (shrubby basil); and O. sanctum L. (sacred basil) was
attempted using synthetic seed technology. Synthetic seeds were produced by encapsulating axillary vegetative buds
harvested from garden-grown plants of these four Ocimum species in calcium alginate gel. The gel contained Murashige
and Skoog (MS) nutrients and 1.14.4 mM benzyladenine (BA). Shoots emerged from the encapsulated buds on all six
planting media tested. However, the highest frequency shoot emergence and maximum number of shoots per bud were
recorded on media containing BA. Of the six planting media tested, both shoot and root emergence from the encapsulated
buds in a single step was recorded on growth regulator-free MS medium as well as on vermi-compost moistened with half-
strength MS medium. Rooted shoots were retrieved from the encapsulated buds of O. americanum, O. basilicum, and O.
sanctum on these two media, whereas shoots of O. gratissimum failed to root. The encapsulated buds could be stored for
60 d at 48C. Plants retrieved from the encapsulated buds were hardened off and established in soil.
Key words: medicinal plants; axillary buds; synthetic seeds; plant regeneration; Ocimum species.

Introduction propagation of rare hybrids, elite genotypes, and genetically

Synthetic seed technology has developed considerably in recent
years. The concept was first published by Murashige (1977), and
the first report on the construction of synthetic seeds was published
in 1982 (Kitto and Janick, 1982), which described the coating of
multiple carrot somatic embryos with a water-soluble resin,
polyethyleneoxide (Polyox). Later, Redenbaugh et al. (1984) were
successful in producing monoembryonic synthetic seeds for alfalfa
by encapsulating somatic embryos with alginate hydrogel. Subse-
quently this technology was extended to a wide variety of plant
species, including cereals, vegetables, fruits, ornamentals, medic-
inal aromatic plants, and conifers (Redenbaugh, 1993; Ghosh and
Sen, 1994; Fowke et al., 1994; Onay et al., 1996; Patnaik and
Debata, 1996; Janeiro et al., 1997; Castillo et al., 1998). However,
it was restricted mostly to plants in which somatic embryogenesis
has been documented. In response to this problem, the possibility of
using non-embryogenic vegetative propagules such as apical shoot
buds/axillary buds/nodal segments for synthetic seed production
has been explored by several workers, with varying degrees of
success (Bapat et al., 1987; Mathur et al., 1989; Sharma et al.,
1994; Pattnaik et al., 1995; Ballester et al., 1997; Sarkar and Naik,
1998). High volume, low production costs, and ease of storage and
transport are the major advantages of synthetic seed technology
(Ghosh and Sen, 1994). The technology offers excellent scope for
*Author to whom correspondence should be addressed: Fax 91-674-
509037; Email pkchanduubot@hotmail.com
engineered plants for which the seeds are either very expensive
or are not available. In recent years the use of synthetic seeds for ex
situ conservation of the germplasm of elite and endangered plant
species has also been emphasized (Gray et al., 1995; Maruyama
et al., 1997). The present method of conservation of these species as
living plants in field genebanks is expensive and subject to loss due
to environmental disasters. This problem could be overcome using
synthetic seeds. In addition, synthetic seed technology allows in
vitro conservation of clonal germplasm of a greater number of
genotypes, as the space problem is reduced (Gray et al., 1995).
Four different Ocimum species, O. americanum, O. basilicum, O.
gratissimum and O. sanctum, are highly valued for their
pharmaceutical properties. These species possess in common
aromatic, carminative, diaphoretic, expectorant, and stimulant
properties, as well as other exclusive medicinal attributes. The
conventional method of propagation of these species is only through
seeds, but the germination of seeds under natural conditions is
season-dependent, and in some species (e.g. O. sanctum) the
seedlings are susceptible to seedling blight and root rot (Anon.,
1966). Synthetic seeds could therefore be useful for propagation as
well as ex situ conservation of these important medicinal herbs.
However, there appear to be no reports to date on the use of this
technology for propagation of Ocimum species. The present
communication is the first report to describe the encapsulation of
axillary buds harvested from garden-grown plants of O. amer-
icanum, O. basilicum, O. gratissimum and O. sanctum in calcium
alginate hydrogel, and to evaluate the in vitro response of the

287
288 MANDAL ET AL.

alginate-encapsulated buds to various media. In addition, the

influence of low-temperature storage on the viability of alginate-
encapsulated buds was investigated.

Materials and Methods

Explant source. Plants of Ocimum americanum L., O. basilicum L., O.

gratissimum L. and O. sanctum L. growing in the experimental garden of the
Postgraduate Department of Botany, Utkal University, served as the source
of explants. Axillary buds collected from the garden-grown plants were
washed with tap water for 30 min, then immersed in an aqueous solution of
5% (v/v) Laboline, a liquid detergent (Qualigens, India) and 7% (vol/vol)
sodium hypochlorite (NaOCl) (BDH, India) for 5 min and rinsed five or six
times with distilled water. The explants were surface-disinfected with an
aqueous solution of 0.1% (wt/vol) HgCl2 (BDH) for 5 min, and rinsed with
autoclaved distilled water (five or six changes).
Encapsulation. Sodium alginate from two sources (Central Drug House,
India; Sigma, USA) was added at 2, 4 or 6% to MS liquid medium
(Murashige and Skoog, 1962) containing 100 mg l21 myoinositol, 30 g l21
sucrose and 1.1 (O. americanum), 2.2 (O. gratissimum) or 4.4 mM (O.
basilicum and O. sanctum) benzyladenine (BA). For complexation, 75
100 mM CaCl2 solution was prepared using MS liquid medium of the same
composition as described above. The pH of the gel matrix and the
complexing agent was adjusted to 5.8 prior to autoclaving at 1218C for
15 min. Encapsulation was accomplished by mixing the axillary buds with
sodium alginate solution and with a pipette dropping these into calcium
chloride solution. Calcium alginate capsules containing the axillary buds
were retrieved from the solution with a tea strainer and rinsed twice with
autoclaved distilled water to remove the traces of calcium chloride. A set of
encapsulated buds (100 encapsulated buds per species) was stored in sterile
Petri dishes (20 to a dish) lined with moist paper towel at 48C for up to 60 d,
and the frequency of shoot emergence on MS medium supplemented with
1.14.4 mM BA was noted. Another set of unencapsulated buds was stored
under the same conditions to serve as a control.
Planting media and culture conditions. In vitro response of the
encapsulated axillary buds was studied by transferring them to 300 ml
screw-capped glass jars containing the following media: (i) MS med-
ium + 1.1, 2.2 or 4.4 mM BA + 30 g l21 sucrose + 8 g l21 agar; (ii) MS
medium + 30 g l21 sucrose + 8 g l21 agar; (iii) tap water + 8 g l21 agar;
(iv) vermi-compost (Ranjan Agrotech, Bhubaneswar) + half-strength liquid
MS medium; (v) Soilrite mix (Kel-perlite, Karnataka Explosives, Bangalore)
+ half-strength liquid MS medium; (vi) garden soil + half-strength liquid MS
medium. Cultures were maintained at 25 ^ 18C and under continuous
illumination of 35 mmol m22 s21 photon flux density provided by white
fluorescent tubes. Media were autoclaved at 1218C for 15 min prior to use.
Each treatment consisted of 10 replicates (culture vessels) and the
experimental unit was six to eight seeds per vessel. Unencapsulated buds
were plated onto the above media and maintained under similar conditions
as a control. Response was evaluated in terms of the percentage of
encapsulated buds showing shoot emergence, and the percentage of shoots
that developed roots after 30 d culture. Each experiment was run three
times. Data presented here are percentage values alone, or pooled for
planting media or plant species. Data were subjected to analysis of variance
(ANOVA) using a factorial design. Percentage data were subjected to arcsin
transformation before analysis. Fisher's least significant difference was used
to separate the means for significant effects.

Results and Discussion

Of the two different commercial sources of sodium alginate used,

the best results with respect to capsule formation were obtained
with the product from Sigma. An optimal ion exchange between Na+
and Ca2+, producing firm, clear, isodiametric beads, was achieved
using a 4% solution of sodium alginate and 75 mM calcium
chloride (Fig. 1a). Lower concentrations of sodium alginate were not
Fig. 1. (a) Calcium alginate beads containing axillary shoot buds of
suitable because the capsules were too soft to handle, whilst at Ocimum sanctum. (b) A rooted shoot retrieved from an encapsulated axillary
higher concentrations the capsules were hard enough to cause bud of O. sanctum on vermi-compost moistened with half-strength MS
considerable delay in shoot emergence. The influence of the medium. (c) Plants of O. sanctum 4 wk after transfer to garden soil.
 ALGINATE ENCAPSULATION OF OCIMUM AXILLARY BUDS 289

TABLE 1A

PERCENTAGE OF SHOOT EMERGENCE FROM THE ENCAPSULATED AXILLARY BUDS OF OCIMUM SPECIES ON VARIOUS MEDIA

Ocimum species (percentage of shoot emergence)

Planting mediaa O. americanum O. basilicum O. gratissimum O. sanctum
MS BA 97 98 95 99
MS basal 43 32 15 52
TW AG 15 11 6 9
VC 12 MS 37 31 23 39
SM 12 MS 21 14 8 25
GS 12 MS 5 7 0 6

a
TW, tap water; AG, agar; VC, vermi-compost; SM, Soilrite mix; GS, garden soil; MS, Murashige and Skoog (1962) medium; MS BA; MS with
benzyladenine.

TABLE 1B

ANALYSIS OF VARIANCE SUMMARY

Source of variation d.f. Mean sum of squares F value

Species 3 2247.8 541.4**
Media 5 45761.8 11026.9**
Species  media 15 328.1 79.0**
Error 216 4.15

** Significant at P # 0:001.

commercial source from which alginate was obtained, as well as its

concentration on capsule quality and plant retrieval, has been
reported earlier (Mathur et al., 1989; Ghosh and Sen, 1994; Castillo Fig. 2. Percentage shoot emergence (pooled for four Ocimum species)
et al., 1998). from the encapsulated axillary buds on various planting media. TW, tap
water; AG, agar; VC, vermi-compost; SM, Soilrite mix; GS, garden soil; MS,
In vitro response of the encapsulated buds. The surface steriliza-
Murashige and Skoog (1962) medium; MS BA; MS with benzyladenine.
tion procedure followed yielded 9095% of axillary buds free of Bars with different letters are statistically significant at P 0:05:
contaminants. In vitro response of the encapsulated axillary buds of
the four Ocimum species to various media is documented in Table 1
and Figs. 2 and 3. Of the six different media tested, the frequency of
shoot emergence from the encapsulated buds was highest (9599%)
in MS medium supplemented with 1.14.4 mM BA, and the lowest
(07%) on garden soil moistened with half-strength MS medium
(Fig. 2). This held true for all the four Ocimum species selected for
the present study (Table 1). Shoots emerged from the encapsulated
buds, breaking the capsule wall within 914 d on BA-supplemen-
ted media (Fig. 4a,b). In O. americanum the maximum number of
encapsulated buds responded to a medium containing 1.1 mM BA,
whereas in O. gratissimum the best results with respect to shoot
emergence were recorded on a medium supplemented with 2.2 mM
BA. On the other hand, in O. basilicum and O. sanctum the
concentration of BA at which the maximum number of encapsulated
buds exhibited shoot development was 4.4 mM (Fig. 5a). Encapsu- Fig. 3. Percentage shoot emergence (pooled for six planting media)
lated buds of all four Ocimum species showed multiple shoot from encapsulated axillary buds of O. americanum (Oa), O. basilicum (Ob),
O. gratissimum (Og), and O. sanctum (Os). Bars with different letters are
development on BA-supplemented medium, but the maximum statistically significant at P 0:05:
number of shoots per bud (1214 shoots) was recorded in O.
sanctum (Fig. 5b). Shoot emergence took 1214 d on a growth-
regulator-free MS medium, and 1821 d on agar-solidified tap 39% on vermi-compost, 825% on Soilrite mix, and 07% on
water. The time required for shoot emergence was 1420 d on garden soil (Table 1). From each encapsulated bud a single shoot
vermi-compost moistened with half-strength MS medium, and 16 developed on these media regardless of Ocimum species. Of the four
30 d on Soilrite mix or garden soil moistened with half-strength MS species studied, O. americanum and O. sanctum were more
medium. The frequency of shoot emergence was 1552% on growth responsive and did not differ significantly from each other with
regulator-free MS medium, 615% on agar-solidified water, 23 respect to the frequency of shoot emergence. Ocimum gratissimum
290
Fig. 4. (a) Sprouting of an encapsulated axillary bud of O.
americanum after 10 d culture on MS BA (1.1 mM) bar 250 mm: (b)
Shoot emergence from an encapsulated axillary bud of O. gratissimum after
12 d culture on MS BA (2.2 mM) bar 250 mm:
was least responsive, and the frequency of shoot emergence was
significantly lower than in the other three Ocimum species (Fig. 3).
The unencapsulated buds (control) of the four Ocimum species
exhibited shoot development only on two media: MS BA and
growth-regulator-free MS. The frequency of shoot development was
8286% on BA-supplemented medium and 1239% on MS
medium devoid of BA. The unencapsulated buds failed to respond
morphogenically to the other four media and turned necrotic within
810 d of inoculation.
The encapsulated buds of the four Ocimum species could be
stored at 48C for 60 d. However, a decline in the frequency of shoot
emergence was observed following storage at low temperature. This
trend was noticed in all four Ocimum species. The frequency of
shoot emergence was 31, 22, 13 and 18% in O. americanum, O.
basilicum, O. gratissimum and O. sanctum, respectively, at the end
of 60 d (Fig. 6). The unencapsulated buds, on the other hand, did
not respond morphogenically even after 1 wk of storage.
Of the six media tested, root development from the sprouted buds
was observed only on MS medium free of growth regulators and on
vermi-compost moistened with half-strength MS medium. The time
required for root development and the percentage of shoots that
developed roots were dependent on the type of medium used
MANDAL ET AL.
Fig. 5. (a) Percentage shoot development from the encapsulated buds
of Ocimum species on media containing various concentrations of BA. (b)
Number of shoots per encapsulated bud of different Ocimum species on
media containing various concentrations of BA.
Fig. 6. Percentage shoot emergence from the encapsulated axillary
buds of different Ocimum species after 0, 30 and 60 d storage at 48C.
(Table 2). Of the shoots tested, 2135% rooted on a growth
regulator-free MS medium within 20 d, and two to three roots
developed from each shoot. Root development took 2530 d on
vermi-compost (Fig. 1b) and the percentage of rooting was 1521%.
Rooted shoots were retrieved from the encapsulated buds of O.
americanum, O. basilicum and O. sanctum on these two media,
whereas those of O. gratissimum failed to root (Fig. 7). Of the three
Ocimum species, the percentage of rooting was highest in O.
basilicum and lowest in O. sanctum (Fig. 7).
Hardening and field establishment. Rooted plantlets with four to
five fully expanded leaves, retrieved from encapsulated shoot buds
on a growth-regulator-free MS medium, were successfully hardened
off in vermi-compost inside the plant growth chamber for 2 wk
followed by a week in natural soil under shade, and finally
 ALGINATE ENCAPSULATION OF OCIMUM AXILLARY BUDS
TABLE 2
ANALYSIS OF VARIANCE SUMMARY FOR PERCENTAGE ROOTING
OF ENCAPSULATED BUDS OF OCIMUM SPECIES ON DIFFERENT
MEDIA
Source of variation d.f. Mean sum of squares F value
Species 2 501.7 58.3**
Media 1 1601.7 186.2**
Species  medium 1 81.6 9.4**
Error 54 8.6
** Significant at P # 0:001:
Fig. 7. Rooting response of shoots derived from encapsulated axillary
buds of O. americanum (Oa), O. basilicum (Ob), O. gratissimum (Og), and O.
sanctum (Os) on different media. Comparisons are made only between like-
patterned bars; bars with different letters are statistically significant at P
0:05:
transferred to the experimental garden. Plantlets retrieved from
encapsulated vegetative buds on vermi-compost were acclimatized
in natural soil for 3 wk (Fig. 1c) and successfully transplanted in
the garden. Mortality was ,5% in plants retrieved on vermi-
compost as against ,10% in the case of plants retrieved on MS
medium. Daughter plants were similar to donor plants with respect
to morphological features and growth characteristics.
The objective of the present investigation was to evaluate the
potential of the axillary buds for alginate encapsulation, and to use
the alginate-encapsulated buds for propagation as well as ex situ
conservation of these medicinal herbs. Axillary buds were used for
the encapsulation process because somatic embryogenesis has not
yet been documented in any Ocimum species. In the present study,
encapsulation of the axillary buds in calcium alginate gel was
successfully achieved, with subsequent growth on various media.
Encapsulation of axillary buds or shoot tips in calcium alginate has
been reported in a few herbaceous (Mathur et al., 1989; Sharma
et al., 1994) and woody (Bapat et al., 1987; Piccioni and Standardi,
1995) species. However, in all the above cited reports, plantlets
raised in vitro were the source of explants for encapsulation. In the
present investigation, explants taken directly from garden-grown
plants were used for synthetic seed production. Therefore, our
procedure is much simpler than those previously reported because
it bypasses the need to establish axenic cultures and thereby
reduces the production cost of synthetic seeds. High-frequency
conversion of synthetic seeds into plantlets on simple planting
substrates such as sand/soil/Soilrite is necessary for their use in
291
commercial-scale propagation. However, to date, only a few reports
are available on the conversion of encapsulated somatic embryos or
shoot buds on such substrates (Redenbaugh et al., 1986; Mathur
et al., 1989; Pattnaik et al., 1995). In the present study, the
conversion frequency of alginate-encapsulated buds was higher on
an agar-based, nutrient-rich medium than on media with reduced
nutrients (vermi-compost/Soilrite/soil). The major limiting factor for
continued development of the encapsulated plant tissue into
complete plantlets could be the availability of nutrients. Therefore,
it is necessary to build up a nutrient reservoir for the encapsulated
plant tissue, either endogenously or exogenously, in order to
improve the conversion frequency (Fujii et al., 1987). The use of
alginate-encapsulated somatic embryos or shoot buds for the
purpose of germplasm conservation has been attempted for a few
plant species (Ghosh and Sen, 1994; Pattnaik et al., 1995; Janeiro
et al., 1997; Maruyama et al., 1997). The conversion frequency of
encapsulated somatic embryos of Asparagus cooperi (Ghosh and
Sen, 1994) and Camellia japonica (Janeiro et al., 1997) or shoot
buds of Morus species (Pattnaik et al., 1995) declined markedly
following storage at low temperature, whereas alginate-encapsulated
shoot tips of Cedrela odorata, Guazama crinita and Jacaranda
mimosaefolia could be stored for 612 mo. at 12258C without
marked loss of viability (Maruyama et al., 1997). In the present
investigation alginate-encapsulated buds could retain their viability
for up to 60 d, although frequency of shoot emergence declined
markedly. Research is now in progress to enhance the frequency of
plant retrieval from the encapsulated buds on natural planting
substrates and to preserve them at low temperature for longer
durations without markedly affecting viability.
Acknowledgments
Jaydip Mandal wishes to express gratitude to the Director,
NCERT, New Delhi for grant of study leave from Regional Institute
of Education, Bhubaneswar to conduct Ph.D. research work.
References
Anon. Wealth of India. A dictionary of Indian raw materials and industrial
products, Vol. VII (NPe). New Delhi, India: CSIR; 1966; 7989.
Ballester, A.; Janeiro, L. V.; Vieitez, A. M. Storage of shoot cultures and
alginate encapsulation of shoot tips of Camellia japonica and C.
reticulata Lindley. Sci. Hortic. 71:6778; 1997.
Bapat, V. A.; Mhatre, M.; Rao, P. S. Propagation of Morus indica L.
(mulberry) by encapsulated shoot buds. Plant Cell Rep. 6:393395;
1987.
Castillo, B.; Smith, M. A. L.; Yadava, U. L. Plant regeneration from
encapsulated somatic embryos of Carica papaya L. Plant Cell Rep.
17:172176; 1998.
Fowke, L. C.; Attree, S. M.; Pomeroy, M. K. Production of vigorous
desiccation tolerant white spruce (Picea glauca [Moench] Voss.)
synthetic seeds in a bioreactor. Plant Cell Rep. 13:601606; 1994.
Fujii, J. A.; Slade, D.; Redenbaugh, K.; Walker, K. A. Artificial seeds for
plant propagation. Trends Biotech. 5:335339; 1987.
Ghosh, B.; Sen, S. Plant regeneration from alginate encapsulated somatic
embryos of Asparagus cooperi Barker. Plant Cell Rep. 13:381385;
1994.
Gray, D. J.; Compton, M. E.; Harrell, R. C.; Cantliffe, D. J. Somatic
embryogenesis and the technology of synthetic seed. In: Bajaj, Y. P. S.,
ed. Biotechnology in agriculture and forestry. Somatic embryo-
genesis and synthetic seeds I. Vol. 30. Berlin, Germany: Springer-
Verlag; 1995; 126151.
Janeiro, L. V.; Ballester, A.; Vieitez, A. M. In vitro response of encapsulated
292 MANDAL ET AL.
somatic embryos of camellia. Plant Cell Tiss. Org. Cult. 51:119126;
1997.
Kitto, S. L.; Janick, J. Polyox as an artificial seed coat for a sexual embryos.
Hort. Sci. 17:448; 1982.
Maruyama, E.; Kinoshita, I.; Ishii, K.; Ohba, K.; Saito, A. Germplasm
conservation of the tropical forest trees, Cedrela odorata L.,
Guazuma crinita Mart. and Jacaranda mimosaefolia D. Don., by
shoot tip encapsulation in calcium alginate and storage at 12258C.
Plant Cell Rep. 16:393396; 1997.
Mathur, J.; Ahuja, P. S.; Lal, N.; Mathur, A. K. Propagation of Valeriana
wallichii DC using encapsulated apical and axial shoot buds. Plant
Sci. 60:111116; 1989.
Murashige, T. Plant cell and organ cultures as horticultural practices. Acta
Hort. 78:17; 1977.
Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays
with tobacco tissue cultures. Physiol. Plant. 15:473497; 1962.
Onay, A.; Jeffree, C. E.; Yeoman, M. M. Plant regeneration from
encapsulated embryoids and embryogenic mass of pistachio,
Pistachia vera L. Plant Cell Rep. 15:723726; 1996.
Patnaik, J.; Debata, B. K. Germination of the artificial seeds of Cymbopogon
martinii. In: Pank, F., ed. Proceedings of an international
symposium: breeding research on medicinal and aromatic plants,
Quedlinburg, Germany; 1996; 330333.
Pattnaik, S. K.; Sahoo, Y.; Chand, P. K. Efficient plant retrieval from
alginate encapsulated vegetative buds of mature mulberry trees. Sci.
Hortic. 61:227239; 1995.
Piccioni, E.; Standardi, A. Encapsulation of micropropagated buds of six
woody species. Plant Cell Tiss. Org. Cult. 42:221226; 1995.
Redenbaugh, K. Synseeds: application of synthetic seeds for crop
improvement. Boca Raton, USA: CRC Press; 1993; .
Redenbaugh, K.; Nichol, J.; Kossler, M. E.; Paasch, B. D. Encapsulation of
somatic embryos for artificial seed production. In Vitro Cell Dev.
Biol. 20:256257; 1984.
Redenbaugh, K.; Paasch, B. D.; Nichol, J. W.; Kossler, M. E.; Viss, P. R.;
Walker, K. A. Somatic seeds: encapsulation of asexual plant
embryos. Bio/Technol. 4:797801; 1986.
Sarkar, D.; Naik, P. S. Synseeds in potato: an investigation using nutrient
encapsulated in vitro nodal segments. Sci. Hortic. 73:179184;
1998.
Sharma, T. R.; Singh, B. M.; Chauhan, R. S. Production of disease free
encapsulated buds of Zingiber officinale Rosc. Plant Cell Rep.
13:300302; 1994.