Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol84.Issue1-2.Apr2000:
Scientia Horticulturae 84 (2000) 15±26
Effects of sugar concentration and strength of
basal medium on conversion of somatic
embryos in Asparagus of®cinalis L.
Kanji Mamiyaa,*, Yuji Sakamotob
a
Central Laboratories for Key Technology, Kirin Brewery Co. Ltd., 3377 Kitsuregawa-machi,
Shioya-gun, Tochigi-ken 329-1491, Japan
b
Applied Research Center, Kirin Brewery Co. Ltd., 3 Miyaharacho, Takasaki 370-1202, Japan
Accepted 28 July 1999
Abstract
The effects of sugar concentration and strength of basal medium were studied to produce plants
from somatic embryos in Asparagus of®cinalis L. There was a signi®cant difference among
concentrations of sugar but not among kinds of sugar tested in the present experiment in growth of
shoots and roots. When the sucrose concentrations were 10, 30, or 50 g lÿ1, the fresh weight of
shoots were 31.5, 14.9, or 8.6 mg per plant and the fresh weight of roots were 14.5, 33.7, or 46.3 mg
per plant, respectively. There was a signi®cant difference among strength of basal medium in shoot
growth but not in root growth. When somatic embryos were cultured in a half, full, or twice the
strength of basal medium, the fresh weight of shoots were 8.9, 31.0, or 60.0 mg per plant,
respectively.
The effects of sugar concentration and strength of basal medium were also studied in the postculture process of somatic embryos to produce encapsulatable units, and in the conversion process
of them. Not only the sugar concentration in the conversion medium but also the concentration in
the post-culture medium had signi®cant effects on growth of shoots and roots. No signi®cant
difference was observed among strength of basal medium in the post-culture process. # 2000
Elsevier Science B.V. All rights reserved.
Keywords: Asparagus of®cinalis L.; Encapsulatable unit; Post-culture; Root; Shoot; Somatic
embryo; Synthetic seed
*
Corresponding author. Tel.: 81-28-686-4511; fax: 81-28-686-5060.
E-mail address: k-mamiya@kirin.co.jp (K. Mamiya).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 9 8 - 9
16
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
1. Introduction
Somatic embryogenesis occurs in many species. Many clonal propagation
methods using somatic embryogenesis have been tried in Asparagus of®cinalis L.
(Saito et al., 1991; Delbreil et al., 1994; Odake et al., 1993; Kohmura et al.,
1994), because this plant is dioecious and has a high genetic heterogeneity within
cultivars. However, many studies focused on the production of somatic embryos
and little was studied about the factors which affect growth of plants from
somatic embryos. Sugar and basal medium are necessary components of plant
tissue culture medium. Sugar concentration was studied to produce asparagus
plants in minicrown (Conner and Falloon, 1993) and in organ formation from
shoot segments (Harada and Yakuwa, 1983). Levi and Sink (1990) studied kind
and concentration of sugar in medium for initiation of embryogenic calli and subculture of them, but they did not study in conversion medium for production of
plants. There is no report about the effects of sugar or basal medium in conversion
process of somatic embryos in Asparagus of®cinalis L.
Improvement in somatic embryogenesis will lead to synthetic seed technology.
Encapsulatable units (EUs) with high conversion ability is indispensable for
synthetic seed (Redenbaugh et al., 1986). When post-cultured somatic embryos
are used as EUs, synthetic seeds have a high conversion ability in celery (Onishi
et al., 1992) and in carrot (Onishi et al., 1994). Although asparagus is one target
species for propagation by synthetic seed technology, no reports exist of the
synthetic seeds or the post-culture method in Asparagus of®cinalis L.
First, we studied sugar concentration and strength of basal medium to produce
plants from somatic embryos, and second we studied their effects in the postculture medium to produce EUs and discussed about the application to synthetic
seed technology in Asparagus of®cinalis L.
In this paper, we have used the following abbreviations: 2,4-D, 2,4dichlorophenoxyacetic acid; EU, encapsulatable unit; MES, 2-(N-morpholino)
ethanesulfonic acid; MS, Murashige and Skoog.
2. Materials and methods
2.1. Production of somatic embryos
Asparagus cultivar ``Fest'' (bred by Hokkai Seikan, Japan) was used as plant
material. Fest is a clonal cultivar of a selected male plant with superior agronomic
characteristics. In vitro plants were initiated by meristem culture (Reuther, 1984)
and they were maintained on MS medium (Murashige and Skoog, 1962)
containing 30 g lÿ1 sucrose, 1 g lÿ1 MES and 4 g lÿ1 gelrite. The temperature was
258C and the photosynthetic photon ¯ux was 60 mmol mÿ2 sÿ1 at 16 h
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
17
photoperiod. The pH of all kinds of media was adjusted to 6.0 before autoclaving.
The suspension cultures were initiated by inoculating two to three pieces of in
vitro storage roots in 70 ml MS liquid medium containing 40 g lÿ1 sucrose, 30 g lÿ1
mannitol, 9.0 mM 2,4-D, 12 mM proline, 0.1 g lÿ1 casein acid hydrolysate and
1 g lÿ1 MES. The roots were 3±4 cm long and about 1 mm in diameter and had
several lateral roots. Erlenmeyer ¯asks of 500 ml total volume were used as
culture vessels. After inducing the suspensions, 0.5 g of the cell clusters that
passed through a 1.0±1.4 mm mesh were sub-cultured every 3 weeks in 70 ml MS
medium containing 30 g lÿ1 sucrose, 30 g lÿ1 sorbitol, 4.5 mM 2,4-D, 0.9 mM
kinetin, 0.1 g lÿ1 casein acid hydrolysate and 1 g lÿ1 MES. To induce and proliferate the suspensions, the temperature was 308C and the ¯asks were kept under
5±10 mmol mÿ2 sÿ1 photosynthetic photon ¯ux at 120 rpm on a rotary shaker.
The cell clusters remaining on the 1.0±1.4 mm mesh were used to produce
somatic embryos by inoculating 0.02 g to 40 ml MS liquid medium containing
10 g lÿ1 sucrose, 30 g lÿ1 sorbitol, 2 g lÿ1 casein acid hydrolysate and 1 g lÿ1
MES. The temperature was 308C and the ¯asks were kept at 90 rpm on a rotary
shaker. Bipolar, rod-shaped embryos were collected after 3 weeks and 6±8 g of
them were dehydrated on 2±3 layers of sterilized ®lter paper in 9 cm petri dishes
for 3±5 days until the surfaces of the embryos became dry. During dehydration,
the temperature was 258C and the photosynthetic photon ¯ux was
60 mmol mÿ2 sÿ1 at 16 h photoperiod. These somatic embryos were used as
materials hereafter.
2.2. Effects of kind and concentration of sugar
Somatic embryos were cultured in MS liquid media with glucose, fructose, or
sucrose, with each sugar at the three concentrations of 10, 30, or 50 g lÿ1. Only
these media were ®lter sterilized because toxic compounds are produced from
glucose or fructose by autoclaving (Sawyer and Hsiao, 1992). In the experiments
hereafter, one somatic embryo was cultured in a well of multiple well plate
(Corning, 24 wells, 16 mm in diameter) containing 1 ml medium by static culture.
The temperature was 258C and the photosynthetic photon ¯ux was
60 mmol mÿ2 sÿ1 at 16 h photoperiod.
To study osmotic effects, somatic embryos were cultured in MS media with
10 g lÿ1 sucrose and 0, 10, or 20 g lÿ1 of sorbitol. The fresh weight of shoots and
roots were measured after three weeks.
2.3. Effects of strength of basal medium
Somatic embryos were cultured in a half, full or twice the strength of MS
medium with 30 g lÿ1 sucrose. The fresh weight of shoots and roots were
measured after three weeks.
18
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
2.4. Effects of sugar concentration and strength of basal medium in the postculture process and the conversion process
The somatic embryos were post-cultured to produce EUs in MS media
containing 10, 30, or 50 g lÿ1 of sucrose for one week. The conversion ability
of the EUs was evaluated by transferring the embryos to MS media containing
0, 10, 30, or 50 g lÿ1 of sucrose to produce plants. Two weeks later, the weight
of the shoots and roots were measured. In another experiment, the somatic
embryos were post-cultured in a half, full or twice the strength of MS medium
with 30 g lÿ1 sucrose for one week. The conversion ability of the EUs was
evaluated by transferring to a half, full or twice the strength of MS medium
with 30 g lÿ1 sucrose. These media were simulators of synthetic seed coats
with nutrients. A sterile tissue culture medium was ideal to evaluate the
maximum ability of EUs because of no microbial contamination or out¯ow
of nutrients.
2.5. Sugar quanti®cation
One post-cultured somatic embryo was squashed and sugar was extracted three
times in 80% hot ethanol at 808C for 10 min. The extracts were combined
together and lyophilized. The samples were stored at ÿ208C until analysis. Five
hundred ml of distilled water was added and the concentrations of sucrose,
glucose and fructose were measured using Boehringer-Mannheim sucrose/
glucose/fructose test kits.
3. Results
3.1. Effects of kind and concentration of sugar
By our method, 1 g suspension cell clusters produced 5000±7000 bipolar, rodshaped embryos, which were used as materials. The percent conversion of these
embryos was an average 90% in this study. Conversion means that plants develop
healthy shoots and roots. Little callus was induced on somatic embryos on the
conversion medium.
Fig. 1 shows the effects of kind and concentration of sugar on the growth of
shoots and roots. The effects were concentration dependent and were similar
among the three sugars. Analysis of variance showed a highly signi®cant
difference among the concentrations of sugar in shoots and roots. However,
neither a signi®cant difference among the kinds of sugar nor a signi®cant
interaction was shown. The shoots grew less, but the roots grew more with
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
19
Fig. 1. Fresh weight of shoots and roots after three weeks in media with different kinds and
concentrations of sugar. In each treatment 24 plants were studied: shoot (darkly shaded); root (block
marked with diagonal lines). Analysis of variance in shoot: kind of sugar (not signi®cant), concentration of sugar (p < 0.001), interaction between kind and concentration (not signi®cant). LSD
(0.05) for shoot was 12.80. Analysis of variance in root: kind of sugar (not signi®cant), concentration of sugar (p < 0.001), interaction between kind and concentration (not signi®cant). LSD
(0.05) for root was 12.46.
increase in the concentrations. The appearance of shoots showed no difference
among all media. The roots that developed in media with 30 or 50 g lÿ1 sugar
were thick and we considered them to be storage roots, but those in media with
10 g lÿ1 of sugar were thin.
Part of the effects might be attributed to osmotic pressure. Then sorbitol was
used to change osmotic pressure. Fig. 2 shows the effects of sorbitol. Analysis of
variance showed a highly signi®cant difference among sorbitol concentrations in
shoot growth, but not in root growth. Shoots grew less with increase in sorbitol
concentrations.
No difference was shown in the effects of concentration among the three
sugars. Therefore only sucrose was used in the following experiments of 3.2, 3.3
and 3.4.
3.2. Effects of strength of basal medium
Fig. 3 shows the effects of strength of basal medium on the growth of shoots
and roots. Analysis of variance showed a highly signi®cant difference among
strength of basal medium on shoot growth, but not in root growth. Shoots grew
more with increase in the strength.
20
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
Fig. 2. Fresh weight of shoots and roots after three weeks in media with different concentrations of
sorbitol. All media contained 10 g lÿ1 of sucrose. In each treatment 24 somatic embryos were
studied: shoot (darkly shaded); root (block marked with diagonal lines). Analysis of variance in
shoot (p < 0.001). LSD (0.05) for shoot was 13.28. Analysis of variance in root (not signi®cant).
3.3. Effects of sugar concentration and strength of basal medium in the postculture process and the conversion process
The effects of sugar in the post-culture process and the conversion process on
the growth of shoots and roots were shown in Fig. 4A and B. Both in shoots and
in roots, highly signi®cant differences were shown among the concentrations
Fig. 3. Fresh weight of shoots and roots after three weeks in media with different strength of basal
medium. All media contained 30 g lÿ1 of sucrose. In each treatment 24 plants were studied: shoot
(darkly shaded); root (block marked with diagonal lines). Analysis of variance in shoot (p < 0.001).
LSD (0.05) for shoot was 21.12. Analysis of variance in root (not signi®cant).
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
21
Fig. 4. Growth of shoots and roots after one week of post-culture in three sucrose concentrations
followed by two weeks in conversion media at four sucrose concentrations. X-axis shows ``sucrose
concentration in the post-culture media Ð the concentration in the conversion media''. (A) Fresh
weight of shoots. (B) Fresh weight of roots. In each treatment 24 plants were studied. Analysis of
variance in shoot: post-culture media (p < 0.01), conversion media (p < 0.001), interaction between
post-culture media and conversion media (p < 0.05). LSD (0.05) for shoot was 15.31. Analysis of
variance in root: post-culture media (p < 0.001), conversion media (p < 0.001), interaction between
post-culture media and conversion media (not signi®cant). LSD (0.05) for root was 15.77.
22
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
in the post-culture process and the conversion process. A signi®cant interaction
was shown in shoots, but not in roots. These results show that sugar concentration in the post-culture medium affected the growth of plants in the conversion
process. The effects of sugar in the post-culture medium on shoot growth
differed depending on the presence of sugar in the conversion process. Shoots
grew more with increase in sugar concentrations in the post-culture medium
when no sugar was in the conversion process. However, the growth of shoots
was suppressed with increase in the concentrations when sugar was in the
conversion process. Roots grew more with increase in the concentrations in
the post-culture medium regardless of the presence of sugar in the conversion
process.
The sugar supplied in the conversion process also affected the growth of plants.
When no sugar was in the conversion process, the growth of plants was poor. The
effects of concentration in the conversion process were similar to the results in
Fig. 1. The optimal concentration for shoot growth was 10 g lÿ1 and those for
roots were 30±50 g lÿ1, regardless of the sucrose concentration in the post-culture
process. When 50 g lÿ1 of sucrose was added in the post-culture process and the
conversion process, the color of some shoots became pale pink and the shoots did
not grow well.
Fig. 5. Growth of shoots after one week of post-culture in three strength of basal medium followed
by two weeks in conversion media at three strength of basal medium. X-axis shows ``strength of
basal medium in the post-culture media Ð the strength in the conversion media''. In each treatment
24 plants were studied. Analysis of variance: post-culture media (not signi®cant), conversion media
(p < 0.001), interaction between post-culture media and conversion media (not signi®cant). LSD
(0.05) was 50.46.
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
23
Fig. 6. Sugar content in somatic embryos after one week of post-culture in media with different
sucrose concentrations. In each treatment 22 somatic embryos were studied: Fructose (darkly
shaded), sucrose (block marked with diagonal lines), glucose (lightly shaded). Analysis of variance
in total sugar content (p < 0.001). LSD (0.05) was 3.04.
Fig. 5 shows the effects of strength of basal medium in the post-culture process
and the conversion process on growth of shoots. Only the growth of shoots was
shown because the effects on roots was not signi®cant as shown in Fig. 3. In
growth of shoots, there was a highly signi®cant difference among the treatments
in the conversion process, but not in the post-culture process.
3.4. Sugar quanti®cation
The content of sucrose, glucose and fructose in the post-cultured somatic
embryos were shown in Fig. 6. When somatic embryos were post-cultured in
media with higher sucrose concentrations, they contained more sugar per fresh
weight. About 2/3 of the sugar was sucrose, and the rests were glucose and
fructose. The amount of glucose was slightly higher than that of fructose.
4. Discussion
The results of this study show that the growth of shoots and roots from somatic
embryos could be controlled by sugar concentration and strength of basal
medium. Lower concentrations of sugar and higher strength of basal medium
promoted the growth of shoots, but higher concentrations of sugar promoted the
growth of roots and inhibited the growth of shoots. The strength of basal medium
tested in this study did not affect the growth of roots. Our results of sugar
24
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
concentration in somatic embryos were similar to reports of sugar concentrations
in the minicrown (Conner and Falloon, 1993) and in the organ formation from
shoot segments (Harada and Yakuwa, 1983), although their reports did not show
numerical and statistical data. The kind of sugar tested in our study did not
affected the growth of shoots and roots. This result differed from the results of
Levi and Sink (1990). They studied the medium for initiation of embryogenic
calli and sub-culture of them; they found that glucose promoted root growth,
fructose promoted shoot growth, sucrose promoted both shoot and root growth.
However, their report did not show numerical and statistical data either, and
they did not study the conversion medium. From the results in Fig. 2, it was
suggested that increased osmotic pressure inhibited the shoot growth, but the
growth of roots was not affected by osmotic pressure tested in this study. We
studied sucrose concentration in the conversion process of somatic embryos in
carrot, but we could not observe the effects shown in asparagus (Mamiya,
unpublished results).
Storage roots are indispensable for acclimatization of in vitro plants in
asparagus (Conner et al., 1992). In our study, plants produced in media with 30±
50 g lÿ1 of sugar survived after acclimatization, but most plants produced in a
medium with 10 g lÿ1 of sugar did not survive (Mamiya, unpublished results).
The conditions suitable for growth was different between shoots and roots.
Selecting a condition suitable for root growth, namely a higher concentration of
sugar, is better.
When the ability of EUs are evaluated after encapsulation, problems that
disturb the evaluation will arise. Those are physical inhibition of conversion
from capsules, dif®culty to supply suf®cient nutrition without microbial
contamination in capsules. By using a tissue culture medium as a simulator of
synthetic seed coat with nutrients, the maximum ability of the EUs can be
evaluated. Our results show that sugar concentration in the post-culture process
affects the growth of plants in the conversion process, and that media with
30±50 g lÿ1 of sugar are suitable to produce EUs that can develop more roots.
The EUs produced in media with more sucrose contained more glucose, fructose
and sucrose than those produced in media with less sucrose. The sugar in the EUs
might affect the later growth in the conversion process. The strength of basal
medium in the post-culture process did not affect the growth of shoots in the
conversion process.
When sugar was not added in the conversion process, the growth of plants was
poor, which is reasonable because the seeds of asparagus are albuminous.
Therefore during the conversion process, sugar is necessary for synthetic seeds
of asparagus. Suitable microcapsules exist that release sucrose or MS salts
gradually (Sakamoto et al., 1991). When nutrient microcapsules are used for
synthetic seeds of asparagus, our results will be useful to determine the
concentration and to predict the growth pattern of the plants. In the germination
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
25
of natural seeds, storage roots develop ®rst and shoots grow later. This growth
pattern is mimicked in synthetic seeds by regulating the sugar and strength of
basal medium.
In conclusion, we studied the effects of sugar concentration and strength of
basal medium, and found that these factors affected the growth of shoots and
roots. We also found that the sugar concentration in the post-culture medium
affected the growth of plants in the conversion process. The results of this study
will be useful to produce plants or synthetic seeds from somatic embryos in
Asparagus of®cinalis L.
Acknowledgements
We thank K. Watanabe, Hokkai Seikan Co. Ltd., for providing the cultivar
`Fest'. We also thank many members in Plant Laboratory, Kirin Brewery Co. Ltd.
for their kind help.
References
Conner, A.J., Falloon, P.G., 1993. Osmotic versus nutritional effects when rooting in vitro asparagus
minicrown on high sucrose media. Plant Sci. 89, 101±106.
Conner, A.J., Aberneithy, D.J., Falloon, P.G., 1992. Importance of in vitro storage root development
for the successful transfer of micropropagated asparagus plants to greenhouse. NZ. J. Crop Hort.
Sci. 20, 477±481.
Delbreil, B., Goebel-Tourand, I., Lefranc,ois, C., Jullien, M., 1994. Isolation and characterization of
long-term embryogenic lines in Asparagus of®cinalis L.. J. Plant Physiol. 144, 194±200.
Harada, T., Yakuwa, T., 1983. Studies on the morphogenesis of Asparagus VI. Effect of sugar on
callus and organ formation in the in vitro culture of shoot segments of the seedlings. J. Fac. Agr.
Hokkaido Univ. 61, 307±314.
Kohmura, H., Chokyu, S., Harada, T., 1994. An effective micro-propagation system using
embryogenic calli induced from bud clusters in Asparagus of®cinalis L. J. Jpn. Soc. Hort. Sci.
63, 51±59.
Levi, A., Sink, K.C., 1990. Differential effects of sucrose, glucose and fructose during somatic
embryogenesis in asparagus. J. Plant Physiol. 137, 184±189.
Murashige, T., Skoog, F., 1962. A revised medium for rapid growth bioassay with tobacco tissue
cultures. Physiol. Plant. 15, 473±497.
Odake, Y., Udagawa, A., Saga, H., Mii, M., 1993. Somatic embryogenesis of tetraploid plants from
internodal segments of diploid cultivar of Asparagus of®cinalis L. grown in liquid culture. Plant
Sci. 94, 173±177.
Onishi, N., Mashiko, T., Okamoto, A., 1992. Culture system producing encapsulatable units of
synthetic seeds in celery. Acta Horti. 319, 113±118.
Onishi, N., Sakamoto, Y., Hirosawa, T., 1994. Synthetic seeds as a application of mass production
of somatic embryos.. Plant Cell Tiss Org. Cult. 39, 137±145.
Redenbaugh, K., Paasch, B.D., Nichol, J.W., Kossler, M.E., Viss, P.R., Walker, K.A., 1986. Somatic
seed: encapsulation of asexual plant embryos. Biotechnology 4, 797±801.
26
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
Reuther, G., 1984. Asparagus. In: Sharp, W.R., Evans, D.A., Ammirato, P.V., Yamada, Y. (Eds.),
Handbook of Plant Cell Culture, vol. 2. Macmillan, New York, pp. 211±242.
Saito, T., Nishizawa, S., Nishimura, S., 1991. Improved culture conditions for somatic
embryogenesis from Asparagus of®cinalis L. using an aseptic ventilative ®lter. Plant Cell
Rep. 10, 230±234.
Sakamoto, Y., Umeda, S., Ogishima, H., 1991. Arti®cial seed comprising a sustained-release
granule. United States Patent 5 010 865.
Sawyer, H., Hsiao, K-C., 1992. Effects of autoclave-induced carbohydrate hydrolysis on the growth
of Beta vulgaris cells in suspension. Plant Cell Tiss Org. Cult. 31, 81±86.
Effects of sugar concentration and strength of
basal medium on conversion of somatic
embryos in Asparagus of®cinalis L.
Kanji Mamiyaa,*, Yuji Sakamotob
a
Central Laboratories for Key Technology, Kirin Brewery Co. Ltd., 3377 Kitsuregawa-machi,
Shioya-gun, Tochigi-ken 329-1491, Japan
b
Applied Research Center, Kirin Brewery Co. Ltd., 3 Miyaharacho, Takasaki 370-1202, Japan
Accepted 28 July 1999
Abstract
The effects of sugar concentration and strength of basal medium were studied to produce plants
from somatic embryos in Asparagus of®cinalis L. There was a signi®cant difference among
concentrations of sugar but not among kinds of sugar tested in the present experiment in growth of
shoots and roots. When the sucrose concentrations were 10, 30, or 50 g lÿ1, the fresh weight of
shoots were 31.5, 14.9, or 8.6 mg per plant and the fresh weight of roots were 14.5, 33.7, or 46.3 mg
per plant, respectively. There was a signi®cant difference among strength of basal medium in shoot
growth but not in root growth. When somatic embryos were cultured in a half, full, or twice the
strength of basal medium, the fresh weight of shoots were 8.9, 31.0, or 60.0 mg per plant,
respectively.
The effects of sugar concentration and strength of basal medium were also studied in the postculture process of somatic embryos to produce encapsulatable units, and in the conversion process
of them. Not only the sugar concentration in the conversion medium but also the concentration in
the post-culture medium had signi®cant effects on growth of shoots and roots. No signi®cant
difference was observed among strength of basal medium in the post-culture process. # 2000
Elsevier Science B.V. All rights reserved.
Keywords: Asparagus of®cinalis L.; Encapsulatable unit; Post-culture; Root; Shoot; Somatic
embryo; Synthetic seed
*
Corresponding author. Tel.: 81-28-686-4511; fax: 81-28-686-5060.
E-mail address: k-mamiya@kirin.co.jp (K. Mamiya).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 9 8 - 9
16
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
1. Introduction
Somatic embryogenesis occurs in many species. Many clonal propagation
methods using somatic embryogenesis have been tried in Asparagus of®cinalis L.
(Saito et al., 1991; Delbreil et al., 1994; Odake et al., 1993; Kohmura et al.,
1994), because this plant is dioecious and has a high genetic heterogeneity within
cultivars. However, many studies focused on the production of somatic embryos
and little was studied about the factors which affect growth of plants from
somatic embryos. Sugar and basal medium are necessary components of plant
tissue culture medium. Sugar concentration was studied to produce asparagus
plants in minicrown (Conner and Falloon, 1993) and in organ formation from
shoot segments (Harada and Yakuwa, 1983). Levi and Sink (1990) studied kind
and concentration of sugar in medium for initiation of embryogenic calli and subculture of them, but they did not study in conversion medium for production of
plants. There is no report about the effects of sugar or basal medium in conversion
process of somatic embryos in Asparagus of®cinalis L.
Improvement in somatic embryogenesis will lead to synthetic seed technology.
Encapsulatable units (EUs) with high conversion ability is indispensable for
synthetic seed (Redenbaugh et al., 1986). When post-cultured somatic embryos
are used as EUs, synthetic seeds have a high conversion ability in celery (Onishi
et al., 1992) and in carrot (Onishi et al., 1994). Although asparagus is one target
species for propagation by synthetic seed technology, no reports exist of the
synthetic seeds or the post-culture method in Asparagus of®cinalis L.
First, we studied sugar concentration and strength of basal medium to produce
plants from somatic embryos, and second we studied their effects in the postculture medium to produce EUs and discussed about the application to synthetic
seed technology in Asparagus of®cinalis L.
In this paper, we have used the following abbreviations: 2,4-D, 2,4dichlorophenoxyacetic acid; EU, encapsulatable unit; MES, 2-(N-morpholino)
ethanesulfonic acid; MS, Murashige and Skoog.
2. Materials and methods
2.1. Production of somatic embryos
Asparagus cultivar ``Fest'' (bred by Hokkai Seikan, Japan) was used as plant
material. Fest is a clonal cultivar of a selected male plant with superior agronomic
characteristics. In vitro plants were initiated by meristem culture (Reuther, 1984)
and they were maintained on MS medium (Murashige and Skoog, 1962)
containing 30 g lÿ1 sucrose, 1 g lÿ1 MES and 4 g lÿ1 gelrite. The temperature was
258C and the photosynthetic photon ¯ux was 60 mmol mÿ2 sÿ1 at 16 h
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
17
photoperiod. The pH of all kinds of media was adjusted to 6.0 before autoclaving.
The suspension cultures were initiated by inoculating two to three pieces of in
vitro storage roots in 70 ml MS liquid medium containing 40 g lÿ1 sucrose, 30 g lÿ1
mannitol, 9.0 mM 2,4-D, 12 mM proline, 0.1 g lÿ1 casein acid hydrolysate and
1 g lÿ1 MES. The roots were 3±4 cm long and about 1 mm in diameter and had
several lateral roots. Erlenmeyer ¯asks of 500 ml total volume were used as
culture vessels. After inducing the suspensions, 0.5 g of the cell clusters that
passed through a 1.0±1.4 mm mesh were sub-cultured every 3 weeks in 70 ml MS
medium containing 30 g lÿ1 sucrose, 30 g lÿ1 sorbitol, 4.5 mM 2,4-D, 0.9 mM
kinetin, 0.1 g lÿ1 casein acid hydrolysate and 1 g lÿ1 MES. To induce and proliferate the suspensions, the temperature was 308C and the ¯asks were kept under
5±10 mmol mÿ2 sÿ1 photosynthetic photon ¯ux at 120 rpm on a rotary shaker.
The cell clusters remaining on the 1.0±1.4 mm mesh were used to produce
somatic embryos by inoculating 0.02 g to 40 ml MS liquid medium containing
10 g lÿ1 sucrose, 30 g lÿ1 sorbitol, 2 g lÿ1 casein acid hydrolysate and 1 g lÿ1
MES. The temperature was 308C and the ¯asks were kept at 90 rpm on a rotary
shaker. Bipolar, rod-shaped embryos were collected after 3 weeks and 6±8 g of
them were dehydrated on 2±3 layers of sterilized ®lter paper in 9 cm petri dishes
for 3±5 days until the surfaces of the embryos became dry. During dehydration,
the temperature was 258C and the photosynthetic photon ¯ux was
60 mmol mÿ2 sÿ1 at 16 h photoperiod. These somatic embryos were used as
materials hereafter.
2.2. Effects of kind and concentration of sugar
Somatic embryos were cultured in MS liquid media with glucose, fructose, or
sucrose, with each sugar at the three concentrations of 10, 30, or 50 g lÿ1. Only
these media were ®lter sterilized because toxic compounds are produced from
glucose or fructose by autoclaving (Sawyer and Hsiao, 1992). In the experiments
hereafter, one somatic embryo was cultured in a well of multiple well plate
(Corning, 24 wells, 16 mm in diameter) containing 1 ml medium by static culture.
The temperature was 258C and the photosynthetic photon ¯ux was
60 mmol mÿ2 sÿ1 at 16 h photoperiod.
To study osmotic effects, somatic embryos were cultured in MS media with
10 g lÿ1 sucrose and 0, 10, or 20 g lÿ1 of sorbitol. The fresh weight of shoots and
roots were measured after three weeks.
2.3. Effects of strength of basal medium
Somatic embryos were cultured in a half, full or twice the strength of MS
medium with 30 g lÿ1 sucrose. The fresh weight of shoots and roots were
measured after three weeks.
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K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
2.4. Effects of sugar concentration and strength of basal medium in the postculture process and the conversion process
The somatic embryos were post-cultured to produce EUs in MS media
containing 10, 30, or 50 g lÿ1 of sucrose for one week. The conversion ability
of the EUs was evaluated by transferring the embryos to MS media containing
0, 10, 30, or 50 g lÿ1 of sucrose to produce plants. Two weeks later, the weight
of the shoots and roots were measured. In another experiment, the somatic
embryos were post-cultured in a half, full or twice the strength of MS medium
with 30 g lÿ1 sucrose for one week. The conversion ability of the EUs was
evaluated by transferring to a half, full or twice the strength of MS medium
with 30 g lÿ1 sucrose. These media were simulators of synthetic seed coats
with nutrients. A sterile tissue culture medium was ideal to evaluate the
maximum ability of EUs because of no microbial contamination or out¯ow
of nutrients.
2.5. Sugar quanti®cation
One post-cultured somatic embryo was squashed and sugar was extracted three
times in 80% hot ethanol at 808C for 10 min. The extracts were combined
together and lyophilized. The samples were stored at ÿ208C until analysis. Five
hundred ml of distilled water was added and the concentrations of sucrose,
glucose and fructose were measured using Boehringer-Mannheim sucrose/
glucose/fructose test kits.
3. Results
3.1. Effects of kind and concentration of sugar
By our method, 1 g suspension cell clusters produced 5000±7000 bipolar, rodshaped embryos, which were used as materials. The percent conversion of these
embryos was an average 90% in this study. Conversion means that plants develop
healthy shoots and roots. Little callus was induced on somatic embryos on the
conversion medium.
Fig. 1 shows the effects of kind and concentration of sugar on the growth of
shoots and roots. The effects were concentration dependent and were similar
among the three sugars. Analysis of variance showed a highly signi®cant
difference among the concentrations of sugar in shoots and roots. However,
neither a signi®cant difference among the kinds of sugar nor a signi®cant
interaction was shown. The shoots grew less, but the roots grew more with
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
19
Fig. 1. Fresh weight of shoots and roots after three weeks in media with different kinds and
concentrations of sugar. In each treatment 24 plants were studied: shoot (darkly shaded); root (block
marked with diagonal lines). Analysis of variance in shoot: kind of sugar (not signi®cant), concentration of sugar (p < 0.001), interaction between kind and concentration (not signi®cant). LSD
(0.05) for shoot was 12.80. Analysis of variance in root: kind of sugar (not signi®cant), concentration of sugar (p < 0.001), interaction between kind and concentration (not signi®cant). LSD
(0.05) for root was 12.46.
increase in the concentrations. The appearance of shoots showed no difference
among all media. The roots that developed in media with 30 or 50 g lÿ1 sugar
were thick and we considered them to be storage roots, but those in media with
10 g lÿ1 of sugar were thin.
Part of the effects might be attributed to osmotic pressure. Then sorbitol was
used to change osmotic pressure. Fig. 2 shows the effects of sorbitol. Analysis of
variance showed a highly signi®cant difference among sorbitol concentrations in
shoot growth, but not in root growth. Shoots grew less with increase in sorbitol
concentrations.
No difference was shown in the effects of concentration among the three
sugars. Therefore only sucrose was used in the following experiments of 3.2, 3.3
and 3.4.
3.2. Effects of strength of basal medium
Fig. 3 shows the effects of strength of basal medium on the growth of shoots
and roots. Analysis of variance showed a highly signi®cant difference among
strength of basal medium on shoot growth, but not in root growth. Shoots grew
more with increase in the strength.
20
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
Fig. 2. Fresh weight of shoots and roots after three weeks in media with different concentrations of
sorbitol. All media contained 10 g lÿ1 of sucrose. In each treatment 24 somatic embryos were
studied: shoot (darkly shaded); root (block marked with diagonal lines). Analysis of variance in
shoot (p < 0.001). LSD (0.05) for shoot was 13.28. Analysis of variance in root (not signi®cant).
3.3. Effects of sugar concentration and strength of basal medium in the postculture process and the conversion process
The effects of sugar in the post-culture process and the conversion process on
the growth of shoots and roots were shown in Fig. 4A and B. Both in shoots and
in roots, highly signi®cant differences were shown among the concentrations
Fig. 3. Fresh weight of shoots and roots after three weeks in media with different strength of basal
medium. All media contained 30 g lÿ1 of sucrose. In each treatment 24 plants were studied: shoot
(darkly shaded); root (block marked with diagonal lines). Analysis of variance in shoot (p < 0.001).
LSD (0.05) for shoot was 21.12. Analysis of variance in root (not signi®cant).
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
21
Fig. 4. Growth of shoots and roots after one week of post-culture in three sucrose concentrations
followed by two weeks in conversion media at four sucrose concentrations. X-axis shows ``sucrose
concentration in the post-culture media Ð the concentration in the conversion media''. (A) Fresh
weight of shoots. (B) Fresh weight of roots. In each treatment 24 plants were studied. Analysis of
variance in shoot: post-culture media (p < 0.01), conversion media (p < 0.001), interaction between
post-culture media and conversion media (p < 0.05). LSD (0.05) for shoot was 15.31. Analysis of
variance in root: post-culture media (p < 0.001), conversion media (p < 0.001), interaction between
post-culture media and conversion media (not signi®cant). LSD (0.05) for root was 15.77.
22
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
in the post-culture process and the conversion process. A signi®cant interaction
was shown in shoots, but not in roots. These results show that sugar concentration in the post-culture medium affected the growth of plants in the conversion
process. The effects of sugar in the post-culture medium on shoot growth
differed depending on the presence of sugar in the conversion process. Shoots
grew more with increase in sugar concentrations in the post-culture medium
when no sugar was in the conversion process. However, the growth of shoots
was suppressed with increase in the concentrations when sugar was in the
conversion process. Roots grew more with increase in the concentrations in
the post-culture medium regardless of the presence of sugar in the conversion
process.
The sugar supplied in the conversion process also affected the growth of plants.
When no sugar was in the conversion process, the growth of plants was poor. The
effects of concentration in the conversion process were similar to the results in
Fig. 1. The optimal concentration for shoot growth was 10 g lÿ1 and those for
roots were 30±50 g lÿ1, regardless of the sucrose concentration in the post-culture
process. When 50 g lÿ1 of sucrose was added in the post-culture process and the
conversion process, the color of some shoots became pale pink and the shoots did
not grow well.
Fig. 5. Growth of shoots after one week of post-culture in three strength of basal medium followed
by two weeks in conversion media at three strength of basal medium. X-axis shows ``strength of
basal medium in the post-culture media Ð the strength in the conversion media''. In each treatment
24 plants were studied. Analysis of variance: post-culture media (not signi®cant), conversion media
(p < 0.001), interaction between post-culture media and conversion media (not signi®cant). LSD
(0.05) was 50.46.
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
23
Fig. 6. Sugar content in somatic embryos after one week of post-culture in media with different
sucrose concentrations. In each treatment 22 somatic embryos were studied: Fructose (darkly
shaded), sucrose (block marked with diagonal lines), glucose (lightly shaded). Analysis of variance
in total sugar content (p < 0.001). LSD (0.05) was 3.04.
Fig. 5 shows the effects of strength of basal medium in the post-culture process
and the conversion process on growth of shoots. Only the growth of shoots was
shown because the effects on roots was not signi®cant as shown in Fig. 3. In
growth of shoots, there was a highly signi®cant difference among the treatments
in the conversion process, but not in the post-culture process.
3.4. Sugar quanti®cation
The content of sucrose, glucose and fructose in the post-cultured somatic
embryos were shown in Fig. 6. When somatic embryos were post-cultured in
media with higher sucrose concentrations, they contained more sugar per fresh
weight. About 2/3 of the sugar was sucrose, and the rests were glucose and
fructose. The amount of glucose was slightly higher than that of fructose.
4. Discussion
The results of this study show that the growth of shoots and roots from somatic
embryos could be controlled by sugar concentration and strength of basal
medium. Lower concentrations of sugar and higher strength of basal medium
promoted the growth of shoots, but higher concentrations of sugar promoted the
growth of roots and inhibited the growth of shoots. The strength of basal medium
tested in this study did not affect the growth of roots. Our results of sugar
24
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
concentration in somatic embryos were similar to reports of sugar concentrations
in the minicrown (Conner and Falloon, 1993) and in the organ formation from
shoot segments (Harada and Yakuwa, 1983), although their reports did not show
numerical and statistical data. The kind of sugar tested in our study did not
affected the growth of shoots and roots. This result differed from the results of
Levi and Sink (1990). They studied the medium for initiation of embryogenic
calli and sub-culture of them; they found that glucose promoted root growth,
fructose promoted shoot growth, sucrose promoted both shoot and root growth.
However, their report did not show numerical and statistical data either, and
they did not study the conversion medium. From the results in Fig. 2, it was
suggested that increased osmotic pressure inhibited the shoot growth, but the
growth of roots was not affected by osmotic pressure tested in this study. We
studied sucrose concentration in the conversion process of somatic embryos in
carrot, but we could not observe the effects shown in asparagus (Mamiya,
unpublished results).
Storage roots are indispensable for acclimatization of in vitro plants in
asparagus (Conner et al., 1992). In our study, plants produced in media with 30±
50 g lÿ1 of sugar survived after acclimatization, but most plants produced in a
medium with 10 g lÿ1 of sugar did not survive (Mamiya, unpublished results).
The conditions suitable for growth was different between shoots and roots.
Selecting a condition suitable for root growth, namely a higher concentration of
sugar, is better.
When the ability of EUs are evaluated after encapsulation, problems that
disturb the evaluation will arise. Those are physical inhibition of conversion
from capsules, dif®culty to supply suf®cient nutrition without microbial
contamination in capsules. By using a tissue culture medium as a simulator of
synthetic seed coat with nutrients, the maximum ability of the EUs can be
evaluated. Our results show that sugar concentration in the post-culture process
affects the growth of plants in the conversion process, and that media with
30±50 g lÿ1 of sugar are suitable to produce EUs that can develop more roots.
The EUs produced in media with more sucrose contained more glucose, fructose
and sucrose than those produced in media with less sucrose. The sugar in the EUs
might affect the later growth in the conversion process. The strength of basal
medium in the post-culture process did not affect the growth of shoots in the
conversion process.
When sugar was not added in the conversion process, the growth of plants was
poor, which is reasonable because the seeds of asparagus are albuminous.
Therefore during the conversion process, sugar is necessary for synthetic seeds
of asparagus. Suitable microcapsules exist that release sucrose or MS salts
gradually (Sakamoto et al., 1991). When nutrient microcapsules are used for
synthetic seeds of asparagus, our results will be useful to determine the
concentration and to predict the growth pattern of the plants. In the germination
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
25
of natural seeds, storage roots develop ®rst and shoots grow later. This growth
pattern is mimicked in synthetic seeds by regulating the sugar and strength of
basal medium.
In conclusion, we studied the effects of sugar concentration and strength of
basal medium, and found that these factors affected the growth of shoots and
roots. We also found that the sugar concentration in the post-culture medium
affected the growth of plants in the conversion process. The results of this study
will be useful to produce plants or synthetic seeds from somatic embryos in
Asparagus of®cinalis L.
Acknowledgements
We thank K. Watanabe, Hokkai Seikan Co. Ltd., for providing the cultivar
`Fest'. We also thank many members in Plant Laboratory, Kirin Brewery Co. Ltd.
for their kind help.
References
Conner, A.J., Falloon, P.G., 1993. Osmotic versus nutritional effects when rooting in vitro asparagus
minicrown on high sucrose media. Plant Sci. 89, 101±106.
Conner, A.J., Aberneithy, D.J., Falloon, P.G., 1992. Importance of in vitro storage root development
for the successful transfer of micropropagated asparagus plants to greenhouse. NZ. J. Crop Hort.
Sci. 20, 477±481.
Delbreil, B., Goebel-Tourand, I., Lefranc,ois, C., Jullien, M., 1994. Isolation and characterization of
long-term embryogenic lines in Asparagus of®cinalis L.. J. Plant Physiol. 144, 194±200.
Harada, T., Yakuwa, T., 1983. Studies on the morphogenesis of Asparagus VI. Effect of sugar on
callus and organ formation in the in vitro culture of shoot segments of the seedlings. J. Fac. Agr.
Hokkaido Univ. 61, 307±314.
Kohmura, H., Chokyu, S., Harada, T., 1994. An effective micro-propagation system using
embryogenic calli induced from bud clusters in Asparagus of®cinalis L. J. Jpn. Soc. Hort. Sci.
63, 51±59.
Levi, A., Sink, K.C., 1990. Differential effects of sucrose, glucose and fructose during somatic
embryogenesis in asparagus. J. Plant Physiol. 137, 184±189.
Murashige, T., Skoog, F., 1962. A revised medium for rapid growth bioassay with tobacco tissue
cultures. Physiol. Plant. 15, 473±497.
Odake, Y., Udagawa, A., Saga, H., Mii, M., 1993. Somatic embryogenesis of tetraploid plants from
internodal segments of diploid cultivar of Asparagus of®cinalis L. grown in liquid culture. Plant
Sci. 94, 173±177.
Onishi, N., Mashiko, T., Okamoto, A., 1992. Culture system producing encapsulatable units of
synthetic seeds in celery. Acta Horti. 319, 113±118.
Onishi, N., Sakamoto, Y., Hirosawa, T., 1994. Synthetic seeds as a application of mass production
of somatic embryos.. Plant Cell Tiss Org. Cult. 39, 137±145.
Redenbaugh, K., Paasch, B.D., Nichol, J.W., Kossler, M.E., Viss, P.R., Walker, K.A., 1986. Somatic
seed: encapsulation of asexual plant embryos. Biotechnology 4, 797±801.
26
K. Mamiya, Y. Sakamoto / Scientia Horticulturae 84 (2000) 15±26
Reuther, G., 1984. Asparagus. In: Sharp, W.R., Evans, D.A., Ammirato, P.V., Yamada, Y. (Eds.),
Handbook of Plant Cell Culture, vol. 2. Macmillan, New York, pp. 211±242.
Saito, T., Nishizawa, S., Nishimura, S., 1991. Improved culture conditions for somatic
embryogenesis from Asparagus of®cinalis L. using an aseptic ventilative ®lter. Plant Cell
Rep. 10, 230±234.
Sakamoto, Y., Umeda, S., Ogishima, H., 1991. Arti®cial seed comprising a sustained-release
granule. United States Patent 5 010 865.
Sawyer, H., Hsiao, K-C., 1992. Effects of autoclave-induced carbohydrate hydrolysis on the growth
of Beta vulgaris cells in suspension. Plant Cell Tiss Org. Cult. 31, 81±86.