Effects of sugar concentration and strength of
basal medium on conversion of somatic
embryos in
Asparagus of®cinalis
L.
Kanji Mamiya
a,*, Yuji Sakamoto
ba
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 post-culture 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).
1. Introduction
Somatic embryogenesis occurs in many species. Many clonal propagation methods using somatic embryogenesis have been tried inAsparagus of®cinalisL. (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 sub-culture 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 inAsparagus 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 inAsparagus 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 post-culture medium to produce EUs and discussed about the application to synthetic seed technology inAsparagus of®cinalis L.
In this paper, we have used the following abbreviations: D, 2,4-dichlorophenoxyacetic acid; EU, encapsulatable unit; MES, 2-(N-morpholino) ethanesulfonic acid; MS, Murashige and Skoog.
2. Materials and methods
2.1. Production of somatic embryos
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, 9.0mM 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, 4.5mM 2,4-D, 0.9mM kinetin, 0.1 g lÿ1
casein acid hydrolysate and 1 g lÿ1
MES. To induce and pro-liferate the suspensions, the temperature was 308C and the ¯asks were kept under 5±10mmol 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 60mmol 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 60mmol 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
2.4. Effects of sugar concentration and strength of basal medium in the post-culture 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 atÿ208C 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, rod-shaped 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.
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.
3.3. Effects of sugar concentration and strength of basal medium in the post-culture 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. 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).
Fig. 3. Fresh weight of shoots and roots after three weeks in media with different strength of basal medium. All media contained 30 g lÿ1of sucrose. In each treatment 24 plants were studied: shoot
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 concen-tration 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 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
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.
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.
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