- Email: [email protected]
Nurse culture of Japanese radish (Raphanus sativus L.) mesophyll protoplasts
Plant Science, 86 (1992) 105-113 Elsevier Scientific Publishers Ireland Ltd.
105
Nurse culture of Japanese radish (Raphanus sativus L.) mesophyll protoplasts Manabu Hagimori* and Masaaki Nagaoka National Research Institute of Vegetables, Ornamental Plants and Tea, Ano, Mie 514-23 (Japan) (Received March 27th, 1992; revision received June 25th, 1992; accepted July 1st, 1992)
Mesophyll protoplasts of 11 cultivars of Japanese radish (Raphanus sativus L.) were cultured. Using protoplasts or cultured cells of different species as nurse, the efficiency and reproducibility of colony formation were improved and protoplasts of all cultivars grew into colonies. Cells of Nicotiana, Brassica, Daucus, Lactuca and Asparagus were effective as nurses, while those of Oryza were not. N. tabacum protoplasts just isolated and those cultured for more than 50 days showed equal effectiveness as nurses. The number of nurse cells correlated with the efficiency of colony formation of the nursed protoplasts: generally highest efficiency was achieved when the numbers of nurse cells and the nursed protoplasts were nearly equal. Although cell division and colony formation were improved by the nurse culture method, viability and cell wall synthesis were unaffected. Conditioned medium showed less promotive effect than nurse culture.
Key words: nurse culture; protoplast; Raphanus sativus
Introduction
Certain kinds of protoplasts, such as protoplasts of some recalcitrant species or of specific organs or tissues are very difficult to culture. In some cases, nurse culture or the use of a conditioned medium is useful in helping such protoplasts multiply. Protoplasts of Gramineae species such as maize [1-3], rice [4,5], wheat [6] or oat [7] which had been very difficult to culture can be cultured efficiently by nurse culture or on a conditioned medium. However, the mechanism of the nursing effect is still unknown. Japanese radish (Raphanus sativus L.) is a very important vegetable crop occupying the most widely planted area of any vegetable crop in Correspondence to: Dr. Manabu Hagimori, Japan Tobacco Inc., Applied Plant Research Laboratory, 1900 Idei, Oyama, Tochigi 323, Japan. Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; NAA, l-naphthaleneacetic acid; BA, 6-benzylaminopurine; MS, Murashige & Skoog's medium. *Present address: Japan Tobacco Inc., Applied Plant Research Laboratory, 1900 Idei, Oyama, Tochigi 323, Japan.
Japan. There are few reports on culturing of Japanese radish protoplasts. Leaf mesophyll protoplasts of the species are very difficult to culture [8] while hypocotyl [9] and cotyledon [10] protoplasts can be readily cultured. To obtain enough protoplasts for gene transfer or protoplast fusion experiments, the leaf is the most suitable source of protoplasts. In the present study, reliable nurse culture methods for callus formation from mesophyll protoplasts of 11 cultivars of R. sativus were established. In addition, some experiments were done to clarify the mechanism of the nursing effect. Materials and Methods
Plant materials Eleven cultivars of Japanese radish (Raphanus sativus L.) were used, these were Minowase, Everest, Sofutori-miyashige, Shirofutori-miyashige-shirimaru, Uchiki-gensuke, Akizumari, Ohkura, Horyo, Wakayama, Sushirazu-shogoin and Sakurajima-ohmaru. The seeds of these cultivars were all purchased from Takii Seed Co., Kyoto,
0168-9452/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
106 Japan. They were cultivated in a Phytotron under 12-h light (30 klux, metal halide + mercuryvapor lamp, 4.4 : 3.0 install wattage, 23°C) 12-h dark (20°C) cycles. True leaves 5-10 cm in length were excised from young plants 10-15 days after germination and used for isolation of protoplasts. For the source of nurse cells, aseptic plants of tobacco (Nicotiana tabacum L. cv BY-4), Nicotiana benthamiana Domin, Nicotiana suaveolens Lehm., kale (Brassica oleracea L. var. acephala DC, Takii Seed Co.), cabbage (Brassica oleracea L. var. capitata L. cv Murasaki, Yamato Nouen Co., Ltd., Tenri, Japan), cauliflower (Brassica oleracea L. var. botrytis L. cv Nozakiwase, Takii Seed Co.), Chinese cabbage (Brassica campestris L. cv Matsushima-shin-nigo, Takii Seed Co.) and lettuce (Lactuca sativa L. var. capitata L. cv Green Wave, Takii Seed Co.) were grown in vitro on MS solid medium [11] supplemented with 3% (w/v) sucrose. Other conditions used were omission of growth substances, 25°C under 12-h light (ca. 5000 lux, fluorescent lamps) 12-h dark cycles. Mesophyll protoplasts were isolated from the leaves of these plants and used as nurse cells. In addition, 3-7-day-old etiolated aseptic seedlings of the cabbage and the cauliflower, which were germinated and grown in vitro under the same conditions but in the dark, were used to isolate hypocotyl protoplasts. Two lines of suspension cultured cells of Oryza sativa L. cv. Nihonbare, kpt-1 and kpt-10, both of which were established according to Ohira et al. [12] and subcultured every 2 weeks in R-2 medium [12] containing 10 /~M 2,4-D, 0.3% (w/v) casamino acids and 3% (w/v) sucrose, were kindly provided by Dr. K. Ohno of the National Institute of Agrobiological Resources, Tsukuba, Japan. A cell suspension culture of Asparagus officinalis L. var, altilis L. cv Mary Washington 500 W which was established by Saito et al. [13] and subcultured every week in Linsmaier and Skoog's medium [14] containing 10 #M 2,4-D and 2% (w/v) sucrose and a cell suspension culture of Daucus carota L. var. sativa DC which was established according to Fujimura and Komamine [15] and subcultured every 2 weeks in MS medium containing 0.4 mg/1 2,4-D and 3% (w/v) sucrose were kindly provided by Mr. T. Saito and Mr. K. Tsuji of the National Research Institute of
Vegetables, Ornamental Plants and Tea, Ano, Japan, respectively.
Isolation of protoplasts A true leaf of Japanese radish was surface sterilized by a 30-s immersion in 70% (v/v) ethanol followed by a 5-min immersion in a sodium hypochlorite solution in which available chlorine was no less than 2%. It was then rinsed 3 times with sterile distilled water. The leaf was cut into strips of 1-2 mm in width and mixed with a filter sterilized enzyme solution (pH 5.8) containing 1% (w/v) Cellulase Onozuka R-10, 0.25% (w/v) Macerozyme R-10, 0.7 M mannitol and 5 mM CaC12. The reaction mixture was allowed to settle overnight at 25°C, and was then filtered through six layers of gauze. The protoplasts in the filtrate were collected and washed 3 times with a washing solution containing 0.7 M mannitol and 5 mM CaC12 at pH 5.8, by centrifugation at 100 x g for 2 min. Finally the protoplasts were suspended in NT5 medium containing 0.7 M mannitol (Table I), a modification of Ref. 16. Protoplasts of the species other than Japanese radish were isolated in a similar way to that described above except for the following: surface sterilization was omitted; hypocotyls were cut
Table I.
Compositionof NT5 medium.
Mineral salts (mg/I) KNO3 NHaNO3 KH2PO4 CaC12 . 2H20 MgSO 4 . 7H20 FeSO 4 . 7H20
Na2EDTA
950 825 680 220 1233
27.8 37.3
MnSO4 . 4H20 22.3 ZnSO4 . 7H20 8.6 H3PO3 6.2 CuSO4 . 5H20 0.025 Na2MoO 4 . 2H20 0.25 KI 0.83 CoC12. 6H20 0.025
Organic constituents (mg/l)a myo-lnositol 100 Pyrioxine . HCI 0.5 Thiamine . HCI 1.0 Nicotinic acid 0.5 Glycine 2.0 Coconut water 10% (v/v)
Sucrose Casaminoacids 2,4-D NAA Seatin Mannitol
10 000 250 0.2 1.0 0.5 0-0.7 M
The pH was adjusted with KOH to 5.6. aThe units for the figures were mg/l except for those for coconut water and mannitol.
107
longitudinally; mannitol concentration in the enzyme solution and in the washing solution was 0.5 M (except for hypocotyl where the washing solution contained 0.25 M KCI in place of 0.5 M mannitol, although the enzyme solution remained the same); and isolated protoplasts were suspended in an NT5 medium containing 0.5 M mannitol and used as nurse cells immediately or else after being cultured for some days (except in the case of lettuce). Protoplasts of lettuce were isolated from cotyledons and cultured in a Y2 medium (pH 5.6) which contained the MS organic constituents [11], 2% (w/v) glucose, 1 mg/1 NAA, 0.5 mg/1 BA, 0.5 M mannitol and half-concentrations of the inorganic salts of the MS medium, except that NHaNO 3 was reduced to 200 mg/l.
Culture of protoplasts, standard method Five milliliters of the protoplast suspension was put into a sterile plastic dish 53 mm in inside diameter at a density of 3 x 104-5 × 10 4 protoplasts/ml. The density of protoplast suspension was measured with Neubauer's hemocytometer. All of the liquid media used for culture of protoplasts were filter sterilized. The dishes were sealed with parafilm and incubated at 25°C under weak light (fluorescent lamp, ca. 500 lux).
Culture of protoplasts, nurse culture method Millicell-HA units (Millipore Co.,) 30 mm in diameter (plastic cylinders with bottoms made of nitrocellulose membrane with 0.45-/~m pores) were used for nurse culture. A Millicell was settled in a plastic dish 53 mm in inside diameter. Two milliliters of nurse protoplast suspension (the cell density of which was adjusted to about 5 x 104/ml except in the case of the experiments on the effect of the nurse cell number) was put into the Millicell. In the case in which suspension cultured cells were used as nurse, 2 ml of cell suspension containing about 0.1 g fresh weight of cells was put into the Millicell. Then 5 ml of Japanese radish protoplast suspension at a density of 3 x 104-5 X 104/ml was put into the dish outside the Millicell. Then the dish was sealed with parafilm and incubated under the same conditions as the standard method (Fig. 1). When cells cultured in media other than NT5 (i.e. rice, asparagus and lettuce cells) were used as a nurse, they were collected by centrifugation at 100 x g, 2 min and suspended before use in fresh NT5 containing 0.7 M mannitol. Occasionally, a 12-mm Millicell was used in combination with a 35-mm dish, in which case the volumes of the nurse cell suspension and of Japanese radish protoplast suspension used were 0.5 ml and 1.5 ml, respectively.
N P
D ,
.,
M
Fig. 1. Schematic diagram of nurse culture using MillicellHA. D, Plastic dish; M, Millicell-HA;N, nurse cell suspension; P, nursed protoplast suspension; F, membrane filter with 0.45 /zm pores. The inside diameter of the plastic dish is 53 mm and the outside diameter of the Millicell-HAis 30 mm. The depth of the dish is 12 ram.
Conditioned medium Conditioned medium was prepared just before use by mixing equal volumes of fresh NT5 medium containing 0.7 M mannitol and a filtrate of NT5 medium in which tobacco or Chinese cabbage mesophyll protoplasts had been cultured for 1-2 weeks. Filtration of the used medium was performed as follows: the protoplast suspension was first filtered through a filter paper, then the filtrate was filtered through a sterilized disposable syringe filter (Corning, New York).
Staining of the cells The cells were stained with fluorescein diacetate according to Nadel [17] for the determination of viability and with Calcofluor White [18] for the determination of cell wall synthesis.
108 Results
Culture of mesophyll protoplasts of Japanese radish by the standard method in various media and at various cell densities Several media were examined for their effectiveness in culture of mesophyll protoplasts of two cultivars of Japanese radish, M i n o w a s e a n d Everest. These media were K M 8 P by K a o a n d Michayluk [191, B by Pelletier et al. [20], modified MS a n d N T 5 with various c o m b i n a t i o n of p l a n t growth substances. A m o n g the media, only the N T 5 sporadically supported cell division a n d colony f o r m a t i o n of M i n o w a s e protoplasts. Mesophyll protoplasts o f b o t h cultivars were cultured with N T 5 m e d i u m at cell densities ranging from 103 to 106 protoplasts/ml. Those of Everest did n o t divide at a n y density. Sporadic colony f o r m a t i o n from M i n o w a s e protoplasts was observed at densities between 104 a n d 105/ml (data n o t shown).
standard, nurse culture a n d c o n d i t i o n e d m e d i u m methods. Mesophyll protoplasts o f tobacco or Chinese cabbage cultured for 7 - 1 4 days were used as nurse cells a n d the culture filtrates from these tobacco a n d Chinese cabbage protoplast cultures were also used to prepare the c o n d i t i o n e d media. Table II shows the best results o f 3 replicated experiments. The protoplasts of all cultivars formed colonies u n d e r the nurse culture method (Fig. 2). C o n d i t i o n e d m e d i u m also showed a promotive effect b u t the effect was far less a n d also inconsistent t h a n that of nurse culture. Protoplasts of cultivars, M i n o w a s e a n d Uchiki-gensuke occasionally formed 1 0 - 5 0 colonies in the s t a n d a r d m e t h o d w i t h o u t a nurse a n d those of five other cultivars occasionally formed up to 10 colonies. The nurse culture m e t h o d increased n o t only the n u m b e r o f colonies formed but also their reproducibility. Protoplasts o f four cultivars formed colonies only when cultured with a nurse.
Effect of the nurse cell number Colony formation from mesophyll protoplasts of 11 cultivars of Japanese radish Mesophyll protoplasts o f 11 cultivars of Japanese radish were cultured by 3 methods, i.e.
Effect of the n u m b e r of nurse cells o n the ciency of colony f o r m a t i o n o f mesophyll toplasts of R. sativus in nurse culture examined. Zero to 1.6 × l0 s mesophyll
effiprowas pro-
Table II. Colony formation from mesophyll protoplasts of 11 cultivars of Japanese radish cultured by three different methods. Cultivars
Standard method a
Nurse cult.
Cond. med.
N.t b
B.c. c
N.t. d
B.c. e
Akizumari
± f
+++
+++
+
+
Everest Horyo Minowase Ohkura Sakurajima-ohmaru Shirofutori-miyashige-shirimaru Sofutori-miyashige Sushirazu-shogoin Uchiki-gensuke Wakayama
± + ± ± + ±
++ ++ +++ ++ + ++ +++ + +++ ++
+++ +++ ++ + + + ++ + +++ ++
+ ++ + ++ + +
++ ++ ++ + +
aCultures by the standard method. b,CCultureswith a nurse of (b), Nicotianatabacumor (c), Brassicacampestrismesophyllprotoplasts cultured for 7-14 days before use. ~'eCultures with conditioned medium prepared from culture broth of mesophyil protoplasts of (d), 31. tabacumor (e), R campestris which had been cultured for 7-14 days before use. fApproximate numbers of colonies were expressed by positive and negative signs. The signs correspond to actual number (x) per 60-mm dish as follows: - , x = 0 ; ±,1 < x < 10; +, 10 < x < 50; ++, 50 < x < 200; +++,200 < x. Colonies were counted 3 weeks after initiation of culture. The best results from three independent experiments are shown here.
109 Table IV. Effect of age of the nurse cells on colony formation from mesophyll protoplasts of Japanese radish cultivar Horyo.
Fig. 2. Nurse culture with Millicell-HA. Mesophyll protoplasts of the Japanese radish cultivar Horyo were cultured for 3 weeks with a nurse of Nicotiana suaveolens mesophyll protoplasts (right dish), or of cabbage hypocotyl protoplasts (middle), or without a nurse (left). Many colonies were observed in the right and middle dishes. Only a few colonies appeared in the left dish. The outside diameter of each dish is 59 mm.
t o p l a s t s o f t o b a c c o w e r e p u t i n t o a M i l l i c e l l as nurse and a fixed number of mesophyll protoplasts of Japanese radish was put into the dish outside t h e Millicell. T a b l e I I I s h o w s t h e r e s u l t s f r o m t w o experiments each replicated 3 times. Though the t h r e s h o l d n u m b e r o f n u r s e cells v a r i e d w i t h cultivars and with experiments, a dose effect of the
Table IlL
1.6 4.8 1.6 1.6
0 × x x x
103 103 104 105
Number of colonies
0 20 57 Control**
39 30 47 0
a a a b
*Nicotiana tabacum mesophyll protoplasts cultured for 0, 20 or 57 days were used as a nurse. **Control was the culture without a nurse. In this experiment, ~ 35-mm dishes and 4~ 12-mm Millicells were used. Colonies were counted 3 weeks after initiation of culture. Values represent means of 3 replicates. Means within a column not sharing the same letter are significantly different by Duncan's multiple range test at P = 0.05.
n u m b e r o f n u r s e cells o n c o l o n y f o r m a t i o n o f n u r s ed protoplasts was observed. These results suggest that Japanese radish prot o p l a s t s r e q u i r e s o m e a m o u n t o f n u r s e cells t o m u l t i p l y . T h e t h r e s h o l d n u m b e r t h a t m e e t s t h e requirements of each protoplast might differ from protoplast to protoplast even among protoplasts i s o l a t e d f r o m a s i n g l e leaf. T h u s , a m o n g p r o t o p l a s t s in a n y c u l t u r e , s o m e m a y d i v i d e w i t h a s m a l l a m o u n t o f n u r s e cells w h i l e t h e o t h e r s d o not.
Effect of nurse cell number on the colony formation from mesophyll protoplasts of Japanese radish cultivars.
Exp. 1 Nurse cell No.**
Age of Nurse cells (days)*
Exp. 2 Colony
No.*
Horyo
Wakayama
Ob 0b 0b 0b 390 a
28b 486 a 396 a 510 a 390 a
Nurse cell No.**
0.5 0.5 0.5 3.3
0 x x x x
102 103 104 104
Colony
No.*
Horyo
Ohkura
Oc 8c 15 bc 47 a 30 ab
3b 0b 1b 16 b 160 a
*Number of colonies per dish. **Inoculated number of nurse cells. In Exp. 1, the inoculated number of protoplasts of Horyo and Wakayama were both 1.5 × 105 per dish. In Exp. 2, ~ 35-mm dishes and ¢ 12-mm Millicells were used. The inoculated number of protoplasts of Horyo and Ohkora were 3.9 x 104 and 5.7 × 104, respectively. In both experiments, Nicotiana tabacum mesophyU protoplasts were used as a nurse. The number of colonies of Japanese radish was counted 3 weeks after initiation of culture. Values represent means of 3 replicates. Means within a column not sharing the same letter are significantly different by Duncan's multiple range test at P = 0.05.
110
Effect of the age of the nurse cells on their efficiency The efficiencies of N. tabacum protoplasts just isolated was compared with those cultured for 20 or 57 days in the nursing of mesophyll protoplasts of R. sativus (Table IV). All had the capacity for vigorous growth and showed nearly equal efficiencies as nurse cells regardless of age. Nursing activities of several species of plants Protoplasts or cultured cells of 10 species, the Table V.
taxonomic positions of which were variously distant from Raphanus, were examined for their ability to nurse mesophyll protoplasts of Raphanus. Three, cauliflower, kale and cabbage, belong to the same family as Raphanus, Cruciferae. Five,
Nicotiana tabacum, N. benthamiana, N. suaveolens, carrot and lettuce, belong to the same subclass, Dicotyledoneae, but different infraclasses. The remaining two, rice and asparagus, belong to a different subclass, Monocotyledoneae. Table V
Effect of cells of several plant species as a nurse on colony formation from mesophyll protoplasts of Japanese radish cultivars.
Exp. 1 Nurse cells
Col. No. a Horyo
Ohkura
Cauliflower hypocotyl protoplasts cultured for 22 days Cabbage hypocotyl protoplasts cultured for 22 days Lettuce cotyledon protoplasts cultured for 104 days N. suaveolens mesophyll protoplasts cultured for 20 days N. benthamiana mesophyll protoplasts cultured for 20 days N. tabacum mesophyll protoplasts cultured for 20 days None
NT b 108 4574630 477 4144 494-
Exp. 2 Nurse cells
Col. No. a Sof. M. c
Cauliflower mesophyll protoplasts Kale mesophyll protoplasts Lettuce cotyledon protoplasts cultured for 89 days N. suaveolens mesophyll protoplasts cultured for 5 days N. benthamiana mesophyll protoplasts cultured for 5 days N. tabacum mesophyll protoplasts cultured for 5 days None
3434450 4516 4414 + 228 484-
Exp. 3 Nurse cells
Col. No. a Wakayana
Nurse growth d
Kale mesophyll protoplasts N. tabacum mesophyll protoplasts None
324 4- 120 185 4- 70 24 4- 13
++ ++
Exp. 4 Nurse cells
Col. No. a Horyo
Sof. M. c
0+ 0 243 397 4- 148 425 4- 215 313 4- 91 040
040 442 158 4- 70 236 4- 106 94 4- 33 O± 0
Rice suspension culture cells, kpt 1 Rice suspension culture cells, kpt 10 Asparagus suspension culture cells Carrot suspension culture cells N. tabacurn mesophyll protoplasts cultured for 14 days None
43 14 177 20 37 8
4 2 108 60 130 84 6
114 4105 4124360 4NT NT 54-
24 35 7 73
5
Nurse growth d
++ ++ ++ +
aNumber of colonies. bNT means Not tested. cSofutori-Miyashige, a cultivar of Japanese radish. dGrowth of the nurse cells. Colonies were counted 2 weeks (Exp. 1), 3 weeks (Exps. 3 and 4) and 4 weeks (Exp. 2) after initiation of culture. Values represent means + S.D. of 3 replicates.
111
shows the results. All dicot species examined, regardless of taxonomic distance from Raphanus, were effective as nurses, so far as the nurse protoplasts or cells divided and grew during the culture period. As for monocots, Asparagus cells were effective but rice cells were not, though both of them appeared to be vigorous at the end of the culture (Table V, Exp. 4).
Effect of nursing on the viability and cell wall synthesis of Japanese radish mesophyll protoplasts How does nursing affect Japanese radish mesophyll protoplasts and allow them to form colonies? Three possibilities were considered: improvement of the viability of protoplasts, promotion of cell wall synthesis and induction and/or promotion of cell division. All three were observed in cultures with and without a nurse for a 14-day culture period (Fig. 3). The protoplasts or cells stainable with fluorescein diacetate were regarded as viable. The cell wall was identified by being stained with Calcofluor White. The proportion of viable protoplasts decreased over time, but no significant difference was observed between cultures with and without the nurse. Cells with cell walls were observed on the third day and their pro100
u~
~ 5o .c 0 u3
J~ h
,,~S ~ , 1 3 4
i
1~
Culture period (days)
11
Fig. 3. Time course of the proportion of viable cells and cells with cell walls in the cultures of mesophyll protoplasts of the Japanese radish cultivar Sofutori-miyashige with and without a nurse. Nicotiana suaveolens mesophyll protoplasts cultured for 14 days were used as nurses. On the indicated days, aliquots of the cultures were taken out and stained with fluorescein diacetate or Calcofluor White. The proportion of the stained cells among the total cells was then determined and expressed as follows: the proportion of cells stained with fluorescein diacetate in the cultures with a nurse, O; and without a nurse, O; the proportion of ceils stained with Calcofluor White in the cultures with a nurse, A; and without a nurse, A. The circles and the tliangles are connected with solid lines and broken lines, respectively.
portion reached about 50% on the fourth day, keeping this level thereafter till the end of culture. No significant difference in the proportion of the cells with cell walls was observed between cultures with or without a nurse. In the cultures with a nurse, dividing cells were observed on and after the seventh day and colonies were observed at the end of the culture period. By contrast, no dividing cells, or colonies, were observed in the cultures without a nurse. Thus, nursing makes Japanese radish mesophyll protoplasts form colonies by inducing or promoting cell division, but not by improving viability or by promoting cell wall synthesis. This is in accord with the results that have been reported for the effect of conditioned medium on maize protoplasts [2]. Discussion There are three possible mechanisms by which nurse cells or the conditioned medium promote division and colony formation in nursed protoplasts. The first supposes that the nurse cells or conditioned medium supply the nursed protoplasts with primary metabolites which leak out from the protoplasts. In this case, nurse cells or conditioned medium could be replaced with a supply of various primary metabolites [19], or else by reduction of culture volume to a microdroplet scale [21]. The second supposes that the nurse cells or conditioned medium supply the nursed protoplasts with some substance(s), which the nursed protoplasts cannot provide for themselves, necessary to induce or promote cell division. Such substances, we call them 'nurse substances', have been partially purified and characterized [22-24] but isolation and identification of such substances has not yet been achieved. The third supposes that the nurse cells or conditioned medium detoxify harmful substances in the medium. Caboche [25] reported that a high concentration of free NAA added to the medium was inhibitory to tobacco protoplasts cultured at low cell density and that nurse cells functioned to reduce free NAA drastically. Nursed protoplasts can be divided into two classes. One class includes those that can grow autonomously given sufficient cell density, but need a nurse at low cell density - - for example, cultures of heterokaryons produced by protoplast fusion
112
[26,27] or of injected protoplasts [28]. In this class, nurse cells seem to function by the first mechanism described above. The other class cannot divide without a nurse no matter what the cell density. Protoplasts of rice [4,5], wheat [61, soybean [29] and beet [30] belong to this class. In this class, nurse cells seem to function according to the second mechanism, though the possibility that the third mechanism is also relevant cannot be excluded. As for mesophyll protoplasts of Japanese radish, those of Everest did not divide even at high cell density of 106/ml while those of Minowase sporadically formed a few colonies at I04 and 105/ml but not at 106/ml. From these results, mesophyll protoplasts of Japanese radish are considered to belong to the latter class. The protoplasts of monocot species, such as rice [4], maize [3] and oat [7], that need the help of nurse or feeder cells, accept only the cells of the same species or of closely related species of mono¢ots as a nurse, not the cells of dicots. From the experiments on the effect of conditioned medium on the colony formation of maize protoplasts, Sornerset al. [2] suggested that the conditioning factor might be different and specific, for monocots and dicots. By contrast, the Japanese radish protoplasts accepted the cells of a very wide range of species, even the monocot Asparagus, as a nurse. This result together with the result reported by Waiters and Earle [31] that protoplasts of a recalcitrant genotype of Brassica oleracea accepted cell suspensions of Brassica campestris and tomato as nurse, suggests that there might be a spectrum of nurse substances which have somewhat common structures among angiosperms and that the specificity of the Japanese radish protoplasts for nurse substances may be lower than that of monocot species. Shoot differentiation from the colonies of the Japanese radish has been tried extensively using various media, but no shoots have been obtained yet. Kishimoto [10] reported that only 2 colonies from cotyledon protoplasts of Japanese radish regenerated shoots. Our nurse culture system is being used in protoplast fusion experiments between Japanese radish and Brassica oleracea the protoplasts of which regenerate readily.
Acknowledgment We thank Miss assistance. We also Saito and Mr. K. suspension cultured
J. Kitazumi for technical thank Dr. K. Ohno, Mr. T. Tsuji for providing us with cell lines.
References 1 C.A. Rhodes, K.S. Lowe and K.L. Ruby, Plant regeneration from protoplasts isolated from embryogenic maize cell cultures. Bio/technology, 6 (1988) 56-60. 2 D.A. Somers, P.R. Birnberg, W.L. Petersen and M.L. Brenner, The effect of conditioned medium on colony formation from Black mexican sweet corn protoplasts. Plant Sci., 53 (1987) 249-256. 3 J.A. Smith, C.E. Green and B.G. Gengenbach, Feeder layer support of low density populations of Zea mays L. suspension cells. Plant Sci. Lett., 36 (1984) 67-72. 4 J. Kyozuka, Y. Hayashi and K. Shimamoto, High frequency plant regeneration from rice protoplasts by novel nurse culture methods. Mol. Gen. Genet., 206 (1987) 408 -413. 5 L. Lee, R.E. Schroll, H.D. Grimes and T.K. Hodges, Plant regeneration from indica rice (Oryza sativa L.) protoplasts. Planta, 178 (1989) 325-333. 6 Y. Hayashi and K. Shimamoto, Wheat protoplast culture: embryogenic colony formation from protoplasts. Plant Cell Rep., 7 (1988) 414-417. 7 B. Hahne, H. L6rz and G. Hahne, Oat mesophyl protoplasts: their response to various feeder cultures. Plant Cell Rep., 8 (1990) 590-593. 8 P.S. Jourdan and E.D. Earle, Genotypic variability in the frequency of plant regeneration from leaf protoplasts of four Brassica-spp and of Raphanus sativus. J. Am. Soc. Hort. Sci., 114 (1989) 343-349. 9 T. Nishio, Y. Sakata, H. Yamagishi, Y. Tabei, T. Sato and K. Takayanagi, Protoplast culture in vegetable crops. Development and improvement of culture procedure. Bull. Natl. Res. Inst. Veg. Ornamental Plants Tea, A3 (1989) 67-96. 10 N. Kishimoto, Shoot regeneration from cotyledon protoplast of Japanese radish (Raphanus sativus L.). Plant Tiss. Cult. Lett., 7 (1990) 114-115. 11 T. Murashige and F. Skoog, A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol. Plant. 15 (1962) 473-497. 12 K. Ohira, K. Ojima and A. Fujiwara, Studies on the nutrition of rice culture I. A simple, defined medium for rapid growth in suspension culture. Plant Cell Physiol., 14 (1973) 1113-1121. 13 T. Saito, S. Nishizawa and S. Nishimura, Improved culture conditions for somatic embryogenesis from Asparagus officinalis L. using an aseptic ventilative filter. Plant Cell Reports, 10 (1991) 230-234.
113 14
15
16
17
18
19
20
21
22
23
E.M. Linsmaier and F. Skoog, Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant., 18 (1965) 100-126. T. Fujimura and A. Komamine, Synchronization of somatic embryogenesis in a carrot cell suspension culture. Plant Physiol., 64 (1979) 162-164. T. Nagata and I. Takebe, Plating of isolated tobacco mesophyll protoplasts on agar medium. Planta, 99 (1971) 12-20. B.L. Nadel, Use of fluorescein diacetate in citrus tissue cultures for the determination of cell viability and the selection of mutants. Sci. Hort., 39 (1989) 15-21. T. Nagata and I. Takebe, Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts. Planta, 92 (1970) 301-308. K.N. Kao and M.R. Michayluk, Nutritional requirements for growth of Vicia hajastana cells and protoplasts at a very low population density in liquid media. Planta, 126 (1975) 105-110. G. Pelletier, C. Primard, F. Vedel, P. Chetrit, R. Remy, X.X. Rousselle and M. Renard, Intergeneric cytoplasmic hybridization in Cruciferae by protoplast fusion. Mol. Gen. Genet., 191 (1983) 244-250. H.U. Koop, G. Weber and H.G. Schweiger, Individual culture of selected single cells and protoplasts of higher plants in microdroplets of defined media. Z. Pflanzenphysiol., 112 (1983) 21-34. R. Stuart and H.E. Street, Studies on the growth of plant cells X. Further studies on the conditioning of culture media by suspensions of Acer pseudoplatanus L. cells. J. Exp. Bot., 22 (1971) 96-106. F. Kohler and G. Wenzel, Regeneration of isolated barley
microspores in conditioned media and trials to characterize the responsible factor. J. Plant Physiol., 121 (1985) 181-191. 24 P.R. Birnberg, D.A. Somers and M.L. Brenner, Characterization of conditioning factors that increase colony formation from Black mexican sweet corn protoplasts. J. Plant Physiol., 132 (1988) 316-321. 25 M. Caboche, Nutritional requirements of protoplastderived, haploid tobacco cells grown at low densities in liquid medium. Planta, 149 (1980) 7-18. 26 H. Morikawa, Y. Hayashi, N. Ohnishi, Y. Yamamoto, F. Sato and Y. Yamada, Culture of electrically induced heterokaryons of plant cells rich in secondary metabolites. Plant Cell Physiol., 29 (1988) 1201-1206. 27 M.R. Thomas and R.J. Rose, Enrichment for Nicotiana heterokaryons after protoplast fusion and subsequent growth in agarose microdrops. Planta, 175 (1988) 396-402. 28 W.A. Lawrence and D.R. Davies, A method for the microinjection and culture of protoplasts at very low densities. Plant Cell Rep., 4 (1985) 33-35. 29 D.M. Tricoli, M.B. Hein and M.G. Carnes, Culture of soybean mesophyll protoplasts in alginate beads. Plant Cell Reports, 5 (1986) 334-337. 30 S.R. Bhat, B.V. Ford-Lloyd and J.A. Callow, Isolation and culture of mesophyll protoplasts of garden, fodder and sugar beets using a nurse culture system: Callus formation and organogenesis. J. Plant Physiol., 124 (1986) 419-423. 31 T.W. Waiters and E.D. Earle, A simple, versatile feeder layer system for Brassica oleracea protoplast culture. Plant Cell Rep., 9 (1990) 316-319.
105
Nurse culture of Japanese radish (Raphanus sativus L.) mesophyll protoplasts Manabu Hagimori* and Masaaki Nagaoka National Research Institute of Vegetables, Ornamental Plants and Tea, Ano, Mie 514-23 (Japan) (Received March 27th, 1992; revision received June 25th, 1992; accepted July 1st, 1992)
Mesophyll protoplasts of 11 cultivars of Japanese radish (Raphanus sativus L.) were cultured. Using protoplasts or cultured cells of different species as nurse, the efficiency and reproducibility of colony formation were improved and protoplasts of all cultivars grew into colonies. Cells of Nicotiana, Brassica, Daucus, Lactuca and Asparagus were effective as nurses, while those of Oryza were not. N. tabacum protoplasts just isolated and those cultured for more than 50 days showed equal effectiveness as nurses. The number of nurse cells correlated with the efficiency of colony formation of the nursed protoplasts: generally highest efficiency was achieved when the numbers of nurse cells and the nursed protoplasts were nearly equal. Although cell division and colony formation were improved by the nurse culture method, viability and cell wall synthesis were unaffected. Conditioned medium showed less promotive effect than nurse culture.
Key words: nurse culture; protoplast; Raphanus sativus
Introduction
Certain kinds of protoplasts, such as protoplasts of some recalcitrant species or of specific organs or tissues are very difficult to culture. In some cases, nurse culture or the use of a conditioned medium is useful in helping such protoplasts multiply. Protoplasts of Gramineae species such as maize [1-3], rice [4,5], wheat [6] or oat [7] which had been very difficult to culture can be cultured efficiently by nurse culture or on a conditioned medium. However, the mechanism of the nursing effect is still unknown. Japanese radish (Raphanus sativus L.) is a very important vegetable crop occupying the most widely planted area of any vegetable crop in Correspondence to: Dr. Manabu Hagimori, Japan Tobacco Inc., Applied Plant Research Laboratory, 1900 Idei, Oyama, Tochigi 323, Japan. Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; NAA, l-naphthaleneacetic acid; BA, 6-benzylaminopurine; MS, Murashige & Skoog's medium. *Present address: Japan Tobacco Inc., Applied Plant Research Laboratory, 1900 Idei, Oyama, Tochigi 323, Japan.
Japan. There are few reports on culturing of Japanese radish protoplasts. Leaf mesophyll protoplasts of the species are very difficult to culture [8] while hypocotyl [9] and cotyledon [10] protoplasts can be readily cultured. To obtain enough protoplasts for gene transfer or protoplast fusion experiments, the leaf is the most suitable source of protoplasts. In the present study, reliable nurse culture methods for callus formation from mesophyll protoplasts of 11 cultivars of R. sativus were established. In addition, some experiments were done to clarify the mechanism of the nursing effect. Materials and Methods
Plant materials Eleven cultivars of Japanese radish (Raphanus sativus L.) were used, these were Minowase, Everest, Sofutori-miyashige, Shirofutori-miyashige-shirimaru, Uchiki-gensuke, Akizumari, Ohkura, Horyo, Wakayama, Sushirazu-shogoin and Sakurajima-ohmaru. The seeds of these cultivars were all purchased from Takii Seed Co., Kyoto,
0168-9452/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
106 Japan. They were cultivated in a Phytotron under 12-h light (30 klux, metal halide + mercuryvapor lamp, 4.4 : 3.0 install wattage, 23°C) 12-h dark (20°C) cycles. True leaves 5-10 cm in length were excised from young plants 10-15 days after germination and used for isolation of protoplasts. For the source of nurse cells, aseptic plants of tobacco (Nicotiana tabacum L. cv BY-4), Nicotiana benthamiana Domin, Nicotiana suaveolens Lehm., kale (Brassica oleracea L. var. acephala DC, Takii Seed Co.), cabbage (Brassica oleracea L. var. capitata L. cv Murasaki, Yamato Nouen Co., Ltd., Tenri, Japan), cauliflower (Brassica oleracea L. var. botrytis L. cv Nozakiwase, Takii Seed Co.), Chinese cabbage (Brassica campestris L. cv Matsushima-shin-nigo, Takii Seed Co.) and lettuce (Lactuca sativa L. var. capitata L. cv Green Wave, Takii Seed Co.) were grown in vitro on MS solid medium [11] supplemented with 3% (w/v) sucrose. Other conditions used were omission of growth substances, 25°C under 12-h light (ca. 5000 lux, fluorescent lamps) 12-h dark cycles. Mesophyll protoplasts were isolated from the leaves of these plants and used as nurse cells. In addition, 3-7-day-old etiolated aseptic seedlings of the cabbage and the cauliflower, which were germinated and grown in vitro under the same conditions but in the dark, were used to isolate hypocotyl protoplasts. Two lines of suspension cultured cells of Oryza sativa L. cv. Nihonbare, kpt-1 and kpt-10, both of which were established according to Ohira et al. [12] and subcultured every 2 weeks in R-2 medium [12] containing 10 /~M 2,4-D, 0.3% (w/v) casamino acids and 3% (w/v) sucrose, were kindly provided by Dr. K. Ohno of the National Institute of Agrobiological Resources, Tsukuba, Japan. A cell suspension culture of Asparagus officinalis L. var, altilis L. cv Mary Washington 500 W which was established by Saito et al. [13] and subcultured every week in Linsmaier and Skoog's medium [14] containing 10 #M 2,4-D and 2% (w/v) sucrose and a cell suspension culture of Daucus carota L. var. sativa DC which was established according to Fujimura and Komamine [15] and subcultured every 2 weeks in MS medium containing 0.4 mg/1 2,4-D and 3% (w/v) sucrose were kindly provided by Mr. T. Saito and Mr. K. Tsuji of the National Research Institute of
Vegetables, Ornamental Plants and Tea, Ano, Japan, respectively.
Isolation of protoplasts A true leaf of Japanese radish was surface sterilized by a 30-s immersion in 70% (v/v) ethanol followed by a 5-min immersion in a sodium hypochlorite solution in which available chlorine was no less than 2%. It was then rinsed 3 times with sterile distilled water. The leaf was cut into strips of 1-2 mm in width and mixed with a filter sterilized enzyme solution (pH 5.8) containing 1% (w/v) Cellulase Onozuka R-10, 0.25% (w/v) Macerozyme R-10, 0.7 M mannitol and 5 mM CaC12. The reaction mixture was allowed to settle overnight at 25°C, and was then filtered through six layers of gauze. The protoplasts in the filtrate were collected and washed 3 times with a washing solution containing 0.7 M mannitol and 5 mM CaC12 at pH 5.8, by centrifugation at 100 x g for 2 min. Finally the protoplasts were suspended in NT5 medium containing 0.7 M mannitol (Table I), a modification of Ref. 16. Protoplasts of the species other than Japanese radish were isolated in a similar way to that described above except for the following: surface sterilization was omitted; hypocotyls were cut
Table I.
Compositionof NT5 medium.
Mineral salts (mg/I) KNO3 NHaNO3 KH2PO4 CaC12 . 2H20 MgSO 4 . 7H20 FeSO 4 . 7H20
Na2EDTA
950 825 680 220 1233
27.8 37.3
MnSO4 . 4H20 22.3 ZnSO4 . 7H20 8.6 H3PO3 6.2 CuSO4 . 5H20 0.025 Na2MoO 4 . 2H20 0.25 KI 0.83 CoC12. 6H20 0.025
Organic constituents (mg/l)a myo-lnositol 100 Pyrioxine . HCI 0.5 Thiamine . HCI 1.0 Nicotinic acid 0.5 Glycine 2.0 Coconut water 10% (v/v)
Sucrose Casaminoacids 2,4-D NAA Seatin Mannitol
10 000 250 0.2 1.0 0.5 0-0.7 M
The pH was adjusted with KOH to 5.6. aThe units for the figures were mg/l except for those for coconut water and mannitol.
107
longitudinally; mannitol concentration in the enzyme solution and in the washing solution was 0.5 M (except for hypocotyl where the washing solution contained 0.25 M KCI in place of 0.5 M mannitol, although the enzyme solution remained the same); and isolated protoplasts were suspended in an NT5 medium containing 0.5 M mannitol and used as nurse cells immediately or else after being cultured for some days (except in the case of lettuce). Protoplasts of lettuce were isolated from cotyledons and cultured in a Y2 medium (pH 5.6) which contained the MS organic constituents [11], 2% (w/v) glucose, 1 mg/1 NAA, 0.5 mg/1 BA, 0.5 M mannitol and half-concentrations of the inorganic salts of the MS medium, except that NHaNO 3 was reduced to 200 mg/l.
Culture of protoplasts, standard method Five milliliters of the protoplast suspension was put into a sterile plastic dish 53 mm in inside diameter at a density of 3 x 104-5 × 10 4 protoplasts/ml. The density of protoplast suspension was measured with Neubauer's hemocytometer. All of the liquid media used for culture of protoplasts were filter sterilized. The dishes were sealed with parafilm and incubated at 25°C under weak light (fluorescent lamp, ca. 500 lux).
Culture of protoplasts, nurse culture method Millicell-HA units (Millipore Co.,) 30 mm in diameter (plastic cylinders with bottoms made of nitrocellulose membrane with 0.45-/~m pores) were used for nurse culture. A Millicell was settled in a plastic dish 53 mm in inside diameter. Two milliliters of nurse protoplast suspension (the cell density of which was adjusted to about 5 x 104/ml except in the case of the experiments on the effect of the nurse cell number) was put into the Millicell. In the case in which suspension cultured cells were used as nurse, 2 ml of cell suspension containing about 0.1 g fresh weight of cells was put into the Millicell. Then 5 ml of Japanese radish protoplast suspension at a density of 3 x 104-5 X 104/ml was put into the dish outside the Millicell. Then the dish was sealed with parafilm and incubated under the same conditions as the standard method (Fig. 1). When cells cultured in media other than NT5 (i.e. rice, asparagus and lettuce cells) were used as a nurse, they were collected by centrifugation at 100 x g, 2 min and suspended before use in fresh NT5 containing 0.7 M mannitol. Occasionally, a 12-mm Millicell was used in combination with a 35-mm dish, in which case the volumes of the nurse cell suspension and of Japanese radish protoplast suspension used were 0.5 ml and 1.5 ml, respectively.
N P
D ,
.,
M
Fig. 1. Schematic diagram of nurse culture using MillicellHA. D, Plastic dish; M, Millicell-HA;N, nurse cell suspension; P, nursed protoplast suspension; F, membrane filter with 0.45 /zm pores. The inside diameter of the plastic dish is 53 mm and the outside diameter of the Millicell-HAis 30 mm. The depth of the dish is 12 ram.
Conditioned medium Conditioned medium was prepared just before use by mixing equal volumes of fresh NT5 medium containing 0.7 M mannitol and a filtrate of NT5 medium in which tobacco or Chinese cabbage mesophyll protoplasts had been cultured for 1-2 weeks. Filtration of the used medium was performed as follows: the protoplast suspension was first filtered through a filter paper, then the filtrate was filtered through a sterilized disposable syringe filter (Corning, New York).
Staining of the cells The cells were stained with fluorescein diacetate according to Nadel [17] for the determination of viability and with Calcofluor White [18] for the determination of cell wall synthesis.
108 Results
Culture of mesophyll protoplasts of Japanese radish by the standard method in various media and at various cell densities Several media were examined for their effectiveness in culture of mesophyll protoplasts of two cultivars of Japanese radish, M i n o w a s e a n d Everest. These media were K M 8 P by K a o a n d Michayluk [191, B by Pelletier et al. [20], modified MS a n d N T 5 with various c o m b i n a t i o n of p l a n t growth substances. A m o n g the media, only the N T 5 sporadically supported cell division a n d colony f o r m a t i o n of M i n o w a s e protoplasts. Mesophyll protoplasts o f b o t h cultivars were cultured with N T 5 m e d i u m at cell densities ranging from 103 to 106 protoplasts/ml. Those of Everest did n o t divide at a n y density. Sporadic colony f o r m a t i o n from M i n o w a s e protoplasts was observed at densities between 104 a n d 105/ml (data n o t shown).
standard, nurse culture a n d c o n d i t i o n e d m e d i u m methods. Mesophyll protoplasts o f tobacco or Chinese cabbage cultured for 7 - 1 4 days were used as nurse cells a n d the culture filtrates from these tobacco a n d Chinese cabbage protoplast cultures were also used to prepare the c o n d i t i o n e d media. Table II shows the best results o f 3 replicated experiments. The protoplasts of all cultivars formed colonies u n d e r the nurse culture method (Fig. 2). C o n d i t i o n e d m e d i u m also showed a promotive effect b u t the effect was far less a n d also inconsistent t h a n that of nurse culture. Protoplasts of cultivars, M i n o w a s e a n d Uchiki-gensuke occasionally formed 1 0 - 5 0 colonies in the s t a n d a r d m e t h o d w i t h o u t a nurse a n d those of five other cultivars occasionally formed up to 10 colonies. The nurse culture m e t h o d increased n o t only the n u m b e r o f colonies formed but also their reproducibility. Protoplasts o f four cultivars formed colonies only when cultured with a nurse.
Effect of the nurse cell number Colony formation from mesophyll protoplasts of 11 cultivars of Japanese radish Mesophyll protoplasts o f 11 cultivars of Japanese radish were cultured by 3 methods, i.e.
Effect of the n u m b e r of nurse cells o n the ciency of colony f o r m a t i o n o f mesophyll toplasts of R. sativus in nurse culture examined. Zero to 1.6 × l0 s mesophyll
effiprowas pro-
Table II. Colony formation from mesophyll protoplasts of 11 cultivars of Japanese radish cultured by three different methods. Cultivars
Standard method a
Nurse cult.
Cond. med.
N.t b
B.c. c
N.t. d
B.c. e
Akizumari
± f
+++
+++
+
+
Everest Horyo Minowase Ohkura Sakurajima-ohmaru Shirofutori-miyashige-shirimaru Sofutori-miyashige Sushirazu-shogoin Uchiki-gensuke Wakayama
± + ± ± + ±
++ ++ +++ ++ + ++ +++ + +++ ++
+++ +++ ++ + + + ++ + +++ ++
+ ++ + ++ + +
++ ++ ++ + +
aCultures by the standard method. b,CCultureswith a nurse of (b), Nicotianatabacumor (c), Brassicacampestrismesophyllprotoplasts cultured for 7-14 days before use. ~'eCultures with conditioned medium prepared from culture broth of mesophyil protoplasts of (d), 31. tabacumor (e), R campestris which had been cultured for 7-14 days before use. fApproximate numbers of colonies were expressed by positive and negative signs. The signs correspond to actual number (x) per 60-mm dish as follows: - , x = 0 ; ±,1 < x < 10; +, 10 < x < 50; ++, 50 < x < 200; +++,200 < x. Colonies were counted 3 weeks after initiation of culture. The best results from three independent experiments are shown here.
109 Table IV. Effect of age of the nurse cells on colony formation from mesophyll protoplasts of Japanese radish cultivar Horyo.
Fig. 2. Nurse culture with Millicell-HA. Mesophyll protoplasts of the Japanese radish cultivar Horyo were cultured for 3 weeks with a nurse of Nicotiana suaveolens mesophyll protoplasts (right dish), or of cabbage hypocotyl protoplasts (middle), or without a nurse (left). Many colonies were observed in the right and middle dishes. Only a few colonies appeared in the left dish. The outside diameter of each dish is 59 mm.
t o p l a s t s o f t o b a c c o w e r e p u t i n t o a M i l l i c e l l as nurse and a fixed number of mesophyll protoplasts of Japanese radish was put into the dish outside t h e Millicell. T a b l e I I I s h o w s t h e r e s u l t s f r o m t w o experiments each replicated 3 times. Though the t h r e s h o l d n u m b e r o f n u r s e cells v a r i e d w i t h cultivars and with experiments, a dose effect of the
Table IlL
1.6 4.8 1.6 1.6
0 × x x x
103 103 104 105
Number of colonies
0 20 57 Control**
39 30 47 0
a a a b
*Nicotiana tabacum mesophyll protoplasts cultured for 0, 20 or 57 days were used as a nurse. **Control was the culture without a nurse. In this experiment, ~ 35-mm dishes and 4~ 12-mm Millicells were used. Colonies were counted 3 weeks after initiation of culture. Values represent means of 3 replicates. Means within a column not sharing the same letter are significantly different by Duncan's multiple range test at P = 0.05.
n u m b e r o f n u r s e cells o n c o l o n y f o r m a t i o n o f n u r s ed protoplasts was observed. These results suggest that Japanese radish prot o p l a s t s r e q u i r e s o m e a m o u n t o f n u r s e cells t o m u l t i p l y . T h e t h r e s h o l d n u m b e r t h a t m e e t s t h e requirements of each protoplast might differ from protoplast to protoplast even among protoplasts i s o l a t e d f r o m a s i n g l e leaf. T h u s , a m o n g p r o t o p l a s t s in a n y c u l t u r e , s o m e m a y d i v i d e w i t h a s m a l l a m o u n t o f n u r s e cells w h i l e t h e o t h e r s d o not.
Effect of nurse cell number on the colony formation from mesophyll protoplasts of Japanese radish cultivars.
Exp. 1 Nurse cell No.**
Age of Nurse cells (days)*
Exp. 2 Colony
No.*
Horyo
Wakayama
Ob 0b 0b 0b 390 a
28b 486 a 396 a 510 a 390 a
Nurse cell No.**
0.5 0.5 0.5 3.3
0 x x x x
102 103 104 104
Colony
No.*
Horyo
Ohkura
Oc 8c 15 bc 47 a 30 ab
3b 0b 1b 16 b 160 a
*Number of colonies per dish. **Inoculated number of nurse cells. In Exp. 1, the inoculated number of protoplasts of Horyo and Wakayama were both 1.5 × 105 per dish. In Exp. 2, ~ 35-mm dishes and ¢ 12-mm Millicells were used. The inoculated number of protoplasts of Horyo and Ohkora were 3.9 x 104 and 5.7 × 104, respectively. In both experiments, Nicotiana tabacum mesophyU protoplasts were used as a nurse. The number of colonies of Japanese radish was counted 3 weeks after initiation of culture. Values represent means of 3 replicates. Means within a column not sharing the same letter are significantly different by Duncan's multiple range test at P = 0.05.
110
Effect of the age of the nurse cells on their efficiency The efficiencies of N. tabacum protoplasts just isolated was compared with those cultured for 20 or 57 days in the nursing of mesophyll protoplasts of R. sativus (Table IV). All had the capacity for vigorous growth and showed nearly equal efficiencies as nurse cells regardless of age. Nursing activities of several species of plants Protoplasts or cultured cells of 10 species, the Table V.
taxonomic positions of which were variously distant from Raphanus, were examined for their ability to nurse mesophyll protoplasts of Raphanus. Three, cauliflower, kale and cabbage, belong to the same family as Raphanus, Cruciferae. Five,
Nicotiana tabacum, N. benthamiana, N. suaveolens, carrot and lettuce, belong to the same subclass, Dicotyledoneae, but different infraclasses. The remaining two, rice and asparagus, belong to a different subclass, Monocotyledoneae. Table V
Effect of cells of several plant species as a nurse on colony formation from mesophyll protoplasts of Japanese radish cultivars.
Exp. 1 Nurse cells
Col. No. a Horyo
Ohkura
Cauliflower hypocotyl protoplasts cultured for 22 days Cabbage hypocotyl protoplasts cultured for 22 days Lettuce cotyledon protoplasts cultured for 104 days N. suaveolens mesophyll protoplasts cultured for 20 days N. benthamiana mesophyll protoplasts cultured for 20 days N. tabacum mesophyll protoplasts cultured for 20 days None
NT b 108 4574630 477 4144 494-
Exp. 2 Nurse cells
Col. No. a Sof. M. c
Cauliflower mesophyll protoplasts Kale mesophyll protoplasts Lettuce cotyledon protoplasts cultured for 89 days N. suaveolens mesophyll protoplasts cultured for 5 days N. benthamiana mesophyll protoplasts cultured for 5 days N. tabacum mesophyll protoplasts cultured for 5 days None
3434450 4516 4414 + 228 484-
Exp. 3 Nurse cells
Col. No. a Wakayana
Nurse growth d
Kale mesophyll protoplasts N. tabacum mesophyll protoplasts None
324 4- 120 185 4- 70 24 4- 13
++ ++
Exp. 4 Nurse cells
Col. No. a Horyo
Sof. M. c
0+ 0 243 397 4- 148 425 4- 215 313 4- 91 040
040 442 158 4- 70 236 4- 106 94 4- 33 O± 0
Rice suspension culture cells, kpt 1 Rice suspension culture cells, kpt 10 Asparagus suspension culture cells Carrot suspension culture cells N. tabacurn mesophyll protoplasts cultured for 14 days None
43 14 177 20 37 8
4 2 108 60 130 84 6
114 4105 4124360 4NT NT 54-
24 35 7 73
5
Nurse growth d
++ ++ ++ +
aNumber of colonies. bNT means Not tested. cSofutori-Miyashige, a cultivar of Japanese radish. dGrowth of the nurse cells. Colonies were counted 2 weeks (Exp. 1), 3 weeks (Exps. 3 and 4) and 4 weeks (Exp. 2) after initiation of culture. Values represent means + S.D. of 3 replicates.
111
shows the results. All dicot species examined, regardless of taxonomic distance from Raphanus, were effective as nurses, so far as the nurse protoplasts or cells divided and grew during the culture period. As for monocots, Asparagus cells were effective but rice cells were not, though both of them appeared to be vigorous at the end of the culture (Table V, Exp. 4).
Effect of nursing on the viability and cell wall synthesis of Japanese radish mesophyll protoplasts How does nursing affect Japanese radish mesophyll protoplasts and allow them to form colonies? Three possibilities were considered: improvement of the viability of protoplasts, promotion of cell wall synthesis and induction and/or promotion of cell division. All three were observed in cultures with and without a nurse for a 14-day culture period (Fig. 3). The protoplasts or cells stainable with fluorescein diacetate were regarded as viable. The cell wall was identified by being stained with Calcofluor White. The proportion of viable protoplasts decreased over time, but no significant difference was observed between cultures with and without the nurse. Cells with cell walls were observed on the third day and their pro100
u~
~ 5o .c 0 u3
J~ h
,,~S ~ , 1 3 4
i
1~
Culture period (days)
11
Fig. 3. Time course of the proportion of viable cells and cells with cell walls in the cultures of mesophyll protoplasts of the Japanese radish cultivar Sofutori-miyashige with and without a nurse. Nicotiana suaveolens mesophyll protoplasts cultured for 14 days were used as nurses. On the indicated days, aliquots of the cultures were taken out and stained with fluorescein diacetate or Calcofluor White. The proportion of the stained cells among the total cells was then determined and expressed as follows: the proportion of cells stained with fluorescein diacetate in the cultures with a nurse, O; and without a nurse, O; the proportion of ceils stained with Calcofluor White in the cultures with a nurse, A; and without a nurse, A. The circles and the tliangles are connected with solid lines and broken lines, respectively.
portion reached about 50% on the fourth day, keeping this level thereafter till the end of culture. No significant difference in the proportion of the cells with cell walls was observed between cultures with or without a nurse. In the cultures with a nurse, dividing cells were observed on and after the seventh day and colonies were observed at the end of the culture period. By contrast, no dividing cells, or colonies, were observed in the cultures without a nurse. Thus, nursing makes Japanese radish mesophyll protoplasts form colonies by inducing or promoting cell division, but not by improving viability or by promoting cell wall synthesis. This is in accord with the results that have been reported for the effect of conditioned medium on maize protoplasts [2]. Discussion There are three possible mechanisms by which nurse cells or the conditioned medium promote division and colony formation in nursed protoplasts. The first supposes that the nurse cells or conditioned medium supply the nursed protoplasts with primary metabolites which leak out from the protoplasts. In this case, nurse cells or conditioned medium could be replaced with a supply of various primary metabolites [19], or else by reduction of culture volume to a microdroplet scale [21]. The second supposes that the nurse cells or conditioned medium supply the nursed protoplasts with some substance(s), which the nursed protoplasts cannot provide for themselves, necessary to induce or promote cell division. Such substances, we call them 'nurse substances', have been partially purified and characterized [22-24] but isolation and identification of such substances has not yet been achieved. The third supposes that the nurse cells or conditioned medium detoxify harmful substances in the medium. Caboche [25] reported that a high concentration of free NAA added to the medium was inhibitory to tobacco protoplasts cultured at low cell density and that nurse cells functioned to reduce free NAA drastically. Nursed protoplasts can be divided into two classes. One class includes those that can grow autonomously given sufficient cell density, but need a nurse at low cell density - - for example, cultures of heterokaryons produced by protoplast fusion
112
[26,27] or of injected protoplasts [28]. In this class, nurse cells seem to function by the first mechanism described above. The other class cannot divide without a nurse no matter what the cell density. Protoplasts of rice [4,5], wheat [61, soybean [29] and beet [30] belong to this class. In this class, nurse cells seem to function according to the second mechanism, though the possibility that the third mechanism is also relevant cannot be excluded. As for mesophyll protoplasts of Japanese radish, those of Everest did not divide even at high cell density of 106/ml while those of Minowase sporadically formed a few colonies at I04 and 105/ml but not at 106/ml. From these results, mesophyll protoplasts of Japanese radish are considered to belong to the latter class. The protoplasts of monocot species, such as rice [4], maize [3] and oat [7], that need the help of nurse or feeder cells, accept only the cells of the same species or of closely related species of mono¢ots as a nurse, not the cells of dicots. From the experiments on the effect of conditioned medium on the colony formation of maize protoplasts, Sornerset al. [2] suggested that the conditioning factor might be different and specific, for monocots and dicots. By contrast, the Japanese radish protoplasts accepted the cells of a very wide range of species, even the monocot Asparagus, as a nurse. This result together with the result reported by Waiters and Earle [31] that protoplasts of a recalcitrant genotype of Brassica oleracea accepted cell suspensions of Brassica campestris and tomato as nurse, suggests that there might be a spectrum of nurse substances which have somewhat common structures among angiosperms and that the specificity of the Japanese radish protoplasts for nurse substances may be lower than that of monocot species. Shoot differentiation from the colonies of the Japanese radish has been tried extensively using various media, but no shoots have been obtained yet. Kishimoto [10] reported that only 2 colonies from cotyledon protoplasts of Japanese radish regenerated shoots. Our nurse culture system is being used in protoplast fusion experiments between Japanese radish and Brassica oleracea the protoplasts of which regenerate readily.
Acknowledgment We thank Miss assistance. We also Saito and Mr. K. suspension cultured
J. Kitazumi for technical thank Dr. K. Ohno, Mr. T. Tsuji for providing us with cell lines.
References 1 C.A. Rhodes, K.S. Lowe and K.L. Ruby, Plant regeneration from protoplasts isolated from embryogenic maize cell cultures. Bio/technology, 6 (1988) 56-60. 2 D.A. Somers, P.R. Birnberg, W.L. Petersen and M.L. Brenner, The effect of conditioned medium on colony formation from Black mexican sweet corn protoplasts. Plant Sci., 53 (1987) 249-256. 3 J.A. Smith, C.E. Green and B.G. Gengenbach, Feeder layer support of low density populations of Zea mays L. suspension cells. Plant Sci. Lett., 36 (1984) 67-72. 4 J. Kyozuka, Y. Hayashi and K. Shimamoto, High frequency plant regeneration from rice protoplasts by novel nurse culture methods. Mol. Gen. Genet., 206 (1987) 408 -413. 5 L. Lee, R.E. Schroll, H.D. Grimes and T.K. Hodges, Plant regeneration from indica rice (Oryza sativa L.) protoplasts. Planta, 178 (1989) 325-333. 6 Y. Hayashi and K. Shimamoto, Wheat protoplast culture: embryogenic colony formation from protoplasts. Plant Cell Rep., 7 (1988) 414-417. 7 B. Hahne, H. L6rz and G. Hahne, Oat mesophyl protoplasts: their response to various feeder cultures. Plant Cell Rep., 8 (1990) 590-593. 8 P.S. Jourdan and E.D. Earle, Genotypic variability in the frequency of plant regeneration from leaf protoplasts of four Brassica-spp and of Raphanus sativus. J. Am. Soc. Hort. Sci., 114 (1989) 343-349. 9 T. Nishio, Y. Sakata, H. Yamagishi, Y. Tabei, T. Sato and K. Takayanagi, Protoplast culture in vegetable crops. Development and improvement of culture procedure. Bull. Natl. Res. Inst. Veg. Ornamental Plants Tea, A3 (1989) 67-96. 10 N. Kishimoto, Shoot regeneration from cotyledon protoplast of Japanese radish (Raphanus sativus L.). Plant Tiss. Cult. Lett., 7 (1990) 114-115. 11 T. Murashige and F. Skoog, A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol. Plant. 15 (1962) 473-497. 12 K. Ohira, K. Ojima and A. Fujiwara, Studies on the nutrition of rice culture I. A simple, defined medium for rapid growth in suspension culture. Plant Cell Physiol., 14 (1973) 1113-1121. 13 T. Saito, S. Nishizawa and S. Nishimura, Improved culture conditions for somatic embryogenesis from Asparagus officinalis L. using an aseptic ventilative filter. Plant Cell Reports, 10 (1991) 230-234.
113 14
15
16
17
18
19
20
21
22
23
E.M. Linsmaier and F. Skoog, Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant., 18 (1965) 100-126. T. Fujimura and A. Komamine, Synchronization of somatic embryogenesis in a carrot cell suspension culture. Plant Physiol., 64 (1979) 162-164. T. Nagata and I. Takebe, Plating of isolated tobacco mesophyll protoplasts on agar medium. Planta, 99 (1971) 12-20. B.L. Nadel, Use of fluorescein diacetate in citrus tissue cultures for the determination of cell viability and the selection of mutants. Sci. Hort., 39 (1989) 15-21. T. Nagata and I. Takebe, Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts. Planta, 92 (1970) 301-308. K.N. Kao and M.R. Michayluk, Nutritional requirements for growth of Vicia hajastana cells and protoplasts at a very low population density in liquid media. Planta, 126 (1975) 105-110. G. Pelletier, C. Primard, F. Vedel, P. Chetrit, R. Remy, X.X. Rousselle and M. Renard, Intergeneric cytoplasmic hybridization in Cruciferae by protoplast fusion. Mol. Gen. Genet., 191 (1983) 244-250. H.U. Koop, G. Weber and H.G. Schweiger, Individual culture of selected single cells and protoplasts of higher plants in microdroplets of defined media. Z. Pflanzenphysiol., 112 (1983) 21-34. R. Stuart and H.E. Street, Studies on the growth of plant cells X. Further studies on the conditioning of culture media by suspensions of Acer pseudoplatanus L. cells. J. Exp. Bot., 22 (1971) 96-106. F. Kohler and G. Wenzel, Regeneration of isolated barley
microspores in conditioned media and trials to characterize the responsible factor. J. Plant Physiol., 121 (1985) 181-191. 24 P.R. Birnberg, D.A. Somers and M.L. Brenner, Characterization of conditioning factors that increase colony formation from Black mexican sweet corn protoplasts. J. Plant Physiol., 132 (1988) 316-321. 25 M. Caboche, Nutritional requirements of protoplastderived, haploid tobacco cells grown at low densities in liquid medium. Planta, 149 (1980) 7-18. 26 H. Morikawa, Y. Hayashi, N. Ohnishi, Y. Yamamoto, F. Sato and Y. Yamada, Culture of electrically induced heterokaryons of plant cells rich in secondary metabolites. Plant Cell Physiol., 29 (1988) 1201-1206. 27 M.R. Thomas and R.J. Rose, Enrichment for Nicotiana heterokaryons after protoplast fusion and subsequent growth in agarose microdrops. Planta, 175 (1988) 396-402. 28 W.A. Lawrence and D.R. Davies, A method for the microinjection and culture of protoplasts at very low densities. Plant Cell Rep., 4 (1985) 33-35. 29 D.M. Tricoli, M.B. Hein and M.G. Carnes, Culture of soybean mesophyll protoplasts in alginate beads. Plant Cell Reports, 5 (1986) 334-337. 30 S.R. Bhat, B.V. Ford-Lloyd and J.A. Callow, Isolation and culture of mesophyll protoplasts of garden, fodder and sugar beets using a nurse culture system: Callus formation and organogenesis. J. Plant Physiol., 124 (1986) 419-423. 31 T.W. Waiters and E.D. Earle, A simple, versatile feeder layer system for Brassica oleracea protoplast culture. Plant Cell Rep., 9 (1990) 316-319.