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Factors which Enhance in vitro Morphogenesis of Arabidopsis thaliana
Laboratorium voor Plantengenetica, Free University of Brussels, Belgium
Factors which Enhance in vitro Morphogenesis of Arabidopsis thaliana lOAN NEGRUTIU
1
)
and
MICHEL JACOBS
Received June 26, 1978 . Accepted July 22, 1978
Summary The regeneration efficiency of Arabidopsis callus cultures can be influenced by several factors as the culture medium (solid or liquid medium, method of sterilization, nitrogen source), the inoculum size, the light and low temperature treatment, and the interval between subcultures. These factors, alone or in combination, were shown to enhance at various degrees the morphogenetic response of callus cultures. Subculture intervals of 6 to 8 weeks in continuous low light or darkness, 4 DC cold treatment for 3 days prior to regeneration, and the use of a low inoculum size in liquid, filter-sterilized regeneration medium exert a positive effect on the regeneration of Arabidopsis calluses.
Key words: Arabidopsis thaliana, in vitro morphogenesis, inoculum size, light and low temperature treatment, interval between subcultures.
Introduction
Studies of in vitro regeneration of Arabidopsis callus cultures have elucidated some of the conditions which permit successful and efficient plantlet formation (GRESSHOFF, 1973; NEGRUTIU, 1976). By using defined geographical races, callus cultures derived from anthers were shown to be the best material for in vitro morphogenesis. The isolation of morphologically distinct callus lines was shown to provide a useful method for selecting highly morphogenetic material. Defined sequences of culture media have been used in order to establish the optimal hormonal balance leading to leaf regeneration (NEGRUTIU et aI., 1978 a; NEGRUTIU et aI., 1978 b). In this paper we consider other factors which might affect the induction of morphogenesis, i. e. the culture medium (its physical nature, the method of sterilization, the nitrogen source present in media used prior to regeneration); the inoculum size, the light and low temperature treatments, the interval between subcultures. These factors, individually or in combination, are shown to enhance at various degrees the morphogenetic response of Arabidopsis callus cultures. 1) Fellow of the Roumanian Government.
Z. PJlanzenphysiol. Bd. 90. S. 423-430. 1978.
424
lOAN NEGRUTIU and MICHEL JACOBS
Material and Methods Materials Seeds, anthers, and leaf explants were used to initiate callus cultures. Regeneration experiments were performed on several established cultures (Est 1075 A, F, Sand S/SB, Est 0876 A and A/SB, Col 0974 S, Col 0575 S and Co 1175 S) obtained from the geographical races Estland, Columbia and Colmbra. Nutrient media and culture conditions Media and culture conditions have been described elsewhere (NEGRUTIU et aI., 1978 a). Callus cultures are maintained on solidified PG 2 medium (5 X 10-6 M 2,4 D + 2.5 X 10-7 M kinetin) in the dark at 25°C. The calluses are subcultured at 4 week intervals. Regeneration of leaves and roots is obtained on solid or liquid PG s (5 X 10-6 M kinetin + 1.5 X 10-4 M lAA) with a 16/8 hrs photoperiod. All phytohormones are filter-sterilised through 0.2 flm Millipore filters. All the other components are sterilised by autoc1aving, except where otherwise stated. Estimation of the morphogenetic response The morphogenetic response of Arabidopsis callus cultures is considered as the expression of the morphogenetic potential of a population of cells under a given set of inductive culture conditions. It is most frequently referred to as leaf formation and expressed as the number of calluses which regenerate leaves as a percentage of the total number of calluses transferred to the regeneration medium. Where root formation is considered, the percentage of roots producing calluses is considered. Whole plant development is achieved by transferring the newly formed rosettes to a medium lacking growth substances.
Results
1. The physical nature of the culture medium and the method of sterilization Calluses agitated at low speed (40-50 min.) in liquid PG 3 medium, gave higher frequencies of leaf formation than on solid medium (Table 1). Filter sterilization of the hormones and glucose doubles the morphogenetic response as compared to filter-sterilization of the hormones only. Regeneration in an all filter-sterilized, liquid medium gave very high regeneratlon frequencies, thus combining the advantages of filter-sterilization and liquid culture. Table 1: The effect of sterilization method of various culture medium components and of the physical nature of the culture medium on leaf formation frequency of Est 0876 A callus cultures (25 weeks in culture, subcultured at 6 week intervals). Regeneration on PGa.
Frequency of leaf formation Range of variation
Solid culture, hormones filtersterilized (control)
Liquid culture, hormones filtersterilized
Solid culture, hormones + gl ucose filtersterilized
Liquid culture, complete medium filter-sterilized
28 (16-38)
41 32-60)
60 (48-66)
89 (80-100)
Z. Pflanzenphysiol. Bd. 90. S. 423-430. 1978.
Morphogenesis in Arabidopsis thaliana
425
2. Inoculum size
Different inoculum weights were used to study the influence of inoculum size on leaf formation after three weeks culture in liquid PG 3 medium (Table 2). The best results were obtained with the smallest inoculum tested. It seems that, once leaf formation is initiated, the biomass accumulation is slowed down at low inoculum weight-to-volume of culture medium ratios. On the other hand, large inoculum size favours rapid callus proliferation at the expense of morphogenetic responses.
Table 2: The effect of inoculum size on leaf formation in Est 0876 A/SB (36 weeks in culture, subcultured at 6 week intervals). Regeneration in liquid, filter-sterilized PG 3 , 20 mil petridish. The range of variation is given in brackets.
Frequency of leaf formation (010) Fresh weight increase after 21 days (g)
Inoculum size (g) 0.075 0.125
0.25
90 (80-100) 0.16 (0.14-0.185)
15 (0-43) 0.70 (0.30-1.30)
53 (20-90) 0.30 (0.25-0.33)
3. The nitrogen source prior to regeneration
Calluses were transferred for the last passage prior to regeneration, from standard PG 2 to similar media containing 0.6 gil urea, 0.36 gil ammonium, supplied as (NH 4 )2S04, or 1.5 gil glutamine as the sole source of nitrogen. Kinetin and zeatin were used as alternative cytokinins in the regeneration medium PG 3 (Table 3). The highest frequency of leaf formation and the highest number of regeneration centres per callus were obtained after the passage on glutamine. Urea produced a high frequency of morphologically normal plantlets, while ammonium markedly Table 3: The effect of nitrogen source on leaf formation frequency (010) in Est 0876 A, 36 weeks in culture, subcultured at 6 week intervals. Calluses were transfered to different nitrogen sources for one passage before regeneration. Regeneration in filter-sterilized, liquid PGs. Inoculum size: 0.125 g per 20 ml medium. Kinetin or zeatin were used as cytokinin sources in PGs . The range of variation is given in brackets. Cytokinin used in PG a
Kinetin Zeatin
Nitrogen source prior to regeneration Standard PG 2 (control) NO a-/NH 4+
Urea (0.6 gil)
Glutamine (1.5 gil)
Ammonium (1.32 gil) (NH4hS04
33 (20-40) 39 '(26-60)
33 (20-50) 21 (0-60)
46 (33-80) 41 (26-53)
3 (0-6) 16 (6-33)
Z. P/lanzenphysiol. Bd. 90. S. 423-430. 1978.
426
lOAN NEGRUTIU
and
MICHEL JACOBS
decreased subsequent leaf formation. In the latter case, the regenerants were arrested at the leaf-primordium stage. 4. The light treatment prior to regeneration
Calluses derived from seeds and anthers initiated and maintained under various light regimes, were regenerated on PG a (Table 4). Calluses cultured in continuous low light intensity (L. 1000 lux), or transferred from the dark to continuous low light, showed a higher frequency of leaf formation than dark-grown callus. A 16 hour light-8 hour dark cycle, or continuous high light intensity during callus initiation or proliferation, reduced the percentage of regenerating calluses. However, the low light regime does not prevent the progressive decline in leaf-forming response that occurs during prolonged callus culwre. Table 4: The effect of light treatment during callus proliferation on frequency of leaf formation. Calluses were subcultured at 4 week intervals. Regeneration on solid PGa. Callus culture
Est 1075 S
Est 1075 A
Light treatment during callus proliferation Time in culture (weeks)
continuous dark (control)
continuous low light intensity «1000 lux)
continuous light (>4000 lux)
16/8hrs photoperiod
5 10 25 35 25
50 18 2 0 9
75 31 4 1 23
3 0
55 15
5. Low-temperature treatment prior to regeneration A 4 DC cold treatment lasting 3 or 6 days was given to different callus cultures before they were transferred to the regeneration medium. A general improvement in morphogenetic responses was observed. Leaf formation (Table 5) was significantly enhanced as compared to the control. Occasionally, the cold treatment enabled us to «restore» leaf formation in older cultures which no longer regenerate under standard conditions. As a rule, a 3 day treatment appeared to be effective enough. Rhizogenesis was strongly stimulated by the cold shock (Table 6). A 6 day treatment seems to be optimal for the 9 months old callus tested. Both the number of calluses producing roots, as well as the weight of roots per callus were markedly increased by the temperature shock.
6. The interval between subcultures The influence of the subculture interval on the efficiency of leaf regeneration was determined in callus cultures derived from seeds, anthers and leaves (Table 7). An
z.
PJlanzenphysiol. Bd. 90. S. 423-430. 1978.
Morphogenesis in Arabidopsis thaliana
427
Table 5: The effect of 4 DC cold treatment on leaf formation frequency in different callus cultures. The cold shock is given before the transfer of calluses to the regeneration medium PG s. Callus culture
Time in culture (weeks)
Duration of 4 DC treatment ( days) 0 3 6
Co 1175 S
15 30 5 35 15 25
20 0 .21 4 38 0
Col 0575 S Est 1975 S
17 6 53 0 46 8
31 0 4 49 2
Table 6: The effect of 4 DC cold treatment on root formation frequency (% RR) and root biomass in Col 0974 S (35 weeks in culture, subcultured at 4 week intervals). Root formation after 6 weeks on PG s . Root formation frequency and root biomass accumulation
o
4
6
14
Ofo RR
40
53
71
43
11.7 0.72
37 1.6
35 2.1
Root biomass per root forming callus (mg)
Duration of 4 DC treatment (days) (control)
fresh weight dry weight
3 0.25
Table 7: The effect of subculture interval on leaf formation. Calluses were subcultured at the intervals shown below during the last 12 weeks before regeneration on PGs. Callus culture and time in culture (weeks) Est 1075 A (25) Est 1075 S (25) Est 1075 F (25) Est 1075 A (35)
Percentage of leaf regeneration after different subculturing regimes (weeks) 4+4+4 4+8 8+4 12 13
36
15
0
2
4
2
0
10
19
12
2
3
31
eight week passage immediately before regeneration gives at least twice the frequency of leaf formation than a four week one. However, if an eight week passage is followed by a four week one before regeneration, there is no net improvement. The regeneration frequency remains similar to that of the control on a four week
z.
Pjlanzenphysiol. Bd. 90. S. 423-430. 1978.
428
lOAN NEGRUTIU
and
MICHEL JACOBS
subculture regime. Passages of more than 8 weeks are not satisfactory because the growth rate decreased, most of the calluses turn brown and do not regenerate when they are transferred to the regeneration medium.
Discussion The present work describes some of the culture conditions which can influence the efficiency of regeneration in in vitro cultures. Several of the factors studied e. g. light treatment, temperature shock, nitrogen source, and interval between subcultures were found to be of considerable importance for the subsequent induction of organogenesis. The results indicate that several parameters of a cell culture should be strictly controlled already before the «switch on» of morphogenetic response by any of the inductive factors. In other words, the inductive factors cannot replace or act in the absence of a «state of readiness» for organ formation. Filter-sterilization of the complete medium combined with the use of a liquid culture method during regeneration, greatly improved leaf formation in all our callus cultures. Sterilization of the carbon source by filtration seems to be of particular importance. The need to use specific inoculum sizes indicates the existence of a balance between morphogenetic process and callus proliferation. This balance in its turn probably reflects the evolution of the endogenous auxin-cytokinin balance either towards morphogenesis (low inoculum size) or towards callus growth (large inoculum size). The control of differentiation by nitrogenous compounds has been studied in Arabidopsis callus cultures in terms of nitrogen source and nitrogen-IAA interaction (GRESSHOFF, 1973). He reported that nitrate concentration was critical (30 mM) and that it cannot be replaced by ammonium, glutamine or casein hydrolysate. Our results indicate that a stimulation of leaf formation can be obtained when glutamine is used as the sole nitrogen source prior to regeneration. We have some evidence that glutamine has no effect on leaf formation frequency when added to the standard PG a regeneration medium. WETHERELL and DOUGAL (1976) reported that glutamine, alanine and possibly glutamic acid can serve as sole source of nitrogen supporting both good growth and embryogenesis in carrot cultures. On the other hand, with ammonium as the sole source of nitrogen before regeneration, there was a pronounced negative effect on the leaf formation frequency and the morphology of regenerants. The low temperature treatment which was shown to have a general, positive influence on morphogenetic processes, may act by synchronizing small groups of cells (DEVREUX et al., 1975). There is some evidence which suggests that bud primordia of certain species can originate from several cell acting in a coordinated manner (TRAN THANH VAN et al., 1974; STREE RAMULU et al., 1976). Thus it may be that initiation of cell division centers is stimulated by low temperature treatment.
z.
P/lanzenphysiol. Bd. 90. S. 423-430. 1978.
Morphogenesis in Arabidopsis thaliana
429
Studies on the effect of light on in vitro regeneration of tobacco have shown that wavelength and intensity are equally important parameters in determining morphogenetic processes (SEIBERT et aI., 1975). Our results provide further evidence regarding the influence of light pre-treatments on subsequent regeneration. High intensities reduced leaf formation while continuous low light intensity enhanced morphogenesis. GRESSHOFF (1973) suggested that light may act through an auxin-mediated developmental process, based on the photo-lability of auxin. Maize seedlings grown in the light were shown to absorb significantly less auxin than their dark-grown counterparts (NAQUI, 1975). Passage times which were longer than those used for routine maintenance of callus cultures, proved more satisfactory for subsequent regeneration. MEYER-TEUTER and REINERT (1973) have proposed that loss of the ability of cultured cells to redifferentiate can be correlated with the number of division cycles which the cells have undergone prior to the induction. In Arabidopsis callus cultures the number of division cycles was 1,5-fold greater when calluses were subcultured every four weeks as compared to 8 weeks. Leaf formation was enhanced by a factor 2 to 3 after only one passage of 8 weeks instead of 4 weeks. The effect of an 8 week interval was not apparent when a 4 week interval followed the 8 week one; the 4 week + 8 week reported conclusions (NEGRUTIU et aI., 1978 a) and may indicate that the endogenous hormone levels become unbalanced as a consequence of subculturing at 4 week intervals. At least a part of the enhanced morphogenetic response obtained by using longer subculture intervals can be attributed to a correction of the hormonal balance. KOCHBA and SPIEGEL-Roy (1977) have also suggested that increased embryogenesis in ovular callus of orange as a consequence of «ageing» could be due to a reduction in endogenous auxin levels. We have also observed that if calluses from a single source were subcultured for several passages at four or eight week intervals, the short intervals resulted in more friable callus, usually with lower morphogenetic ability, while longer intervals favoured the proliferation of calluses with higher morphogenetic capacity (NEGRUTIU et aI., in the press). In conclusion, an enhanced frequency of leaf formation can be obtained by using a defined set of physical and chemical culture conditions such as subculture intervals of 6 to 8 weeks in continuous low light or darkness, 4 °C treatments for 3 days prior to regeneration, and a specific low inoculum size in a liquid and filter-sterilised regeneration medium. Acknowledgements This work was supported by a grant from the I.W.O.N.L. (krediet 2383 A).
References DEVREUX, M., U. LANERI, and P. DE MARTINIS: Giornale Botanico Italiano, 109, 335 (1975). GRESSHOFF, P. M.: Ph. D. Thesis. University of Canberra, Oct. 1973. KOCHBA, ]. and P. SPIEGEL-Roy: Z. Pflanzenphysiol. 81, 283 (1977).
Z. Pjlanzenphysiol. Ed. 90. S. 423-430. 1978.
430
lOAN NEGRUTIU and MICHEL JACOBS
MEYER-TEUTER, H. and]. REINERT: Protoplasma, 78,273 (1973). NAQUI, S. M.: Z. Pflanzenphysiol. 76, 379 (1975). NEGRUTIU, 1.: Arabidopsis Inf. Service, 13, 180 (1976). NEGRUTIU, 1., M. JACOBS, and D. CACHITA: Z. Pflanzenphysiol. 86, 113 (1978 a). NEGRUTIU, 1., M. JACOBS, and W. DE GREEF: Z. Pflanzenphysiol. 90,363 (1978 b). SEIBERT, M., P. J. WETHERBEE, and D. D. JOB: Plant Physiol., 56, 130 (1975). STREE RAMULU, K., M. DEVREUX, G. ANCORA, and V. LANERI: Z. Pflanzenziicht. 76, 299
(1976).
TRAN THANHVAN, M., H. CHLYAH, and A. CHLYAH: Regulation of organogenesis in thin layer of epidermal and sub-epidermal cells. In: STREET H. E. (Ed.): Tissue culture and plant science, 101-239, Academic Press, London, 1974. WETHERELL, D. 1. and D. K. DOUGALL: Physiol. Plant. 37, 97 (1976).
M. JACOBS, Laboratorium voor Plantengenetica, Free University of Brussels, Sint-Genesius-Rode, Brussels, Belgium.
z. Pflanzenphysiol. Bd. 90. S. 423-430.
1978.
Factors which Enhance in vitro Morphogenesis of Arabidopsis thaliana lOAN NEGRUTIU
1
)
and
MICHEL JACOBS
Received June 26, 1978 . Accepted July 22, 1978
Summary The regeneration efficiency of Arabidopsis callus cultures can be influenced by several factors as the culture medium (solid or liquid medium, method of sterilization, nitrogen source), the inoculum size, the light and low temperature treatment, and the interval between subcultures. These factors, alone or in combination, were shown to enhance at various degrees the morphogenetic response of callus cultures. Subculture intervals of 6 to 8 weeks in continuous low light or darkness, 4 DC cold treatment for 3 days prior to regeneration, and the use of a low inoculum size in liquid, filter-sterilized regeneration medium exert a positive effect on the regeneration of Arabidopsis calluses.
Key words: Arabidopsis thaliana, in vitro morphogenesis, inoculum size, light and low temperature treatment, interval between subcultures.
Introduction
Studies of in vitro regeneration of Arabidopsis callus cultures have elucidated some of the conditions which permit successful and efficient plantlet formation (GRESSHOFF, 1973; NEGRUTIU, 1976). By using defined geographical races, callus cultures derived from anthers were shown to be the best material for in vitro morphogenesis. The isolation of morphologically distinct callus lines was shown to provide a useful method for selecting highly morphogenetic material. Defined sequences of culture media have been used in order to establish the optimal hormonal balance leading to leaf regeneration (NEGRUTIU et aI., 1978 a; NEGRUTIU et aI., 1978 b). In this paper we consider other factors which might affect the induction of morphogenesis, i. e. the culture medium (its physical nature, the method of sterilization, the nitrogen source present in media used prior to regeneration); the inoculum size, the light and low temperature treatments, the interval between subcultures. These factors, individually or in combination, are shown to enhance at various degrees the morphogenetic response of Arabidopsis callus cultures. 1) Fellow of the Roumanian Government.
Z. PJlanzenphysiol. Bd. 90. S. 423-430. 1978.
424
lOAN NEGRUTIU and MICHEL JACOBS
Material and Methods Materials Seeds, anthers, and leaf explants were used to initiate callus cultures. Regeneration experiments were performed on several established cultures (Est 1075 A, F, Sand S/SB, Est 0876 A and A/SB, Col 0974 S, Col 0575 S and Co 1175 S) obtained from the geographical races Estland, Columbia and Colmbra. Nutrient media and culture conditions Media and culture conditions have been described elsewhere (NEGRUTIU et aI., 1978 a). Callus cultures are maintained on solidified PG 2 medium (5 X 10-6 M 2,4 D + 2.5 X 10-7 M kinetin) in the dark at 25°C. The calluses are subcultured at 4 week intervals. Regeneration of leaves and roots is obtained on solid or liquid PG s (5 X 10-6 M kinetin + 1.5 X 10-4 M lAA) with a 16/8 hrs photoperiod. All phytohormones are filter-sterilised through 0.2 flm Millipore filters. All the other components are sterilised by autoc1aving, except where otherwise stated. Estimation of the morphogenetic response The morphogenetic response of Arabidopsis callus cultures is considered as the expression of the morphogenetic potential of a population of cells under a given set of inductive culture conditions. It is most frequently referred to as leaf formation and expressed as the number of calluses which regenerate leaves as a percentage of the total number of calluses transferred to the regeneration medium. Where root formation is considered, the percentage of roots producing calluses is considered. Whole plant development is achieved by transferring the newly formed rosettes to a medium lacking growth substances.
Results
1. The physical nature of the culture medium and the method of sterilization Calluses agitated at low speed (40-50 min.) in liquid PG 3 medium, gave higher frequencies of leaf formation than on solid medium (Table 1). Filter sterilization of the hormones and glucose doubles the morphogenetic response as compared to filter-sterilization of the hormones only. Regeneration in an all filter-sterilized, liquid medium gave very high regeneratlon frequencies, thus combining the advantages of filter-sterilization and liquid culture. Table 1: The effect of sterilization method of various culture medium components and of the physical nature of the culture medium on leaf formation frequency of Est 0876 A callus cultures (25 weeks in culture, subcultured at 6 week intervals). Regeneration on PGa.
Frequency of leaf formation Range of variation
Solid culture, hormones filtersterilized (control)
Liquid culture, hormones filtersterilized
Solid culture, hormones + gl ucose filtersterilized
Liquid culture, complete medium filter-sterilized
28 (16-38)
41 32-60)
60 (48-66)
89 (80-100)
Z. Pflanzenphysiol. Bd. 90. S. 423-430. 1978.
Morphogenesis in Arabidopsis thaliana
425
2. Inoculum size
Different inoculum weights were used to study the influence of inoculum size on leaf formation after three weeks culture in liquid PG 3 medium (Table 2). The best results were obtained with the smallest inoculum tested. It seems that, once leaf formation is initiated, the biomass accumulation is slowed down at low inoculum weight-to-volume of culture medium ratios. On the other hand, large inoculum size favours rapid callus proliferation at the expense of morphogenetic responses.
Table 2: The effect of inoculum size on leaf formation in Est 0876 A/SB (36 weeks in culture, subcultured at 6 week intervals). Regeneration in liquid, filter-sterilized PG 3 , 20 mil petridish. The range of variation is given in brackets.
Frequency of leaf formation (010) Fresh weight increase after 21 days (g)
Inoculum size (g) 0.075 0.125
0.25
90 (80-100) 0.16 (0.14-0.185)
15 (0-43) 0.70 (0.30-1.30)
53 (20-90) 0.30 (0.25-0.33)
3. The nitrogen source prior to regeneration
Calluses were transferred for the last passage prior to regeneration, from standard PG 2 to similar media containing 0.6 gil urea, 0.36 gil ammonium, supplied as (NH 4 )2S04, or 1.5 gil glutamine as the sole source of nitrogen. Kinetin and zeatin were used as alternative cytokinins in the regeneration medium PG 3 (Table 3). The highest frequency of leaf formation and the highest number of regeneration centres per callus were obtained after the passage on glutamine. Urea produced a high frequency of morphologically normal plantlets, while ammonium markedly Table 3: The effect of nitrogen source on leaf formation frequency (010) in Est 0876 A, 36 weeks in culture, subcultured at 6 week intervals. Calluses were transfered to different nitrogen sources for one passage before regeneration. Regeneration in filter-sterilized, liquid PGs. Inoculum size: 0.125 g per 20 ml medium. Kinetin or zeatin were used as cytokinin sources in PGs . The range of variation is given in brackets. Cytokinin used in PG a
Kinetin Zeatin
Nitrogen source prior to regeneration Standard PG 2 (control) NO a-/NH 4+
Urea (0.6 gil)
Glutamine (1.5 gil)
Ammonium (1.32 gil) (NH4hS04
33 (20-40) 39 '(26-60)
33 (20-50) 21 (0-60)
46 (33-80) 41 (26-53)
3 (0-6) 16 (6-33)
Z. P/lanzenphysiol. Bd. 90. S. 423-430. 1978.
426
lOAN NEGRUTIU
and
MICHEL JACOBS
decreased subsequent leaf formation. In the latter case, the regenerants were arrested at the leaf-primordium stage. 4. The light treatment prior to regeneration
Calluses derived from seeds and anthers initiated and maintained under various light regimes, were regenerated on PG a (Table 4). Calluses cultured in continuous low light intensity (L. 1000 lux), or transferred from the dark to continuous low light, showed a higher frequency of leaf formation than dark-grown callus. A 16 hour light-8 hour dark cycle, or continuous high light intensity during callus initiation or proliferation, reduced the percentage of regenerating calluses. However, the low light regime does not prevent the progressive decline in leaf-forming response that occurs during prolonged callus culwre. Table 4: The effect of light treatment during callus proliferation on frequency of leaf formation. Calluses were subcultured at 4 week intervals. Regeneration on solid PGa. Callus culture
Est 1075 S
Est 1075 A
Light treatment during callus proliferation Time in culture (weeks)
continuous dark (control)
continuous low light intensity «1000 lux)
continuous light (>4000 lux)
16/8hrs photoperiod
5 10 25 35 25
50 18 2 0 9
75 31 4 1 23
3 0
55 15
5. Low-temperature treatment prior to regeneration A 4 DC cold treatment lasting 3 or 6 days was given to different callus cultures before they were transferred to the regeneration medium. A general improvement in morphogenetic responses was observed. Leaf formation (Table 5) was significantly enhanced as compared to the control. Occasionally, the cold treatment enabled us to «restore» leaf formation in older cultures which no longer regenerate under standard conditions. As a rule, a 3 day treatment appeared to be effective enough. Rhizogenesis was strongly stimulated by the cold shock (Table 6). A 6 day treatment seems to be optimal for the 9 months old callus tested. Both the number of calluses producing roots, as well as the weight of roots per callus were markedly increased by the temperature shock.
6. The interval between subcultures The influence of the subculture interval on the efficiency of leaf regeneration was determined in callus cultures derived from seeds, anthers and leaves (Table 7). An
z.
PJlanzenphysiol. Bd. 90. S. 423-430. 1978.
Morphogenesis in Arabidopsis thaliana
427
Table 5: The effect of 4 DC cold treatment on leaf formation frequency in different callus cultures. The cold shock is given before the transfer of calluses to the regeneration medium PG s. Callus culture
Time in culture (weeks)
Duration of 4 DC treatment ( days) 0 3 6
Co 1175 S
15 30 5 35 15 25
20 0 .21 4 38 0
Col 0575 S Est 1975 S
17 6 53 0 46 8
31 0 4 49 2
Table 6: The effect of 4 DC cold treatment on root formation frequency (% RR) and root biomass in Col 0974 S (35 weeks in culture, subcultured at 4 week intervals). Root formation after 6 weeks on PG s . Root formation frequency and root biomass accumulation
o
4
6
14
Ofo RR
40
53
71
43
11.7 0.72
37 1.6
35 2.1
Root biomass per root forming callus (mg)
Duration of 4 DC treatment (days) (control)
fresh weight dry weight
3 0.25
Table 7: The effect of subculture interval on leaf formation. Calluses were subcultured at the intervals shown below during the last 12 weeks before regeneration on PGs. Callus culture and time in culture (weeks) Est 1075 A (25) Est 1075 S (25) Est 1075 F (25) Est 1075 A (35)
Percentage of leaf regeneration after different subculturing regimes (weeks) 4+4+4 4+8 8+4 12 13
36
15
0
2
4
2
0
10
19
12
2
3
31
eight week passage immediately before regeneration gives at least twice the frequency of leaf formation than a four week one. However, if an eight week passage is followed by a four week one before regeneration, there is no net improvement. The regeneration frequency remains similar to that of the control on a four week
z.
Pjlanzenphysiol. Bd. 90. S. 423-430. 1978.
428
lOAN NEGRUTIU
and
MICHEL JACOBS
subculture regime. Passages of more than 8 weeks are not satisfactory because the growth rate decreased, most of the calluses turn brown and do not regenerate when they are transferred to the regeneration medium.
Discussion The present work describes some of the culture conditions which can influence the efficiency of regeneration in in vitro cultures. Several of the factors studied e. g. light treatment, temperature shock, nitrogen source, and interval between subcultures were found to be of considerable importance for the subsequent induction of organogenesis. The results indicate that several parameters of a cell culture should be strictly controlled already before the «switch on» of morphogenetic response by any of the inductive factors. In other words, the inductive factors cannot replace or act in the absence of a «state of readiness» for organ formation. Filter-sterilization of the complete medium combined with the use of a liquid culture method during regeneration, greatly improved leaf formation in all our callus cultures. Sterilization of the carbon source by filtration seems to be of particular importance. The need to use specific inoculum sizes indicates the existence of a balance between morphogenetic process and callus proliferation. This balance in its turn probably reflects the evolution of the endogenous auxin-cytokinin balance either towards morphogenesis (low inoculum size) or towards callus growth (large inoculum size). The control of differentiation by nitrogenous compounds has been studied in Arabidopsis callus cultures in terms of nitrogen source and nitrogen-IAA interaction (GRESSHOFF, 1973). He reported that nitrate concentration was critical (30 mM) and that it cannot be replaced by ammonium, glutamine or casein hydrolysate. Our results indicate that a stimulation of leaf formation can be obtained when glutamine is used as the sole nitrogen source prior to regeneration. We have some evidence that glutamine has no effect on leaf formation frequency when added to the standard PG a regeneration medium. WETHERELL and DOUGAL (1976) reported that glutamine, alanine and possibly glutamic acid can serve as sole source of nitrogen supporting both good growth and embryogenesis in carrot cultures. On the other hand, with ammonium as the sole source of nitrogen before regeneration, there was a pronounced negative effect on the leaf formation frequency and the morphology of regenerants. The low temperature treatment which was shown to have a general, positive influence on morphogenetic processes, may act by synchronizing small groups of cells (DEVREUX et al., 1975). There is some evidence which suggests that bud primordia of certain species can originate from several cell acting in a coordinated manner (TRAN THANH VAN et al., 1974; STREE RAMULU et al., 1976). Thus it may be that initiation of cell division centers is stimulated by low temperature treatment.
z.
P/lanzenphysiol. Bd. 90. S. 423-430. 1978.
Morphogenesis in Arabidopsis thaliana
429
Studies on the effect of light on in vitro regeneration of tobacco have shown that wavelength and intensity are equally important parameters in determining morphogenetic processes (SEIBERT et aI., 1975). Our results provide further evidence regarding the influence of light pre-treatments on subsequent regeneration. High intensities reduced leaf formation while continuous low light intensity enhanced morphogenesis. GRESSHOFF (1973) suggested that light may act through an auxin-mediated developmental process, based on the photo-lability of auxin. Maize seedlings grown in the light were shown to absorb significantly less auxin than their dark-grown counterparts (NAQUI, 1975). Passage times which were longer than those used for routine maintenance of callus cultures, proved more satisfactory for subsequent regeneration. MEYER-TEUTER and REINERT (1973) have proposed that loss of the ability of cultured cells to redifferentiate can be correlated with the number of division cycles which the cells have undergone prior to the induction. In Arabidopsis callus cultures the number of division cycles was 1,5-fold greater when calluses were subcultured every four weeks as compared to 8 weeks. Leaf formation was enhanced by a factor 2 to 3 after only one passage of 8 weeks instead of 4 weeks. The effect of an 8 week interval was not apparent when a 4 week interval followed the 8 week one; the 4 week + 8 week reported conclusions (NEGRUTIU et aI., 1978 a) and may indicate that the endogenous hormone levels become unbalanced as a consequence of subculturing at 4 week intervals. At least a part of the enhanced morphogenetic response obtained by using longer subculture intervals can be attributed to a correction of the hormonal balance. KOCHBA and SPIEGEL-Roy (1977) have also suggested that increased embryogenesis in ovular callus of orange as a consequence of «ageing» could be due to a reduction in endogenous auxin levels. We have also observed that if calluses from a single source were subcultured for several passages at four or eight week intervals, the short intervals resulted in more friable callus, usually with lower morphogenetic ability, while longer intervals favoured the proliferation of calluses with higher morphogenetic capacity (NEGRUTIU et aI., in the press). In conclusion, an enhanced frequency of leaf formation can be obtained by using a defined set of physical and chemical culture conditions such as subculture intervals of 6 to 8 weeks in continuous low light or darkness, 4 °C treatments for 3 days prior to regeneration, and a specific low inoculum size in a liquid and filter-sterilised regeneration medium. Acknowledgements This work was supported by a grant from the I.W.O.N.L. (krediet 2383 A).
References DEVREUX, M., U. LANERI, and P. DE MARTINIS: Giornale Botanico Italiano, 109, 335 (1975). GRESSHOFF, P. M.: Ph. D. Thesis. University of Canberra, Oct. 1973. KOCHBA, ]. and P. SPIEGEL-Roy: Z. Pflanzenphysiol. 81, 283 (1977).
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MEYER-TEUTER, H. and]. REINERT: Protoplasma, 78,273 (1973). NAQUI, S. M.: Z. Pflanzenphysiol. 76, 379 (1975). NEGRUTIU, 1.: Arabidopsis Inf. Service, 13, 180 (1976). NEGRUTIU, 1., M. JACOBS, and D. CACHITA: Z. Pflanzenphysiol. 86, 113 (1978 a). NEGRUTIU, 1., M. JACOBS, and W. DE GREEF: Z. Pflanzenphysiol. 90,363 (1978 b). SEIBERT, M., P. J. WETHERBEE, and D. D. JOB: Plant Physiol., 56, 130 (1975). STREE RAMULU, K., M. DEVREUX, G. ANCORA, and V. LANERI: Z. Pflanzenziicht. 76, 299
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M. JACOBS, Laboratorium voor Plantengenetica, Free University of Brussels, Sint-Genesius-Rode, Brussels, Belgium.
z. Pflanzenphysiol. Bd. 90. S. 423-430.
1978.