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Polyamine Changes during in vitro Morphogenesis of Asparagus Cloning
]. Plant P/rysiol. Vol. 138. pp. 172 -175 (1991)
Polyamine Changes during in vitro Morphogenesis of Asparagus Cloning V.
FIALA*,
Y.
QUEROU*, D. GEORGES*\
and C.
DORE**
* Laboratoire de Metabolisme et de la Nutrition des Plantes ** Station de Genetique et d' Amelioration des Plantes, LN.R.A., 78026 Versailles Cedex, France Received October 11, 1990 . Accepted February 7,1991
Summary Putrescine and spermidine are the most represented free polyamines in asparagus tissues. The variability in polyamine composition in relation to genotype and in vitro plant morphogenesis was observed. Higher quantities of putrescine and spermidine in some genotypes promoted in vitro initiation of buds and formation of crowns. A determined level of spermidine may be considered as a threshold level that is necessary to reach the micropropagation stage of asparagus culture.
Key words: Asparagus officinalis, in vitro culture, micropropagation, arginine, polyamine, putrescine, spero midine. Introduction Some physiological processes such as growth, differentiation and development of higher plants are regulated by free polyamines or by their conjugated form as the phenolamides. As of yet little is known about their role in regulation of growth and plant morphogenesis (Biondi et al., 1990). It is generally accepted that polyamines are associated with cell division and active growth (Bagni et al., 1982). The role of polyamines (putrescine, spermidine) in micropropagation and adventitious root formation in wild cherry was reported by Biondi et al. (1990). Correlation between polyamine accumulation and adventitious root formation have been also observed in Passi/lora leaf discs (Desai and Mehta, 1985), in cell layers of tobacco (Torrigiani et al., 1989) and in Datura innoxia leaf explants (Chriqui et al., 1986). Several studies have shown that a rise in polyamine titres occurs in cells undergoing a change of their state of differentiation, such as during somatic embryogenesis in carrot, mango and alfalfa (Feirer et al., 1984; Fienberg et al., 1984; Litz and Shaffer, 1987; Meijer and Simonds, 1988; Mengoli et al., 1989). Polyamines may also be the limiting factor in somatic embryogenesis of Hevea (Hadrami et al., 1989). The inhibition of polyamine synthesis by specific inhibitors led © 1991 by Gustav Fischer Verlag, Stuttgan
to no or reduced morphogenesis. Previous results from our laboratory have shown a correlation between micropropagation of asparagus in vitro and the endogenous arginine and polyamine levels. Asparagus is a dioecious species, the cloning of which in horticultural conditions is very limited. Therefore, micropropagation appeared as a solution to clone different breeding material and has allowed the release of several clonal hybrid varieties, the parents of which are micropropagated (Don!, 1977; Benson and Takatori, 1980; Falavigna et al., 1982; Ellison, 1984). Our observations have shown that asparagus' behaviour during in vitro cloning presents several analogies with ligneous plants, probably because of their perennial common character (Dore, 1988). Asparagus in vitro micropropagation has been characterized by three stages: 1) preliminary rejuvenation, during which it is very difficult to propagate the plant material, 2) the propagation stage, when propagation becomes possible and 3) the one-rooting stage, with root initiation and crown formation. It was of interest to investigate whether the level of endogenous free arginine and polyamines varies during the different stages of in vitro asparagus propagation and particularly during the passage from the preliminary stage to the multiplication stage.
Polyamines during in vitro Morphogenesis
173
Table 1: Free amino acid and polyamine levels in asparagus tissues in relation to extraction method (in JLmoles . g-l FW). metabolite
5 % PCA
EtOH/HCI
lysine argmme putrescine spermidine
1.49 ±0.05 32.40 ± 1.14 0.089 ± 0.008 0.134±0.011
1.25 ±0.04 32.37 ±0.90 0.127 ± 0.013 0.122±0.005
(numbers represent means ± SE at least four replicates)
DEESSE JUVENILE 40r------------------------------,
Fig. 1: Different steps in asparagus in vitro cloning.
Material and methods Plant material
30
u..: ~ 20 CD
'0
E:i. 10
Three genotypes were used as donor plants, a pure line L 627 and two female heterozygous clones, Deesse and Eugenie. The donor plants were less than 1 year old, which means before the first flowering stage. They were grown in a greenhouse (22°C day, 15°C night) and were chosen for their various typical behaviours in micropropagation. For L 627, plandets were also used that originated from seeds germinating under the same greenhouse conditions. In vitro cloning method
The method has been already described elsewhere (Dore, 1988, 1990). Samples were harvested at each subculture during the first two stages of micropropagation. They consisted of fragments of the future crown platform with basal parts of stems and initiated buds (Fig.1). Extraction and analysis
;:
0/polyamines
About 200 mg fresh weight of tissue was ground in a mortar with 5mL of a EtOH 96%: HCIN mixture (90: 10, v/v) or in 5 %PCA. After 30 min of shaking the extract was centrifuged at 20,000gx 20 min, and purified with a Sep-Pak C 18 cartridge (Waters) to eliminate the pigments that disturb the chromatographic determination. The temperature of extraction was less than 5°C. One part (2 mL) of the supernatant was hydrolyzed in 6 N HCI during 18 h at 110°C. The free polyamine (putrescine, cadaverine, spermidine, agmatine and spermine) and free amino acid (lysine and arginine) contents were determinated by ion exchange liquid chromatography (Biotronik LC 6000) by use of fluorometric detection (Villanueva et aI., 1977) with o-phthalaldehyde. Diaminoheptane (DAH) was used as internal standard. All analyses were repeated twice.
Results The quantities of free amino acids and polyamines obtained by the two extraction methods used are approximately
3
6
9
~
Arginine
•
Lysine
12
15
18
21
24 27 WEEKS
Fig. 2: Evolution of free arginine and lysine levels in asparagus tissue during micropropagation (DEESSE JUVENILE). (F.W. = fresh weight).
the same (Table 1). The fact that putrescine and spermidine levels after hydrolysis did not increase shows the absence of ami des in the asparagus tissue at this state of evolution. The increase of free arginine during the first multiplication stage (6 weeks) was very rapid, attaining a value of 22 - 30 /-tmoles g-l FW, which then remained relatively stable (Fig. 2). The rate of two endogenous free polyamines (putrescine and spermidine) detected in the tissue showed a regular increase during the successive subcultures of the preliminary phase (Fig. 3). In some samples very low quantities of diaminopropane (DAP) were detected. The formation period of the crown corresponded to this increase. Transition from the preliminary stage to the propagation stage was characterized by a threshold value of the spermidine level. The spermidine level was enhanced more or less rapidly according to genotypes. For the three studied genotypes a good adequacy could be observed between their behaviour at cloning and evolution of spermidine level during the preliminary stage (Fig. 4). The evolution of polyamines in the plantlets originated from seeds shows some differences in relation to plantlets issued from micropropagation, particularly by the absence of putrescine during the 6 months after germination.
174
V. FIALA, Y. QUEROU, D. GEORGES, and C. DORE
DEESSE JUVENILE 300r-----------------~------------~
~
200
IL til
Ui ~
o E
100
c
3
6
9
12
15
18
21
24
27
Weeks ---
Spermidine
--0--
Putrescine
Fig.3: Evolution of endogenous spermidine and putrescine levels during the preliminary (1) and propagation stages (2) (DEESSE JUVENILE).
Discussion Our results have shown that free arginine, the precursor of putrescine synthesis, is very quickly accumulated in asparagus tissue and attains a threshold value of 30/-tmoles g - 1 FW. This increase of arginine is not related to genotype. We have recently found approximately the same levels of arginine in tulip and iris bulbs (Le Nard and Fiala, 1990).
300
300
2
~
EUGENIE
DEESSE JUVENILE
L 627 300
Putrescine and spermidine are the most represented free polyamines. The higher level of polyamines was also observed in callus culture of Picea abies during somatic embryogenesis (Santanen et aI., 1990). It is possible that these polyamines are related to RNA metabolism and are correlated with root initiation, or that they may be essential for root formation Qarvis et aI., 1985). The capacity of polyamines to stimulate nucleic acid metabolism may be in relation to accelerated cell proliferation and rhizogenesis (Bagni, 1986). The polyamines are probably the biochemicals that transduce external signals into information of biochemical and developmental relevance. Thus, they may play a role in the control of some basic developmental functions such as flowering and lateral organ initiation (Fiala et aI., 1988; Martin-Tanguy et aI., 1990). The analysis of biochemical variations of the tissue during these two stages and its relation to genotype show a variability in polyamine composition. The variability existing between the clones was also observed in Hevea culture (Hadrami et aI., 1989). The observation of three different genotypes has allowed the identification of three distinct behaviour patterns of micropropagation. The quick increase of free putrescine was observed in the L 627 clone, which has the shortest passage from the preliminary to the propagation stage. The amounts of endogenous polyamines confirm the observed differences. It is possible that the higher quantities of polyamines in some genotypes promote in vitro initiation of buds and formation of crowns ready to be divided. An interaction between in vitro apple rooting, putrescine level and IBA treatment was also observed (Burak, 1990). The comparison of morphologic characteristics with results of biochemical analysis has allowed to specify the nature of the preliminary stage. On the contrary, tissue issued from seed germination has a very low putrescine level.
2
i
1
200
200
200
100
100
100
~
2
IL. til
iii CII
0
E
c
OL.&-L.&-L.&-I0....1-&....I.JL...L...JL...L...JL...L.............
o
3 6 9 121518212427
good
o ............... o 3 _6
..L..L...A-.I..............A-.I.................................J
9 1 21 5 1 8 2 1 2 4 2 7
medium
0 .....................................................................................
o
3 6 9 121518212427
poor
weeks
Fig. 4: Evolution of spermidine level in three asparagus genotypes (L 627, DEESSE JUVENILE, EUGENIE) with different behaviour in micropropagation (good, medium and poor) during two phases of cloning: 1) preliminary stage, 2) propagation stage.
Polyamines during in vitro Morphogenesis The addition of exogenous arginine to the culture medium has no effect on asparagus behaviour during micropropagation. We suggest that the level of endogenous free arginine in the tissue is high and, therefore, may not be a limiting factor for putrescine and spermidine biosynthesis under these conditions. A determined level of spermidine may be considered as a threshold level that is necessary to reach the micropropagation stage of asparagus culture. A morphological evolution of asparagus plants grown in vitro is accompanied by biochemical changes in free arginine, putrescine and spermidine levels. Spermidine appears as a biochemical marker of the preliminary phases during which crown formation is the place of higher cellular activity and presents juvenile characteristics.
References BAGNI, N.: The function and metabolism of polyamines in plants. Acta Hortic. 179, 95-103 (1986). BAGNI, N., D. SERAFINI-FRACASSINI, and P. TORRIGIANI: Polyamines and cellular growth in higher plants. In: VAREING, P. F., ed., Plant growth substances. Academic Press, New York, 473 -482 (1982). BENSON, B. and F. TAKATORI: Foundation Asparagus Seed program and the release of UC 157 parental clones to the seed industry. Asparagus Research, 1979-1980, Univer. of Calif., Davis: 25-26 (1980). BIONDI, S., T. DIAZ, 1. IGLESIAS, G. GAMBERINI, and N. BAGNI: Polyamine and ethylene in relation to adventitious root formation in Prunus avium shoot cultures. Physiol. Plant. 78, 474 - 483 (1990). BURAK, M.: The interaction between putrescine and IBA on in vitro rooting of MM 106 apple (Malus communis) clone. Abstracts VlIth International Congress on Plant Tissue and Cell Culture, Amsterdam, p. 277 (1990). CHRIQUI, D., D. D'ORAZI, and N. BAGNI: Ornithine and arginine decarboxylases and polyamine involvement during in vivo differentiation and in vitro dedifferentiation of Datura inoxia leaf explants. Physiol. Planta. 68, 589-596 (1986). DESAI, H. V. and A. R. MEHTA: Changes in polyamine levels during shoot formation, root formation and callus induction in cultured Passiflora leaf discs. J. Plant Physiol. 119, 45 - 53 (1985). DORE, c.: In vitro techniques as an efficient tool in Asparagus breeding. Acta Hortic. 78, 89-93 (1977). - Nouveau regard sur Ie comportement de l'asperge, (Asparagus of ficinalis) mUltiplie in vitro. Agronomie 8,843-850 (1988). - Asparagus cloning revisited. Acta Hortic. 271, 101-108 (1990). ELLISON, H.: Release of Jersey Giant and Greenwich. Asparagus Research Newsletters 2, p. 23 (1984).
175
FALAVIGNA, A., P. CASALI, and G. SORESSI: Diego: first italian hybrid of asparagus from heterozygous clones. Genet. agrar. 162 -163 (1982). FEIRER, R. B., G. MIGNON, andJ. D. LITVAY: Arginine decarboxylase and polyamines required for embryogenesis in wild carrot. Science 223,1433-1435 (1984). FIALA, V., M. LE NARD, Y. QUEROU, and E. JOLIVET: La spermidine, marqueur moleculaire de I'induction florale chez Ie bulbe d'Iris hollandica. C.R. Acad. Sci. Paris, 306, Serie III: 579-582 (1988). FIENBERG, A. A., J. H. CHOI, W. P. LUBICH, and Z. R. SUNG: Developmental regulation of polyamine metabolism in growth and differentiation of carrot culture. Planta 162,532-539 (1984). HADRAMI, 1. L., N. MICHAUX-FERRlERE, M. P. CARRON, and J. D' AUZAc: Les polyamines, facteur limit ant possible de I'embryogenese somatique chez I'Hevea brasiliensis. C.R. Acad. Sci. Paris, 308, Serie III: 205 - 211 (1989). JARVIS, B. c., S. YASMIN, and M. T. COLEMAN: RNA and protein metabolism during adventitious root formation in stem cuttings of Phaseolus aureus cultivar berkin. Physiol. Plant. 64, 53 - 59 (1985). LE NARD, M. and V. FIALA: Post-harvest variation of free arginine in basal plate tissues of tulip bulbs; relation to bulb physiological evolution. Acta Hortic. 266, 293 - 298 (1990). LITz, R. E. and B. SCHAFFER: Polyamines in adventitious and somatic embryogenesis in mango (Mangifera indica L.). J. Plant Physiol. 128, 251-258 (1987). MARTIN-TANGUY, J., D. TEPFER, M. PAYNOT, D. BURTIN, L. HEISLER, and C. MARTIN: Inverse relationship between polyamine levels and the degree of phenotypic alteration induced by the root-inducing, left-hand transferred DNA from Agrobacterium rhi· zogenes. Plant Physiol. 92, 912-918 (1990). MEIJER, E. T. M. and J. SIMONDS: Polyamine levels in relation to growth and somatic embryogenesis in tissue cultures of Medicago sativa L. J. Exp. Bot. 39, 787 -794 (1988). MENGOLI, M., N. BAGNI, G. LUCCARINI, V. NUTI-RoNCHI, and D. SERAFINI-FRACASSINI: Daucus carota cell cultures: polyamines and effect of polyamines biosynthesis inhibitors in the preembryogenic phase and different embryo stages. J. Plant Physiol. 134, 389-394 (1989). SANTANEN, A., L. K. SIMOLA, and V. R. VILLANUEVA: Polyamine changes during somatic embryogenesis in callus cultures of Picea abies. Abstracts VIIth International Congress on Plant Tissue and Cell Culture, Amsterdam, p. 267 (1990). TORRIGIANI, P., M. M. ALTAMURA, F. CAPITANI, D. SERAFINI-FRACASSINI, and N. BAGNI: De novo root formation in thin cell layers of tobacco: changes in free and bond polyamines. Physiol. Plant. 77,294-301 (1989). VILLANUEVA, V. R., C. R. ADLAKHA, and A. M. CANTERA-SOLER: Determination rapide de polyamines et de quelques mono- et diamines dans les extraits vegetaux. J. Chromatogr. 139, 381-385 (1977).
Polyamine Changes during in vitro Morphogenesis of Asparagus Cloning V.
FIALA*,
Y.
QUEROU*, D. GEORGES*\
and C.
DORE**
* Laboratoire de Metabolisme et de la Nutrition des Plantes ** Station de Genetique et d' Amelioration des Plantes, LN.R.A., 78026 Versailles Cedex, France Received October 11, 1990 . Accepted February 7,1991
Summary Putrescine and spermidine are the most represented free polyamines in asparagus tissues. The variability in polyamine composition in relation to genotype and in vitro plant morphogenesis was observed. Higher quantities of putrescine and spermidine in some genotypes promoted in vitro initiation of buds and formation of crowns. A determined level of spermidine may be considered as a threshold level that is necessary to reach the micropropagation stage of asparagus culture.
Key words: Asparagus officinalis, in vitro culture, micropropagation, arginine, polyamine, putrescine, spero midine. Introduction Some physiological processes such as growth, differentiation and development of higher plants are regulated by free polyamines or by their conjugated form as the phenolamides. As of yet little is known about their role in regulation of growth and plant morphogenesis (Biondi et al., 1990). It is generally accepted that polyamines are associated with cell division and active growth (Bagni et al., 1982). The role of polyamines (putrescine, spermidine) in micropropagation and adventitious root formation in wild cherry was reported by Biondi et al. (1990). Correlation between polyamine accumulation and adventitious root formation have been also observed in Passi/lora leaf discs (Desai and Mehta, 1985), in cell layers of tobacco (Torrigiani et al., 1989) and in Datura innoxia leaf explants (Chriqui et al., 1986). Several studies have shown that a rise in polyamine titres occurs in cells undergoing a change of their state of differentiation, such as during somatic embryogenesis in carrot, mango and alfalfa (Feirer et al., 1984; Fienberg et al., 1984; Litz and Shaffer, 1987; Meijer and Simonds, 1988; Mengoli et al., 1989). Polyamines may also be the limiting factor in somatic embryogenesis of Hevea (Hadrami et al., 1989). The inhibition of polyamine synthesis by specific inhibitors led © 1991 by Gustav Fischer Verlag, Stuttgan
to no or reduced morphogenesis. Previous results from our laboratory have shown a correlation between micropropagation of asparagus in vitro and the endogenous arginine and polyamine levels. Asparagus is a dioecious species, the cloning of which in horticultural conditions is very limited. Therefore, micropropagation appeared as a solution to clone different breeding material and has allowed the release of several clonal hybrid varieties, the parents of which are micropropagated (Don!, 1977; Benson and Takatori, 1980; Falavigna et al., 1982; Ellison, 1984). Our observations have shown that asparagus' behaviour during in vitro cloning presents several analogies with ligneous plants, probably because of their perennial common character (Dore, 1988). Asparagus in vitro micropropagation has been characterized by three stages: 1) preliminary rejuvenation, during which it is very difficult to propagate the plant material, 2) the propagation stage, when propagation becomes possible and 3) the one-rooting stage, with root initiation and crown formation. It was of interest to investigate whether the level of endogenous free arginine and polyamines varies during the different stages of in vitro asparagus propagation and particularly during the passage from the preliminary stage to the multiplication stage.
Polyamines during in vitro Morphogenesis
173
Table 1: Free amino acid and polyamine levels in asparagus tissues in relation to extraction method (in JLmoles . g-l FW). metabolite
5 % PCA
EtOH/HCI
lysine argmme putrescine spermidine
1.49 ±0.05 32.40 ± 1.14 0.089 ± 0.008 0.134±0.011
1.25 ±0.04 32.37 ±0.90 0.127 ± 0.013 0.122±0.005
(numbers represent means ± SE at least four replicates)
DEESSE JUVENILE 40r------------------------------,
Fig. 1: Different steps in asparagus in vitro cloning.
Material and methods Plant material
30
u..: ~ 20 CD
'0
E:i. 10
Three genotypes were used as donor plants, a pure line L 627 and two female heterozygous clones, Deesse and Eugenie. The donor plants were less than 1 year old, which means before the first flowering stage. They were grown in a greenhouse (22°C day, 15°C night) and were chosen for their various typical behaviours in micropropagation. For L 627, plandets were also used that originated from seeds germinating under the same greenhouse conditions. In vitro cloning method
The method has been already described elsewhere (Dore, 1988, 1990). Samples were harvested at each subculture during the first two stages of micropropagation. They consisted of fragments of the future crown platform with basal parts of stems and initiated buds (Fig.1). Extraction and analysis
;:
0/polyamines
About 200 mg fresh weight of tissue was ground in a mortar with 5mL of a EtOH 96%: HCIN mixture (90: 10, v/v) or in 5 %PCA. After 30 min of shaking the extract was centrifuged at 20,000gx 20 min, and purified with a Sep-Pak C 18 cartridge (Waters) to eliminate the pigments that disturb the chromatographic determination. The temperature of extraction was less than 5°C. One part (2 mL) of the supernatant was hydrolyzed in 6 N HCI during 18 h at 110°C. The free polyamine (putrescine, cadaverine, spermidine, agmatine and spermine) and free amino acid (lysine and arginine) contents were determinated by ion exchange liquid chromatography (Biotronik LC 6000) by use of fluorometric detection (Villanueva et aI., 1977) with o-phthalaldehyde. Diaminoheptane (DAH) was used as internal standard. All analyses were repeated twice.
Results The quantities of free amino acids and polyamines obtained by the two extraction methods used are approximately
3
6
9
~
Arginine
•
Lysine
12
15
18
21
24 27 WEEKS
Fig. 2: Evolution of free arginine and lysine levels in asparagus tissue during micropropagation (DEESSE JUVENILE). (F.W. = fresh weight).
the same (Table 1). The fact that putrescine and spermidine levels after hydrolysis did not increase shows the absence of ami des in the asparagus tissue at this state of evolution. The increase of free arginine during the first multiplication stage (6 weeks) was very rapid, attaining a value of 22 - 30 /-tmoles g-l FW, which then remained relatively stable (Fig. 2). The rate of two endogenous free polyamines (putrescine and spermidine) detected in the tissue showed a regular increase during the successive subcultures of the preliminary phase (Fig. 3). In some samples very low quantities of diaminopropane (DAP) were detected. The formation period of the crown corresponded to this increase. Transition from the preliminary stage to the propagation stage was characterized by a threshold value of the spermidine level. The spermidine level was enhanced more or less rapidly according to genotypes. For the three studied genotypes a good adequacy could be observed between their behaviour at cloning and evolution of spermidine level during the preliminary stage (Fig. 4). The evolution of polyamines in the plantlets originated from seeds shows some differences in relation to plantlets issued from micropropagation, particularly by the absence of putrescine during the 6 months after germination.
174
V. FIALA, Y. QUEROU, D. GEORGES, and C. DORE
DEESSE JUVENILE 300r-----------------~------------~
~
200
IL til
Ui ~
o E
100
c
3
6
9
12
15
18
21
24
27
Weeks ---
Spermidine
--0--
Putrescine
Fig.3: Evolution of endogenous spermidine and putrescine levels during the preliminary (1) and propagation stages (2) (DEESSE JUVENILE).
Discussion Our results have shown that free arginine, the precursor of putrescine synthesis, is very quickly accumulated in asparagus tissue and attains a threshold value of 30/-tmoles g - 1 FW. This increase of arginine is not related to genotype. We have recently found approximately the same levels of arginine in tulip and iris bulbs (Le Nard and Fiala, 1990).
300
300
2
~
EUGENIE
DEESSE JUVENILE
L 627 300
Putrescine and spermidine are the most represented free polyamines. The higher level of polyamines was also observed in callus culture of Picea abies during somatic embryogenesis (Santanen et aI., 1990). It is possible that these polyamines are related to RNA metabolism and are correlated with root initiation, or that they may be essential for root formation Qarvis et aI., 1985). The capacity of polyamines to stimulate nucleic acid metabolism may be in relation to accelerated cell proliferation and rhizogenesis (Bagni, 1986). The polyamines are probably the biochemicals that transduce external signals into information of biochemical and developmental relevance. Thus, they may play a role in the control of some basic developmental functions such as flowering and lateral organ initiation (Fiala et aI., 1988; Martin-Tanguy et aI., 1990). The analysis of biochemical variations of the tissue during these two stages and its relation to genotype show a variability in polyamine composition. The variability existing between the clones was also observed in Hevea culture (Hadrami et aI., 1989). The observation of three different genotypes has allowed the identification of three distinct behaviour patterns of micropropagation. The quick increase of free putrescine was observed in the L 627 clone, which has the shortest passage from the preliminary to the propagation stage. The amounts of endogenous polyamines confirm the observed differences. It is possible that the higher quantities of polyamines in some genotypes promote in vitro initiation of buds and formation of crowns ready to be divided. An interaction between in vitro apple rooting, putrescine level and IBA treatment was also observed (Burak, 1990). The comparison of morphologic characteristics with results of biochemical analysis has allowed to specify the nature of the preliminary stage. On the contrary, tissue issued from seed germination has a very low putrescine level.
2
i
1
200
200
200
100
100
100
~
2
IL. til
iii CII
0
E
c
OL.&-L.&-L.&-I0....1-&....I.JL...L...JL...L...JL...L.............
o
3 6 9 121518212427
good
o ............... o 3 _6
..L..L...A-.I..............A-.I.................................J
9 1 21 5 1 8 2 1 2 4 2 7
medium
0 .....................................................................................
o
3 6 9 121518212427
poor
weeks
Fig. 4: Evolution of spermidine level in three asparagus genotypes (L 627, DEESSE JUVENILE, EUGENIE) with different behaviour in micropropagation (good, medium and poor) during two phases of cloning: 1) preliminary stage, 2) propagation stage.
Polyamines during in vitro Morphogenesis The addition of exogenous arginine to the culture medium has no effect on asparagus behaviour during micropropagation. We suggest that the level of endogenous free arginine in the tissue is high and, therefore, may not be a limiting factor for putrescine and spermidine biosynthesis under these conditions. A determined level of spermidine may be considered as a threshold level that is necessary to reach the micropropagation stage of asparagus culture. A morphological evolution of asparagus plants grown in vitro is accompanied by biochemical changes in free arginine, putrescine and spermidine levels. Spermidine appears as a biochemical marker of the preliminary phases during which crown formation is the place of higher cellular activity and presents juvenile characteristics.
References BAGNI, N.: The function and metabolism of polyamines in plants. Acta Hortic. 179, 95-103 (1986). BAGNI, N., D. SERAFINI-FRACASSINI, and P. TORRIGIANI: Polyamines and cellular growth in higher plants. In: VAREING, P. F., ed., Plant growth substances. Academic Press, New York, 473 -482 (1982). BENSON, B. and F. TAKATORI: Foundation Asparagus Seed program and the release of UC 157 parental clones to the seed industry. Asparagus Research, 1979-1980, Univer. of Calif., Davis: 25-26 (1980). BIONDI, S., T. DIAZ, 1. IGLESIAS, G. GAMBERINI, and N. BAGNI: Polyamine and ethylene in relation to adventitious root formation in Prunus avium shoot cultures. Physiol. Plant. 78, 474 - 483 (1990). BURAK, M.: The interaction between putrescine and IBA on in vitro rooting of MM 106 apple (Malus communis) clone. Abstracts VlIth International Congress on Plant Tissue and Cell Culture, Amsterdam, p. 277 (1990). CHRIQUI, D., D. D'ORAZI, and N. BAGNI: Ornithine and arginine decarboxylases and polyamine involvement during in vivo differentiation and in vitro dedifferentiation of Datura inoxia leaf explants. Physiol. Planta. 68, 589-596 (1986). DESAI, H. V. and A. R. MEHTA: Changes in polyamine levels during shoot formation, root formation and callus induction in cultured Passiflora leaf discs. J. Plant Physiol. 119, 45 - 53 (1985). DORE, c.: In vitro techniques as an efficient tool in Asparagus breeding. Acta Hortic. 78, 89-93 (1977). - Nouveau regard sur Ie comportement de l'asperge, (Asparagus of ficinalis) mUltiplie in vitro. Agronomie 8,843-850 (1988). - Asparagus cloning revisited. Acta Hortic. 271, 101-108 (1990). ELLISON, H.: Release of Jersey Giant and Greenwich. Asparagus Research Newsletters 2, p. 23 (1984).
175
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