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Photosynthesis and transpiration of in vitro cultured asparagus plantlets
Scientia Horticulturae, 49 ( 1992 ) 9- ! 6 Elsevier Science Publishers B.V., Amsterdam
Photosynthesis and transpiration of in vitro cultured asparagus plantlets De Yue, Yves Desjardinsl, Michel Lamarre and Andr6 Gosselin Centre de recherche en horticulture. Dbpartement de phytologie, Facuitb des sciences de I'agriculture et de l'alimentation, Universitb Laval. Ste-Foy, Qub., G IK 7P4, Canada (Accepted l I July 1991 )
ABSTRACT Yue, D., Desjardins, Y., Lamarre, M. and Gosselin, A., 1992. Photosynthesis and transpiration of in vitro cultured asparagus plantlets. Scientia Hortlc., 49: 9-16. Nodal sections of asparagus (Asparagus officinalis L. ) were cultured and rooted on a modified Murashige and Skoog (MS) medium for 10 weeks. These plants were then acclimatized for 5 weeks. Photosynthesis and transpiration of in vitro plantlets, acclimatized plantlets and control seedlings were measured with an open gas exchange system. Rates of photosynthesis of in vitro-cultured plantiers were as high as those of seedlings grown in a greenhouse, while their rates of transpiration were much higher. Photosynthetic capacity of in vitro-cultured asparagus plantlets was sufficiently high to support autotrophic growth during the period of acclimatization. Photosynthesis of acclimatized plantlets was always considerably lower than that of in vitro plantlets except at high concentrations of CO2. Evapotranspiration of acclimatized plantlets was comparable to that of seedlings. These results suggest that high water loss incurred by in vitro shoots imposes severe limitations on newly formed shoots in acclimatization thus reducing whole plant photosynthesis. Protecting in vitro plantlets from water stress is the most important factor to consider in order to ensure their survival during the acclimatization period. Keywords: ,4sparagus of.ficinalis: micropropagation; photosynthesis: trar~spiration. Abbreviations: DW=dry weight~ MS=Murashige and Skoog: NAA=naphthaleneacetic acid; PPF= photosynthetic photon flux.
INTRODUCTION
Leaves of many crops cultured in vitro under conditions of high humidity and low light intensity are characterized by a reduced deposition of epicuticular wax (Grout and Aston, 1977), by malfunctioning stomata (Brainerd and Fuchigami, 1982 ) and by a lower photosynthetic ability (Grout and Aston, 1978). Such characteristics affect their survival after transplanting to greenhouse or field conditions. ~Author to whom correspondence should be addressed.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4238/92/$05.00
10
D. YU E ET AL.
For asparagus, the survival of in vitrc~cultured plantlets has been a problem and recovery has been unusually slow after removal from culture. Hasegawa et al. (1973) reported that plantlets grown under high light had relatively more cladophylls and survived better to acclimatization. Since photosynthesis in asparagus is carried out mainly by cladophylls (Inagaki et al., 1989 ) the results reported by Hasegawa et al. ( 1973 ) suggest that one of the factors affecting the sur,,iva! of in vitro olantlets is the photosynthetic capacity. In addition, Yang and Clore (1974) reported that growing transplanted plantlets under mist improved their survival, suggesting that water stress is also an important factor. Li ( 1985 ) showed that plant growth regulators and inorganic nutrition affected the survival of in vitro cultured asparagus plantlets. However, little research has been carried out to investigate the basic physiological characteristics of in vitro cultured plantlets of asparagus and to determine the cause of slow recovery or death of transplanted plantlets. In our experiments, the photosynthesis and transpiration of in vitro cultured asparagus plantlets were measured with an open gas exchange system. The objective of the experiment was to determine how these physiological characteristics may affect acclimatization and growth of micropropagated asparagus plantlets. MATERIALS AND METHODS
In vitro cultured plantlets. - A superior clonal selection (G- 171 ) made at the
University of Guelph from cultivar Viking-2G was used. Branched shoots at the base of spears were excised and cultured on a modified Murashige and Skoog (MS) medium containing 0.1 mg I- ~naphthaleneacetic acid (NAA), 0.1 mg 1-t kinetin, 30 g 1-i sucrose, 8 g 1-t agar, and 6.4 mg I-~ ancymidol. Cultures were maintained in a growth room kept at 23 +__1°C under cool-white fluorescent tubes at 48/~mol m-2s-! and a 16-h photoperiod. Ten weeks after culture, well-developed shoots were selected for measurement of photosynthesis. Acclimatized plants. - G-I 71 plantlets cultured in vitro for 11 weeks were
washed free of agar, transplanted in 12.5-cm plastic pots containing pro-mix BX artificial substrate (Premier Peat-moss, Rivi~re-du-Loup, Qu6., and transfered to acclimatization. Temperature in the greenhouse was maintained at 24_+2°C, and the relative humidity was about 45%. During the first week, the pots were covered with polyethylene film bags and the humidity surrounding the plantlets was near saturation. During the second week, the bags were partly opened and they were removed by Week 3. After 5 weeks, plantlets with actively growing new shoots, that is the shoots formed in vivo, were selected for measurement of photosynthesis and transpiration.
PHOTOSYNTHESIS AND TRANSPIRATION OF ASPARAGUS
I1
Seedlings. - Asparagus seedlings cultivar 'Viking' were grown in the green-
house for 5 weeks, and photosynthesis and transpiration were measured on the most recently developed mature shoot. All plant materials used in this experiment were 10-15 mm in height, and their ferns had fully developed cladophylls. M e a s u r e m e n t o f photosynthesis a n d transpiration. - An open gas exchange
system was developed to measure photosynthesis and transpiration of in vitro cultured plantlets (Fig. 1). Two ADC MK225 infra-red gas analyzers (Hoddsdon, UK) were used in this system for measurement of CO2 and water vapour concentration. A controller (MT 1000, Measurement Technology Inc., Stoughton, MA ) and a personal computer (IBM-clone) were used to monitor and record data. Roots with rooting medium were put into a small plastic tube, the tube apperture was closed with paraffin film and sealed at the base of the stem with silicon vacuum grease. The plantlet was placed in an assimilation chamber which was made out of a test tube (20 mm diameter× 150 mm long) closed with a rubber stopper equipped with an air inlet and outlet. The assimilation chamber was immersed in a water bath where temperature was precisely maintained at 26 °C. The relative humidity of inlet gas stream at this temperature was 70%. Light intensity had no influence on plant temperature. A period of 5-30 min was provided to establish steady state of CO2 and water pressure conditions in the outlet gas stream before measurements
t L
1
2 B
f
Fig. 1. Schematic view of the system used to measure photosynthesis and transpiration. The desired CO2 concentration is produced by using valves 1 and 2 to mix CO2-free dry air (A) with 5% CO~ in N2 (C). Valves 3 and 4 split the resultant air stream, so that a portion is humidified in a temperature controlled bubbler (B) and subsequently re-mixed with dry air. The air flow passes in part through the reference tubes oftwo infrared gas analyzers (IC for CO2 and IW for water vepor) and in part in the assimilation chamber (AC) suspended in a temperature controlled water bath (WB). The air exiting the chamber passes through the analysis tubes and finally through a flowmeter (F). Lighting of the chamber is supplied by high-pressure sodium vapor lamps (L) suspended above a 3-cm deep water filter (W).
12
D. YUE ET AL.
were taken. Measurements were first made at high photosynthetic photon flux (PPF) followed by gradual lowering of light intensity. Photosynthesis and transpiration rates were calculated according to the equations developed by Moon and Flore (1986) and took into account inlet gas flow rate, temperature and delta CO2 and H20. Owing to the difficulty of measuring leaf area of asparagus cladophylls, photosynthesis measurements were calculated on a dry weight (DW) basis. RESULTS
Notwithstanding the origin of the plant materials used in this experiment, net photosynthesis increased with PPF increases (Fig. 2 ). Plantlets of different origin had almost the same rate of dark respiration at 0.06-0.09 #mol CO2 g-~ DW s-t. In vitro-cultured plantlets exhibited similar rates of photosynthesis as seedlings and both their light compensation points were below 50 gmol m-2 s-l. For these two types of plantlets, rates of photosynthesis increased significantly when PPF increased from 0 to 150/zmol m-2 s-t, and remained essentially steady from 150 to 450/zmol m -2 s -!. At a PPF of 450 /zmol m -2 s-t, their net photosynthesis was about 0.19 gmol CO2 g-i DW s-i. In contrast, the shoots of acclimatized plants had much lower net photosynthesis than that of in vitro cultured plantlets or seedlings. Their photosynthesis increased gradually with PPF increases, and the light compensation point was about 450 #mol m - 2 s- t. The net photosynthesis of in vitro cultured plantlets was higher than that of acclimatized plants at all concentrations of CO: set up in this experiment '7
0.2
t., (~
0.1
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|
0.0 .
.
.
.
.
.
.
.
.
.
.
.
-0 1
J I 3 z
-
~ Acclimated plantlets -'-[~-- Seedlings 0
0
. 100
2 200
~ 300
400
500
Photosynthetic Photon Flux (~mol. m"2. s'l )
Fig. 2. Photosynthetic rates of in vitro cultured plantlets, acclimatized plantlets and seedlings of asparagus under different PPF. The ambient CO2 concentration was 300/~11-2. Vertical bars represent SE of the mean of five replications.
PHOTOSYNTHESIS AND TRANSPIRATION OF ASPARAGUS
13
(Fig. 3). Saturation concentration of CO2 was about 600 ld 1-~. The CO2 compensation concentration of in vitro cultured plantlets was lower than 150 /111-I. No significant differences in photosynthesis were observed between in vitro and acclimatized plantlets at high CO2 concentration ( 1200/tl 1- I ). In vitro cultured plantlets had much higher transpiration rates than acclimatized plants or seedlings (Fig. 4). Under dark conditioi~s, transpiration of in vitro cultured plantlets was 21/zmol H20 g-~DW s -i, 8.3 times and 1.7 times higher than acclimatized plants and seedlings, respectively. At a PPF of 450/zmol m -2 s-~, the transpiration rate of in vitro cultured plantlets was 32
'7~ ool
0.1
~
0.0
i .
.
.
.
.
._~ ID
0
~
IQ.
Acclimatized
.0.1
z
•
0
|
|
200
i
400
600
|
800
CO 2 Concentration
!
|
1000 1200 1400
(!~1I "1)
Fig. 3. Photosynthetic rates of in vitro cultured plantlets, acclimatized plantlets of asparagus at different CO: concentrations. The ambient PFF during measurement was 150/~moi m - : s - ~. Vertical bars represent SE of the mean of five replications.
.,~
40
~
In
vitro plantlets ~Acclimatized plantlets
T
";"~ ~~ O30 _---O--Seedllng,.'~ : ~
o E
20
c .9
o. w t.-
,m
I-
10
0
0
100
200
Photosynthetic Photon
300
400
500
Flux (l~mol-rn"2- s "1)
Fig. 4. Transpiration rates of in vitro cultured plantlets, acclimatized plantlets and seedlings of asparagus at different PPF. Vertical bars represent SE of the mean of fi,,.e replications.
14
D. YUE ET AL.
/tmol H20 g-~ DW s-i, that is 3.4 times and 1.8 times higher than acclimatized plants and seedlings, respectively. DISCUSSION
Our results show that in vitro cultured asparagus plantlets have as high a photosynthetic rate as seedlings (Fig. 2). In previous experiments, we measured photosynthesis of plantlets cultured for 20 weeks (data not shown ) and found that their photosynthetic rates were as high as those measured by Inagaki et al. (1989) for comparable greenhouse-grown plantlets. Therefore, we believe that in vitro plantlets cultured as in this experiment have a high enough rate of photosynthesis to support their growth at the time of removal from culture. Based on these results, asparagus can be considered as photosynthetically competent in vitro. According to Grout and Donkin ( 1987 ) species can be regrouped i~ two categories on the basis of their photosynthetic competence. Several species have been shown to possess low photosynthetic rates in vitro (e.g. cauliflower (Grout and Aston, 1978), strawberry (Grout and Millam, 1985 ) ). The recovery of non-competent species during acclimatization is difficult. In the case of asparagus, slow recovery or even death of plantlets during the period of acclimatization appears to be related to the severe water stress they suffer at that time. Since asparagus plantlets were able to carry out photosynthesis, the conditions during and after culture may affect their growth. Increasing the PPF from 50 to 100 and 150 #molm - 2 s - I promoted photosynthesis by 85% and 180°o, respectively (Fig. 2). Increasing the CO., from 150 jtl i-i to 390 or 600 /tl l -I promoted photosynthesis by 155% or 331%, respectively (Fig. 3 ). These data suggested that increasing the light intensity and CO2 concentration in vitro or ex vitro should promote the growth of aspara,-us. Such results have been reported with other crops (Desjardins et al., I o~', Kozai and lwanami, 1988; Laforge et al., 1990). Moreover, the converging photosynthetic activity of in vitro and acclimatized plantlets at high CO., concentration ( 1200 #l I- i ) is indicative of stomatal limitations to photosynthesis. Stomatal closure of acclimatized plantlets appears to be responsible for the low photosynthetic rate of these plantlets. The transpiration rates of in vitro cultured plantlets were much higher than that of acclimatized plants or seedlings (Fig. 4). Llnder dark conditions, the transpiration rates of in vitro cultured plantlets were 8.3 times or 1.7 times higher, respectively, than acclimatized plants or seedlings, suggesting that their epicuticular layer was very thin or absent. Moreover, their stomata might be partly opened even under dark conditions. Both low leaf resistance and poor system of water transfer are some of the reasons explaining the sensitivity of in vitro cultured plantlets to water stress (Grout and Aston, 1977). In vitro cultured plantlets of asparagus lose water very quickly and may wilt in a few
PHOTOSYNTHESIS AND TRANSPIRATION OF ASPARAGUS
15
minutes under normal room or greenhouse conditions. These facts suggested that the main factor causing death and slow recovery of micropropagated asparagus daring the ~,~.-....'~,-'~-aof. zec!imatization__ may be water loss. It is also important to notice that the poor adaptation of in vitro leaves may have an extended effect on the photosynthetic efficiency of the newly formed leaves during acclimatization. ~t is quite possible that acclimatized plantlets possess the capacity for higher photosynthesis but the severe stress imposed at the whole plant level, resulting from the important transpiration of in vitro leaves, may reduce stomatal opening leading to reduced photosynthesis in newly formed leaves. Indeed, low transpiration observed with acclimatized plantlets and their correspondingly low photosynthesis strongly suggest that stomata are closed. This however remains to be verified with porometric or anatomic measurements. To insure high rates of survival, the most important factor may be protecting tissue-cultured asparagus plantlets from water stress. Preliminary results in our laboratory demonstrate that it is possible to reduce the transpiration rate of in vitro plantlets of asparagus by removing the cap for some time before transfer as was presented by Sutter ( 1988 ). Therefore, pre-acclimatization of asparagus plantlets during in vitro culture may be one promising way to ensure high survival rates during the period of acclimatization. Experiments are underway to assess this possibility. ACKNOWLEDGMENTS
We gratefully acknowledge Conseil de la recherche en p&heries et alimentation du Qu6bec (CORPAQ) and Agriculture Canada for their financial support. We also acknowledge Dr. Michael E.D. Graham and France Crocheti~re for their helpful contribution to the photosynthetic measurements and reviewing the manuscript, respectively.
REFERENCES Rrainerd, K.E. and Fuchigami, L.H., 1982. Stomatai functioning of in vitro and greenhouse apple leaves in darkness, mannitol, ABA and CO2. J. Exp. Bot., 33: 388-392. Desjardins, Y., Gosselin, A. and Yelle, S., 1987. Acclimatization ofex vitro strawberry plantlets in CO2-enriched environments and supplementary lighting. J. Am. Soc. Hortic. Sci., 112: 846-851. Grout, B.W.W. and Aston, M.J., 1977. Transpiration of cauliflower plants regenerated from meristem culture. I. Water loss and water transfer related to changes in leaf wax and to xylem regeneration. Hortic. Res., 17: 1-17. Grout, B.W.W. and Aston, M.J., 1978. Transpiration of cauliflower plants regenerated from meristem culture. 1. Carbon dioxide fixation antt the development of photosynthetic activity. Hortic. Res., 17:65-71.
16
D. YUE ET AL.
Grout, B.W.W. and Donkin, M.E., 1987. Photosynthetic activity of cauliflower meristem cultures in vitro and at transplanting into soil. Acta Hortic., 212: 323-327. Grout, B.W.W. and Millam, S., 1985. Photosynthetic development of micropropagated strawberry plantlets following transplanting. Ann. Bot., 55:129-131. Hasegawa, P.M., Murashige, T. and Takatori, F.H., 1973. Propagation of asparagus through shoot apex culture. II. Light and temperature requirements, transplantability of plants, and cytohistolog~cai characteristics. J. Am. Soc. Hortic. Sci., 98:143-148. Inagaki, N., Tsuda, K., Maekawa, S. and Terabun, M., 1989. Effects of light intensity, CO2 concentration, and temperature on photosynthesis of Asparagus officinalis L. J. Jpn. Soc. Hortic. Sci., $8: 369-376. Kozai, T. and lwanami, Y., 1988. Effects of CO2 enrichment and sucrose concentration under high photon fluxes on plantlet growth of carnation (lJianthus caryophyllus L. ) in tissue culture during the preparation stage. J. Jpn. Soc. Ho~tic. Sci., 57: 279-288. Laforge, F., Lussier, C., Desjardins, Y. and Gosselin, A., 1990. Effect of light intensity and CO2 enrichment during in vitro rooting on subsequent growth of p|antlets of strawberry, rasoberry and asparagus in acclimatization. Scientia Hortic., 47: 259-269. Li, S.Z., 1985. Investigation of the survival rate after transplanting of asparagus plantlets raised in vitro. Zhejiang Agric. Sci., No. l: 47-48. Moon Jr., J. and Flore, J.A., 1986. A basic computer program for calculation of photosynthesis, stomatai conductance, and related parameters in an open gas exchange system. Photosynth. Res., 7: 269-279. Sutter, E., 1988. Stomatal and cuticular wmer io:~ from apple, cherry and sweetgum after removal from in vitro culture. J. Am. Soc. Hortic Sci., 113: 234-238. Yang, H.J. and Clore, W.J., 1974. Improving the survival of aseptically-cultured asparagus plants in transplanting. HortScience~ o: 235-236.
Photosynthesis and transpiration of in vitro cultured asparagus plantlets De Yue, Yves Desjardinsl, Michel Lamarre and Andr6 Gosselin Centre de recherche en horticulture. Dbpartement de phytologie, Facuitb des sciences de I'agriculture et de l'alimentation, Universitb Laval. Ste-Foy, Qub., G IK 7P4, Canada (Accepted l I July 1991 )
ABSTRACT Yue, D., Desjardins, Y., Lamarre, M. and Gosselin, A., 1992. Photosynthesis and transpiration of in vitro cultured asparagus plantlets. Scientia Hortlc., 49: 9-16. Nodal sections of asparagus (Asparagus officinalis L. ) were cultured and rooted on a modified Murashige and Skoog (MS) medium for 10 weeks. These plants were then acclimatized for 5 weeks. Photosynthesis and transpiration of in vitro plantlets, acclimatized plantlets and control seedlings were measured with an open gas exchange system. Rates of photosynthesis of in vitro-cultured plantiers were as high as those of seedlings grown in a greenhouse, while their rates of transpiration were much higher. Photosynthetic capacity of in vitro-cultured asparagus plantlets was sufficiently high to support autotrophic growth during the period of acclimatization. Photosynthesis of acclimatized plantlets was always considerably lower than that of in vitro plantlets except at high concentrations of CO2. Evapotranspiration of acclimatized plantlets was comparable to that of seedlings. These results suggest that high water loss incurred by in vitro shoots imposes severe limitations on newly formed shoots in acclimatization thus reducing whole plant photosynthesis. Protecting in vitro plantlets from water stress is the most important factor to consider in order to ensure their survival during the acclimatization period. Keywords: ,4sparagus of.ficinalis: micropropagation; photosynthesis: trar~spiration. Abbreviations: DW=dry weight~ MS=Murashige and Skoog: NAA=naphthaleneacetic acid; PPF= photosynthetic photon flux.
INTRODUCTION
Leaves of many crops cultured in vitro under conditions of high humidity and low light intensity are characterized by a reduced deposition of epicuticular wax (Grout and Aston, 1977), by malfunctioning stomata (Brainerd and Fuchigami, 1982 ) and by a lower photosynthetic ability (Grout and Aston, 1978). Such characteristics affect their survival after transplanting to greenhouse or field conditions. ~Author to whom correspondence should be addressed.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4238/92/$05.00
10
D. YU E ET AL.
For asparagus, the survival of in vitrc~cultured plantlets has been a problem and recovery has been unusually slow after removal from culture. Hasegawa et al. (1973) reported that plantlets grown under high light had relatively more cladophylls and survived better to acclimatization. Since photosynthesis in asparagus is carried out mainly by cladophylls (Inagaki et al., 1989 ) the results reported by Hasegawa et al. ( 1973 ) suggest that one of the factors affecting the sur,,iva! of in vitro olantlets is the photosynthetic capacity. In addition, Yang and Clore (1974) reported that growing transplanted plantlets under mist improved their survival, suggesting that water stress is also an important factor. Li ( 1985 ) showed that plant growth regulators and inorganic nutrition affected the survival of in vitro cultured asparagus plantlets. However, little research has been carried out to investigate the basic physiological characteristics of in vitro cultured plantlets of asparagus and to determine the cause of slow recovery or death of transplanted plantlets. In our experiments, the photosynthesis and transpiration of in vitro cultured asparagus plantlets were measured with an open gas exchange system. The objective of the experiment was to determine how these physiological characteristics may affect acclimatization and growth of micropropagated asparagus plantlets. MATERIALS AND METHODS
In vitro cultured plantlets. - A superior clonal selection (G- 171 ) made at the
University of Guelph from cultivar Viking-2G was used. Branched shoots at the base of spears were excised and cultured on a modified Murashige and Skoog (MS) medium containing 0.1 mg I- ~naphthaleneacetic acid (NAA), 0.1 mg 1-t kinetin, 30 g 1-i sucrose, 8 g 1-t agar, and 6.4 mg I-~ ancymidol. Cultures were maintained in a growth room kept at 23 +__1°C under cool-white fluorescent tubes at 48/~mol m-2s-! and a 16-h photoperiod. Ten weeks after culture, well-developed shoots were selected for measurement of photosynthesis. Acclimatized plants. - G-I 71 plantlets cultured in vitro for 11 weeks were
washed free of agar, transplanted in 12.5-cm plastic pots containing pro-mix BX artificial substrate (Premier Peat-moss, Rivi~re-du-Loup, Qu6., and transfered to acclimatization. Temperature in the greenhouse was maintained at 24_+2°C, and the relative humidity was about 45%. During the first week, the pots were covered with polyethylene film bags and the humidity surrounding the plantlets was near saturation. During the second week, the bags were partly opened and they were removed by Week 3. After 5 weeks, plantlets with actively growing new shoots, that is the shoots formed in vivo, were selected for measurement of photosynthesis and transpiration.
PHOTOSYNTHESIS AND TRANSPIRATION OF ASPARAGUS
I1
Seedlings. - Asparagus seedlings cultivar 'Viking' were grown in the green-
house for 5 weeks, and photosynthesis and transpiration were measured on the most recently developed mature shoot. All plant materials used in this experiment were 10-15 mm in height, and their ferns had fully developed cladophylls. M e a s u r e m e n t o f photosynthesis a n d transpiration. - An open gas exchange
system was developed to measure photosynthesis and transpiration of in vitro cultured plantlets (Fig. 1). Two ADC MK225 infra-red gas analyzers (Hoddsdon, UK) were used in this system for measurement of CO2 and water vapour concentration. A controller (MT 1000, Measurement Technology Inc., Stoughton, MA ) and a personal computer (IBM-clone) were used to monitor and record data. Roots with rooting medium were put into a small plastic tube, the tube apperture was closed with paraffin film and sealed at the base of the stem with silicon vacuum grease. The plantlet was placed in an assimilation chamber which was made out of a test tube (20 mm diameter× 150 mm long) closed with a rubber stopper equipped with an air inlet and outlet. The assimilation chamber was immersed in a water bath where temperature was precisely maintained at 26 °C. The relative humidity of inlet gas stream at this temperature was 70%. Light intensity had no influence on plant temperature. A period of 5-30 min was provided to establish steady state of CO2 and water pressure conditions in the outlet gas stream before measurements
t L
1
2 B
f
Fig. 1. Schematic view of the system used to measure photosynthesis and transpiration. The desired CO2 concentration is produced by using valves 1 and 2 to mix CO2-free dry air (A) with 5% CO~ in N2 (C). Valves 3 and 4 split the resultant air stream, so that a portion is humidified in a temperature controlled bubbler (B) and subsequently re-mixed with dry air. The air flow passes in part through the reference tubes oftwo infrared gas analyzers (IC for CO2 and IW for water vepor) and in part in the assimilation chamber (AC) suspended in a temperature controlled water bath (WB). The air exiting the chamber passes through the analysis tubes and finally through a flowmeter (F). Lighting of the chamber is supplied by high-pressure sodium vapor lamps (L) suspended above a 3-cm deep water filter (W).
12
D. YUE ET AL.
were taken. Measurements were first made at high photosynthetic photon flux (PPF) followed by gradual lowering of light intensity. Photosynthesis and transpiration rates were calculated according to the equations developed by Moon and Flore (1986) and took into account inlet gas flow rate, temperature and delta CO2 and H20. Owing to the difficulty of measuring leaf area of asparagus cladophylls, photosynthesis measurements were calculated on a dry weight (DW) basis. RESULTS
Notwithstanding the origin of the plant materials used in this experiment, net photosynthesis increased with PPF increases (Fig. 2 ). Plantlets of different origin had almost the same rate of dark respiration at 0.06-0.09 #mol CO2 g-~ DW s-t. In vitro-cultured plantlets exhibited similar rates of photosynthesis as seedlings and both their light compensation points were below 50 gmol m-2 s-l. For these two types of plantlets, rates of photosynthesis increased significantly when PPF increased from 0 to 150/zmol m-2 s-t, and remained essentially steady from 150 to 450/zmol m -2 s -!. At a PPF of 450 /zmol m -2 s-t, their net photosynthesis was about 0.19 gmol CO2 g-i DW s-i. In contrast, the shoots of acclimatized plants had much lower net photosynthesis than that of in vitro cultured plantlets or seedlings. Their photosynthesis increased gradually with PPF increases, and the light compensation point was about 450 #mol m - 2 s- t. The net photosynthesis of in vitro cultured plantlets was higher than that of acclimatized plants at all concentrations of CO: set up in this experiment '7
0.2
t., (~
0.1
tO
|
0.0 .
.
.
.
.
.
.
.
.
.
.
.
-0 1
J I 3 z
-
~ Acclimated plantlets -'-[~-- Seedlings 0
0
. 100
2 200
~ 300
400
500
Photosynthetic Photon Flux (~mol. m"2. s'l )
Fig. 2. Photosynthetic rates of in vitro cultured plantlets, acclimatized plantlets and seedlings of asparagus under different PPF. The ambient CO2 concentration was 300/~11-2. Vertical bars represent SE of the mean of five replications.
PHOTOSYNTHESIS AND TRANSPIRATION OF ASPARAGUS
13
(Fig. 3). Saturation concentration of CO2 was about 600 ld 1-~. The CO2 compensation concentration of in vitro cultured plantlets was lower than 150 /111-I. No significant differences in photosynthesis were observed between in vitro and acclimatized plantlets at high CO2 concentration ( 1200/tl 1- I ). In vitro cultured plantlets had much higher transpiration rates than acclimatized plants or seedlings (Fig. 4). Under dark conditioi~s, transpiration of in vitro cultured plantlets was 21/zmol H20 g-~DW s -i, 8.3 times and 1.7 times higher than acclimatized plants and seedlings, respectively. At a PPF of 450/zmol m -2 s-~, the transpiration rate of in vitro cultured plantlets was 32
'7~ ool
0.1
~
0.0
i .
.
.
.
.
._~ ID
0
~
IQ.
Acclimatized
.0.1
z
•
0
|
|
200
i
400
600
|
800
CO 2 Concentration
!
|
1000 1200 1400
(!~1I "1)
Fig. 3. Photosynthetic rates of in vitro cultured plantlets, acclimatized plantlets of asparagus at different CO: concentrations. The ambient PFF during measurement was 150/~moi m - : s - ~. Vertical bars represent SE of the mean of five replications.
.,~
40
~
In
vitro plantlets ~Acclimatized plantlets
T
";"~ ~~ O30 _---O--Seedllng,.'~ : ~
o E
20
c .9
o. w t.-
,m
I-
10
0
0
100
200
Photosynthetic Photon
300
400
500
Flux (l~mol-rn"2- s "1)
Fig. 4. Transpiration rates of in vitro cultured plantlets, acclimatized plantlets and seedlings of asparagus at different PPF. Vertical bars represent SE of the mean of fi,,.e replications.
14
D. YUE ET AL.
/tmol H20 g-~ DW s-i, that is 3.4 times and 1.8 times higher than acclimatized plants and seedlings, respectively. DISCUSSION
Our results show that in vitro cultured asparagus plantlets have as high a photosynthetic rate as seedlings (Fig. 2). In previous experiments, we measured photosynthesis of plantlets cultured for 20 weeks (data not shown ) and found that their photosynthetic rates were as high as those measured by Inagaki et al. (1989) for comparable greenhouse-grown plantlets. Therefore, we believe that in vitro plantlets cultured as in this experiment have a high enough rate of photosynthesis to support their growth at the time of removal from culture. Based on these results, asparagus can be considered as photosynthetically competent in vitro. According to Grout and Donkin ( 1987 ) species can be regrouped i~ two categories on the basis of their photosynthetic competence. Several species have been shown to possess low photosynthetic rates in vitro (e.g. cauliflower (Grout and Aston, 1978), strawberry (Grout and Millam, 1985 ) ). The recovery of non-competent species during acclimatization is difficult. In the case of asparagus, slow recovery or even death of plantlets during the period of acclimatization appears to be related to the severe water stress they suffer at that time. Since asparagus plantlets were able to carry out photosynthesis, the conditions during and after culture may affect their growth. Increasing the PPF from 50 to 100 and 150 #molm - 2 s - I promoted photosynthesis by 85% and 180°o, respectively (Fig. 2). Increasing the CO., from 150 jtl i-i to 390 or 600 /tl l -I promoted photosynthesis by 155% or 331%, respectively (Fig. 3 ). These data suggested that increasing the light intensity and CO2 concentration in vitro or ex vitro should promote the growth of aspara,-us. Such results have been reported with other crops (Desjardins et al., I o~', Kozai and lwanami, 1988; Laforge et al., 1990). Moreover, the converging photosynthetic activity of in vitro and acclimatized plantlets at high CO., concentration ( 1200 #l I- i ) is indicative of stomatal limitations to photosynthesis. Stomatal closure of acclimatized plantlets appears to be responsible for the low photosynthetic rate of these plantlets. The transpiration rates of in vitro cultured plantlets were much higher than that of acclimatized plants or seedlings (Fig. 4). Llnder dark conditions, the transpiration rates of in vitro cultured plantlets were 8.3 times or 1.7 times higher, respectively, than acclimatized plants or seedlings, suggesting that their epicuticular layer was very thin or absent. Moreover, their stomata might be partly opened even under dark conditions. Both low leaf resistance and poor system of water transfer are some of the reasons explaining the sensitivity of in vitro cultured plantlets to water stress (Grout and Aston, 1977). In vitro cultured plantlets of asparagus lose water very quickly and may wilt in a few
PHOTOSYNTHESIS AND TRANSPIRATION OF ASPARAGUS
15
minutes under normal room or greenhouse conditions. These facts suggested that the main factor causing death and slow recovery of micropropagated asparagus daring the ~,~.-....'~,-'~-aof. zec!imatization__ may be water loss. It is also important to notice that the poor adaptation of in vitro leaves may have an extended effect on the photosynthetic efficiency of the newly formed leaves during acclimatization. ~t is quite possible that acclimatized plantlets possess the capacity for higher photosynthesis but the severe stress imposed at the whole plant level, resulting from the important transpiration of in vitro leaves, may reduce stomatal opening leading to reduced photosynthesis in newly formed leaves. Indeed, low transpiration observed with acclimatized plantlets and their correspondingly low photosynthesis strongly suggest that stomata are closed. This however remains to be verified with porometric or anatomic measurements. To insure high rates of survival, the most important factor may be protecting tissue-cultured asparagus plantlets from water stress. Preliminary results in our laboratory demonstrate that it is possible to reduce the transpiration rate of in vitro plantlets of asparagus by removing the cap for some time before transfer as was presented by Sutter ( 1988 ). Therefore, pre-acclimatization of asparagus plantlets during in vitro culture may be one promising way to ensure high survival rates during the period of acclimatization. Experiments are underway to assess this possibility. ACKNOWLEDGMENTS
We gratefully acknowledge Conseil de la recherche en p&heries et alimentation du Qu6bec (CORPAQ) and Agriculture Canada for their financial support. We also acknowledge Dr. Michael E.D. Graham and France Crocheti~re for their helpful contribution to the photosynthetic measurements and reviewing the manuscript, respectively.
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Grout, B.W.W. and Donkin, M.E., 1987. Photosynthetic activity of cauliflower meristem cultures in vitro and at transplanting into soil. Acta Hortic., 212: 323-327. Grout, B.W.W. and Millam, S., 1985. Photosynthetic development of micropropagated strawberry plantlets following transplanting. Ann. Bot., 55:129-131. Hasegawa, P.M., Murashige, T. and Takatori, F.H., 1973. Propagation of asparagus through shoot apex culture. II. Light and temperature requirements, transplantability of plants, and cytohistolog~cai characteristics. J. Am. Soc. Hortic. Sci., 98:143-148. Inagaki, N., Tsuda, K., Maekawa, S. and Terabun, M., 1989. Effects of light intensity, CO2 concentration, and temperature on photosynthesis of Asparagus officinalis L. J. Jpn. Soc. Hortic. Sci., $8: 369-376. Kozai, T. and lwanami, Y., 1988. Effects of CO2 enrichment and sucrose concentration under high photon fluxes on plantlet growth of carnation (lJianthus caryophyllus L. ) in tissue culture during the preparation stage. J. Jpn. Soc. Ho~tic. Sci., 57: 279-288. Laforge, F., Lussier, C., Desjardins, Y. and Gosselin, A., 1990. Effect of light intensity and CO2 enrichment during in vitro rooting on subsequent growth of p|antlets of strawberry, rasoberry and asparagus in acclimatization. Scientia Hortic., 47: 259-269. Li, S.Z., 1985. Investigation of the survival rate after transplanting of asparagus plantlets raised in vitro. Zhejiang Agric. Sci., No. l: 47-48. Moon Jr., J. and Flore, J.A., 1986. A basic computer program for calculation of photosynthesis, stomatai conductance, and related parameters in an open gas exchange system. Photosynth. Res., 7: 269-279. Sutter, E., 1988. Stomatal and cuticular wmer io:~ from apple, cherry and sweetgum after removal from in vitro culture. J. Am. Soc. Hortic Sci., 113: 234-238. Yang, H.J. and Clore, W.J., 1974. Improving the survival of aseptically-cultured asparagus plants in transplanting. HortScience~ o: 235-236.