Planta (Berl.) 102, 11-25 (1972) 9 by Springer-Verlag 1972

Photosynthesis by Carrot Tissue Cultures A. D. HA~so~ and J. EDV,L~rA~ Biology Department, Queen Elizabeth College, London Received July 12/30, 1971

Summary. 14C02-fixation rates in green carrot callus cultures (about 35 ~g ehlorophyll/g fresh wt) were determined in gaseous and liquid media using a range of light intensities and COS concentrations. Main products of light-dependent COgfixation were sucrose, alanine, glutamine, serine/glycine and malie acid. In darkness, glutamine and malie acid were formed. Light CO2-fixation rates were about ten times higher than dark fixation rates and reached 50-90 ~mol/mg ehlorophyll/h in 10000 lux, 1% COS in air. Net 02-evolution by the tissue was demonstrated polarographically under these conditions. Light C02-fixation rates were linearly related to chlorophyll levels while dark fixation was independent of chlorophyll content. Lowered 02 partial pressures in gaseous conditions increased 14CO2-fixationrates. Ribulose diphosphate carboxylase ~nd phosphoenol pyruvate earboxylase activities and their distribution in subcellular fractions were examined. When carrot tissue cultures were grown for two or four weeks on agar media lacking a carbohydrate source, in 10000 lux and 1% CO2 in either air or N2, dry weight increases were obtained although chlorophyll levels eventually declined. Introduction Green plant tissue cultures often possess moderately well developed chloroplasts (Laetsch and Stetler, 1965; Sunderland and Wells, 1968; Blackwell et al., 1969; Edelman and Hanson, 1971) and the capacity for photosynthetic COn-fixation (Laetsch and Stetler, 1965; Venketeswaran, 1965; McLaren and Thomas, 1967; Hanson and Edelman, 1971) and O nevolution (Naef, 1968). Factors known to affect final chlorophyll concentrations in tissue cultures include iron-availability, auxins, sugars, complex nutrients such as coconut milk, and light regimes, but the chlorophyll levels so far attained are generally far lower than those of mature leaves, resulting in poor carbon fixation in the light and correspondingly poor growth in the absence of exogenous carbohydrate. Using relatively low light intensities and atmospheric COn levels several authors have failed to obtain autotrophic growth (for example: Venketeswaran, 1965; F u k a m i and Hildebrandt, 1967; Sunderland and Wells, 1968) although two workers who employed higher light intensities and enriched COn atmospheres have claimed satisfactory growth rates

12

A.D. Hanson and J. Edelman:

with tobacco suspensions (Bergmann, 1967) a n d Rula graveolens calluses (Corduan, 1970). W e have i n v e s t i g a t e d the p h o t o s y n t h e t i c physiology of two carrot callus strains, C ~ T 1 a n d C R T 2 , to d e t e r m i n e conditions o p t i m a l for photosynthesis. This i n f o r m a t i o n , together with t h a t o n respiratory rates, was used to provide a pictm'e of the carbon balance of the tissues on which were based p r e l i m i n a r y a u t o t r o p h i c growth experiments. These studies form p a r t of a p r o g r a m on the growth in culture of green p l a n t tissues. Materials and Methods

Callus Cultures. Carrot callus strains CRT 1 and CRT 2 were grown and harvested routinely as described by Hanson and Edelman (1971). In antotrophic growth trials, CRT1 calluses were cultured in tubes on 5 ml of normal CRT1 nutrient medium from which sugar was omitted, in 10000 lux of incandescent or mercury vapour light. The tubes were held in one or two 1 capacity transparent gas-tight vessels which were continually flushed with 1% CO2 in air or 1% C02 in N2 (B.O.C. Special Gases, London, S.W. 19) at flow rates of 25 or 50 ml/min. The temperature was maintained at 264-2 ~ by circulating cooling water. Callus Dry Weights. Small pieces were dried to constant weight at 70~. 14C02 Exposure Conditions. After a 30 rain preillumination in air, C02-fixation rates over 30 or 60 rain periods were determined either in liquid media as previously described (Hanson and Edelman, 1971) or as follows: replicate callus pieces (weighing about 200 mg each) were placed on moistened filter paper in 25 ml tubular glass vessels equipped with side arms containing 0.2 ml lactic acid. After closing the vessels with serum caps, Na214C0~ of known specific activity was delivered to the lactic acid to give the required C02 level. When low CO2 concentrations were used, the tubes were initially flushed with C02-free air. Flushing with 1% C02 in air followed by addition of 50 ~Ci Na214C03 (57 mCi/m~ol) was used for routine assay of photosynthetic rates in 1% CO~. Various light intensities were obtained either by positioning the tubes in relation to the tungsten illumination or by tinen neutral density filters. Dark control tubes were wrapped in alumininm foil. Extraction and Assay o/ Chlorophyll, Fructose, and 14C. Tissues were killed by grinding in cold ethanol, and extracted sequentially in ethanol and 30 % ethanol as detailed elsewhere (Hanson and Edelman, 1971). Chlorophyll, total fructose and ethanol-soluble and -insoluble 14C were determined as described previously. Soluble 14C was assayed in a liquid scintillation spectrometer; insoluble 14C by thin endwindow counting. Chromatography. Ethanolic tissue extracts were fractionated into cationic, anionic and neutral fractions using Dowex columns and chromatography of the fractions was carried out using the following solvents: a) n-butanol:acetic acid: water, 4:1:5, upper phase; b) iso-butanol: 3 % NH4OH, 3:1; e) n-propanol:ethyl acetate: water, 7:1 : 2; d) ethyl acetate: pyridine: water, 8: 2:1 ; e) water--saturated diethylether: 98% formic acid, 7:1; f) 96% ethanol:water: 25% NHaOH, 100: 12: t6. Solvents a)-d) were used in a one-dimensional descending system on Whatman No. 1 paper, and solvents e) and f) on Eastman prepared silica gel plates; developed chromatograms and TLC plates were autoradiographed. Respiratory Rates. Warburg manometry was carried out in darkness at 25~ Polarography. O~-exchange in illuminated callus pieces at 25~ was measured polarographically with Clark-type electrodes.

Photosynthesis by Tissue Cultures

13

Cell Fractionation. CRT2 tissue (20 g) was ground for 10 see in a domestic blendor in 200 ml semi-frozen medium containing sucrose 0.33 M, tris-HC1 0.1 M pH 8, MgCl~ 5 raM, 2-mereaptoethanol 1 m_~ (Baldry et al., 1970). The resultant brei was filtered through muslin and separated by differential centrifugation into four fractions: a 4000g, 2 min pellet (P1); an 18000g, 20rain pellet (P2); a 100000 g, 60 min pellet, (P3) and a supernatant fraction. Pellets were resuspended in a small volume of grinding medium, examined microscopically and sonieated before enzyme assay. Chlorophyll was determined in 80% acetone solution using the equations of Arnon (1949). Protein was isolated by precipitation with 80% acetone, washed with further 80% acetone, taken up in i N NaOH and estimated by the method of Lowry et al. (1951) using a bovine plasma albumin standard in 1 N Na0H. Enzyme Assay. Phosphoenol pyruvate carboxylase (PEP earboxylase, EC 4.1.1.31) was assayed at 30~ by measuring the incorporation of laC into acid-stable compounds in reaction mixtures containing 100 ~moles tris-HC1, ptI 8.3; 1 ~mole MgCl~; 0.1 ~mole 2-mercaptoethanol; 10 ~mo]es L-glutamic acid; 1 ~mole phosphoenol pyruvate; 1.35 p.moles NaHC0~ containing 20 ~Ci 14C and 0.1 ml enzyme preparation; total volume 0.5 ml. Reactions were stopped after 30 rain by addition of 0.1 ml 24% trichloroaeetie acid and aliquots of the reaction mixture evaporated to dryness and taken up in 0.2 ml water before 14C assay by liquid scintillation counting. Ribulose diphosphate carboxylase (RuDP carboxylase, EC 4.1.1.39) was assayed in a similar reaction mixture which included 30 tzmoles NattCO 3 containing 50 ~Ci 14C, 0.5 ~zmoles ribulose-l,5-diphosphate and 0.2 ml enzyme preparation. Results and Discussion

Chlorophyll Content and C02-tZixatio~ Rates Strain C R T 1 tissues contained a b o u t 30 [zg chlorophyll/g fresh wt and C R T 2 tissues about 50 ~g/g fresh wt. Chlorophyll a/b ratios were 2.0 and 1.8 respectively. A certain a m o u n t of heterogeneity in chlorophyll distribution within calluses occurred in b o t h strains; this lack of uniformity of chlorophyll distribution was used to test for correlation between light-dependent COs-fixation , dark fixation and tissue chlorophyll levels. Fig. 1 demonstrates a linear relationship between the chlorophyll content of callus pieces and their rate of C02-fixation in the light; dark fixation rate was independent of chlorophyll content. Several authors have assumed t h a t increases in the chlorophyll contents of plant tissue cultures would lead to increased photosynthesis (Fukami and Hildebrandt, 1967; Stobart et al., 1967) b u t this has n o t been substantiated. The d a t a of Fig. 1 show t h a t this assumption is valid for carrot calluses.

P E P Carboxylase and R u D P Carboxylase Activities Fig. 1 implies a non-chloroplast location for the dark carboxylation system as dark COs-fixation was n o t related to tissue chlorophyll concentration. Table 1 summarises the results of assay of two major

14

A.D. Hanson and J. Edelman:

oo/

40

o

30

2o

o/ /o

10

I

I

l

I

I

2

4

6

8

10

I

12

Chlorophyl[(IJ-gper 200rag piece) Fig. 1. The relationship between callus chlorophyll content and CO2-fixation rates in light (o) and darkness (.). CRT1 callus pieces, each weighing 200 mg but with various chlorophyll contents, were exposed for 30 rain to 0.08 % 14C02, in 10000 lux or darkness, 25~

carboxylation enzymes in CRT2 callus brei and in various subcellular fractions of the brei isolated b y differential centrifugation. P E P carboxylase activity in the brei exceeded R u D P carboxylase activity 13-fold. Although only 24% of R u D P carboxylase activity was recovered in fractions containing intact chloroplasts (1)1 and P2), less than 7% of P E P carboxylase activity was located in these fractions. I n addition, the specific activity of P E P carboxylase in the supernatant was more than six times higher t h a n in any particulate fraction, in contrast to R u D P carboxylase. These results suggest t h a t P E P carboxylase was not associated with intact chloroplasts. This enzyme, operating with malate dehydrogenase, is known to catalyse dark COs-fixation in plants (Walker, 1962). Consistent with the presence of such a system in carrot caUuses, malate was the major product of dark COs-fixation (Table 4) and very high N A D H linked malate dehydrogenase activities (about 2000nmol N A D H consumed/min/mg protein) were detectable in callus brei. When expressed on a chlorophyll basis, the R u D P carboxylase activity values of Table 1 are insufficient to account for the maximal photosynthetic rates obtained with intact calluses. Although this result

-

0

6

63

68

154

Chlorophyll distribution (~g/fraction)

298 (91.2)

6 (2.0)

12 (3.6)

11 (3.3) b

397

8.58

1.30

1.30

1.30

--

20.8 (71.9)

1.2 (4.1)

3.5 (12.1)

3.4 (11.9)

31

0.60

0.24

0.39

0.42

--

Specific activity (nmol CO~/min/mg protein)

Enzyme units (nmol CO~/min) per fraction

Enzyme units (nmol CO~/min) per fraction

Specific activity (nmol CO~/min/mg protein)

R u D P Carboxylase

P E P Carboxylase

a Fraction composition was determined with a Zeiss phase-contrast microscope 9 No viable cells (i.e. showing cyclosis) were observed in the brci or pellets. b % distributions of enzymes among fractions are given in parentheses.

-

--

P3

Supernatant

Small intact ehloroplasts, chloroplast fragments and mitochondria

P2

-

Intact chloroplasts. Broken tracheids and other cell debris

-

Contents a

P1

Brei

Fraction

Table 1. Subcellular distribution o / P E P carboxylase and R u D P carboxylase activities in CRT2

c~

k

I/l

Y

N. ~ ~

b

~

I~

I

I

~

~

,

- -

\

~-

,,

g

oj. (,.)xZ / IX /

,,,

o

a~

7,,

~o ~ ,,

~ o

~

/ \

\

3.

z

~

C02- fixation rate (IJ-mol/mg chlorophyll/h) o~

e

~

\

\

k

/IX>--.._ \

.4": ~176

.~.~o/_A/ / / A

~.:~

/

o

b

C02- fixation rate (iJ.mol/mg chlorophyll/h)

Photosynthesisby Tissue Cultures

17

resembles that reported for tropical grasses showing the C4.dicarboxylic acid pathway of photosynthesis (Slack and Hatch, 1967), it is unlikely that carrot calluses, derived from a plant with Calvin-type photosynthesis possess this C4-acid pathway. The anomalous relative activities of the two carboxylases are probably due to the high phenoloxidase levels and high phenol concentrations present in callus brei; it has been shown recently that phenols and their oxidation products are more inhibitory to RuDP carboxylase than to PEP carboxylase (Baldry et al., 1970).

E//ect o/Light Intensity and CO2 Concentration on C02-Fixation Rates of CO~-fixation by CRT 1 in a range of COs concentrations and hght intensities are shown for gaseous conditions in Fig. 2a and for submerged conditions in Fig. 2 b. The curves indicate considerable interaction in the determination of photosynthetic rate between the two factors varied. High rates of COs-fixation (> 50 ~mol/mg chlorophyll/h) occurred in both gaseous and submerged conditions at higher light intensities and COS levels while at the lower levels of these two factors, COs-fixation rates were extremely low (about 2 ~mol/mg chlorophyll/h). These results would explain the failure of attempts to grow plant tissue cultures autotrophically when high light intensities and enriched COs atmospheres have not been used together (Venketeswaran, 1965; Fukami and Hildebrandt, 1967; Sunderland and Wells, 1968). Rates of dark fixation of COS in high COS concentrations in liquid media were considerably greater than in air. Nature o/Products o/C02-Fixation The labelled products of light laCO2-fixation under gaseous and submerged conditions, at high and low COs levels by CRT 1 and CRT2 were fractionated on Dowex columns (Table 2); the compositions of these fractions are summarised in Table 3. The data of Tables 2 and 3 show the following features: a) CRT2 has a greater tendency to form laC-cationic products than CRT 1 ; b) the proportion of laC fixed into insoluble products is always less than 30 %, and less than 20 % in CRT 1 ;

Fig. 2. The effect of light intensity and COs concentration on COs-fixation by CRT 1 tissue, a) in air: callus pieces were exposed to 14C0s for 30 min, 25~ b) immersed in 5.0 ml Murashige and Skoog mineral element solution in 10 ml vessels. Exposure to Na 214C03 was for 30 min, 25 ~ with continuous reciprocal shaking at 80 eycles/min. CO2 concentration was adjusted with carrier Na2CO 3 and is shown as an approximate percentage for comparison with Fig. 1 a 2 Planta (Berl.), Bd. 102

Gaseous Gaseous Submerged Submerged

Gaseous Gaseous Submerged Submerged

CRT1

CRT2

Incubation conditions

Strain

0.03 1.0 0.03 1.0

0.03 1.0 0.03 1.0

CO s conch. (% )

1.9 33.6 0.6 11.4

3.2 50.1 2.0 45.4

COs-fixation rate (tLmol/mg chlorophyll/h)

22.3 26.7 27.8 20.2

8.4 11.6 18.3 11.3

~ac recovered in E t O H insoluble residue ( % t o t a l fixation)

22 21 32 35

38 44 56

33 33 46 66

Anionic

42

14 13 21 18

Cationic

37 41 25 9

53 55 33 17

Neutral

~C recovered in fractions of E t O t t e x t r a c t / t o t a l ~aC in E t O H e x t r a c t ( % )

Table 2. Distribution o] products o/light laco2-/ixation among #actions o] ethanol extracts and ethanol-insoluble material CRT 1 or CRT 2 tissue was exposed to 0.03% or 1.0% 14C02, 10,000 lux for 60 rain a t 25 ~ in gaseous or submerged conditions.

~

~o

Photosynthesis by Tissue Cultures Table 3. Products The fractions matography. No CRT1 or CRT2 conditions.

19

o/light 14CO~-fixation identiiied in extracts o] C R T 1 and CR T 2 shown in Table 2 were analysed by paper and thin-layer chroclear differences were found among the fractions from either exposed to high or low COx levels in gaseous or submerged

Fraction

14C-labelled products identified

Cationic

Glutamine, alanine, serine/glycine; lesser amounts of glutamic acid and histidine

Anionic

Malic acid, lml~nown organic acida; traces of succinie acid and organic phosphates

Neutral

Sucrose; traces of glucose and fructose

a The unknown acid ran with malate in ether: formic acid and slightly ahead of glycollate in ethanol: water: ~IHaOH and in n-butanol: acetic acid: water. Table 4. Distribution o] dark 14C02-]ixation products o] CRT1 among ~factions o] ethanol extracts and ethanol-insoluble material CRT 1 tissue was exposed to 0.03 % or 1.0 % ~4C02 for 60 rain at 25 ~ in darkness. Incubation conditions

C02 concn. (%)

14C recovered in fractions of 14C recovered in EtOtt extract/total laC EtOH-insoluble residue in EtOH extract (%) (% total fixation) Cationic a Anionicb Neutral

Gaseous Gaseous Submerged Submerged

0.03 1.0 0.03 1.0

4.8 11.9 7.1 8.9

28 25 29 20

70 74 69 80

2 1 2 0

a Mainly glutamine. b Mainly malic acid.

C) CO z c o n c e n t r a t i o n does n o t significantly affect 14C-product distrib u t i o n in C R T 1 a n d C R T 2 exposed to laC02 in gaseous conditions, b u t high 1aC02 c o n c e n t r a t i o n s in s u b m e r g e d conditions l e a d to r e d u c e d I~Ci n c o r p o r a t i o n into n e u t r a l p r o d u c t s (sucrose) ; d) b o t h strains p h o t o s y n t h e s i s i n g in gaseous conditions i n c o r p o r a t e a higher p r o p o r t i o n of 14C i n t o n e u t r a l p r o d u c t s (sucrose) t h a n when p h o t o s y n t h e s i s i n g in s u b m e r g e d conditions. Similar low l a c - i n c o r p o r a t i o n i n t o sucrose has been shown in e x t r a c t s of HCO3-fed Kalanchog callus (McLaren a n d T h o m a s , 1967) a n d of C02-fed t o b a c c o callus (Laetsch a n d Stetler, 1965). Since, in c a r r o t tissue cultures, gaseous conditions r e s u l t in g r e a t e r s y n t h e s i s of h i g h l y - r e d u c e d c a r b o n (sucrose) a n d since t h e y 2*

20

A.D.

Hanson and J. Edelman:

also favour higher fight-dependent CO~-fixation rates at higher COs concentrations, they appeared more suitable for the investigation of autotrophic growth. The dark J4CO2-fixation products of CRT 1 were also fraetionated and ehromatographed (Table 4). Almost no 14C-activity was recovered in neutral products; cationic fractions contained mainly glutamine, anionic fractions mainly mafie acid.

02- Uptake

and Evolution

In darkness at 25 ~ the Os-uptake rate of CRT 1 tissue was between 50 and 75 ~l/g fresh wt/h throughout a passage of six weeks duration (Table 5). CRT2 showed an appreciably higher 02-uptake rate.

Table 5. Os-uptake rates/or CRT1 and CRT2 Rates were determined manometrically during a 6h period in darkness at 25~ Strain

Callus age (weeks)

CRT 1

2 3 4 6 2

CLOT2

O~-uptakerate (tA/g fresh wt/h) 55 75 65 50 112

Assuming a chlorophyll content of 40 f~g/g fresh wt, a C0~-fixation rate of 70 ~mol/mg chlorophyll/h in 10000 lux, 1% COs and a photosynthetic quotient of unity, an 03 output rate of about 70 ~l/g fresh wt/h would be predicted, making net 02-evolution from the tissue a possibility. An overaU rate of Os-evolution of only 20 91/g fresh wt/h would be required for a substantial dry weight increase in a one month passage. Net 02-evolution was observed in several short-term experiments with CRT 1 and CRT2; on some oeeasions, however, Orevolution in the fight only balanced O~-consumption (Fig. 3). DCMU (10-e M) inhibited 02-evolution. E]]ect o / 0 s Partial Pressure on COs-Fixation

Fig. 4a and b demonstrate that increasing Os partial pressure in O~/Ns mixtures markedly inhibited COs-fixation by CRT 1, the inhibition being greatest at high light intensity and lo~ COs concentration.

Photosynthesis by Tissue Cultures

21

T

Dark X

0.1llmole|

Dirk

\

X

"\ L!ght "~ark Start ~

~.

\

%%

Finish

Fig. 3. Representative tracings showing 02-electrode responses. About 300 mg of callus tissue broken into 1 mma fragments was incubated in 3.0 ml liquid medium. 25~ 10000 lux, 1% COs . In experiment I (solid lines) net 02-evolution was observed; in experiment II (dotted lines) O2-evolution balanced 02-consumption

Inhibition of photosynthesis by 02 has been observed in many higher plants showing Calvin-type COs-fixation and in algae and is frequently greater at high light intensities and low COs concentrations (Coombs and Whittingham, 1966). In both higher plants (e.g. Zelitch, 1968) and algae (e.g. Coombs and Whittingham, 1966) 02-inhibition of photosynthesis is accompanied by an increase in the synthesis of glycollie acid and glycine at the expense of sucrose. Although we found a reduction of laC-sucrose synthesis and an increase of J4C-ineorporation into organic and amino acids under conditions of 02-inhibition (Table 6), 14C-glycollie acid was not detected in the 02-treatment. The glyeine/serine zone of chromatograms was, however, heavily labelled in the 02-treatment but almost unlabe]led in the N2-treatment. Since we have obtained preliminary evidence t h a t 14COs-evolution from calluses fed 14C-glucose under COsfree conditions is stimulated by 02 and high light intensities, it seems probable that in photosynthesising carrot calluses glyeollic acid is oxidised or metabolised further as rapidly as it is formed. Table 6. Distribution o/product8 o/14CO~-fixatiou in 0.03 % COs, 10000 lux among /factions o/ethanol extracts

Carrier 14C recovered in fractions (%) gas Cationic Anionic Neutral N2 0s

12 26

25 41

62 33

22

A.D. Hanson and J. Edelman:

9~

0.03%C02

1.0%C02 I00

~6 o

~5

\oooo,o, E

60

~3 P,

~ 0 . 0 0 0

.------//-----'x

lux ~

oc 4O

~2 c~ (.9

u

0

I i 1 20 02 concentration(%)

I 100

20

,--'--,I'-'-'~1.5oo o

"

lux

I 20 02 concentration(%) b

Fig. 4. The effect of partial pressure of O3 in N 2 on !~C02-fixation rates (corrected for dark fixation) in 15001ux (.) and 100001ux (o). Strain CRT1, 30rain exposure to laC02, a) 0.03% CO2; b) 1.0% C02

An increased rate of apparent photosynthesis in lowered Os levels could account for the successful report of autotrophic growth of garden rue callus in 1% CO s in N s (Corduan, 1970).

Autotrophic Growth Trials Strain CRT1 was chosen for autotrophic growth experiments in preference to CRT2 because it grew faster on defined media, showed a higher photosynthetic efficiency, better incorporation of fixed carbon into sucrose and a lower Os-uptake rate than CRT2 (Tables 2 and 5). Gaseous, rather than submerged, conditions were employed as they favoured sucrose as opposed to organic acid production (Table 2); the light intensity/CO 2 concentration combination of 10000 lux, 1% CO s was selected with reference to Fig. 2 a.

a) 1% CO2 in Air; lO000 Lux Continuous Mercury Valour Light. Table 7 outlines the results obtained using these conditions. Initial inoeula were relatively large (300mg). During the first 14 days of the experiment, substantial dry weight increases occurred and COs-fixation

100

Photosynthesis by Tissue Cultures

23

Table 7. Characteristics o / C R T 1 tissues grown under autotrophic conditions. I. Tissues were maintained at 26~ on sugar-free medium for one or two 32-day passages in 10000 lux continuous mercury vapour light in 1% COSin air. Oa-uptake rate was determined manometrically in air in darkness at 25~ COs-fixation rate in 1% COs in air, 10000 lux, 25~ Values in the Table are means of four replicates. Control tissues grown on sugar-free medium in 1300 lux in air lost turgor and showed necrotic zones after a single 32-day passage. Passage Day of Fresh harwt vesting (mg)

Dry wt (mg)

Chloro- Total phyll fructose content content (~g/g (mg/g fresh wt) fresh wt)

02-uptake C02-fixarate (tzl/g tion rate fresh wt/h) (~mol/mg chlorophyll/h)

Fkst

0 14 32

300 720 670

16 30 32

34 16 13

3.7 1.4 1.7

65 45 40

45 84 52

Second 32

440

19

15

2.0

13

33

Table 8. Characteristics o/C•T1 tissues grown under autotrophic conditions. H Tissues were maintained at 26~ on sugar-free medium under various gas mixtures and tungsten light regimes. O~-uptake and CO~-fixation rates were determined as in Table 7. Values in the Table are means of four replicates. Control tissues incubated in an air stream in 10000 lux of continuous illumination were necrotic after 15 days. Gas mixture

Tungsten Day of Fresh illumination harwt (10000 lux) vesting (rag)

Dry Chlorowt phyll ( r a g ) content (t~g/g fresh wt)

02-uptake CO~-fixarate (tzl/g tion rate fresh wt/h) (~zmol/mg chlorophyll/h)

- -

- -

0

1% COs in air

Continuous

15

150

7.6

47

78

140

6.5

7

23

1% C02 in air

22

16 h light/8 h 15 dark cycles

160

7.8

15

34

65

1% C02 in N 2

Continuous

180

8.9

16

26

91

15

75

rate per u n i t chlorophyll rose; O2-uptake rate fell a n d chlorophyll c o n t e n t declined sharply. F o r the r e m a i n i n g 18 days of the first passage, d r y weight, O~-uptake rate a n d chlorophyll c o n t e n t did n o t change further, b u t CO~-fixation rate decreased. W h e n 300 mg pieces of the a u t o t r o p h i c a l l y grown tissue were s u b c u l t u r e d a n d grown for a f u r t h e r 32 days i n the same conditions, dry weight did n o t increase significantly a n d respiratory a n d p h o t o s y n t h e t i c rates fell to v e r y low levels.

b) 1% CO 2 in A i r or N~; 10000 L u x Continuous or Discontinuous Tungsten Light. I n this trial, t u n g s t e n light was s u b s t i t u t e d for m e r c u r y

24

A. D. Hanson and J. Edelman:

vapour light. Smaller inocula were taken to lessen nutrient carry-over effects. A 1 To CO S in N2 gas mixture was employed because high photosynthetic rates were observed in short term experiments with this substrate (Fig. 4b). A regime of 16 h light and 8 h darkness was also tested because of its stimulatory effect on heterotrophie callus growth (Bfinning and Welte, 1954). Results are given in Table 8; after 15 days changes in dry weight in all treatments were insignificant, presumably because of the smaller inocula used compared with the previous experiment (Table 7). Oz-uptake rates and chlorophyll levels fell markedly in all cases, although photosynthetic rates per unit chlorophyll remained high in at least two treatments. Supplementing the medium with high levels of chelated iron and with organic acids known to improve chlorophyll synthesis in other systems did not delay chlorophyll degradation.

Conclusion Our results on the effects of fight intensity, CO 2 concentration and 02-tension on carrot tissue culture photosynthesis generally resemble those obtained with whole plants, isolated leaves and leaf disks of species showing Calvin-type COs-fixation; carrot tissue cultures behave photosynthetically as normal Calvin plants despite the absence of stomata and other structural speciafisations. Using large inocula, carrot callus cultures may be grown autotrophically for short periods (e.g. two weeks); declining chlorophyll levels, however, eventually check growth. The problem of rapid chlorophyll loss from illuminated callus tissues is currently under investigation in our laboratory. This work was supported in part by a grant from Tare and Lyle Ltd.

References Arnon, D. I.: Copper enzymes in isolated chloroplasts: polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1-15 (1949). Baldry, C. W., Bucke, C., Coombs, J.: Effects of some phenoloxidase inhibitors on chloroplasts and carboxylating enzymes of sugar cane and spinach. Planta (Berl.) 94, 124-133 (1970). Bergmann, L.: Waehstum griiner Suspensionkulturen yon Nicotiana tabacum ear. ,,Samsun" mit C02 als Kohlenstoffquelle. Planta (Berl.) 74, 243-249 (1967). Blackwell, S. J., Laetsch, W. M., Hyde, B. B. : Development of chloroplast fine structure in aspen tissue culture. Amer. J. Bot. 56, 457-463 (1969). Biinning, E., Welte, H.: Photoperiodische Reaktionen an pflanzlichen Gewebekulturen. Physiol. Plantarum (Cph.) 7, 197-203 (1954). Coombs, J., Whittingham, C. P. : The mechanism of inhibition of photosynthesis by high partial pressures of oxygen in Chlorella. Proc. roy. Soc. B 164, 511-520 (1966). Corduan, G. : Autotrophe Gewebekulturen yon Ruta graveolens und deren 14CO2Markierungsprodukte. Planta (Berl.) 91, 291-301 (1970).

Photosynthesis by Tissue Cultures

25

Edelman, J., Hanson, A. D. : Sucrose suppression of chlorophyll synthesis in carrot callus cultures. Planta (Berl.) 98, 150-156 (1971). Fukami, T., Hildebrandt, A. C. : Growth and chlorophyll formation in edible green plant callus tissues in vitro on media with limited sugar supplements. Bot. Mag. (Tokyo) 80, 199-212 (1967). Hanson, A. D., Edelman, J. : Secretion of photosynthetic products by carrot tissue cultures. Planta (Berl.) 98, 97-108 (1971). Laetsch, W.M., Stetler, D.A.: Chloroplast structure and function in cultured tobacco tissue. Amer. J. Bot. 5~, 798-804 (1965). Lowry, O. H., Rosenbrough, N. J., Farr, A. L., Randall, R. J. : Protein measurement with Folin-Phenol reagent. J. biol. Chem. 198, 265-275 (1951). McLaren, I., Thomas~ D. R. : CO2 fixation, organic acids and some enzymes in green and colourless tissue cultures of Kalanchog crenata. New Phytologist 66, 683=695 (1967). Naef, J. : Action combinde de la lumi~re et du glucose sur les souches tissulalres de carotte. Les Cultures des Tissus de Plantes, p. 301-314, ed. R. J. Gautheret, M. L. Hirth. Strasbourg: C.N.R.S. 1968. Slack, C. R., Hatch, M. D. : Comparative studies on the activity of carboxylases and other enzymes in relation to the new pathway of photosynthetic carbon dioxide fixation in tropical grasses. Biochem. J. 103, 660-665 (1967). Stobart, A.K., MeLaren, I., Thomas, D.R.: Chlorophylls and carotenoids of colourless callus, green callus and leaves of Kalancho~ crenata. PhyCochem. 6, 1467-1474 (1967). Sunderland, N., Wells, B. : Plastid structure and development in green callus tissues of Oxalis dispar. Ann. Bot. 32, 327-346 (1968). Venketeswaran, S.: Isolation of green pigmented callus tissue and its continued maintenance in suspension cultures. Physiol. Plantarum (Cph.) 18, 767-789 (1965). Walker, D.A.: Pyruvate carboxylation and plant metabolism. Biol. Rev. 37, 215-256 (1962). Zeliteh, I.: Investigations on photorespiration with a sensitive 14C-assay. Plant Physiol. 43, 1829-1837 (1968). Professor J. Edelman Biology Department, Queen Elizabeth College Atkins Building Campden Hill, London, W.8, U.K.