Plant Cell Reports

Plant Cell Reports (1986) 5:247-251

© Springer-Verlag 1986

Condensed tannins in the tissue culture of sainfoin (Onob chis viciifolia Scop.) and birdsfoot trefoil (Lotus corniculatus L.)* G.L. Lees Agriculture Canada Research Station, 107 Science Cres., Saskatoon, Saskatchewan, Canada S7N OX2 Received February 19, 1986 / Revised version received April 24, 1986 - Communicated by F. Constabel

ABSTRACT Two forage legumes, birdsfoot trefoil (Lotus corniculatus L.) and sainfoin (Onobrychis viciifolia Scop.), containing condensed tannins in their leaves and stems were used as source material to study condensed tannins in tissue culture. More protoplasts were isolated from mesophyll tissue of a low tannin-containing strain of birdsfoot trefoil than from a high tannin-containing strain, but more tannin-filled protoplasts were observed in the latter. Growth rates of leaf explant-derived callus tissue were greater for the high-tannin than for the low-tannin strain. In sainfoin, callus cultures from leaf explants produced numerous tannin-filled cells by 21 days. Explants from sainfoin cotyledons and roots, tissues which normally do not contain tannins, also formed callus with tannin-filled cells in 21 days but in almost every case, a cytokinin was required for tannin formation to occur. The occurrence of tanninfilled cells in callus from root and cotyledon explants was variable and genotype specific. These results show that endogenous tannins can affect protoplast isolation and possibly callus growth in birdsfoot trefoil, and that the formation of condensed tannins in sainfoin callus culture can be influenced by a growth regulator. Abbreviations: BAP, benzylaminopurine; KIN, kinetin; NAA, naphthaleneacetic acid; PAR, photosynthetically active radiation INTRODUCTION Condensed tannins are a class of water-soluble phenolic compounds which have the ability to precipitate proteins (Swain and Bate-Smith, 1962). These secondary metabolites are known to occur in many, but not all herbaceous legumes (Marshall et al., 1979; Sarkar et al., 1976). Their significance is not completely understood but they have been associated with disease resistance in crops (Van Sumere et al., 1975) and they are considered to be an important component of the seed coat conferring water impermeability and resistance to mechanical damage in the seeds of many species (Crofts, 1979). Condensed tannins are potent antibiotics and in sufficient concentrations can lead to an almost negligible decay in leaf litter (Swain, 1977). They have been implicated as inhibitors of microbial digestion in herbivores (Becker, 1984; Martin and Martin, 1983) and in our laboratory, we associate condensed tannins in the herbage of a farage legume with the inability of that legume to cause pas-

ture bloat when grazed by ruminant animals et al., 1979).

Howarth

The significance and occurrence of tannins in tissue culture is less clear. Studies show that these secondary metabolites accumulate in greater quantity in the stationary growth phase of cell culture (Constabel, 1965; Baur and Walkinshaw, 1974; Jalal et al., 1979) and that there is an antagonism between tannin synthesis and nitrogen metabolism (Westcott and Henshaw, 1976). Yeoman et al. (1982) found that in Dioscorea composita suspension cultures, the accumulation of secondary products occurs during the phase of decreasing cell growth rate and increasing differentiation. In Prunus tissue culture, the addition of catechins (tannin monomers) to the culture medium results in a highly significant promotion of callus proliferation (Feucht and Schmid, 1980; Feucht and Nachit, 1977). Callus cultures of pine, a tannin-containing species, accumulate tannins as they age (Barnett, 1978). In other species the switch to secondary metabolism can be influenced by growthpromoting substances such as a cytokinin (Yeoman et al. 1982; Mantell and Smith, 1983). Sainfoin (Onobryehis vieiifolia Scop.) and birdsfoot trefoil (Lotus corniculatus L.) both produce condensed tannins in their herbage (Sarkar et al., 1976). Sainfoin leaves have a uniformly high tannin content, but workers in our laboratory have developed a high and a very low-tannin strain of birdsfoot trefoil (Dalrymple et al., 1984). In the present study, the high and low strains of birdsfoot trefoil were used to determine some of the effects of endogenous condensed tannins in tissue cultures, and the appearance of condensed tannins was observed in callus cultures from tannincontaining and non tannin-containing sainfoin explants cultured in the presence and absence of a cytokinin. MATERIALS AND METHODS Plant srowth Sainfoin cv. 'Melrose' and the high and low-tannin strains of birdsfoot trefoil cv. 'Cree' were grown in 6" clay pots in a greenhouse. Seeds were sown in soi~ less mix consisting of peat, vermiculite and sand fortified with a controlled release fertilizer, superphosphate, trace elements, and calcium carbonate to control the pH. Moisture was maintained at an optimum level using sub-irrigation. The photoperiod was extended to 16 hr. by using 400 watt high pressure sodium lamps located 80 cm above the canopy as supple-

* Contribution no. 920 of Agriculture Canada Research Station, 107 Science Cres., Saskatoon, Saskatchewan, Canada S7N OX2

248 mentary lighting (200-230 pEs-lm -2 PAR measured with a Li-Cor model LI-185B light meter). Temperatures ranged from 17 - 19°C at night to 24 - 28°C during the day.

were harvested for protoplast isolation. The leaves were dried for 96 h at 80°C and weighed again. Means and standard errors were calculated for each treatment Slide preparatiQns from one duplicate were stained as above and examined for condensed tannins.

Sterile culture Sterile plants were grown from seed under aseptic conditions. The seeds were sterilized for I0 min in 10% Javex, rinsed three times for I min in sterile distilled water and finally left in distilled water for a further 30 min. Sterile seeds were germinated in iO cm high polycarbonate vessels (Magenta Corp., Chicago, Iii., U.S.A.) containing 1 cm of Schenk and Hildebrandt medium (1972) minus growth regulators and incubated at 25°C under fluorescent and incandescent lights (210 pEs-lm -2 PAR). Seedlings were harvested for tissue culture studies when the first true leaves began to develop. Protoplast

isolation

New, fully expanded leaves were harvested from greenhouse-grown plants and surface-sterilized for 5 min in 10% Javex. After rinsing twice for 5 min in distilled water, the abaxial epidermis was peeled from each leaflet and the leaflet floated peeled side down in a 20 x 60 mm petri dish containing 8 ml of protoplast isolation medium. The medium consisted of: i% Onozuka RIO; 1% Rhozyme hemicellulase; 0.2% Pectolyase Y23; 0.5M mannitol; 0.5 g/L CaCI.2H20; 0.25 g/L KH2PO 4 and was adjusted to pH 5.8. The leaves were incubated in darkness at 25°C for three to four hr with occasiona l shaking. Protoplasts were harvested by filtering the isolation medium through a 125 ~m filter to remove coarse debris and then through a 44 ~m filter to eliminate cell clumps. Finally, the protoplasts were gently washed and resuspended in medium A (Kao et al., 1980). Condensed tannin determinations Catechin equivalents in the herbage of high and low tannin birdsfoot trefoil strains were determined by the method of Dalrymple (1982). Protoplasts were stained for the presence of condensed tannins by placing a drop or two of vanillin-HCl reagent (Sarkar and Howarth, 1976) near the edge of a coverslip on a slide containing fresh protoplasts. In the area of stain infiltration, tannin-filled protoplasts could be identified by a bright, cherry red color which filled the entire cell. Tannin-filled cells in callus culture were identified by squashing the callus tissue on a slide, and staining with vanillin-HCl reagent. In all eases, identical protoplast and callus material was stained with a control solution to ensure that there was no false indication from anthocyanins. Birdsfoot trefoil experiments An initial experiment compared the number of mesophyll protoplasts isolated from high-tannin and low-tannin birdsfoot trefoil plants and examined these protoplast preparations for the presence of condensed tannins. Twenty four plants from each strain were divided into two groups to alleviate crowding. For each group, one trifoliolate leaf from each plant was used for protoplast isolation as described above. Duplicate experiments were repeated for three successive days. Isolated protoplasts were counted using a haemocytometer and the average of six readings was used for each duplicate isolation. The results were expressed in terms of protoplasts isolated per gram dry weight of leaf material. A dry weight factor was obtained by harvesting and weighing 6 to 8 g of leaf material from each group of plants on day 3 and at the same time leaves

In the second experiment, callus growth rates from leaf explants of I0 high-tannin and i0 low-tannin plants were compared. Leaves were surface-sterilized as above and discs were cut from individual leaflets using a i mm canula. Three leaflet explants (discs) from one plant were placed in a petri dish containing 3 mL of B5 medium (Gamborg, 1968) supplemented with 2,4-D at 4 mg/L. Four plates were made for each plant so that growth could be measured over four time intervals. The plates were incubated at 25°C with an 18 hr photoperiod using low light (20 pEs-lm-2). On days 5, 13 and 25 callus growth was determined by calculating the average diameter of the three calli in a petri plate. On day 40 the average wet weight of the three calli was used as an indicator of growth. Means and standard errors were calculated for each treatment at each time period. Sainfoin experiments An initial experiment used leaves from i0 greenhousegrown sainfoin plants (cv. 'Melrose') representing iO different genotypes to investigate the production of condensed tannins in callus culture. Three leaflet explants from each genotype were plated in each of three petri dishes containing Blaydes (1966) medium supplemented with 2,4-D, NAA and BAP, each at 2 mg/L. The cultures were incubated at 25°C with a 16 h photoperiod of 20 ~Es-lm -2 PAR). The calli were tested for the presence of condensed tannins at days 19, 27 and 32 as described earlier. The relative number of tannin-filled cells were counted for each treatment and time period. A second experiment used iO sainfoin seedlings grown from seed in sterile culture to observe the influence of eytokinins on condensed tannin production in callus culture. Two root and two cotyledon explants from each genotype were cultured in a 20 X 60 mm petri plate containing one treatment and to save space, explants from two genotypes were placed in the same petri dish. Each treatment medium consisted of a modified B5 media (mB5) (Atanassov and Brown, 1984) with 2 mg/L 2,4-D and one of the following (mg/L): 2 BAP; 4 BAP; 2 NAA + 2 BAP; 2 NAA + 4 BAP; 2 NAA + 2 KIN. Incubation conditions were identical to the previous experiment except the time was for 21 days. Each callus was tested individually for the presence and number of tannin-filled cells using the vanillin-HCl stain. RESULTS AND DISCUSSION Birdsfoot trefoil experiments Protoplasts were isolated easily from leaflets of both the high and low tannin strains of birdsfoot trefoil. When slide preparations were stained with vanillin-HCl, tannin-filled protoplasts, identifiable by their bright, cherry red appearance, were seen scattered throughout the area of stain penetration within 30 sec of stain application. More tannin-containing protoplasts were seen in the slide preparations from the high-tannin than from the low-tannin strain (Figs. IA and IB). This was expected since leaves from the hightannin plants contained four times as much condensed tannin as those from the low-tannin strain (Table i). Even after filtering, the protoplasts isolated from high-tannin leaflets tended to clump together in large aggregations while those from low-tannin plants appeared as single cells (Figs. IA and IB). The clumping

249 1. I s o l a t i o n of p r o t o p l a s t s f r o m l e a v e s of h i g h a n d low tannin-containing birdsfoot trefoil cultivars .................................... ~ ......................... Cultlvar Tannin equivalents protoplasts/g in l e a f t i s s u e dry weight

Table

..............................................................

callus

Low

(~13)

Day

5

1.91

(~.06)

1.88

(~7)

Day

13

3.11

(~.28)

2.62

(~.19)

Day

25

4.53

(~.55)

3.42

(~.30)

Wet

Weight

Day

40

(~75)

64.2

(~20)

Table

High

tannin tannin

0.65

x 10 -3

(~0.1)

2.61

x 10 -3

(~0.2)

124

x 106

77 x 106

.............................................................. N u m b e r s in b r a c k e t s i n d i c a t e s t a n d a r d e r r o r of the m e a n s . *Tannin

equivalent

**n = 24,

= g catechin/g

2.

C a l l u s g r o w t h f r o m l e a f e x p l a n t s of h i g h tannin-containing c u l t i v a r s of b i r d s f o o t

a n d low trefoil

............................................. T ................ Growth High tannin Low t a n n i n .............................................................. diamster

(mm) (~.06)

(mg)

sample. 296.7

* * * n = 6. N u m h e r ~ in b r a c k e t s i n d i c a t e s t a n d a r d i0. E ~ p l a n t s w e r e g r o w n in B5 m e d i u m T = 25°C,

light

e r r o r of the m e a n s , N = c o n t a i n i n g 4 m g / L 2,4-D,

= 18 h at 20 ~ E s - l m - 2

ed growth in culture. Feucht and Nachit (1977) r e p o r ~ ed that the increase in Prunus callus growth due to exogenously applied catchins was the result of the auxin-protecting activity of these phenolics. Another suggestion (Feucht and Schmid, 1980) was that the increased callus growth was due mainly to cell expansion and to a lesser extent, cell division. This may also have occurred in this study but it was not confirmed histologically. Sainfoin experiments

Figure i.

Isolated protoplasts from mesophyll tissue of high-tannin and low-tannin strains of birdsfoot trefoil shortly after staining with vanillin-HCl (X275). The dark cells contain condensed tannin and show a positive reaction to the stain, a) Protoplasts from the high-tannin strain; b) Protoplasts from the low-tannin strain.

Only six of the i0 greenhouse-grown sainfoin plants produced callus from leaf explants in culture, but by day 19 all of these calli contained large numbers of identifiable tannin-filled cells. It should be noted that all sainfoin plants examined to date from this cultivar have had uniformly high amounts of tannins in their leaves. Red-stained areas could be seen without the aid of magnification on the surface of calli stained with vanillin-HCl (Figure 2A) however, when viewed microscopically, the red areas found in the callus tissue were discrete red-colored cells which appeared singly or in groups (Figure 2B). Although

may have been due to the large amounts of tannins present in this strain. Other studies in our laboratory have shown that the same type of aggregation occurs in mesophyll protoplasts isolated from sainfoin leaves which also have a high condensed tannin content (results not shown). In addition, Howarth et al. (1978) reported that during maceration of sainfoin leaves, chloroplast fragments from ruptured cells aggregated unless the condensed tannin inherent in the leaf tissue was removed by using polyvinylpyrrolidone in the extraction mixture. The low tannin strain gave almost twice the protoplast yield when calculated on a dry weight basis (Table i). This observation is consistent with earlier reports that condensed tannins are known inhibitors of cellulases and pectinases (Van Sumere et al., 1975), enzymes used in the protoplast isolation medium.

Figure 2.

Callus cultures from leaflet explants of the hightannin strain showed a higher rate of growth over a period of 40 days (Table 2). There was no difference in explant or callus diameter at day 5, but by day 13 and 25, calli from the high-tannin strain were 25% larger. Most explants grew well forming a whitish friable callus but a few grew slowly and remained small. In the latter weeks of culture, the calli grew vertically more than they increased in diameter, consequently, wet weight was used as a more accurate measure of growth. After 40 days, the callus wet weight of the high tannin strain was 4.6 times greater than that of the low tannin strain. During breeding for the low tannin strain, selection of some other characteristic may have also taken place which affect-

the original leaf explant would have contained tannins, these cells were derived from new callus growth. Protoplast isolations from sainfoin mesophyll tissue also show tannin-filled cells when stained with vanillin-HCl (results not shown) and may be analagous to the cells seen in callus culture. There was no apparent change by day 27, but by day 32 the tannin cells had disappeared from two of the genotypes. In the remaining four, the number of tannin cells had decreased and their color had changed from a cherry red

!i!~i

~!i~]!.i. . .

~

Three week cultures of sainfoin callus stained with vanillin-HCl, a) Dark spots on the callus surface are cells containing condensed tannins (Xil). b) Darkly-stained individual callus cells containing condensed tannin (X350).

250 to a light red or pink, an indication of less tannin present. Constabel (1965, 1974) reported that in juniper tissue culture, tannins were isolated in single or groups of cells scattered throughout the callus and that the tannin concentration passed a peak at the onset of higher growth rates and then declined. Butcher (1977) found that the duration of catechin synthesis in cell suspension cultures from Paul's Scarlet Rose was largely determined by the availability of carbohydrates. The disappearance of tannins at this late stage in callus development may be interpreted as the result of altered metabolism caused by the decreasing nutrient availability from the medium, although Timmerman et al. (1984), using a number of different species, have shown that the stress accompanying declining nutrients usually results in greater concentrations of phenolic compounds, including tannins. Explants from the cotyledons and roots of sterile seedlings were better able to produce callus in culture than were explants from sterile leaflets of the mature plants used in the previous experiment. All IO of the genotypes produced callus from cotyledon and root explants when cultured in mB5 medium plus 2,4-D, with or without cytokinins. The calli did not, however, all produce condensed tannins within the 21 days of culturing. Virtually no tannins were formed when cytokinins were left out of the culture medium, but when present, nine of the genotypes produced cotyledonderived calli with tannin-filled cells, and one genotype showed tannins in callus from a root explant (Table 3). The sainfoin seedlings grown in sterile Table

3.

E f f e c t of c y t o k i n i n s on t h e f o r m a t i o n o f c o n d e n s e d tannin-filled c e l l s in c a l l u s f r o m e x p l a n t s of s a i n f o i n c o t y l e d o n a n d r o o t t i s s u e a f t e r 21 d a y s

.................................. ~........................... Genotype eytokinins added No cytokinins number cotyledon

cotyledon

root

ND

ND

ND

ND

ND

ND

................. ~............................................ 2-BAP 4-BAP 2-BAP 4-BAP 2-KIN Genotype number

2

+

3

+++

+++++

ND

ND

4

+

ND

ND

ND

5

+

ND

ND

ND

+

7 8 9 i0

*See

ND

Culture

text

+

ND

ND

ND

ND

ND

++++

ND

ND

ND

4 for

ND

as

ND

in T a b l e

cytokinlns

4.

E f f e c t of d i f f e r e n t c y t o k i n i n t r e a t m e n t s and levels o n c o n d e n s e d t a n n i n f o r m a t i o n in c a l l u s t i s s u e f r o m s a i n f o i n c o t y l e d o n e x p l a n t s c u l t u r e d f o r 21 d a y s

2-NAA

2-NAA

2-NAA

.............................................................. 1

+

+

++

2

ND

+++++ +

ND

ND

ND ND

3

+++

ND

+++

+++++

++++

4

ND

+++

ND

ND

ND ND

ND

ND

conditions

and Table

ND

++

+++

2,4-D.

Genotypic specificity was very evident in this experiment as the individual genotypes showed striking differences in their ability to produce tannins in culture. Genotype 9 produced calli containing tanninfilled cells in each of the five treatments but number 8 did not show tannins with any of the treatments applied. The remaining genotypes were intermediate in their response (Table 4). Of the iO genotypes, only plant number 3 produced a root explant callus which contained tannin-filled cells but this occurred in every treatment. These results indicate that after 21 days, tannin production under our culture conditions is highly genotype specific however, given more time in,culture, it is possible that tannins may have occurred in more genotypes from both types of tissue explant. Table

++**

6

The effect of different cytokinin and auxin treatments on cotyledon-derived explants is shown in Table 4. Calli with tannin-containing cells were produced by some of the genotypes in each treatment at the end of 21 days, but some treatments were more effective than others. Eight of the IO genotypes produced calli with tannin-containing cells when cultured in the treatment containing 4 mg/L of BAP, but all other treatments showed fewer genotypes producing tannins. BAP was more effective as an inducing agent than KIN, and BAP alone was more effective than a combination of BAP and NAA. The additional auxin added as NAA may have offset the effects of the cytokinins by changing the auxin to cytokinin ratio.

root

.............................................................. 1

callus tissue. A previous study has shown that cytokinins may act as inducing agents. Yeoman and Aitchison (1973) reported that KIN promotes the production of nicotine in callus from the stem pith of tobacco, although nicotine is normally a product of the roots of Nicotiana species.

5

ND

++

ND

ND

6

+

+

ND

ND

ND

7

++

+++

+++

ND

ND

ND

8

ND

ND

ND

9

+++++

+++

ND

+++++

ND

+++

+++

10

+++++

+++

+++

+++++

ND

2.

added.

**Relative numbers of tannin-filled cells/genotype f o u n d in all cytokinin treatments. +, 1-2; ++, 3-5; +++, 6-10; ++++, 11-20; +++++, )20; N D , n o t d e t e c t a b l e .

culture in our laboratory contained condensed tannins in the first true leaves and parts of the hypocotyl, but no tannins were found in either the cotyledons or the roots, the explant tissue used for this experiment. The results show that the source explant material does not have to be producing tannins to allow expression of this secondary metabolite in callus culture, and that cotyledons were better than roots in producing tannin-containing calli after three weeks of culture. Further, the cytokinins acted as inducing agents since the medium with 2,4-D alone supported callus growth but not tannin formation. Secondary metabolite accumulation is known to occur with differentiation (Yeoman 1982) and the cytokinins may have indirectly induced tannin formation by promoting differentiation in the

The basal medium was mB5 media 2 for culture conditions. *2-SAP

=

**Numbers

2 mg/L of

In summary,

EAP

plus

2 mg/L 2,4-D.

See

Table

etc.

tannin-filled

cells

found.

See

Table

3.

the following points can be made:

- High tannin birdsfoot trefoil leaf explants form callus faster than explants from low tannin plants but leaves from high tannin plants do not release protoplasts as easily. - Tannin-filled cells appear after 21 days in callus culture from explants of sainfoin root, leaf and cotyledon tissue. - Tannins can be produced in callus derived from non

251 tannin-containing root and cotyledon sainfoin after 21 days in culture.

tissues of

- BAP or KIN added to the culture medium induces the formation of tannin-filled cells in sainfoin cotyledon or root callus within three weeks of culture. - Tannin formation in sainfoin root or cotyledon callus is genotype specific. Work is continuing in our laboratory to assess the effect of BAP on growth and tannin production in sainfoin callus tissue. ACKNOWLEDGEMENTS Appreciation is expressed to Mr. Neil Suttill technical assistance during this study.

for his

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