Plant Cell Reports

Plant Cell Reports (1992) 10:604-607

9 Springer-Verlag 1992

The role of ethylene and reducing agents on anther culture response of tetraploid potato (Solanum tuberosum L.) Taina Tiainen Agricultural Research Centre, Institute of Plant Breeding, SF-31600 Jokioinen, Finland Received June 11, 1991/Revised version received September 7, 1991 - Communicated by H. L6rz

Summary. The role of ethylene in embryogenesis of cultured potato anthers was studied indirectly by testing various substances known to affect ethylene formation. The reducing agents ascorbic acid and Lcysteine prevented browning of anther cultures and significantly stimulated embryogenesis. Embryogenesis was also promoted by the use of the ethylene inhibitors AgNO 3 and n-propyl-gallate and by the polyamines spermldine and putrescine. The use of the ethylene releasing compound ethrel significantly inhibited embryogenesis. Abbreviations: MS = Murashige & Skoog, PVP = polyvinylpyrrolidone, MW = molecular weight, ACC = laminocyclopropane-l-carboxylic acid, ethrel = 2chloroethylphosphonic acid (ethephon). Key words: Anther culture - Solanum tuberosum reducing agents - ethylene inhibitors - polyamines


Introduction Ethylene is produced by cultured plant cells, tissues and organs. There has been increasing evidence that growth and differentiation of plant cells and tissues in vitro can be affected by ethylene (Chi et al. 1990). Inhibition of ethylene biosynthesis has been shown to enhance plant regeneration from callus cultures of Triticum aestivum (Purnhauser et al. 1987) and Zea maya (Vain et al. 1989), to improve plating efficiency of potato protoplasts (Perl et al. 1988), and to enhance shoot regeneration from cotyledon and hypocotyl cultures of Brassica campestris and Brassica junoea (Chi et al. 1990). Studies on anther culture of some species indicate that ethylene affects microspore embryogenesis (Babbar and Gupta 1986; Cho and Kasha 1989; Reynolds 1987; Dunwell 1979; Biddington et al. 1988). Biddington et al. (1988) have reported silver nitrate to promote embryogenesis in anther cultures of Brussel sprouts by blocking the inhibitory effect of ethylene. Also Cho and Kasha (1989) have shown ethylene production to be related to embryogenesis in barley anther cultures. Browning, which is a serious problem in plant tissue cultures, is often linked to inhibition of growth. It is however a poorly understood phenomenon (Gupta 1986). The percentage of anthers turning brown varies depending on the variety and species (Babbar and Gupta 1982; Cho and Kasha 1989). Mii (1976) has reported that within the first seven days of culture

Present address." Pakilantie 5 A 14, SF-00630 Helslnki, Finland

browning of anthers is associated with inhibition of growth. The browning has been attributed to oxidation of phenols to quinones which are known to inhibit plant cell growth (Monaco et al. 1977). Potato cultivars are tetraploid and highly heterozygous. The availability of a reliable haploidization system would greatly facilitate the production of improved potato cultivars (Powell and Uhrig 1987). However, cultivated tetraploid potato has net yet been readily amenable to anther culture techniques because of the low number of embryoids and their low regeneration rate (Jehansson 1986). Therefore the method is not yet widely used in practical potato breeding. Since potato tissue cultures are extremely sensitive to ethylene, and the growth of potato shoots i~ retarded in closed culture vessels (Hussey and Stacey 1981, 1984), the effects of ethylene on anther cultures were investigated. Furthermore, the objective of the present study was to improve culture response of anthers of potato by analyzing the effects of different reducing agents. The involvement of ethylene in androgenesis in vitro was studied indirectly by incorporating various substances into the medium reported to influence ethylene biosynthesis in various plant systems.

Materials and methods Plant material The commercial tetraploid potato variety Pito, which has been shown to be a responsive variety in previous anther culture experiments (Tiainen 1991), was chosen as a model plant for testing the effects of reducing agents and ethylene inhibitors on anther culture response. Plants were grown from tubers planted in the greenhouse. The anther donor plants were grown in 60 % relative humidity in 18-h daylength. Natural light was supplemented with high pressure sodium lamps. The growth temperature in the greenhouse ranged from 17 ~ to 25 ~ Plants were grown in pots, 21 em in diameter into which 1 % nutrient solution (Kekkil~ Co, Number 5) was applied at each watering. Anther culture procedure Flower buds were harvested when most of the microspores were at the uninueleate or tetrad stage which is normally reached when the buds are 4-6 mm

605 long. The buds were surface sterilized in i0 % sodiumhypochlorite, dipped into 70 % (v/v) ethanol and washed three times with sterile distilled water. Ten anthers were placed on MS medium (made from powder obtained from Flow Co. England) containing 6 % sucrose, 0.5 % activated charcoal and i mg/l 6benzylaminopurine (Wenzel and Uhrig 1981) on 60 mm Petri dishes. The medium was supplemented with various concentrations of filter sterilized reducing agents, ethylene inhibitors, polyamines or ethylenegenerating compounds. PVP, n-propyl-gallate, ethrel, spermidine and putrescine were obtained from Sigma Co. (USA) and L-cysteine-HCl from BDH Co. (England). All other chemicals used were obtained from Merck Co. (Germany). The silver thiosulfate stock solution was made according to Perl et al. (1988). The Petri dishes were placed at 24 ~ and were exposed to low intensity fluorescent light (1500-3000 lux) for 8 weeks after which the number of anthers producing embryoids was counted. Embryoids which did not develop into plantlets were transferred to MS medium supplemented with i0 % (v/v) coconut water (Gibco Co., USA), 0.3 mg/l zeatin and 3 % sucrose for regeneration (Wenzel and Uhrig 1981). Regenerated shoots were placed on MS medium with 0.5 mg/l 6-benzylaminopurine, i00 mg/l casein hydrolysate and 2 % sucrose for rooting. The numbers of chromosomes were counted from root tip cells of regenerated plants according to Karp et al. (1984).

obtained at 1.136 mM ascorbic acid and at 0.634 mM Lcysteine. In this medium 11.7 % of anthers formed embryoids.

Statistical analysis and presentation of the results

Table 2. The combined influence of different ascorbic acid and L-cysteine-HCl concentrations on cv. Pito anther cultures.

Percentage of embryoid formation and plant regeneration was determined by counting the number of anthers producing embryoids and plants in relation to the number of anthers plated. The ethylene experiments were repeated three times with at least ten replications (plates) for each concentration. The results are presented as means of the different treatments and were subjected to t-test analysis.

Table i. The influence of reducing agents and PVP on embryoid formation and plant regeneration in anther cultures of potato cv. Pito. Treatment

Number of anthers plated

Control Polyvinylpyrrolidone (PVP) i0 g/l 20 g / 1 Ascorbic acid 0 , 5 6 8 mM

1.136 mM Glutathione 0.325 mM 0.651 mM

Anthers forming embryoids

Anthers producing plants (%)




540 540



4.07 *~*














L-Cysteine-HC1 0.159 mM

550 5.82*** 2.91"** 0.317 mM 590 10.17"** 5.42*** Note: Difference between control and treatment is significant according to t-test at p = 0.05", p = 0.025** and p = 0.01"**, respectively.


Number of anthers plated

Anthers forming embryoids

Anthers producing plants (%)

(~) Control







Ascorbic acid & L-cysteine-HCl Results

The influence of reducing agents anther culture response of cv. Pito





Three reducing agents (anti-oxidants) at two concentrations were compared. Ascorbic acid and L-cysteine-HCl promoted embryogenesis significantly in the cultures of cv. Pito (Table i). L-Cysteine resulted in a two- to fivefold increase in embryoid yields compared to the control, and addition of ascorbic acid in the medium resulted in a threefold increase in embryoid yield. The highest embryoid production rate (> i0 %) for anthers giving rise to embryoids was obtained with 0.317 mM L-cysteine (Table i). Concerning plant regeneration rates, ascorbic acid and L-cysteine had an even greater effect. Similarly the use of glutathione slightly increased embryoid yields but the concentration of 0.325 mM inhibited plantlet formation. However the concentration of 0.651 mM significantly promoted plantlet yields (Table i). Addition o f l O g / l polyvinylpyrrolidone (PVP) in the medium significantly inhibited embryoid and plantlet yields, whereas the concentration of 20g/i had a slight promoting effect on embryoid and plant formation (Table i). In addition, the use of reducing agents in the media prevented the anthers from browning during the first four weeks in culture. The combined effects of ascorbic acid and Lcysteine-HCl were further tested at five concentrations. The addition of ascorbie acid and Lcysteine together significantly increased embryoid and plant yields (Table 2), the best results being

0 . 5 6 8 mM a s c o r b . + 0 . 3 1 7 mM L - c y s t . 0 . 5 6 8 mM a s c o r b . + 0 . 6 3 4 mM L-cyst.

290 8.28*** 2.07*** 1.136 mM ascorb. + 0.317 mM L-cyst. 280 5.00** 0.71"* 1.136 mM ascorb. + 0.634 mM L-cyst. 240 11.67"** 3.75*** 1.419 mM ascorb. + 0.317 mM L-cyst. 310 10.32"** 4.19"** Note: Difference between control and treatment is significant according to t-test at p = 0.025** and p = 0.01"**, respectively.

The influence of ethylene inhibitors culture response of cv. Pito

on the


The effects of five ethylene inhibitors at two concentrations on anther culture response were compared. Of the five inhibitors tested silver nitrate (AgNO 3) promoted embryoid and plant formation at both concentrations (Table 3). The addition of 0.059 mM doubled the embryoid yield compared to control. A free redical scavenger N-propyl gallate, 0.047 mM, significantly promoted embryoid yield, whereas a concentration of 0.118 mM significantly lowered embryoid yields (Table 3). However, the highest plant yields in this experiment were obtained with i0 gM CoCI 2. Silver thiosulfate, NiCI_2 and CoCI_ were not very effective in increasing em%ryoid an~ plantlet yields (Table 3). Both silver thiosulfate and NiCI 2 had a significant negative effect on

606 embryoid yield at low concentrations but had no .effect on the plant regeneration rate. In contrast, the higher concentration of NiCI~ (50 ~M) significantly stimulated embryoid formation. CoCI 2 at the concentration of i0 pM had no effect on embryoid yield, buz it did stimulate plant regeneration. At the concentration of 50 ~M both embryoid and plant yields were significantly inhibited (Table 3). Table 3. The influence of ethylene inhibitors on embryoid formation and plant regeneration in anther cultures of potato cv. Pito.

The influence of ethrel and methionine on the anther culture response of cv. Pito Ethrel had a significant negative effect on both embryoid and plant yields (Table 5), whereas addition of methionine, a precursor of ethylene biosynthesis, did not significantly affect emhryoid yields (Table 5). Table 5. The influence of ethrel and methionine on anther culture response of potato cv. Pito. Treatment


Number of anthers plated

Anthers forming embryoids

Anthers producing plants (%)

(%) Control 460 2.17 0.22 Silver thiosulfate (STS) 0.001 mM 420 0.95*** 0.24 0.002 mM 420 1.67 0.95*** Silver nitrate (AgNG 3) 0.015 mM 570 2.63 1.40"** 0 . 0 5 9 mM 590 4.41"** 0.68** Cobalt chloride (0oCt~ x 6H O~ i0 ~ mol.~410 2.21 1.46"** 50 ~ mol.l -I 470 0.21"** 0"* Nickel c h l o r ~ e (NiCI 2) i0 ~ mol.l 370 0.81"** 0.27 50 ~ mol.l -I 390 3.31"* 0.51 N-propyl-gallate 0.047 mM 470 4.04*** 0.85*** 0.118 mM 430 1.16"** 0.23 Note: Difference between control and treatment is significant according to t-test at p = 0.025** and p = 0.01"**, respectively.

The influence of polyamines response of cv. Pito





The effects of two polyamines, spermidine and putrescine, were tested at two concentrations. Both polyamines significantly increased embryoid yields and had a small positive effect on plant yields (Table 4). Spermidine was more effective than putrescine. Putrescine inhibited plant formation at the lower concentration; at the higher concentration the plant formation rate was nearly the same as for the control. Table 4. The influence of polyamines on embryoid formation and plant regeneration in anther cultures of potato cv. Pito. Treatment

Number of anthers plated

Anthers forming embryoids

Anthers producing plants (%)

(%) Control 350 2.29 1.14 Spermidine 0.069 mM 380 4.47*** 2.63*** 0.172 mM 370 4.59*** 1.62 Putrescine 0.113 mM 370 3.78** 0.27*** 0.284 mM 280 3.93*** 2.14"** Note: Difference between control and treatment is significant according to t-test at p = 0.025** and p = 0.01"**, respectively.

Number of anthers plated

Anthers producing embryoids

Anthers producing plants (5)

(%) Control 340 2.35 1.18 Ethrel 0.010 mM 310 0.97*** 0"** 0.104 mM 330 0.30*** 0"** L-Methionine 0.168 mM 400 3.00 1.25 0.335 mM 270 1.85 0"** Note: Difference between control and treatment significant according to t-test at p = 0.01"**.


The chromosome numbers of plants regenerated (n = 51) after the different treatments (reducing agents, ethylene inhibitors, polyamines, etc.) have been counted. Chromosome numbers were not influenced by the treatments. About 65 % of the obtained plants were dihaploids and 35 % were tetraploids.

Discussion The presence of several compounds in the culture medium, which have been known to stimulate or to suppress ethylene production and to prevent browning, had varying effects on the embryogenesis in Pito anther cultures. The stimulatory effect of Lcysteine-HCl and PVP on embryoid production reported by Johansson (1986) was confirmed in this study. In Johansson's (1986) experiments optimum results were obtained with 0.095 mM L-cysteine and higher concentrations did not improve the embryoid yields. In this study, embryoid yields were almost doubled when the concentration of L-cysteine concentration was raised from 0.159 mM to 0.317 mM, indicating genotypic differences between the varieties for optimal levels. Johansson (1986) also reported that PVP (MW 44000) had no stimulating effect on anther culture response in potato and that the concentration of 10 g/l inhibited embryogenesis. A similar inhibitory effect of PVP (MW I0 000) at the concentration of i0 g/l was observed also in this study, whereas the concentration of 20 g/l significantly stimulated embryoid formation. Babbar and Gupta (1982) have also reported that incorporation of PVP and L-cysteine enhanced plantlet yields in Datura metel anther cultures. PVP is known to adsorb phenolic compounds (Andersen and Sewers 1968) and L-cysteine curtails the phenolic oxidation by acting as an antioxidant (Monaco et al. 1977). The effects of other reducing agents, ethylene inhibitors and polyamines have not been reported previously in potato anther culture studies. In this study ascorbic acid significantly stimulated embryoid production. Ascorbic acid has been used as a medium supplement to decrease the deleterious effects of browning associated with poor growth (Gupta 1986). Elmore et al. (1990) have reported that since ascorbic acid rapidly decays in plant tissue culture media, it may not exert its effect as an intact molecule, rather its antioxidant/antibrowning role in plant cell cultures may be mediated by a product of

607 further oxidation (Elmore et al. 1990). The use of glutathione in potato anther culture was not very effective compared to ascorbic acid and L-cysteine. The incorporation of ethylene inhibitors in the culture media also improved embryoid and plantlet yields to some extent. The silver ion is known to be an inhibitor of ethylene action (Beyer 1979) and its incorporation into tissue culture media has shown beneficial effects on growth (Williams et al. 1990). The silver ions in the form of silver thiosulfate and silver nitrate had varying effects on anther culture response of ev. Pito. Silver thiosulfate had no stimulatory effect on embryoid formation, and at the lower concentration it was inhibitory to embryoid formation. However at higher concentration it promoted plantlet formation. Silver nitrate, on the other hand, had no stimulatory effect at the lower concentration, but at the higher concentration it significantly stimulated embryoid formation. Similar enhancement by AgNO_ in embryoid formation has been reported in anther cultures of Brasslca oleracea var. gemmifera (Biddington et al. 1988). Embryoid production from anther cultures of three poorly responding Brussel sprout genotypes was increased by the addition of AgNO_. However, it did not improve embryoid yield of a normally responslve genotype to the same extent (Biddington et al. 1988). Cobalt and nickel ions are also inhibitors of ethylene biosynthesis (Lau and Yang 1976). They have been reported to inhibit the enzymatic conversion of ACC to ethylene in a variety of plant systems (Yang and Hoffman 1984). In this study, neither cobalt chloride nor nickel chloride were very effective in stimulating embryoid formation. Reynolds (1987) has reported that the inclusion of CoCI^ in the medium reduced ethylene levels around t~e anthers but retarded embryo production in Solanum carolinense. Also Babbar and Gupta (1986) have reported that increasing levels of silver and cobalt ions, while not affecting the percentage of inductive anthers significantly, inhibited the androgenetic response of Datura metel by decreasing the average number of embryos/plantlets developed per anther. Also in this study the use of n-propyl-gallate, significantly stimulated embryoid formation at the lower concentration but had an inhibitory effect at the higher concentration (0.118 mM). N-propyl-gallate has been reported to decrease ethylene production in barley anther cultures (Cho and Kasha 1989). The ethylene generating compounds tested did not stimulate embryogenesis in ev. Pito anther cultures. The use of ethrel significantly inhibited embryoid formation, but methionine had no effect on embryoid production compared to the control. This may in part be due to the use of only two concentrations which may not have been optimal. Also Reynolds (1987) has reported that methionine had no effect on embryogenie induction in S. carolinense anther cultures. In Solanum carolinense anther culture embryogenesis was promoted by low concentrations (0.069 mM) of ethrel but inhibited at higher (0.692 mM) concentrations (Reynolds 1987). In another study Reynolds (1989) reported that ethrel and ethylene did not stimulate pollen embryogenesis in vitro and he concluded that supraoptimal levels of ethylene inhibit pollen embryogenesis. Babbar and Gupta (1986) have reported that in Datura metel anther cultures methionine and ethrel stimulated embryo formation. In barley anther culture ethrel has been shown to increase ethylene production and it also increased callus production in two varieties at some concentrations and decreased it in one variety (Cho and Kasha 1989). Variation in results is a constant problem in tissue culture work. In this experiments the control treatments produced varying numbers of embryoids and plants. This is because anthers rarely respond

uniformly to culture conditions and batch to batch variation in greenhouse grown plants is evident (Sunderland 1974). However the results from this study show that treatments resulting in lower ethylene levels in cv. Pito anther cultures increased pollen embryogenesis and addition of ethylene releasing compounds inhibited it. Also the different chemical treatments did not affect the chromosome levels of the regenerated plants, and a higher number of plants were dihaploid than obtained in previous studies (Tiainen 1991). Therefore the results indicate that ethylene seems to influence the embryogenesis in potato anther cultures.

Acknowledgements. The author wishes to acknowledge the excellent technical assistance provided by Ms. Kirsti Salmi and Ms. Senja Paajanen. The author also wishes to thank Dr. Eija Pehu for valuable assistance and critical review of the manuscript. This work was supported by a bioteehnology grant from the Ministry of Agriculture and Forestry.


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The role of ethylene and reducing agents on anther culture response of tetraploid potato (Solanum tuberosum L.).

The role of ethylene in embryogenesis of cultured potato anthers was studied indirectly by testing various substances known to affect ethylene formati...
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