Plant Cell Reports
Plant Cell Reports (1989) 8:415-417
© Springer-Verlag 1989
Ethylene production and embyogenesis from anther cultures of barley (Hordeum vulgate) Uh-Haing Cho and Ken J. Kasha Department of Crop Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Received May 10, 1989/Revised version received August 29, 1989 - Communicated by E. D. Earle
Summary. Ethylene production was measured in cultured barley (Hordeum vulqare L.) anthers. The pattern of ethylene production and the content of the ethylene precursor 1-aminocyclopropane-l-carboxylic acid (ACC) were different among cultivars. Ethylene production appeared to be related to embryogenesis (callus and embryo production). In cultivars in which anthers had low amounts of ACC and produced ethylene slowly, the addition of ethylene promotors (Ethrel or ACC) increased embryogenesis. However, in the cultivar Klages, in which anthers had high amounts of ACC and produced ethylene rapidly, the addition of an ethylene production inhibitor (putrescine) increased embryogenesis. Thus, an optimum level of ethylene production appears to be important for embryogenesis. The differences in anther response and callus production among cultivars may be due to both the capacity to produce ethylene and the sensitivity to high ethylene levels. Introduction Ethylene is produced by all higher plants and, in trace amounts, regulates a wide variety of phenomena in plant growth and development (Sisler and Yang 1987). The pollen of many species contains high levels of the ethylene precursor l-aminocyclopropane1-carboxylic acid (Hill et al. 1987; Stead 1985; Whitehead et al. 1983) and ethylene may be associated with some aspects of pollen germination and pollen tube growth. However, in in vitro cultures of anthers or micropsores, the role of ethylene is not known and l i t t l e research has been conducted. Although there are contradictory results, i t has been suggested that ethylene may be involved in microspore embryogenesis. Horner et al. (1977) found that Nicotiana anther cultures produce ethylene throughout the period of culture with the maximum evolution occurring at I-2 weeks after inoculation. However, the role of ethylene on plantlet production was not clear. Complete removal of ethylene by an ethylene absorbent, mercuric perchlorate, or by air flushing did not change plantlet production. However, Dunwell (1979) reported that removal of ethylene from the culture vessel could either enhance or retard embryo production, embryo survival and the number of plantlets produced, depending upon the size of the culture vessel and the age of the anthers. He also showed that silver nitrate (AgNO.) increased embryo induction slightly. Recently Bab~r and Gupta (1986) reported that the ethylene precursor methionine and the ethylene releasing compound Ethrel stimulated Offprint requests to: Uh-Haing Cho
embryo formation, whereas the ethylene antagonists Co++ and Ag+ decreased the average number of embryos/plants developed per anther in Datura anther culture. Reynolds (1987) has also shown that pollen embryogenesis was promoted by Ethrel and the precursor ACC in anther cultures of Solanum carolinensis. He suggested that IAA-induced pollen embryogenesis occured (at least partially) through auxin-mediated ethylene production. He also found a reduction of embryo production by cobalt ions associated with a reduction of ethylene production. Biddington et al. (1988) showed that AgNO3 increased the embryo production from anther cultures of poorly responding Brussels sprout genotypes. The7 suggested that lowering ethylene production by Ag~ increased embryogenesis in that genotype although ethylene production was not measured. The present study was designed to analyze ethylene production and the ACC content in anthers of several genotypes of barley in order to assess the variability in ethylene production and its relationship with embryogenesis. Various substances known to interfere with or promote ethylene synthesis in plant tissues were also included. Materials and Methods Plants. Plants of barley (Hordeum vulqare L. cvs. Klages, Bruce and Elrose) were grown in a growth room (irradiance 320 uEm-z sec"I, photoperiod 17 h, day and night temperatures 22° and 17°C, respectively). Previous studies (Marsolais and Kasha 1985, Szarejko and Kasha 1989) with these genotypes indicated that Klages gave a high % anther response and callus production but poor regeneration, whereas Bruce and Elrose gave a lower response and better regeneration. Seeds were planted in 15 cm diameter pots. A loampeat-sand (2.5-1-2) potting soil was used with approximately 10 g of slow release f e r t i l i z e r pellets (14-14-14) placed on top of the soil. After 2 weeks, water soluble f e r t i l i z e r (20-20-20) was applied once a week. Anther Cultures. Anthers with microspores at the mid-
uninucleate stage were placed on modified BACI medium (Marsolais and Kasha 1985) containing 2 mg/L 2,4-D, 0.5 mg/L zeatin riboside and 750 mg/L glutamine for induction of callus. Replenishments were made every 10 days by withdrawing I ml of induction medium and replacing i t with medium containing 2 mg/L IAA and 0.5 mg/L zeatin riboside. Twenty to t h i r t y anthers were placed per 60 X 15 mm plastic Petri dish
416 (Fisher) containing 3 ml mediumand incubated at 28% in the dark. Four weeks after inoculation, the number of anthers producing call± (anther response %) and total number of call± were counted. Transferable call± (>I mm) were placed on modified MS medium containing 2 mg/L IAA, 0.5 mg/L BA and 0.8% agar. At least 15 replications (plates) per treatment were used and a two-way ANOVAwith Duncan's new multiple range test was performed. Ethylene and ACC Measurement. Twenty mg of fresh anthers (100-150 anthers) was transfered to a 15 ml glass vial containing 3 ml BACl medium. After incubation at designated culture times, the vials were stoppered with a rubber stopper containing a cylindrical half-hole rubber septum. After I hr, I ml of head gas was with-drawn with a syringe and injected into a Hewlett Packard (Model 5880A) gas chromatograph equipped with a flame ionized detector and a 6 f t 2 in X I/8 in Porapack Q- packed, stainless steel column. With an oven temperature of 60°C, an injector temperature of 60%, and a detector temperature of 350%, the average retention time for ethylene was 1.05 min. For the ACC assay, anthers (20 mg) were used and processed by the modified Lizada and Yang method of H i l l et ai.(1987). For the calculation of ACC concentration, the method of Lizada and Yang (1979) which depends on the conversion of ACC to ethylene, was used.
With respect to ACC levels, Klages had a higher concentration (13.6 nmol/g) than the other two cultivars (Bruce 7.9 and Elrose 9.9 nmol/g) at inoculation time. Ethylene production was observed (Table I I ) during the callus induction period ( f i r s t 10 days after inoculation). Ethylene production started before culturing and no production was observed after 48 h. The greatest ethylene production was observed in Klages just after inoculation compared to two other cultivars in which ethylene production was not observed until 2 h of incubation. In fact, the fastest degreening of anthers was observed in Klages (data not shown). This phenomenon was assumed to be due to the more rapid ethylene production and the higher level of ACC. Although the source of ethylene production in anther culture was not well understood, Table I I .
Culture Time (h) 0 2 12 24 36 48
Results and Discussion Ethylene production from anthers at four microspore stages was observed after a 2 h incubation.(Table I ) . Ethylene was produced by cultured anthers at a rate of up to about 80 nl/g/h. Generally, ethylene production increased according to the stage except in the cultivar Bruce. Previously, Horner et al. (1977) had also observed that later-staged anthers produced more ethylene than early ones. Table I.
Ethylene production (nl/h/g) after 2 h incubation of barley (H. vulqare) anthers cultured at various microspore stages. Cultivar Kl aqes El rose Bruce
Staqe Earlyuninucleate Miduninucleate Lateuninucleate Bi-nucleate
18.2 + 1.2
26.2 + 1.4
46.8 + 2.6
30.1 + 2.6
40.5 + 1.8 40.9 + 2.1
55.6 + 4.3 47.6_+4.8
47.6 + 2.8 77.8 +_ 9.6
30.9 + 1.8 50.8 + 3.2
Note: Data are mean ± SE for at least 5 observations.
Ethylene production (nl/h/g) from anthers during callus induction period in barley (H. vulqare) anther cultures Cultivar Elrose
Klaqes
45.2 30.1 47.6 30.9 29.4
+ 2.3 + 3.1 _+ 3.8 + 3.6 + 2.6 0
0 39.7 _+ 4.3 30.1 + 1.8 32.5 + 2.7 47.6 _+ 2.4 0
Bruce 0 40.5 ± 23.0 + 31.3 + 56.3 + 0
Note: Data are mean ± SE for at least 5 observations. i t may be controlled by endogenous precursors (eg. ACC), hormones (eg. IAA), or s e n s i t i v i t y of anther tissues to stress conditions (e.g. dehydration, wounding, medium composition, etc.). The application to the medium of several chemicals which have been known to stimulate or suppress ethylene production influenced ethylene production in Klages (Table I l l ) . The ethylene releasing compound, Ethrel, and the ethylene precursor, ACC, increased ethylene production (only s l i g h t l y by ACC). The polyamine (putrescine) and the free radical scavenger (n-propyl gallate) decreased ethylene production. Ethrel, which increased ethylene production, also increased callus production in Bruce and Elrose at some concentrations. There was a decrease of embryogenesis in Klages (Table IV). ACC, which s l i g h t l y increased ethylene production, also increased callus production in Elrose but decreased call± number in K!ages. Putrescine, w h i c h decreased ethylene production, also decreased embryogenesis in Bruce and Elrose but increased i t in Klages. The free radical
Table I I I . The effects of ethylene inhibitors (n-propyl gallate and putrescine) and promotors (Ethrel and ACC) on ethylene production (nl/g/h) from anthers in barley (H. vulqare cv. Klages) anther cultures Treatment* Control Ethrel (0.1 mM)* ACC (0.1 mM) n-Propyl gallate (0.1 mM) Putrescine (0.1 mM)
0 55.5 + 277.6 + 39.7 + 31.7_+ 31.7 ±
12 8.1 13.1 3.3 1.7 0.3
Note: Data are mean ± SE. * The concentration of chemicals is in BAC1 medium.
3.6 2.4 1.2 6.4
56.3 634.5 63.4 28.7 47.6
+ 7.5 ± 22.7 + 4.7 + 2.2 +_ 0.8
Culture Time (h) 24 39.7 293.5 55.5 27.8 27.8
+ + + + +
3.6 21.3 1.3 1.8 1.0
36 39.6 +_ 3.1 396.6 + 31.3 71.4_+ 2.7 31.7 + 0.9 35.7 +_ 1.1
417 Table IV. The effect of ethylene inhibitors (n-propyl gallate and putrescine) and promotors (Ethrel and ACC) on embryogenesis in barley (H. vulqare) anther cultures. Treatment (M)
Cultivar Elrose
Klaqes
b a c d
38 31 60 72
b b a a
30 25 11 11
a a b b
a a b b
35 57 75 11
c b a d
19 19 8 11
a a b b
a c c b
46 13 38 10
a c b c
13 23 14 28
b a b a
References
d c b a
40 20 38 19
a b a b
24 18 16 5
a b b d
Biddington NL, Sutherland RA, Robinson HT (1988) Ann. Bot. 62:181-185
Bruce
Ethrel 0 98 10-s 127 10.4 65 10.3 47 ACC 0 78 10.5 85 10.4 54 10.3 64 Putrescine 0 51 10.5 13 10.4 12 10.3 24 n-Propyl qallate 0 63 10.5 83 10.4 107 10.3 168 Note:
in anthers and the i n i t i a l concentration of ethylene may be responsible for the high or low callusing response of a genotype. I t may be one reason why optimal donor plant growth conditions are required for maximum response since i t would influence the levels of ethylene precursors and auxins in anthers. Treatments leading to an increase in i n i t i a l concentrations of ethylene can result in higher levels of embryogenesis ( i . e . in Bruce and Elrose) or lowering ethylene level can increase embryogenesis ( i . e . in Klages). Thus, there may be a threshold level at which higher concentrations are deleterious.
Values in each column followed by the same l e t t e r are not s i g n i f i c a n t l y different ~p=O.05) Number of c a l l i per 100 anthers
Acknowledgments. Financial support for this research was provided by the Natural Science and Engineering Research Council of Canada and by the Ontario Ministry of Agriculture and Food, and is gratefully acknowledged.
Babbar SB, Gupta SC (1986) Physiol. Plant. 68:141144
Cho UH (1988) MSc thesis Univ. of Guelph Canada Dunwell JM (1979)
J. Exp. Bot. 30:419-428
H i l l SE, Stead AD, Nichols R (1987) J. Plant Growth Regul. 6:1-13 Horner M, McComb JA, McCombAJ (1977) 28:1365-1372
scavenger, n-propyl gallate increased calli production in Bruce. The difference in embryogenesis (particularly, in callus production) among genotypes could be explained in terms of capacity to produce ethylene and s e n s i t i v i t y of anthers to ethylene. In Elrose, ethylene production gave a positive effect on embryogenesis. Also in Bruce, ethylene was thought to be necessary for callus production because the suppression of ethylene production by putrescine decreased anther response and c a l l i production. In Klages, ethylene production was at least supraoptimal because the application of putrescine increased embryogenesis whereas an increase of ethylene gave obscure results. In previous experiments (Cho 1988; Szarejko and Kasha 1989), the addition of AgNO3 to the medium showed positive effects on anther response and c a l l i production with Klages. The evidence to-date leads us to conclude that ethylene production is one of the factors responsible for genotype differences in anther culture a b i l i t y . The concentrations of ethylene precursors (e.g. ACC)
J. Exp. Bot.
Lizada MCC, Yang SF (1979) Anal. Biochem. 100:140145 Marsolais AA, Kasha KJ (1985) Can. J. Bot. 63:22092212 Reynolds TL (1987) Amer. J. Bot. 74:967-969 Sisler EC, Yang SF (1987) In: NewmanDW, Wilson KG (eds) Models in plant physiology and biochemistry. V o l . I I CRC Press Inc. pp 99-102 Stead AD (1985) In: Tucker G, Roberts J (eds) Ethylene in plant development. Butterworth London pp. 71-81 Szarejko I, Kasha KJ (1989) Proc. FAO/IAEAResearch Coord. Meeting. Katowice, Poland. July 1988 (in press) Whitehead CS, Fujino DW, Reid MS (1983) Horticult. 21:291-297
Sci.
Plant Cell Reports (1989) 8:415-417
© Springer-Verlag 1989
Ethylene production and embyogenesis from anther cultures of barley (Hordeum vulgate) Uh-Haing Cho and Ken J. Kasha Department of Crop Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Received May 10, 1989/Revised version received August 29, 1989 - Communicated by E. D. Earle
Summary. Ethylene production was measured in cultured barley (Hordeum vulqare L.) anthers. The pattern of ethylene production and the content of the ethylene precursor 1-aminocyclopropane-l-carboxylic acid (ACC) were different among cultivars. Ethylene production appeared to be related to embryogenesis (callus and embryo production). In cultivars in which anthers had low amounts of ACC and produced ethylene slowly, the addition of ethylene promotors (Ethrel or ACC) increased embryogenesis. However, in the cultivar Klages, in which anthers had high amounts of ACC and produced ethylene rapidly, the addition of an ethylene production inhibitor (putrescine) increased embryogenesis. Thus, an optimum level of ethylene production appears to be important for embryogenesis. The differences in anther response and callus production among cultivars may be due to both the capacity to produce ethylene and the sensitivity to high ethylene levels. Introduction Ethylene is produced by all higher plants and, in trace amounts, regulates a wide variety of phenomena in plant growth and development (Sisler and Yang 1987). The pollen of many species contains high levels of the ethylene precursor l-aminocyclopropane1-carboxylic acid (Hill et al. 1987; Stead 1985; Whitehead et al. 1983) and ethylene may be associated with some aspects of pollen germination and pollen tube growth. However, in in vitro cultures of anthers or micropsores, the role of ethylene is not known and l i t t l e research has been conducted. Although there are contradictory results, i t has been suggested that ethylene may be involved in microspore embryogenesis. Horner et al. (1977) found that Nicotiana anther cultures produce ethylene throughout the period of culture with the maximum evolution occurring at I-2 weeks after inoculation. However, the role of ethylene on plantlet production was not clear. Complete removal of ethylene by an ethylene absorbent, mercuric perchlorate, or by air flushing did not change plantlet production. However, Dunwell (1979) reported that removal of ethylene from the culture vessel could either enhance or retard embryo production, embryo survival and the number of plantlets produced, depending upon the size of the culture vessel and the age of the anthers. He also showed that silver nitrate (AgNO.) increased embryo induction slightly. Recently Bab~r and Gupta (1986) reported that the ethylene precursor methionine and the ethylene releasing compound Ethrel stimulated Offprint requests to: Uh-Haing Cho
embryo formation, whereas the ethylene antagonists Co++ and Ag+ decreased the average number of embryos/plants developed per anther in Datura anther culture. Reynolds (1987) has also shown that pollen embryogenesis was promoted by Ethrel and the precursor ACC in anther cultures of Solanum carolinensis. He suggested that IAA-induced pollen embryogenesis occured (at least partially) through auxin-mediated ethylene production. He also found a reduction of embryo production by cobalt ions associated with a reduction of ethylene production. Biddington et al. (1988) showed that AgNO3 increased the embryo production from anther cultures of poorly responding Brussels sprout genotypes. The7 suggested that lowering ethylene production by Ag~ increased embryogenesis in that genotype although ethylene production was not measured. The present study was designed to analyze ethylene production and the ACC content in anthers of several genotypes of barley in order to assess the variability in ethylene production and its relationship with embryogenesis. Various substances known to interfere with or promote ethylene synthesis in plant tissues were also included. Materials and Methods Plants. Plants of barley (Hordeum vulqare L. cvs. Klages, Bruce and Elrose) were grown in a growth room (irradiance 320 uEm-z sec"I, photoperiod 17 h, day and night temperatures 22° and 17°C, respectively). Previous studies (Marsolais and Kasha 1985, Szarejko and Kasha 1989) with these genotypes indicated that Klages gave a high % anther response and callus production but poor regeneration, whereas Bruce and Elrose gave a lower response and better regeneration. Seeds were planted in 15 cm diameter pots. A loampeat-sand (2.5-1-2) potting soil was used with approximately 10 g of slow release f e r t i l i z e r pellets (14-14-14) placed on top of the soil. After 2 weeks, water soluble f e r t i l i z e r (20-20-20) was applied once a week. Anther Cultures. Anthers with microspores at the mid-
uninucleate stage were placed on modified BACI medium (Marsolais and Kasha 1985) containing 2 mg/L 2,4-D, 0.5 mg/L zeatin riboside and 750 mg/L glutamine for induction of callus. Replenishments were made every 10 days by withdrawing I ml of induction medium and replacing i t with medium containing 2 mg/L IAA and 0.5 mg/L zeatin riboside. Twenty to t h i r t y anthers were placed per 60 X 15 mm plastic Petri dish
416 (Fisher) containing 3 ml mediumand incubated at 28% in the dark. Four weeks after inoculation, the number of anthers producing call± (anther response %) and total number of call± were counted. Transferable call± (>I mm) were placed on modified MS medium containing 2 mg/L IAA, 0.5 mg/L BA and 0.8% agar. At least 15 replications (plates) per treatment were used and a two-way ANOVAwith Duncan's new multiple range test was performed. Ethylene and ACC Measurement. Twenty mg of fresh anthers (100-150 anthers) was transfered to a 15 ml glass vial containing 3 ml BACl medium. After incubation at designated culture times, the vials were stoppered with a rubber stopper containing a cylindrical half-hole rubber septum. After I hr, I ml of head gas was with-drawn with a syringe and injected into a Hewlett Packard (Model 5880A) gas chromatograph equipped with a flame ionized detector and a 6 f t 2 in X I/8 in Porapack Q- packed, stainless steel column. With an oven temperature of 60°C, an injector temperature of 60%, and a detector temperature of 350%, the average retention time for ethylene was 1.05 min. For the ACC assay, anthers (20 mg) were used and processed by the modified Lizada and Yang method of H i l l et ai.(1987). For the calculation of ACC concentration, the method of Lizada and Yang (1979) which depends on the conversion of ACC to ethylene, was used.
With respect to ACC levels, Klages had a higher concentration (13.6 nmol/g) than the other two cultivars (Bruce 7.9 and Elrose 9.9 nmol/g) at inoculation time. Ethylene production was observed (Table I I ) during the callus induction period ( f i r s t 10 days after inoculation). Ethylene production started before culturing and no production was observed after 48 h. The greatest ethylene production was observed in Klages just after inoculation compared to two other cultivars in which ethylene production was not observed until 2 h of incubation. In fact, the fastest degreening of anthers was observed in Klages (data not shown). This phenomenon was assumed to be due to the more rapid ethylene production and the higher level of ACC. Although the source of ethylene production in anther culture was not well understood, Table I I .
Culture Time (h) 0 2 12 24 36 48
Results and Discussion Ethylene production from anthers at four microspore stages was observed after a 2 h incubation.(Table I ) . Ethylene was produced by cultured anthers at a rate of up to about 80 nl/g/h. Generally, ethylene production increased according to the stage except in the cultivar Bruce. Previously, Horner et al. (1977) had also observed that later-staged anthers produced more ethylene than early ones. Table I.
Ethylene production (nl/h/g) after 2 h incubation of barley (H. vulqare) anthers cultured at various microspore stages. Cultivar Kl aqes El rose Bruce
Staqe Earlyuninucleate Miduninucleate Lateuninucleate Bi-nucleate
18.2 + 1.2
26.2 + 1.4
46.8 + 2.6
30.1 + 2.6
40.5 + 1.8 40.9 + 2.1
55.6 + 4.3 47.6_+4.8
47.6 + 2.8 77.8 +_ 9.6
30.9 + 1.8 50.8 + 3.2
Note: Data are mean ± SE for at least 5 observations.
Ethylene production (nl/h/g) from anthers during callus induction period in barley (H. vulqare) anther cultures Cultivar Elrose
Klaqes
45.2 30.1 47.6 30.9 29.4
+ 2.3 + 3.1 _+ 3.8 + 3.6 + 2.6 0
0 39.7 _+ 4.3 30.1 + 1.8 32.5 + 2.7 47.6 _+ 2.4 0
Bruce 0 40.5 ± 23.0 + 31.3 + 56.3 + 0
Note: Data are mean ± SE for at least 5 observations. i t may be controlled by endogenous precursors (eg. ACC), hormones (eg. IAA), or s e n s i t i v i t y of anther tissues to stress conditions (e.g. dehydration, wounding, medium composition, etc.). The application to the medium of several chemicals which have been known to stimulate or suppress ethylene production influenced ethylene production in Klages (Table I l l ) . The ethylene releasing compound, Ethrel, and the ethylene precursor, ACC, increased ethylene production (only s l i g h t l y by ACC). The polyamine (putrescine) and the free radical scavenger (n-propyl gallate) decreased ethylene production. Ethrel, which increased ethylene production, also increased callus production in Bruce and Elrose at some concentrations. There was a decrease of embryogenesis in Klages (Table IV). ACC, which s l i g h t l y increased ethylene production, also increased callus production in Elrose but decreased call± number in K!ages. Putrescine, w h i c h decreased ethylene production, also decreased embryogenesis in Bruce and Elrose but increased i t in Klages. The free radical
Table I I I . The effects of ethylene inhibitors (n-propyl gallate and putrescine) and promotors (Ethrel and ACC) on ethylene production (nl/g/h) from anthers in barley (H. vulqare cv. Klages) anther cultures Treatment* Control Ethrel (0.1 mM)* ACC (0.1 mM) n-Propyl gallate (0.1 mM) Putrescine (0.1 mM)
0 55.5 + 277.6 + 39.7 + 31.7_+ 31.7 ±
12 8.1 13.1 3.3 1.7 0.3
Note: Data are mean ± SE. * The concentration of chemicals is in BAC1 medium.
3.6 2.4 1.2 6.4
56.3 634.5 63.4 28.7 47.6
+ 7.5 ± 22.7 + 4.7 + 2.2 +_ 0.8
Culture Time (h) 24 39.7 293.5 55.5 27.8 27.8
+ + + + +
3.6 21.3 1.3 1.8 1.0
36 39.6 +_ 3.1 396.6 + 31.3 71.4_+ 2.7 31.7 + 0.9 35.7 +_ 1.1
417 Table IV. The effect of ethylene inhibitors (n-propyl gallate and putrescine) and promotors (Ethrel and ACC) on embryogenesis in barley (H. vulqare) anther cultures. Treatment (M)
Cultivar Elrose
Klaqes
b a c d
38 31 60 72
b b a a
30 25 11 11
a a b b
a a b b
35 57 75 11
c b a d
19 19 8 11
a a b b
a c c b
46 13 38 10
a c b c
13 23 14 28
b a b a
References
d c b a
40 20 38 19
a b a b
24 18 16 5
a b b d
Biddington NL, Sutherland RA, Robinson HT (1988) Ann. Bot. 62:181-185
Bruce
Ethrel 0 98 10-s 127 10.4 65 10.3 47 ACC 0 78 10.5 85 10.4 54 10.3 64 Putrescine 0 51 10.5 13 10.4 12 10.3 24 n-Propyl qallate 0 63 10.5 83 10.4 107 10.3 168 Note:
in anthers and the i n i t i a l concentration of ethylene may be responsible for the high or low callusing response of a genotype. I t may be one reason why optimal donor plant growth conditions are required for maximum response since i t would influence the levels of ethylene precursors and auxins in anthers. Treatments leading to an increase in i n i t i a l concentrations of ethylene can result in higher levels of embryogenesis ( i . e . in Bruce and Elrose) or lowering ethylene level can increase embryogenesis ( i . e . in Klages). Thus, there may be a threshold level at which higher concentrations are deleterious.
Values in each column followed by the same l e t t e r are not s i g n i f i c a n t l y different ~p=O.05) Number of c a l l i per 100 anthers
Acknowledgments. Financial support for this research was provided by the Natural Science and Engineering Research Council of Canada and by the Ontario Ministry of Agriculture and Food, and is gratefully acknowledged.
Babbar SB, Gupta SC (1986) Physiol. Plant. 68:141144
Cho UH (1988) MSc thesis Univ. of Guelph Canada Dunwell JM (1979)
J. Exp. Bot. 30:419-428
H i l l SE, Stead AD, Nichols R (1987) J. Plant Growth Regul. 6:1-13 Horner M, McComb JA, McCombAJ (1977) 28:1365-1372
scavenger, n-propyl gallate increased calli production in Bruce. The difference in embryogenesis (particularly, in callus production) among genotypes could be explained in terms of capacity to produce ethylene and s e n s i t i v i t y of anthers to ethylene. In Elrose, ethylene production gave a positive effect on embryogenesis. Also in Bruce, ethylene was thought to be necessary for callus production because the suppression of ethylene production by putrescine decreased anther response and c a l l i production. In Klages, ethylene production was at least supraoptimal because the application of putrescine increased embryogenesis whereas an increase of ethylene gave obscure results. In previous experiments (Cho 1988; Szarejko and Kasha 1989), the addition of AgNO3 to the medium showed positive effects on anther response and c a l l i production with Klages. The evidence to-date leads us to conclude that ethylene production is one of the factors responsible for genotype differences in anther culture a b i l i t y . The concentrations of ethylene precursors (e.g. ACC)
J. Exp. Bot.
Lizada MCC, Yang SF (1979) Anal. Biochem. 100:140145 Marsolais AA, Kasha KJ (1985) Can. J. Bot. 63:22092212 Reynolds TL (1987) Amer. J. Bot. 74:967-969 Sisler EC, Yang SF (1987) In: NewmanDW, Wilson KG (eds) Models in plant physiology and biochemistry. V o l . I I CRC Press Inc. pp 99-102 Stead AD (1985) In: Tucker G, Roberts J (eds) Ethylene in plant development. Butterworth London pp. 71-81 Szarejko I, Kasha KJ (1989) Proc. FAO/IAEAResearch Coord. Meeting. Katowice, Poland. July 1988 (in press) Whitehead CS, Fujino DW, Reid MS (1983) Horticult. 21:291-297
Sci.
