Plant Cell Reports
Plant Cell Reports (1987) 6:360-362
© Springer-Verlag 1987
Callus induction and plant regeneration from maize mature embryos Andrew S. Wang Sungene Technologies Corp., 3330 Hillview Avenue, Palo Alto, CA 94304, USA Received May 29, 1987 / Revised version received August 7, 1987 - Communicated by E. D. Earle
ABSTRACT
MATERIALS and METHODS
Calli were induced from mature embryos of maize (Ze__aamays L.) inbred lines A632, B73 and Mo17 on MS medium supplemented with 1-2 mg/l 2,4-dichlor~phenoxyacetic acid. Callus induction frequency ranged from 23-100%, with Mo]7 having the highest frequency. Plants were regenerated from 4-5% of the B73 and Mo17 explants. Embryogenic and organegenic calli of B73 were maintained for more than two and one half years without losing regenerability. Of 95 regenerated plants, only one R0 plant with abnormal pollen was detected, and no morphological variants were observed in the R 1 progeny.
Fifty driedmature seeds frcm each of maize inbred lines A632, B73 and Mo17 were surface sterilized for 20 m in with 50 ml of 2.5% sodium hypochlorite plus one drop of liquid detergent as a wetting agent. The sterilized seeds were rinsed four times with autoclaved, deionized water and softened by soaking in the autoclaved water overnight. Mature embryos were removed fremseeds with a scalpel and radicles were then separated from plumu!es on scutellar nodes. The plumule section (2-5 nm) was longitudinally sliced into halves and then plated cut-side down on pH(5.7) preadjustedMSmedium (GIBCO) which contains Murashige and Skoog salts (Murashige and Skoog 1962), i-inositol (I00 mg/l), thiamine HCI (i mg/l, modified), phytion (159mg/l, buffering agent), sucrose (2%), and gelrite (.2%, Kelco), and supplemented with the following hormone combinations: (I) 1 mg/l 2,4-D; (2) 2 mg/l 2,4-D; (3) I mg/l 2,4-D, i0 zM IAA, I0 ~MNAA, I0 ~M Ze; (4) 2 mg/l 2,4-D, i0 ~M IAA, I0 zMNAA, i0 bM Ze.
ABBREVIATIONS Dicamba, 3,6-Dichloro-o-anisic acid; IAA, 3-indoleacetic acid; NAA, l-naphthaleneacetic acid; 2,4-D, 2,4-dichlorophenoxyacetic acid; Ze, zeatin.
INI~RODUCTION Plant regeneration from maize tissue culture has mainly been obtained from callus derived from inmature embryos (Green and Phillips 1975, Duncan et al. 1985, Close and Ludeman 1987). Successful plant regeneration has also been reported from calli initiated from anthers (Ting et al. 1981), glumes (Suprasanna et al. 1986), immature tassels (Rhodes et al. 1986), leaf bases (Chang 1983), mesocotyls (Harm et al. 1976), and seedling segments (Santos et al. 1984). Inmature embryo-derived callus is more embryegenic and efficient for plant regeneration than calli from other explant tissues. All the explant tissues used to date must be obtained from living plants, which require time and space to grow. Utilization of maize mature embryos frown dried seeds could circumvent this requirement. A methed for the induction of embryogenic and organogenic calli frommaizemature embryos is reported in this publication.
Callus was maintained on MS medium supplemented with 2 mg/l dicamba, 25 n~proline and 100mg/l casein hydrolysate. Cultures were subculturedat two week intervals and kept in the dark at 26°C. Plantlets were regenerated by transferring the callus onto hormone-free MS medium. When the plantlets reached 8-10 em, they were transferred to soil and grown to maturity in a greenhouse. Pollen abnormality and sterility of the regenerated (R0 ) plants were determined by a pocket microscope (10X, Nikken, Tokyo). R0 plants were selfpollinated and the resulting R 1 seeds were harvested. The R1 progeny was grown in a field for screening somaclonal variants.
RESULTS and DISCUSSION Callus induction from mature embryos of maize can be observed one week after initiation. Induction frequencies of A632, B73 and Mo17 calli are listed in Table 1. The induction frequency ranged from 23-100%, with Mo17 having the highest induction frequency and B73 the lowest. All of the four media were efficient in callus induction, although MS
361 medium supplemented with 2,4-D alone gave a higher induction frequency than media supplemented with other hormone combinations. Both 1 and 2 ng/l 2,4-D gave similar callus induction frequencies.
Table i. Callus induction frequency of A632, 373 and Mo17 mature embryos on MS medium supplemented with various hormones. Plant Hormones
Callus Induction Frequency (%)a A632 B73 Mo17
I, 2,4-D (img/l) II, 2,4-D(2mg/l) h I + IAA + NAA + Ze-b II + IAA + NAA + Ze a. b.
58(11/19) 49(17/35) 35(12/34) 26(9/34)
52(20/38) 23(9/39) 39(13/33) 24(6/25)
100(26/26) 94(17/18) 65(17/26) 100(17/17)
organogenic sectors were observed in the B73 and Mo17 calli two weeks after induction. The embryogenic calli were isolated and transferred onto maintenance medium supplemented with dicamba (Fig.2). In our experience, most maize calli grow better on medium containing dicamba. The use of 2,4-D in MS medium produced 10% embryogenic calli in B73 and 14% embryogenic calli in Mo17 (Table 2). Plant regeneration frequency was 4% for 373 and 5% for Mo17. Supplementing this medium with other hormones in addition to the 2,4-D also induced callus formation for B73 and Mol7. These calli were embryogenic and organogenic, but no attempt was made to collect these data or maintain these calli. Ten plants were regenerated from two Mo17 callus lines which were derived frem two mature embryos.
Number of embryos showing callus initiation/total number of embryos plated. The concentration of IAA, NAA and Ze was l0 uM.
~'100 z~ w
80
o w
60
z o
4O
~ Q z
20
Figure 2. Compact (a) and friable (b) embryogenic calli derived from B73 mature embryos and maintained on MS medium supplemented with dicamba (2 mg/l).
2
!
!
O
4
6
8
I
10
SUCROSE CONC. (~)
Figure i. Effect of sucrose concentration on frequency of callus induction from mature embryos of 373 and Mo17. Each point represents the average of three determinations; each determination consists of 12 to 20 embryos.
The effect of sucrose concentration on induction frequency was also studied (Fig.l). Induction frequency decreased with increasing sucrose concentration and 2% sucrose was the best level for beth B73 and Mo17. Generally, calli grew slower and were more compact o n M S medium supplemented with higher concentrations of sucrose. A632 callus was whiter soft, fast-growing and non-embryogenic. Therefore, no A632 plantlets were regenerated. Mo17 and B73 calli were compact and grew more slowly than A632 callus. Embryogenic and
Table 2. Embryogenic callus (EF) and plant regeneration (PF) frequencies for B73 and Mo17 mature embryos induced on MS medium supplemented with 2,4-D alone.
2,4-D
B73
Mo17
Conc.
EF(%) a
PF(%) b
EF(%) a
Img/l 2mg/l Total
8(3/38) 13(5/39) 10(8/77)
5(2/38) 3(1/39) 4(3/77)
15(4/26) 11(2/18) 14(6/44)
a. b.
PF(%) b 4(1/26) 5(1/18) 5(2/44)
Number of embryos showing embryogenic callus initiation/total number of embryos plated. Number of embryos showing plant regeneration/ total number of embryos plated.
Three B73 callus lines isolated from three mature embryos have beenmaintained for more than two and one half years without losing regenerability
362 (Fig.3). A total of I0, 80 and 5 R 0 plants were regenerated from B73 calli in the first, second and third year after callus induction, respectively. Of the 95 R0 plants, only one second year R 0 plant had 50% pollen sterility, and variant segregation has not been detected in the R 1 progeny test. Wang et al. (1987) have showed that 29% of 183 B73 R 0 plants regenerated from inmature embryo callus cultures carried various degrees of pollen Sterility; 138 R 0 plants produced enough seed (at least 15 seeds) for evaluation of variant segregation in R 1 progeny tests, 17% showed variant segregation. The results suggest that B73 plants regenerated from
mature embryo callus are more stable than those from /nmature embryo callus in terms of chrc~osomal constitution (reflected by pollen sterility) and somaclonal variation.
ACKNOWLEDGEMENTS I wish to thank A. Miller for her critical reading of this manuscript, M. Hollingsworth and J. Milcic for their technical assistance and P. Brookhouzen for collecting field data.
REFERENCES
Figure 3. Plantlets regenerated from one year old B73 calli initiated frc~mature embryos (Table 2).
Chang YF (1983) Plant Cell Reports. 2:183-185 Close K, Ludeman L. (1987) Plant Sci. (in press) Duncan DR, Williams ME, Zehr BE, widholmJM (1985) Planta 165:322-332 Green CE, Phillips RL (1975) Crop Sci. 15:417-421 Harm CT, Lorz H, Potrykus (1976) Z. Pflangenzuchtg 77:347-351 Suprasanna P, Rao KV, Reddy GM (1986) Theor. Appl. Genet. 72:120-122 Rhodes CA, Green CE, Phillips RL (1986) Plant Sci. 46:225-232 Santos MA, Torne JM, Blanco JL (1984) Plant Sci. Lett. 33:309-315 Ting YC, Yu M, Zheng WZ (1981) Plant Sci. Lett. 23:139-145 Wang AS, HollingsworthMD, Milcic JB (1987) Maize Genetic News Lett. 61:81-83 Murashige T, Skoog F. (1962) Plant Physiol. 15:473-497
Plant Cell Reports (1987) 6:360-362
© Springer-Verlag 1987
Callus induction and plant regeneration from maize mature embryos Andrew S. Wang Sungene Technologies Corp., 3330 Hillview Avenue, Palo Alto, CA 94304, USA Received May 29, 1987 / Revised version received August 7, 1987 - Communicated by E. D. Earle
ABSTRACT
MATERIALS and METHODS
Calli were induced from mature embryos of maize (Ze__aamays L.) inbred lines A632, B73 and Mo17 on MS medium supplemented with 1-2 mg/l 2,4-dichlor~phenoxyacetic acid. Callus induction frequency ranged from 23-100%, with Mo]7 having the highest frequency. Plants were regenerated from 4-5% of the B73 and Mo17 explants. Embryogenic and organegenic calli of B73 were maintained for more than two and one half years without losing regenerability. Of 95 regenerated plants, only one R0 plant with abnormal pollen was detected, and no morphological variants were observed in the R 1 progeny.
Fifty driedmature seeds frcm each of maize inbred lines A632, B73 and Mo17 were surface sterilized for 20 m in with 50 ml of 2.5% sodium hypochlorite plus one drop of liquid detergent as a wetting agent. The sterilized seeds were rinsed four times with autoclaved, deionized water and softened by soaking in the autoclaved water overnight. Mature embryos were removed fremseeds with a scalpel and radicles were then separated from plumu!es on scutellar nodes. The plumule section (2-5 nm) was longitudinally sliced into halves and then plated cut-side down on pH(5.7) preadjustedMSmedium (GIBCO) which contains Murashige and Skoog salts (Murashige and Skoog 1962), i-inositol (I00 mg/l), thiamine HCI (i mg/l, modified), phytion (159mg/l, buffering agent), sucrose (2%), and gelrite (.2%, Kelco), and supplemented with the following hormone combinations: (I) 1 mg/l 2,4-D; (2) 2 mg/l 2,4-D; (3) I mg/l 2,4-D, i0 zM IAA, I0 ~MNAA, I0 ~M Ze; (4) 2 mg/l 2,4-D, i0 ~M IAA, I0 zMNAA, i0 bM Ze.
ABBREVIATIONS Dicamba, 3,6-Dichloro-o-anisic acid; IAA, 3-indoleacetic acid; NAA, l-naphthaleneacetic acid; 2,4-D, 2,4-dichlorophenoxyacetic acid; Ze, zeatin.
INI~RODUCTION Plant regeneration from maize tissue culture has mainly been obtained from callus derived from inmature embryos (Green and Phillips 1975, Duncan et al. 1985, Close and Ludeman 1987). Successful plant regeneration has also been reported from calli initiated from anthers (Ting et al. 1981), glumes (Suprasanna et al. 1986), immature tassels (Rhodes et al. 1986), leaf bases (Chang 1983), mesocotyls (Harm et al. 1976), and seedling segments (Santos et al. 1984). Inmature embryo-derived callus is more embryegenic and efficient for plant regeneration than calli from other explant tissues. All the explant tissues used to date must be obtained from living plants, which require time and space to grow. Utilization of maize mature embryos frown dried seeds could circumvent this requirement. A methed for the induction of embryogenic and organogenic calli frommaizemature embryos is reported in this publication.
Callus was maintained on MS medium supplemented with 2 mg/l dicamba, 25 n~proline and 100mg/l casein hydrolysate. Cultures were subculturedat two week intervals and kept in the dark at 26°C. Plantlets were regenerated by transferring the callus onto hormone-free MS medium. When the plantlets reached 8-10 em, they were transferred to soil and grown to maturity in a greenhouse. Pollen abnormality and sterility of the regenerated (R0 ) plants were determined by a pocket microscope (10X, Nikken, Tokyo). R0 plants were selfpollinated and the resulting R 1 seeds were harvested. The R1 progeny was grown in a field for screening somaclonal variants.
RESULTS and DISCUSSION Callus induction from mature embryos of maize can be observed one week after initiation. Induction frequencies of A632, B73 and Mo17 calli are listed in Table 1. The induction frequency ranged from 23-100%, with Mo17 having the highest induction frequency and B73 the lowest. All of the four media were efficient in callus induction, although MS
361 medium supplemented with 2,4-D alone gave a higher induction frequency than media supplemented with other hormone combinations. Both 1 and 2 ng/l 2,4-D gave similar callus induction frequencies.
Table i. Callus induction frequency of A632, 373 and Mo17 mature embryos on MS medium supplemented with various hormones. Plant Hormones
Callus Induction Frequency (%)a A632 B73 Mo17
I, 2,4-D (img/l) II, 2,4-D(2mg/l) h I + IAA + NAA + Ze-b II + IAA + NAA + Ze a. b.
58(11/19) 49(17/35) 35(12/34) 26(9/34)
52(20/38) 23(9/39) 39(13/33) 24(6/25)
100(26/26) 94(17/18) 65(17/26) 100(17/17)
organogenic sectors were observed in the B73 and Mo17 calli two weeks after induction. The embryogenic calli were isolated and transferred onto maintenance medium supplemented with dicamba (Fig.2). In our experience, most maize calli grow better on medium containing dicamba. The use of 2,4-D in MS medium produced 10% embryogenic calli in B73 and 14% embryogenic calli in Mo17 (Table 2). Plant regeneration frequency was 4% for 373 and 5% for Mo17. Supplementing this medium with other hormones in addition to the 2,4-D also induced callus formation for B73 and Mol7. These calli were embryogenic and organogenic, but no attempt was made to collect these data or maintain these calli. Ten plants were regenerated from two Mo17 callus lines which were derived frem two mature embryos.
Number of embryos showing callus initiation/total number of embryos plated. The concentration of IAA, NAA and Ze was l0 uM.
~'100 z~ w
80
o w
60
z o
4O
~ Q z
20
Figure 2. Compact (a) and friable (b) embryogenic calli derived from B73 mature embryos and maintained on MS medium supplemented with dicamba (2 mg/l).
2
!
!
O
4
6
8
I
10
SUCROSE CONC. (~)
Figure i. Effect of sucrose concentration on frequency of callus induction from mature embryos of 373 and Mo17. Each point represents the average of three determinations; each determination consists of 12 to 20 embryos.
The effect of sucrose concentration on induction frequency was also studied (Fig.l). Induction frequency decreased with increasing sucrose concentration and 2% sucrose was the best level for beth B73 and Mo17. Generally, calli grew slower and were more compact o n M S medium supplemented with higher concentrations of sucrose. A632 callus was whiter soft, fast-growing and non-embryogenic. Therefore, no A632 plantlets were regenerated. Mo17 and B73 calli were compact and grew more slowly than A632 callus. Embryogenic and
Table 2. Embryogenic callus (EF) and plant regeneration (PF) frequencies for B73 and Mo17 mature embryos induced on MS medium supplemented with 2,4-D alone.
2,4-D
B73
Mo17
Conc.
EF(%) a
PF(%) b
EF(%) a
Img/l 2mg/l Total
8(3/38) 13(5/39) 10(8/77)
5(2/38) 3(1/39) 4(3/77)
15(4/26) 11(2/18) 14(6/44)
a. b.
PF(%) b 4(1/26) 5(1/18) 5(2/44)
Number of embryos showing embryogenic callus initiation/total number of embryos plated. Number of embryos showing plant regeneration/ total number of embryos plated.
Three B73 callus lines isolated from three mature embryos have beenmaintained for more than two and one half years without losing regenerability
362 (Fig.3). A total of I0, 80 and 5 R 0 plants were regenerated from B73 calli in the first, second and third year after callus induction, respectively. Of the 95 R0 plants, only one second year R 0 plant had 50% pollen sterility, and variant segregation has not been detected in the R 1 progeny test. Wang et al. (1987) have showed that 29% of 183 B73 R 0 plants regenerated from inmature embryo callus cultures carried various degrees of pollen Sterility; 138 R 0 plants produced enough seed (at least 15 seeds) for evaluation of variant segregation in R 1 progeny tests, 17% showed variant segregation. The results suggest that B73 plants regenerated from
mature embryo callus are more stable than those from /nmature embryo callus in terms of chrc~osomal constitution (reflected by pollen sterility) and somaclonal variation.
ACKNOWLEDGEMENTS I wish to thank A. Miller for her critical reading of this manuscript, M. Hollingsworth and J. Milcic for their technical assistance and P. Brookhouzen for collecting field data.
REFERENCES
Figure 3. Plantlets regenerated from one year old B73 calli initiated frc~mature embryos (Table 2).
Chang YF (1983) Plant Cell Reports. 2:183-185 Close K, Ludeman L. (1987) Plant Sci. (in press) Duncan DR, Williams ME, Zehr BE, widholmJM (1985) Planta 165:322-332 Green CE, Phillips RL (1975) Crop Sci. 15:417-421 Harm CT, Lorz H, Potrykus (1976) Z. Pflangenzuchtg 77:347-351 Suprasanna P, Rao KV, Reddy GM (1986) Theor. Appl. Genet. 72:120-122 Rhodes CA, Green CE, Phillips RL (1986) Plant Sci. 46:225-232 Santos MA, Torne JM, Blanco JL (1984) Plant Sci. Lett. 33:309-315 Ting YC, Yu M, Zheng WZ (1981) Plant Sci. Lett. 23:139-145 Wang AS, HollingsworthMD, Milcic JB (1987) Maize Genetic News Lett. 61:81-83 Murashige T, Skoog F. (1962) Plant Physiol. 15:473-497
