Plant Cell Reports
Plant Cell Reports (1989) 7:701-703
© Springer-Verlag 1989
Analysis of lipid composition and morphological characteristics in soybean regenerantsl D.F. Hildebrand, T. R. Adams, M.L. Dahmer, E. G. Williams, and G.B. Collins Received August 1, 1988/Revised version received January 24, 1989 - Communicated by G. C. Phillips
ABSTRACT A study was conducted to examine the extent of somaclonal variation of soybean plants, Glycine max (L.) Merrill cv. 'McCall', regenerated via somatic embryogenesis from cultured immature cotyledons using two different protocols. The sexual progeny of regenerants were compared with normal, seed-derived populations for morphological characteristics and fatty acid composition of seeds. First generation progeny of regenerants showed greater phenotypic variation than the control population, but this variation was not observed in the second generation. No stable somaclonal variants for fatty acid composition of the seed oil or morphological characteristics were observed, indicating that this somatic embryogenesis system should be adaptable for transformation with minimal generation of unwanted variation. ABBREVIATIONS
N•, l-naphtaleneacetic acid; 2,4-D, 2,4dichlorophenoxyacetic acid; IBA, indolebutyric acid. INTRODUCTION Several reports have described the regeneration of whole soybean plants in which plants did not arise from preexisting meristems (Lazzeri et al, 1985; Barwale et al, 1986; Ranch et al, 1985). Establishment of de novo regeneration in soybean tissue cultures opens the possibility of recovering culture-induced genetic v a r i a b i l i t y or somaclonal variation in regenerated plants (Evans and Sharp, 1983; Larkin et al, 1984; Baertlein and McDaniel, 1987; Grabosch et al, 1987). Such variation may affect traits that could be useful in plant breeding. In soybean breeding, additional variation for fatty acid composition of the seed oil is desirable because natural variation appears to be limited. Mutation breeding and recurrent selection have had some I j / Contribution from the Department of Agronomy, University of Kentucky, Lexington, KY 40546. The research, was supported by the American Soybean Association and the Lubrizol Genetics Company, published with the approval of the director of the Kentucky Agricultural Experiment Station as Journal Article Number 88-3-264.
Offprintrequ~ to.'D.F. Hildebrand
success in developing commercial soybean lines with desired oil compositions (e.g. a 50-70% decrease in linolenic acid), but selection of soybean lines with further changes in seed oii composition is s t i l l desirable (Burton et al, 1983; Hammond and Fehr, 1984; Wilcox and Cavins, ]985). The purpose of this study was to evaluate the nature of variability, particularly for fatty acid composition of seed lipids, in plants regenerated from tissue culture by two protocols. MATERIALS AND METHODS Plant materials Plants of Glycine max (L.) Merrill cv. 'McCall' were grown in the greenhouse with natural illumination supplemented by high pressure sodium lamps to a 14 h photoperiod with 22/28 C night/day temperatures. Three populations were compared: (N) Normal, seed derived control plants. (J) Sexual progeny of nine plants regenerated after ca. 5 months in culture using the protocol of Lazzeri et ai.(1985) with 15 mg/l NAA. First and second generation progeny are designated JR1 and JR2. (T) Sexual progeny of a singl6 plant regenerated by a protocol developed for transformation as follows: An immature zygotic embryo (3.5 mm long) was excised from a surface sterilized pod. The embryo axis was removed and the isolated cotyledons were plated on modified MS medium (Lazzeri et al, 1985) containing 10 mg/L 2,4-D in a 2 cm x 10 cm plastic petri dish. After 10 days the cotyledons were inoculated with an overnight culture ofAgrobacterium tumefaciens washed and resuspended in MS medium without hormones. This Agrobacterium contained pAN/Kan-] (kindly supplied by Don Merlow, Agrigenetics Advanced Research Co., Madison, WI) which is a disarmed octopine-type Ti plasmid with neomycin phosphotransferase as the selectable marker (Facciotti et al, 1985; Umbeck et al,1987). After 2 days exposure to Agrobacterium, the cotyledons were replated on the same medium with the addition of 400 #g/ml Mefoxin (sodium cefoxitin) and 50 #g/ml Kanamycin. After 5 days, a well developed somatic embryo (>2 mm long) was transferred to MS BKZN medium (Lazzeri e t a ] , 1985). This embryo was transferred after
702 another 25 days to the same medium but with gelrite instead of agar, and containing 300 mg/ml Mefoxin and I00 pg/ml Kanamycin. After 229 days on this medium, the plantlet was transferred to a Magenta box containing the same basal medium but with 9 mg/L coumarin, 0.5 mg/L IBA, 100 pg/ml Claforan (sodium cefotaxime), and KAO vitamins (Kao, 1977). The rooted plantlet was placed in potting soil in a growth chamber after 10 days, and the resulting plant was moved to the greenhouse after 34 days. The plant produced 43 seeds, 42 of which germinated and were grown out to produce the TR] population. The second sexual generation is designated TR2 •
Primary regenerants (Rn) were not examined in detail, as these would be e~pected to show direct carry-over effects of tissue culture manipulations on whole-plant physiology. First and second generation selfedprogeny were produced by selfing N, J or T plants. For f i r s t generation comparisons, 44 J RI seeds (from 9 Rn plants) and 42 T R1 seeds were compared with 28 Nl control seeds. For second generation comparisons, 60 J R~ seeds (from 3 R1 plants) and 70 T R) seeds (fro~ 5 R1 plants) wePe compared with 40 N~ seeds (from 3 RI plants). Fatty Acid Analysis Mature seeds were sampled non-destructively by excising a small portion (ca. 25 mg dry weight) of the cotyledon distal to the embryonic axis. Neutral lipids were extracted from this sample with petroleum ether. Fatty acid methyl esters (FAME) were produced from the extracted lipids and quantified using gasliquid chromatography with flame, ionization detection (FID) (Chaven et al, 19827. RESULTS Morpholoqical and reproductive v a r i a b i l i t y Morphological abnormalities were observed in the TRl population at greater frequencies than observed in th~ JRl or control populations. Among the 42 TRi plants~ 19 displayed lateral indentation (lobingJ of the f i r s t unifoliolate leaf (Fig. Ia), two showed sectorial loss of chlorophyll, and two had dual apical meristems with three cotyledons and three unifoliolate leaves (Fig. Ib). The JRl and TRI populations showed similar mean plant height~ typical of McCall, although the TR1 population was more variable. Both TRl and JRI populations produced fewer seeds per plant-than the control population . L i t t l e morphological variation was seen in plants of the TR) and JRp populations. Several TRp plants were Observed-with leaf indentation or sectoring but not in the progeny of RI plants showing these characteristics. One normal TR1 plant gave rise to an Rp individual with three cot)ledons and three unifoliDlate leaves. Plant height and seed set in the R? populations were within the normal range for McCall.- One sterile plant was observed in each of the TR2 and TR3 populations. Variability in fatty acid composition JRI populations produced fewer seeds per plant than the control population . L i t t l e morphological variation was seen in plants of the TR) and JRp populations. Several TR2 plants were Observed-with leaf indentation or sectoring but not in the progeny of RI plants showing these characteristics. One normal TRI plant gave rise to an R) individual with three cotyledons and three unifoliBlate leaves. Plant
Fig. I.
Morphological abnormalities observed in the TR1 population, a. Unifoliate lobed leaf on right (compared to normal leaf on l e f t ) , b. Seedling with three unifoliate leaves and dual meristems.
height and seed set in the R2 populations were within the normal range for McCall. One s t e r i l e plant was observed in each of the TR2 and TR3 populations. Variability in fatty acid composition Fatty acid (FA) compositions of seed lipids for N, J and T populations, and selected individual plants, are shown in Table I. Variability was highest for TR 1 regenerants, intermediate for JR 1 regenerants, and lowest for controls and both second-Oeneration regenerant populations. A single exception was the unexpectedly high variability for oleic acid (18:17 among controls planted with first generation regenerants (asterisk in Table I). This was due to two seeds with abnormally high 18:1 contents. Certain JR 1 and TR i individuals showed unusual FA composition patterns (Table 17, but the R 2 progeny of these plants were normal. DISCUSSION AND CONCLUSION
First-generation, sexual progenies of plants regenerated from tissue culture showed increased variability relative to non-regenerant control populations. The greater variability of the TR i population relative to the JR I population may have resulted from differences in lhe culture history of the primary regenerants (McCoy et al, 19827. The TR 0 embryo was induced on 2,4-D, was exposed to Agrobacterium tumefaciens, was treated with the antibiotics Mefoxin, Claforan and Kanamycin, and spent g months in culture. The JR n embryos were induced with NAA, received no antlbiftic treatments, and
703 Table i.
Variation in f a t t y acid composition (as a percentage of fatty acid methyl esters, FAME) in a normal soybean population (N) and the f i r s t and second generation progenies (R1 & R2) of plants regenerated fro~ tissue culture. Flame ionization detector (FID) response was used to approximate FAME composi t ion.
......................................................
FAME %-Mean ± S . E ,
& (Range) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Populatio~ of plant. 16:0
18:0
18:1
18:2
18:3
NI
16.I +_ 0.3
(13.8-20.1)
4.3_+ 0.1
(8.3- 5.7)
22.4_+ 0.9
('15.7-39.4)
47.9 + 0.8
(36.5-52.2)
9.1 + 0.2
16.4-12.0)
JRI
18.9 +_ 0.4
(8.7-23.9)
4.2 + 0.I
{Z.O- 6.3)
13.0 + 0.3
(8.7-17.5)
54.1 _+ 0.6
(49.2-75.5)
10.0_+ 0.2
(5.1-13.5)
TRI
18.0_+ 0.8
(13.2-42.4)
3.8_+ 0.2
(2.8-i0.2}
21.7 _+ 0.6
(8.6-28.4)
48.1 + 0.7
(25.5-61.6)
8.4 +_ 0.3
(5.3-15.9)
N?
15.6 + 0.1
(14.2-17.4)
3.5_+ 0.0
(0.0- 4.3)
16.3 +. 0.4
(12.6-21.1)
53.8 + 0.2
(51.3-56.5)
10.9 _+ 0.2
(9.1-13.1)
JR2
14.5 _+ 0.i
(12.7-15.4)
3.5 _+ 0.0
(3.3- 4.0)
16.0 + 0.5
(13.2-21.2)
53.8 +_ 0.2
(51.3-55.8)
12.2 +_ 0.3
(8.3-15.1)
TR2
15.1 + 0.1
(13.4-19.9)
3.9 _+ 0.1
[3.1- 5.D)
13.8_+ 0.2
(11.3-17.8)
55.3 + 0.i
(51.9-57.1)
11.9 + 0.i
(9.2-14.9)
N-I
18.0
4.7
20.9
47.2
g.?
JRI-I
8.7
2.0
8.7
75.5
5.1
TRI-I
13.0
2.8
28.9
47.1
8.2
-2
15.9
2.7
8.4
60.1
13.5
-3
37.8
9.0
14.9
32.7
4.7
aNI and N2 are the f i r s t and second generation controls.
JRI, TRI. and JR2, TR2 are the f i r s t and second generation progeny of regenerants.
N-I, JRI-I and
TRI-I, -2 and -3 are individuals selected for specific comparisons of seed oll FAME.compositions.
remained in culture only 5 months. The additional elements designed to impose genetic transformation on a regeneration system may therefore have contributed to increased variation among regenerated plants. Although the variability observed in R~ populations had been transmitted through one meiotic cycle from the Rn parents, this variability had almost entirely disappeared after passage through one further meiotic cycle to the R? generation. The nature of such unstable epigeBetic effects is not understood, but may be due to altered gene expression (Meins and Binns, 1977; Mermod et al, 1983; Sager and Grabowy, 1983). Although cytological analyses were not made in this study, reversion to normality by the R~ generation, and a lack of distinctive aneuploid ph~notypes, indicate that the regenerants were cytologically normal. Graybosch et a] (1987) also observed l i t t l e variation among soybean plants regenerated by shoot organogenesis from nascent meristematic regions within cultured cotyledonary nodes (Wright et al, ]986). The absence of stable, heritable variations among the progeny of regenerants produced from immature soybean cotyledons by somatic embryogenesis indicates that, while this protocol is unlikely to provide useful numbers of somaclonal variants, i t should be suitable for development of transformation systems using elite genotypes for which culture-induced variability is undesirable. ACKNOWLEDGMENTS We are grateful to Dr. P.A. Lazzeri for provision of regenerant plants, to Jeff Berger, Gary Veach and Pierce Flemming for assistance with fatty acid analyses, to Drs. T. Pfeiffer and W. Parrott for helpful discussions, and to Brenda Hays, Mandy Collins and Ann Kupper for typing the manuscript.
REFERENCES Baertlein DA, McDaniel RG (1987) Theor Appl Genet 73:575-580. Barwa]e UB, Kerns HR, Widho]m JM (1986) Planta 16:473-478.
Burton JW, Wilson RF, Brim CA (1983) Crop Sci 23:744-747. Chaven CT, Hymowitz T, Newell CA (1982) J Amer Oil Chem Soc 59:23-25. Evans DA, Sharp WR(]983) Science 221:949-951. Facciotti D, O'Neal JK, Lee S, Shewmaker CK (1985) Biotechnology 3:241-246. Graybosch RA, Edge ME, Delannay X (1987) Crop Sci 27:803-806. Hammond EG, Fehr WR (1984) J Amer Oil ChemSoc 61:1713-1716. Kao KN (1977) Mol Gen Genet 150:225-230. Larkin PJ, Ryan SA, Brettell RIS, Scowcroft WR (1984) Theor Appl Genet 67:443-455. Lazzeri PA, Hildebrand DF, Collins GB (1985) Plant Molec B i o l Rep 3:]60-167. McCoy TJ, Phillips RL, Rines HW (1982) Can J Genet Cytol 24:37-50. Meins F, Binns A (1977) Proc Natl Acad Sci 74:2928-2932. Mermod J-J, Bourgeois S, Defer N, Crepin M (1983) Proc Natl Acad Sci 80:110-114. Ranch JL, Oglesby L, Zielinski AC (1985) In Vitro Cellular & Developmental Biol 21:653-658. Sager R, Grabowy C (1983) Proc Natl Acad Sci 80:3025-3029. Umbeck P, Johnson G, Barton K, Swain W (1987) Biotechnology 5:263-266. Wilcox JR, Cavins JF (1985) Theor Appl Genet 71:74-78. Wright MS, Koehler SM, Hinchee MA, Carries MG (1986) Wright MS, Koehler SM, Hinchee MA, Carnes MG (1986) Plant Cell Rep 5:150-154.
Plant Cell Reports (1989) 7:701-703
© Springer-Verlag 1989
Analysis of lipid composition and morphological characteristics in soybean regenerantsl D.F. Hildebrand, T. R. Adams, M.L. Dahmer, E. G. Williams, and G.B. Collins Received August 1, 1988/Revised version received January 24, 1989 - Communicated by G. C. Phillips
ABSTRACT A study was conducted to examine the extent of somaclonal variation of soybean plants, Glycine max (L.) Merrill cv. 'McCall', regenerated via somatic embryogenesis from cultured immature cotyledons using two different protocols. The sexual progeny of regenerants were compared with normal, seed-derived populations for morphological characteristics and fatty acid composition of seeds. First generation progeny of regenerants showed greater phenotypic variation than the control population, but this variation was not observed in the second generation. No stable somaclonal variants for fatty acid composition of the seed oil or morphological characteristics were observed, indicating that this somatic embryogenesis system should be adaptable for transformation with minimal generation of unwanted variation. ABBREVIATIONS
N•, l-naphtaleneacetic acid; 2,4-D, 2,4dichlorophenoxyacetic acid; IBA, indolebutyric acid. INTRODUCTION Several reports have described the regeneration of whole soybean plants in which plants did not arise from preexisting meristems (Lazzeri et al, 1985; Barwale et al, 1986; Ranch et al, 1985). Establishment of de novo regeneration in soybean tissue cultures opens the possibility of recovering culture-induced genetic v a r i a b i l i t y or somaclonal variation in regenerated plants (Evans and Sharp, 1983; Larkin et al, 1984; Baertlein and McDaniel, 1987; Grabosch et al, 1987). Such variation may affect traits that could be useful in plant breeding. In soybean breeding, additional variation for fatty acid composition of the seed oil is desirable because natural variation appears to be limited. Mutation breeding and recurrent selection have had some I j / Contribution from the Department of Agronomy, University of Kentucky, Lexington, KY 40546. The research, was supported by the American Soybean Association and the Lubrizol Genetics Company, published with the approval of the director of the Kentucky Agricultural Experiment Station as Journal Article Number 88-3-264.
Offprintrequ~ to.'D.F. Hildebrand
success in developing commercial soybean lines with desired oil compositions (e.g. a 50-70% decrease in linolenic acid), but selection of soybean lines with further changes in seed oii composition is s t i l l desirable (Burton et al, 1983; Hammond and Fehr, 1984; Wilcox and Cavins, ]985). The purpose of this study was to evaluate the nature of variability, particularly for fatty acid composition of seed lipids, in plants regenerated from tissue culture by two protocols. MATERIALS AND METHODS Plant materials Plants of Glycine max (L.) Merrill cv. 'McCall' were grown in the greenhouse with natural illumination supplemented by high pressure sodium lamps to a 14 h photoperiod with 22/28 C night/day temperatures. Three populations were compared: (N) Normal, seed derived control plants. (J) Sexual progeny of nine plants regenerated after ca. 5 months in culture using the protocol of Lazzeri et ai.(1985) with 15 mg/l NAA. First and second generation progeny are designated JR1 and JR2. (T) Sexual progeny of a singl6 plant regenerated by a protocol developed for transformation as follows: An immature zygotic embryo (3.5 mm long) was excised from a surface sterilized pod. The embryo axis was removed and the isolated cotyledons were plated on modified MS medium (Lazzeri et al, 1985) containing 10 mg/L 2,4-D in a 2 cm x 10 cm plastic petri dish. After 10 days the cotyledons were inoculated with an overnight culture ofAgrobacterium tumefaciens washed and resuspended in MS medium without hormones. This Agrobacterium contained pAN/Kan-] (kindly supplied by Don Merlow, Agrigenetics Advanced Research Co., Madison, WI) which is a disarmed octopine-type Ti plasmid with neomycin phosphotransferase as the selectable marker (Facciotti et al, 1985; Umbeck et al,1987). After 2 days exposure to Agrobacterium, the cotyledons were replated on the same medium with the addition of 400 #g/ml Mefoxin (sodium cefoxitin) and 50 #g/ml Kanamycin. After 5 days, a well developed somatic embryo (>2 mm long) was transferred to MS BKZN medium (Lazzeri e t a ] , 1985). This embryo was transferred after
702 another 25 days to the same medium but with gelrite instead of agar, and containing 300 mg/ml Mefoxin and I00 pg/ml Kanamycin. After 229 days on this medium, the plantlet was transferred to a Magenta box containing the same basal medium but with 9 mg/L coumarin, 0.5 mg/L IBA, 100 pg/ml Claforan (sodium cefotaxime), and KAO vitamins (Kao, 1977). The rooted plantlet was placed in potting soil in a growth chamber after 10 days, and the resulting plant was moved to the greenhouse after 34 days. The plant produced 43 seeds, 42 of which germinated and were grown out to produce the TR] population. The second sexual generation is designated TR2 •
Primary regenerants (Rn) were not examined in detail, as these would be e~pected to show direct carry-over effects of tissue culture manipulations on whole-plant physiology. First and second generation selfedprogeny were produced by selfing N, J or T plants. For f i r s t generation comparisons, 44 J RI seeds (from 9 Rn plants) and 42 T R1 seeds were compared with 28 Nl control seeds. For second generation comparisons, 60 J R~ seeds (from 3 R1 plants) and 70 T R) seeds (fro~ 5 R1 plants) wePe compared with 40 N~ seeds (from 3 RI plants). Fatty Acid Analysis Mature seeds were sampled non-destructively by excising a small portion (ca. 25 mg dry weight) of the cotyledon distal to the embryonic axis. Neutral lipids were extracted from this sample with petroleum ether. Fatty acid methyl esters (FAME) were produced from the extracted lipids and quantified using gasliquid chromatography with flame, ionization detection (FID) (Chaven et al, 19827. RESULTS Morpholoqical and reproductive v a r i a b i l i t y Morphological abnormalities were observed in the TRl population at greater frequencies than observed in th~ JRl or control populations. Among the 42 TRi plants~ 19 displayed lateral indentation (lobingJ of the f i r s t unifoliolate leaf (Fig. Ia), two showed sectorial loss of chlorophyll, and two had dual apical meristems with three cotyledons and three unifoliolate leaves (Fig. Ib). The JRl and TRI populations showed similar mean plant height~ typical of McCall, although the TR1 population was more variable. Both TRl and JRI populations produced fewer seeds per plant-than the control population . L i t t l e morphological variation was seen in plants of the TR) and JRp populations. Several TRp plants were Observed-with leaf indentation or sectoring but not in the progeny of RI plants showing these characteristics. One normal TR1 plant gave rise to an Rp individual with three cot)ledons and three unifoliDlate leaves. Plant height and seed set in the R? populations were within the normal range for McCall.- One sterile plant was observed in each of the TR2 and TR3 populations. Variability in fatty acid composition JRI populations produced fewer seeds per plant than the control population . L i t t l e morphological variation was seen in plants of the TR) and JRp populations. Several TR2 plants were Observed-with leaf indentation or sectoring but not in the progeny of RI plants showing these characteristics. One normal TRI plant gave rise to an R) individual with three cotyledons and three unifoliBlate leaves. Plant
Fig. I.
Morphological abnormalities observed in the TR1 population, a. Unifoliate lobed leaf on right (compared to normal leaf on l e f t ) , b. Seedling with three unifoliate leaves and dual meristems.
height and seed set in the R2 populations were within the normal range for McCall. One s t e r i l e plant was observed in each of the TR2 and TR3 populations. Variability in fatty acid composition Fatty acid (FA) compositions of seed lipids for N, J and T populations, and selected individual plants, are shown in Table I. Variability was highest for TR 1 regenerants, intermediate for JR 1 regenerants, and lowest for controls and both second-Oeneration regenerant populations. A single exception was the unexpectedly high variability for oleic acid (18:17 among controls planted with first generation regenerants (asterisk in Table I). This was due to two seeds with abnormally high 18:1 contents. Certain JR 1 and TR i individuals showed unusual FA composition patterns (Table 17, but the R 2 progeny of these plants were normal. DISCUSSION AND CONCLUSION
First-generation, sexual progenies of plants regenerated from tissue culture showed increased variability relative to non-regenerant control populations. The greater variability of the TR i population relative to the JR I population may have resulted from differences in lhe culture history of the primary regenerants (McCoy et al, 19827. The TR 0 embryo was induced on 2,4-D, was exposed to Agrobacterium tumefaciens, was treated with the antibiotics Mefoxin, Claforan and Kanamycin, and spent g months in culture. The JR n embryos were induced with NAA, received no antlbiftic treatments, and
703 Table i.
Variation in f a t t y acid composition (as a percentage of fatty acid methyl esters, FAME) in a normal soybean population (N) and the f i r s t and second generation progenies (R1 & R2) of plants regenerated fro~ tissue culture. Flame ionization detector (FID) response was used to approximate FAME composi t ion.
......................................................
FAME %-Mean ± S . E ,
& (Range) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Populatio~ of plant. 16:0
18:0
18:1
18:2
18:3
NI
16.I +_ 0.3
(13.8-20.1)
4.3_+ 0.1
(8.3- 5.7)
22.4_+ 0.9
('15.7-39.4)
47.9 + 0.8
(36.5-52.2)
9.1 + 0.2
16.4-12.0)
JRI
18.9 +_ 0.4
(8.7-23.9)
4.2 + 0.I
{Z.O- 6.3)
13.0 + 0.3
(8.7-17.5)
54.1 _+ 0.6
(49.2-75.5)
10.0_+ 0.2
(5.1-13.5)
TRI
18.0_+ 0.8
(13.2-42.4)
3.8_+ 0.2
(2.8-i0.2}
21.7 _+ 0.6
(8.6-28.4)
48.1 + 0.7
(25.5-61.6)
8.4 +_ 0.3
(5.3-15.9)
N?
15.6 + 0.1
(14.2-17.4)
3.5_+ 0.0
(0.0- 4.3)
16.3 +. 0.4
(12.6-21.1)
53.8 + 0.2
(51.3-56.5)
10.9 _+ 0.2
(9.1-13.1)
JR2
14.5 _+ 0.i
(12.7-15.4)
3.5 _+ 0.0
(3.3- 4.0)
16.0 + 0.5
(13.2-21.2)
53.8 +_ 0.2
(51.3-55.8)
12.2 +_ 0.3
(8.3-15.1)
TR2
15.1 + 0.1
(13.4-19.9)
3.9 _+ 0.1
[3.1- 5.D)
13.8_+ 0.2
(11.3-17.8)
55.3 + 0.i
(51.9-57.1)
11.9 + 0.i
(9.2-14.9)
N-I
18.0
4.7
20.9
47.2
g.?
JRI-I
8.7
2.0
8.7
75.5
5.1
TRI-I
13.0
2.8
28.9
47.1
8.2
-2
15.9
2.7
8.4
60.1
13.5
-3
37.8
9.0
14.9
32.7
4.7
aNI and N2 are the f i r s t and second generation controls.
JRI, TRI. and JR2, TR2 are the f i r s t and second generation progeny of regenerants.
N-I, JRI-I and
TRI-I, -2 and -3 are individuals selected for specific comparisons of seed oll FAME.compositions.
remained in culture only 5 months. The additional elements designed to impose genetic transformation on a regeneration system may therefore have contributed to increased variation among regenerated plants. Although the variability observed in R~ populations had been transmitted through one meiotic cycle from the Rn parents, this variability had almost entirely disappeared after passage through one further meiotic cycle to the R? generation. The nature of such unstable epigeBetic effects is not understood, but may be due to altered gene expression (Meins and Binns, 1977; Mermod et al, 1983; Sager and Grabowy, 1983). Although cytological analyses were not made in this study, reversion to normality by the R~ generation, and a lack of distinctive aneuploid ph~notypes, indicate that the regenerants were cytologically normal. Graybosch et a] (1987) also observed l i t t l e variation among soybean plants regenerated by shoot organogenesis from nascent meristematic regions within cultured cotyledonary nodes (Wright et al, ]986). The absence of stable, heritable variations among the progeny of regenerants produced from immature soybean cotyledons by somatic embryogenesis indicates that, while this protocol is unlikely to provide useful numbers of somaclonal variants, i t should be suitable for development of transformation systems using elite genotypes for which culture-induced variability is undesirable. ACKNOWLEDGMENTS We are grateful to Dr. P.A. Lazzeri for provision of regenerant plants, to Jeff Berger, Gary Veach and Pierce Flemming for assistance with fatty acid analyses, to Drs. T. Pfeiffer and W. Parrott for helpful discussions, and to Brenda Hays, Mandy Collins and Ann Kupper for typing the manuscript.
REFERENCES Baertlein DA, McDaniel RG (1987) Theor Appl Genet 73:575-580. Barwa]e UB, Kerns HR, Widho]m JM (1986) Planta 16:473-478.
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