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Influence of 2,4-D, IAA, and duration of callus induction in anther cultures of spring wheat
Plant Science, 90 (1993) 195-200
195
Elsevier Scientific Publishers Ireland Ltd.
Influence of 2,4-D, IAA, and duration of callus induction in anther cultures of spring wheat* S h a n e T. Ball a, H u a P i n g Z h o u b a n d C a l v i n F. K o n z a k c aAsgrow Seed Company, 634 E. Lincolnway, Ames, 1A 50010, bMailzone GG4H, Monsanto Agriculture Group, 700 ChesterfieM Parkway North, St. Louis, MO 63011 and CDepartment of Crop and Soil Science, Washington State University, Pullman, WA 99164 ( USA ) (Received November 30th, 1992; revision received February 9th, 1993; accepted February 17th, 1993)
Culture media and environmental factors may significantly influence the yield of haploid plants from anther cultures. Our objectives were to identify a combination of 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA) concentrations which produce the maximum number of haploid plants, and to evaluate the effects of duration in induction medium on calli induction, plant regeneration, and green plant production from anther cultures in spring wheat. Significant (P _< 0.01) plant growth regulator concentration effects (2,4-D and IAA) were observed on the number of calli, green plants and albino plants produced, and on direct plant regeneration. Addition of 2,4-D to the induction medium resulted in significantly (P _< 0.01) higher means for all anther culture components compared to IAA. While addition of 2,4-D significantly (P _< 0.01) reduced plant regeneration, it substantially increased green plant percentage at a 0.3-mg 1-l concentration of IAA. Use of response functions to estimate the maximum effective 2,4-D x IAA combination implied that higher 2,4-D levels in the induction medium should be investigated, and that the optimum hormone combination differs for plant regeneration and green plant percentage. Significant (P < 0.01) effects of duration on callus induction medium were observed for plant regeneration and green plant percentage.
Key words: Triticum aestivum; environmental factors; plant breeding; plant growth regulators; response functions
Introduction
Doubled haploid plants are unique materials for genetic studies and plant breeding. However, the yields of haploid plants from cereal crops often are too low for many practical applications. Genetic factors are considered to be the major components responsible for differences in haploid yields [1,2]. Both nuclear and cytoplasmic genes and their interactions are known to influence the production of haploid plants from anther cultures [3-5]. However, prior experiments have shown that environmental factors also may markedly effect the production of haploid plants. These environmental factors include: (i) growth conditions Correspondence to: Shane T. Ball, Asgrow Seed Company, 634 E. Lincolnway, Ames, IA 50010, USA. *College of Agriculture and Home Economics Research Center Project No. 3571.
of'donor plants [5]; (ii) pretreatment of anthers [6]; (iii) the type and concentration of carbon source [7,8]; (iv) chemical and physical properties of culture media [9,10]; and, (v) culture conditions during callus induction and plant regeneration [2]. The type and content of plant growth regulators in media have been recognized as important factors in the production of haploid plants from anther culture [11-13]. Auxins are essential growth regulator components for callus induction in anther culture [14]. The pathways of microspore development in anther culture are regulated by the type and concentration of auxins present in the induction medium. Indoleacetic acid (IAA) may induce direct embryogenesis, whereas 2,4dichlorophenoxyacetic acid (2,4-D) favors rapid cell proliferation and callus formation [15]. When 2,4-D is replaced by naphthaleneacetic acid (NAA) in induction media, haploid plants are pro-
0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
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duced directly in the induction medium without transfer to a regeneration medium [16]. Phenylacetic acid (PAA) was recently identified as an effective auxin for increasing green plant production in barley and wheat anther cultures compared to 2,4D [6]. Auxins also affect the yield of haploid plants in addition to their influence on androgenic pathways [6,16]. The objectives of this study were to identify a combination of 2,4-D and IAA concentrations needed to produce the maximum number of haploid plants, and to evaluate the effects of duration of callus induction on plant regeneration. Materials and Methods
Spring wheat cultivar Pavon 76 was used, because it is one of the most responsive genotypes to anther culture facilitating production of significant numbers of calli and regenerates [7]. Plants were grown in a greenhouse under a 16-h per day photoperiod. The light intensity was 300 /zmol m -2 s -l supplemented with high-pressure sodium lights. Day/night temperatures were approximately 25/15°C. Two to three primary spikes from each plant were sampled when the microspores from the anthers in the central portion of the spikes were at the mid- to late-uninucleate stage. The sampled spikes were placed in flasks containing tap water and stored in a dark refrigerator at 4°C for 2 days. Only those anthers considered to have microspores at the uninucleate stage were excised with fine forceps, after the spikes were surface-sterilized with 800 ml 1-1 ethanol, and plated aseptically on a liquid induction medium. A modified liquid potato 4 (P4) induction medium [17] supplemented with 0.5 mg 1-1 kinetin and 90 g 1-1 sucrose was used. Different levels of 2,4D and IAA were added to the induction medium (in addition to the cytokinin and kinetin) as auxin treatments. Calli induced from the cultured anthers were transferred to a 190-2 semi-solid regeneration medium [2] when they reached 1-2 mm in diameter. The regeneration medium contained 30 g 1-l sucrose and 2.5 g I -l Gelrite gellan gum. No plant growth regulator was included in the regeneration medium. Transfer of the calli to regeneration media was
performed temporally in three groups. The calli were transferred at weekly intervals when they reached 1-2 mm which occurred between 30 and 44 days after anther plating. Calli were cultured at room temperature with 100 tzmol m -2 s -1 of illumination provided by fluorescent lights in a 16-h per day photoperiod. After 30 days of regeneration culture, the number of calli that developed into green or albino plants was recorded. Callus induction was expressed as the number of calli obtained from 100 cultured anthers. Plant regeneration was expressed as a percentage based on the plant number per 100 calli transferred. Plantlets directly regenerated from the induction media were also recorded. The green plant percentage was the number of calli producing green plants divided by the total calli producing either green or albino plants. Experiment 1 was designed to compare the effect of 2,4-D and IAA at four exponential concentrations (0.01, 0.1, 1, and 10 mg 1-1) on anther culture response. Two additional concentrations comparable to standard anther culture applications (0.5 and 2 mg l -l) also were included. Anther culture responses were evaluated using a nested randomized complete block design with four individual petri dishes as replicates. Experiment 2 was designed to examine the effect of four 2,4-D concentrations (0.15, 0.30, 0.60, and 1.20 mg 1-l) and four IAA concentrations (0.30, 0.60, 1.20, and 2.40 mg 1-1) on callus induction, plant regeneration and green plant percentage. The concentrations were chosen from the results of anther culture response in experiment 1. Anther culture response components were evaluated using a two factor experiment arranged in a randomized complete-block design with six individual petri dishes as replicates. The effects of 2,4-D and IAA concentrations were expressed as deviations around their means. Large variations among the treatments were detected and the corrective measure employed was a natural logarithmic [Yijk = ln(Yijk + 1)] transformation [18]. After analysis of the data, the treatment means were converted from natural logarithmic values using the exponential function. The data from experiment 2 were also analyzed for duration of calli growth on the induction
197 than did IAA concentrations (Table II). Exceptions were albino production at the 10-mg 1-1 2,4D level and the direct haploid plant induction numbers for the two plant growth regulators. Direct production of plants was obtained only from the low-level 2,4-D additions to the induction media. On average, the number of calli was about 56, green plants about 10, and albinos about 17 per 100 anthers higher for 2,4-D than for IAA additions. Although the 0.01- and 10-mg 1-1 concentrations of 2,4-D were less productive, no other response pattern was detected from increasing 2,4D and IAA concentrations in the media. However, the IAA compared to 2,4-D additions resulted in means with larger standard errors. The mean squares from the ANOVA for the natural-log transformation of anther culture components showed significant (P < 0.01) differences among 2,4-D concentrations for plant regenerations and green plant percentages in experiment 2 (Table III). Although the IAA effect was significant (P _< 0.05) for green plant percentage, nonsignificant interaction effects were observed for the other anther culture components. Addition of 2,4-D to the induction medium significantly (P ___ 0.01) reduced plant regeneration, and the linear response (r2= 0.97) was inversely related to 2,4-D concentration within the range o f 0.15-1.2 mg 1-l (Fig. 1). Over 42 plants were regenerated per 100 calli from the induction medium containing 0.15 mg 1-1 2,4-D, but below 30 plants were produced by calli from a similar medium containing 1.2 mg 1-l 2,4-D. In contrast to the plant regeneration data, the F-
media. The analysis of variance was partitioned to examine the effects of dates that calli were transferred to regeneration media (30, 37 and 42 days) on plant regeneration and green plant percentage. The anther culture components were evaluated using a single-factor experiment in a randomized complete-block design with six individual petri dishes as replicates. The least square estimators of the parameters in the model, the predicted values, the residuals, sum of squares, and mean squares were obtained using the GLM procedure of SAS/PC [19]. Individual response functions for 2,4-D and IAA concentrations were fitted to polynomials of successively higher order up to the third power in experiment 2. Resolving whether the cubic and quadratic terms could be dropped from the model was determined by partial F-tests (P _< 0.05). The decision whether to use separate regression lines for each IAA concentration was determined by the F-tests (P _< 0.05) from the analysis of variance. Thus, regression lines and their coefficients were derived from the best-fit equations for callus induction, plant regeneration, and green plant percentage. Results
Mean squares from the analysis of variance (ANOVA) showed that the concentrations of 2,4D and IAA had significant (P _< 0.01) influences on anther culture response components (Table I). However, addition of 2,4-D to the induction media generally resulted in significantly (P < 0.01) higher means for all anther culture components
Table I. Meansquares from the analysis of variance for a study of plant growth regulators (PGR), 2,4-D and IAA, in anther culture of wheat in experiment 1 Source of variation
Replication Concentration within PGR Error
df
3 11 32
*Significanceat the 0.05 level of probability. **Significanceat the 0.01 level of probability.
Mean squares Callus
Green plant
Albino
Direct induction
1363" 3090** 391
30 187"* 13
4 321"* 17
1.5 2.5** 0.5
198 Table II. Culture response ( 4- S.E.) of Pavon 76 anthers to different concentrations of 2,4-D and IAA containing induction media in experiment 1. PGR
Conc. (mg 1-1)
Callus
Green plants
2,4-D
0.01 0.1 0.5 1.0 2.0 10.0
78.8 100.7 93.8 91.2 121.8 61.2
± 4± 444-
7.0 10.2 15.7 4.5 13.1 6.8
3.5 14.8 17.0 15.2 19.3 2.3
IAA
0.01 0.1 0.5 1.0 2.0 10.0
42.7 31.7 41.5 51.0 34.3 52.5
+ + 4444-
16.3 7.3 7.1 17.5 4.8 1.7
1.8 1.2 1.5 3.0 2.3 2.5
tests for green plant percentage indicated that a single (0.3 mg 1-1) concentration of IAA had significant (P _< 0.01) effects, while regressions of the higher concentrations (0.5, 1.2, and 2.4 mg 1-1) were nonsignificant. Therefore, a regression analysis at the 0.3 mg 1-1 IAA concentration only was appropriate and used in this experiment. The addition of 2,4-D to the induction medium significantly (P ___ 0.01) increased green plant percentage, and the linear increase was directly related to the concentration of 2,4-D added to the medium (Fig. 1). Over 44 green plants were produced from 100 regenerates of calli induced in the medium containing 1.2 mg 1-l 2,4-D, but below 29 in a medium containing 0.15 mg 1-1 2,4-D. The increment of increase in green plants was proportional to the
444444-
Albino plants
Plants from induction media
1.3 3.6 3.8 1.0 1.4 0.4
2.7 23.2 22.8 15.8 15.6
444440
0.6 2.9 4.6 2.3 4.0
2.3 4- 0.6 0 0 0 0 0
+ 0.9 -4- 0.5 4- 0.5 4- 2.1 4- 0.6 4- 0.4
1.1 1.5 2.0 1.3 0.7 3.7
+ 44444-
0.4 0.9 1.0 0.3 0.3 0.6
1.0 0.3 0.8 1.7 0.5 1.3
± 44444-
0.6 0.2 0.3 1.0 0.2 0.3
concentration of 2,4-D added to the induction medium. Highly significant (P ___ 0.01) differences among duration of callus induction were observed for plant regeneration and green plant percentage (Table IV). A single degree of freedom contrast of dates 1 and 2 vs. date 3 explained most of the variability in plant regeneration. On the other hand, the contrast of date 1 vs. dates 2 and 3 explained most of variability in green plant percentage. Variable means of the original data showed a pattern consistent with the mean squares. Both the means and ANOVA results indicated that the optimum callus duration in the induction medium varied for plant regeneration and green plant percentage.
Table III. Mean squares from the analysis of variance for 2,4-D and IAA levels in anther culture of wheat in experiment 2 (presented in the natural-log scale) Source of variation
Replication 2,4-D IAA 2,4-D × IAA Error
df
5 3 3 9 75
*Significance at the 0.05 level of probability. **Significance at the 0.01 level of probability.
Mean squares Callus induction
Plant regeneration
Green plant percentage
0.315"* 0.062 0.147 0.105 0.075
0.098** 0.763** 0.007 0.035 0.032
0.626** 1.131"* 0.553* 0.197 0.148
199
8
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40
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40
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o
32t
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30
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Discussion
.... . . o 4 4
"~-28 Z
12, ~:
'o13 'o.45' 'o'.6' 'o,7~' 'o19' '1.65' '112 2,4-D CONCENTRATION (mg I-1)
Fig, 1. Responsefunction and sample means of 2,4-D concentrations (mg I-l) plant regeneration (i) and green plant percentage (El) in experiment 2. Data are presented in the original scale. The regression lines were obtained from best-fit (P _< 0.05) equations for plant regeneration (Y = 44.9-12.0X, r 2=0.97.* and n=96) and green plant percentage (Y = 24.4 + 16.4X, r 2 = 0.99** and n = 24).
Table IV. Variablemeans and mean squares from an analysis of variance across transfer dates in experiment 2. (a) Variable means (original data)t Date of callus transfer
Plant regeneration
Green plant percentage~t
1 2 3
52a 45b 17c
36a 24b 22b
(b) Mean squares (logarithmic scale) Source of variation
df
Plant regeneration
Green plant percentage
Replication Date Dates I & 2 vs. date 3 D a t e 1 vs. dates 2 & 3 Error
5 2 1 1 280
0.65** 32.83** 64.56**
3.85** 9.84** -19.28"* 0.96
0.12
**Denote significance at the 0.01 level of probability. 1-Date 1 refers to the first transfer of calli from induction to regeneration media. Dates 2 and 3 refer to subsequent transfers of calli 7 and 14 days after the initial transfer. ~Within columns, means followed by the same letter are not significantly different by LS.D. test (P _< 0.05).
We concluded from the F-tests that an analysis o f plant growth regulator ( P G R ) levels was needed (Table I). These results also indicated that a combined study o f 2,4-D and I A A concentrations was warranted to estimate the combination o f P G R s needed to produce a m a x i m u m yield o f plants from anther culture (Table II). Nonsignificant F-values for callus induction suggested the addition o f 2,4-D and I A A P G R s to the induction media had little effect on these anther culture components (Table III). In addition, significant (P _< 0.01) replication effects were observed, suggesting the need for increased population size in future anther culture experiments or more r a n d o m distribution o f anthers in the petri dish replicates. A preliminary statistical analysis established that a single regression analysis (averaged across I A A concentrations) was appropriate for the plant regeneration data (Table III). The response function showed clearly that plant regeneration was inversely related to 2,4-D concentration in the induction media (Fig. 1). In addition, results suggested that higher 2,4-D levels, in combination with I A A , should be investigated in future experiments. In most anther culture experiments, calli are generally transferred temporally from induction to regeneration media in groups, and the resulting data are subsequently bulked before statistical analyses [17]. However, this practice m a y ignore an important source o f variability researchers can measure and adjust to improve anther culture responses. The results suggest that the regeneration ability of calli is maintained for a longer period over which calli are induced than is their chlorophyllforming ability. More detailed analyses o f calli produced over time and from individual anthers may be needed to understand the basis for the duration of callus induction effects. Results from this study suggested that wheat anther culture can be improved by culture medium modifications. The o p t i m u m 2,4-D and I A A combination, estimated for plant regeneration by polynomial response functions m a y not be opti-
200
mum for green plant production. In addition, these functions indicate that future experiments should include higher 2,4-D concentrations to identify the maximum production of haploid plants and the pathway of plant regeneration. Reduced green plant yields with time also suggested that the currently used procedure of transferring calli to regeneration medium at weekly intervals may not be ideal for the production of green plants. Therefore, adjustments to media and callus transfer procedures may help to optimize the efficiency of culture methods.
Acknowledgements We value the contributions of F.M. Lu for her technical assistance in managing the cultures and collecting the data.
References 1
C.A. Quimio and J. Zapata, Diallel analysis of callus induction and green plant regeneration in rice anther culture. Crop Sci., 30 (1990) 188-192. 2 H. Zhou and C.F. Konzak, Improvement of anther culture methods for haploid production in wheat (T. aestivum L.). Crop Sci., 29 (1989) 817-821. 3 H. Ekiz and C.F. Konzak, Nuclear and cytoplasmic control of anther culture response in wheat. III. Common wheat crosses. Crop Sci., 31 (1991) 1432-1436. 4 E.T. Larsen, I.K.D. Tuvesson and S.B. Andersen, Nuclear genes affecting percentage of green plants in barley (Hordeum vulgate L.) anther culture. Theor. Appl. Genet., 82 (1991) 417-420. 5 M.D. Lazar, G.W. Schaeffer and P.S. Baenziger, Cultivar and cultivar × environment effects on the development of callus and polyhaploid plants from anther cultures of wheat. Theor. Appl. Genet., 67 (1984) 273-277. 6 A. Ziauddin, E. Simion and K.J. Kasha, Improved plant regeneration from shed microspore culture in barley
(Hordeum vulgare L.) cv. igri. Plant Cell Rep., 9 (1991) 69-72. 7 S.T. Ball, H. Zhou and C.F. Konzak, Sucrose concentration and its relationship to anther culture in wheat. Crop Sci., 32 (1992) 149-154. 8 B.R. Orshinsky, L.J. McGregor, G.I.E. Johnson, P. Hucl and K.K. Kartha, Improved embryoid induction and green shoot regeneration from wheat anthers cultured in medium with maltose. Plant Cell Rep., 9 (1990) 365-369. 9 A.M. Jones and J.F. Petolino, Effects of support medium on embryo and plant production from cultured anthers of soft-red winter wheat (Triticum aestivum L.). Plant Cell Tissue Organ Cult., 12 (1988) 253-261. 10 H. Zhou, Y. Zheng and C.F. Konzak, Several medium components affecting albinism in wheat anther culture. Plant Cell Rep., 10 (1991) 63-66. 11 T.L. Reynolds, A possible role for ethylene during IAAinduced pollen embryogenesis in anther cultures of Solanum carolinense L. Am. J. Bot., 74 (1987) 967-969. 12 L.B. Torrizo and F.J. Zapata, Anther culture in rice. IV. The effect of abscisic acid on plant regeneration. Plant Cell Rep., 5 (1986) 136-141. 13 I.K.D. Tuvesson, S. Peterson and S.B. Andersen, Nuclear genes affecting albinism in wheat (Triticum aestivum L.) anther culture. Theor. Appl. Genet., 78 (1989) 879-883. 14 Y.C. Chien and K.N. Kao, Effects of osmolality, cytokinin and organic acids on pollen callus formation in triticale anthers. Can. J. Bot., 61 (1983) 639-641. 15 T.A. Armstrong, S.G. Metz and P.N. Mascia, Two regeneration systems for the production of haploid plants from wheat anther culture. Plant Sci., 51 (1987) 231-237. 16 G.H. Liang, A. Xu and H. Tang, Direct generation of wheat haploids via anther culture. Crop Sci., 27 (1987) 336-339. 17 C.F. Konzak and H. Zhou, Anther culture methods for doubled haploid production in wheat. Cereal Res. Commun., 19 (1991) 147-164. 18 J. Neter, W. Wasserman and M. H. Kutner, Applied Linear Statistical Models. Regression, Analysis of Variance and Experimental Designs. 3rd ed. R.D. Irwin, Inc., Homewood, IL, 1990, pp. 911-940. 19 SAS Institute., SAS/STAT User's Guide, Release 6.03 edition. SAS Institute Inc., Cary, NC, 1988.
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Influence of 2,4-D, IAA, and duration of callus induction in anther cultures of spring wheat* S h a n e T. Ball a, H u a P i n g Z h o u b a n d C a l v i n F. K o n z a k c aAsgrow Seed Company, 634 E. Lincolnway, Ames, 1A 50010, bMailzone GG4H, Monsanto Agriculture Group, 700 ChesterfieM Parkway North, St. Louis, MO 63011 and CDepartment of Crop and Soil Science, Washington State University, Pullman, WA 99164 ( USA ) (Received November 30th, 1992; revision received February 9th, 1993; accepted February 17th, 1993)
Culture media and environmental factors may significantly influence the yield of haploid plants from anther cultures. Our objectives were to identify a combination of 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA) concentrations which produce the maximum number of haploid plants, and to evaluate the effects of duration in induction medium on calli induction, plant regeneration, and green plant production from anther cultures in spring wheat. Significant (P _< 0.01) plant growth regulator concentration effects (2,4-D and IAA) were observed on the number of calli, green plants and albino plants produced, and on direct plant regeneration. Addition of 2,4-D to the induction medium resulted in significantly (P _< 0.01) higher means for all anther culture components compared to IAA. While addition of 2,4-D significantly (P _< 0.01) reduced plant regeneration, it substantially increased green plant percentage at a 0.3-mg 1-l concentration of IAA. Use of response functions to estimate the maximum effective 2,4-D x IAA combination implied that higher 2,4-D levels in the induction medium should be investigated, and that the optimum hormone combination differs for plant regeneration and green plant percentage. Significant (P < 0.01) effects of duration on callus induction medium were observed for plant regeneration and green plant percentage.
Key words: Triticum aestivum; environmental factors; plant breeding; plant growth regulators; response functions
Introduction
Doubled haploid plants are unique materials for genetic studies and plant breeding. However, the yields of haploid plants from cereal crops often are too low for many practical applications. Genetic factors are considered to be the major components responsible for differences in haploid yields [1,2]. Both nuclear and cytoplasmic genes and their interactions are known to influence the production of haploid plants from anther cultures [3-5]. However, prior experiments have shown that environmental factors also may markedly effect the production of haploid plants. These environmental factors include: (i) growth conditions Correspondence to: Shane T. Ball, Asgrow Seed Company, 634 E. Lincolnway, Ames, IA 50010, USA. *College of Agriculture and Home Economics Research Center Project No. 3571.
of'donor plants [5]; (ii) pretreatment of anthers [6]; (iii) the type and concentration of carbon source [7,8]; (iv) chemical and physical properties of culture media [9,10]; and, (v) culture conditions during callus induction and plant regeneration [2]. The type and content of plant growth regulators in media have been recognized as important factors in the production of haploid plants from anther culture [11-13]. Auxins are essential growth regulator components for callus induction in anther culture [14]. The pathways of microspore development in anther culture are regulated by the type and concentration of auxins present in the induction medium. Indoleacetic acid (IAA) may induce direct embryogenesis, whereas 2,4dichlorophenoxyacetic acid (2,4-D) favors rapid cell proliferation and callus formation [15]. When 2,4-D is replaced by naphthaleneacetic acid (NAA) in induction media, haploid plants are pro-
0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
196
duced directly in the induction medium without transfer to a regeneration medium [16]. Phenylacetic acid (PAA) was recently identified as an effective auxin for increasing green plant production in barley and wheat anther cultures compared to 2,4D [6]. Auxins also affect the yield of haploid plants in addition to their influence on androgenic pathways [6,16]. The objectives of this study were to identify a combination of 2,4-D and IAA concentrations needed to produce the maximum number of haploid plants, and to evaluate the effects of duration of callus induction on plant regeneration. Materials and Methods
Spring wheat cultivar Pavon 76 was used, because it is one of the most responsive genotypes to anther culture facilitating production of significant numbers of calli and regenerates [7]. Plants were grown in a greenhouse under a 16-h per day photoperiod. The light intensity was 300 /zmol m -2 s -l supplemented with high-pressure sodium lights. Day/night temperatures were approximately 25/15°C. Two to three primary spikes from each plant were sampled when the microspores from the anthers in the central portion of the spikes were at the mid- to late-uninucleate stage. The sampled spikes were placed in flasks containing tap water and stored in a dark refrigerator at 4°C for 2 days. Only those anthers considered to have microspores at the uninucleate stage were excised with fine forceps, after the spikes were surface-sterilized with 800 ml 1-1 ethanol, and plated aseptically on a liquid induction medium. A modified liquid potato 4 (P4) induction medium [17] supplemented with 0.5 mg 1-1 kinetin and 90 g 1-1 sucrose was used. Different levels of 2,4D and IAA were added to the induction medium (in addition to the cytokinin and kinetin) as auxin treatments. Calli induced from the cultured anthers were transferred to a 190-2 semi-solid regeneration medium [2] when they reached 1-2 mm in diameter. The regeneration medium contained 30 g 1-l sucrose and 2.5 g I -l Gelrite gellan gum. No plant growth regulator was included in the regeneration medium. Transfer of the calli to regeneration media was
performed temporally in three groups. The calli were transferred at weekly intervals when they reached 1-2 mm which occurred between 30 and 44 days after anther plating. Calli were cultured at room temperature with 100 tzmol m -2 s -1 of illumination provided by fluorescent lights in a 16-h per day photoperiod. After 30 days of regeneration culture, the number of calli that developed into green or albino plants was recorded. Callus induction was expressed as the number of calli obtained from 100 cultured anthers. Plant regeneration was expressed as a percentage based on the plant number per 100 calli transferred. Plantlets directly regenerated from the induction media were also recorded. The green plant percentage was the number of calli producing green plants divided by the total calli producing either green or albino plants. Experiment 1 was designed to compare the effect of 2,4-D and IAA at four exponential concentrations (0.01, 0.1, 1, and 10 mg 1-1) on anther culture response. Two additional concentrations comparable to standard anther culture applications (0.5 and 2 mg l -l) also were included. Anther culture responses were evaluated using a nested randomized complete block design with four individual petri dishes as replicates. Experiment 2 was designed to examine the effect of four 2,4-D concentrations (0.15, 0.30, 0.60, and 1.20 mg 1-l) and four IAA concentrations (0.30, 0.60, 1.20, and 2.40 mg 1-1) on callus induction, plant regeneration and green plant percentage. The concentrations were chosen from the results of anther culture response in experiment 1. Anther culture response components were evaluated using a two factor experiment arranged in a randomized complete-block design with six individual petri dishes as replicates. The effects of 2,4-D and IAA concentrations were expressed as deviations around their means. Large variations among the treatments were detected and the corrective measure employed was a natural logarithmic [Yijk = ln(Yijk + 1)] transformation [18]. After analysis of the data, the treatment means were converted from natural logarithmic values using the exponential function. The data from experiment 2 were also analyzed for duration of calli growth on the induction
197 than did IAA concentrations (Table II). Exceptions were albino production at the 10-mg 1-1 2,4D level and the direct haploid plant induction numbers for the two plant growth regulators. Direct production of plants was obtained only from the low-level 2,4-D additions to the induction media. On average, the number of calli was about 56, green plants about 10, and albinos about 17 per 100 anthers higher for 2,4-D than for IAA additions. Although the 0.01- and 10-mg 1-1 concentrations of 2,4-D were less productive, no other response pattern was detected from increasing 2,4D and IAA concentrations in the media. However, the IAA compared to 2,4-D additions resulted in means with larger standard errors. The mean squares from the ANOVA for the natural-log transformation of anther culture components showed significant (P < 0.01) differences among 2,4-D concentrations for plant regenerations and green plant percentages in experiment 2 (Table III). Although the IAA effect was significant (P _< 0.05) for green plant percentage, nonsignificant interaction effects were observed for the other anther culture components. Addition of 2,4-D to the induction medium significantly (P ___ 0.01) reduced plant regeneration, and the linear response (r2= 0.97) was inversely related to 2,4-D concentration within the range o f 0.15-1.2 mg 1-l (Fig. 1). Over 42 plants were regenerated per 100 calli from the induction medium containing 0.15 mg 1-1 2,4-D, but below 30 plants were produced by calli from a similar medium containing 1.2 mg 1-l 2,4-D. In contrast to the plant regeneration data, the F-
media. The analysis of variance was partitioned to examine the effects of dates that calli were transferred to regeneration media (30, 37 and 42 days) on plant regeneration and green plant percentage. The anther culture components were evaluated using a single-factor experiment in a randomized complete-block design with six individual petri dishes as replicates. The least square estimators of the parameters in the model, the predicted values, the residuals, sum of squares, and mean squares were obtained using the GLM procedure of SAS/PC [19]. Individual response functions for 2,4-D and IAA concentrations were fitted to polynomials of successively higher order up to the third power in experiment 2. Resolving whether the cubic and quadratic terms could be dropped from the model was determined by partial F-tests (P _< 0.05). The decision whether to use separate regression lines for each IAA concentration was determined by the F-tests (P _< 0.05) from the analysis of variance. Thus, regression lines and their coefficients were derived from the best-fit equations for callus induction, plant regeneration, and green plant percentage. Results
Mean squares from the analysis of variance (ANOVA) showed that the concentrations of 2,4D and IAA had significant (P _< 0.01) influences on anther culture response components (Table I). However, addition of 2,4-D to the induction media generally resulted in significantly (P < 0.01) higher means for all anther culture components
Table I. Meansquares from the analysis of variance for a study of plant growth regulators (PGR), 2,4-D and IAA, in anther culture of wheat in experiment 1 Source of variation
Replication Concentration within PGR Error
df
3 11 32
*Significanceat the 0.05 level of probability. **Significanceat the 0.01 level of probability.
Mean squares Callus
Green plant
Albino
Direct induction
1363" 3090** 391
30 187"* 13
4 321"* 17
1.5 2.5** 0.5
198 Table II. Culture response ( 4- S.E.) of Pavon 76 anthers to different concentrations of 2,4-D and IAA containing induction media in experiment 1. PGR
Conc. (mg 1-1)
Callus
Green plants
2,4-D
0.01 0.1 0.5 1.0 2.0 10.0
78.8 100.7 93.8 91.2 121.8 61.2
± 4± 444-
7.0 10.2 15.7 4.5 13.1 6.8
3.5 14.8 17.0 15.2 19.3 2.3
IAA
0.01 0.1 0.5 1.0 2.0 10.0
42.7 31.7 41.5 51.0 34.3 52.5
+ + 4444-
16.3 7.3 7.1 17.5 4.8 1.7
1.8 1.2 1.5 3.0 2.3 2.5
tests for green plant percentage indicated that a single (0.3 mg 1-1) concentration of IAA had significant (P _< 0.01) effects, while regressions of the higher concentrations (0.5, 1.2, and 2.4 mg 1-1) were nonsignificant. Therefore, a regression analysis at the 0.3 mg 1-1 IAA concentration only was appropriate and used in this experiment. The addition of 2,4-D to the induction medium significantly (P ___ 0.01) increased green plant percentage, and the linear increase was directly related to the concentration of 2,4-D added to the medium (Fig. 1). Over 44 green plants were produced from 100 regenerates of calli induced in the medium containing 1.2 mg 1-l 2,4-D, but below 29 in a medium containing 0.15 mg 1-1 2,4-D. The increment of increase in green plants was proportional to the
444444-
Albino plants
Plants from induction media
1.3 3.6 3.8 1.0 1.4 0.4
2.7 23.2 22.8 15.8 15.6
444440
0.6 2.9 4.6 2.3 4.0
2.3 4- 0.6 0 0 0 0 0
+ 0.9 -4- 0.5 4- 0.5 4- 2.1 4- 0.6 4- 0.4
1.1 1.5 2.0 1.3 0.7 3.7
+ 44444-
0.4 0.9 1.0 0.3 0.3 0.6
1.0 0.3 0.8 1.7 0.5 1.3
± 44444-
0.6 0.2 0.3 1.0 0.2 0.3
concentration of 2,4-D added to the induction medium. Highly significant (P ___ 0.01) differences among duration of callus induction were observed for plant regeneration and green plant percentage (Table IV). A single degree of freedom contrast of dates 1 and 2 vs. date 3 explained most of the variability in plant regeneration. On the other hand, the contrast of date 1 vs. dates 2 and 3 explained most of variability in green plant percentage. Variable means of the original data showed a pattern consistent with the mean squares. Both the means and ANOVA results indicated that the optimum callus duration in the induction medium varied for plant regeneration and green plant percentage.
Table III. Mean squares from the analysis of variance for 2,4-D and IAA levels in anther culture of wheat in experiment 2 (presented in the natural-log scale) Source of variation
Replication 2,4-D IAA 2,4-D × IAA Error
df
5 3 3 9 75
*Significance at the 0.05 level of probability. **Significance at the 0.01 level of probability.
Mean squares Callus induction
Plant regeneration
Green plant percentage
0.315"* 0.062 0.147 0.105 0.075
0.098** 0.763** 0.007 0.035 0.032
0.626** 1.131"* 0.553* 0.197 0.148
199
8
,,!
°!
40
i
Z/,"
,/""
40
~-
Z
o
32t
,'"
..,/"'""'/"
30
30!~/, "/'D
~ 28o.~5....
Discussion
.... . . o 4 4
"~-28 Z
12, ~:
'o13 'o.45' 'o'.6' 'o,7~' 'o19' '1.65' '112 2,4-D CONCENTRATION (mg I-1)
Fig, 1. Responsefunction and sample means of 2,4-D concentrations (mg I-l) plant regeneration (i) and green plant percentage (El) in experiment 2. Data are presented in the original scale. The regression lines were obtained from best-fit (P _< 0.05) equations for plant regeneration (Y = 44.9-12.0X, r 2=0.97.* and n=96) and green plant percentage (Y = 24.4 + 16.4X, r 2 = 0.99** and n = 24).
Table IV. Variablemeans and mean squares from an analysis of variance across transfer dates in experiment 2. (a) Variable means (original data)t Date of callus transfer
Plant regeneration
Green plant percentage~t
1 2 3
52a 45b 17c
36a 24b 22b
(b) Mean squares (logarithmic scale) Source of variation
df
Plant regeneration
Green plant percentage
Replication Date Dates I & 2 vs. date 3 D a t e 1 vs. dates 2 & 3 Error
5 2 1 1 280
0.65** 32.83** 64.56**
3.85** 9.84** -19.28"* 0.96
0.12
**Denote significance at the 0.01 level of probability. 1-Date 1 refers to the first transfer of calli from induction to regeneration media. Dates 2 and 3 refer to subsequent transfers of calli 7 and 14 days after the initial transfer. ~Within columns, means followed by the same letter are not significantly different by LS.D. test (P _< 0.05).
We concluded from the F-tests that an analysis o f plant growth regulator ( P G R ) levels was needed (Table I). These results also indicated that a combined study o f 2,4-D and I A A concentrations was warranted to estimate the combination o f P G R s needed to produce a m a x i m u m yield o f plants from anther culture (Table II). Nonsignificant F-values for callus induction suggested the addition o f 2,4-D and I A A P G R s to the induction media had little effect on these anther culture components (Table III). In addition, significant (P _< 0.01) replication effects were observed, suggesting the need for increased population size in future anther culture experiments or more r a n d o m distribution o f anthers in the petri dish replicates. A preliminary statistical analysis established that a single regression analysis (averaged across I A A concentrations) was appropriate for the plant regeneration data (Table III). The response function showed clearly that plant regeneration was inversely related to 2,4-D concentration in the induction media (Fig. 1). In addition, results suggested that higher 2,4-D levels, in combination with I A A , should be investigated in future experiments. In most anther culture experiments, calli are generally transferred temporally from induction to regeneration media in groups, and the resulting data are subsequently bulked before statistical analyses [17]. However, this practice m a y ignore an important source o f variability researchers can measure and adjust to improve anther culture responses. The results suggest that the regeneration ability of calli is maintained for a longer period over which calli are induced than is their chlorophyllforming ability. More detailed analyses o f calli produced over time and from individual anthers may be needed to understand the basis for the duration of callus induction effects. Results from this study suggested that wheat anther culture can be improved by culture medium modifications. The o p t i m u m 2,4-D and I A A combination, estimated for plant regeneration by polynomial response functions m a y not be opti-
200
mum for green plant production. In addition, these functions indicate that future experiments should include higher 2,4-D concentrations to identify the maximum production of haploid plants and the pathway of plant regeneration. Reduced green plant yields with time also suggested that the currently used procedure of transferring calli to regeneration medium at weekly intervals may not be ideal for the production of green plants. Therefore, adjustments to media and callus transfer procedures may help to optimize the efficiency of culture methods.
Acknowledgements We value the contributions of F.M. Lu for her technical assistance in managing the cultures and collecting the data.
References 1
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