The effect of auxin-like plant growth regulators and osmotic regulation on induction of somatic embryogenesis from elite maize inbreds

Plant Science, 52 (1987) 81-89 Elsevier Scientific Publishers Ireland Ltd.

81

THE E F F E C T OF A U X I N - L I K E P L A N T GROWTH R E G U L A T O R S A N D OSMOTIC R E G U L A T I O N ON I N D U C T I O N OF SOMATIC E M B R Y O G E N E S I S FROM ELITE MAIZE INBREDS

K.R. CLOSE* and L.A. LUDEMAN** Sungene Technologies Corp., 3330 Hillview Avenue., Palo Alto, CA 94304 (U.S.A.)

(Received December 24th, 1986) (Revision received April 29th, 1987) (Accepted May 6th, 1987) Fourteen public maize (Zea mays L.) inbreds were evaluated for their ability to undergo somatic enbryogenesis. Immature embryos were placed onto solid medium containing 5-20/~M of either 2,4-dichlorophenoxyacetic acid (2,4-D) or one of seven chlorinated benzoic acid derivatives at various concentrations of sucrose with or without abscisic acid (ABA). When 2,4-D was used as the sole growth regulator, there was a pronounced genotypic effect on culture induction. When any of the benzoic acid derivatives were substituted for 2,4-D, the genotypic effect was not as evident. The maximum frequency of somatic embryo formation under optimum conditions was in excess of 70% for most genotypes. In addition to the growth regulator-genotype interactions observed, each genotype was found to have a specific osmotic requirement for obtaining the maximum culture induction frequency. This requirement was fulfilled by high levels of sucrose (9-12%) or combinations of sucrose and mannitol. Inclusion of ABA in the culture medium had an effect on the induction of embryogenesis similar to that produced by high sucrose concentrations. Key words: abscisic acid; somatic embryogenesis; maize; osmotic; plant growth regulator; structure-activity relationship

Introduction N u m e r o u s r e p o r t s e x i s t of p l a n t r e g e n e r a t i o n from t i s s u e c u l t u r e s of v a r i o u s m a i z e inbred lines via somatic embryogenesis. Most e a r l y r e p o r t s c o n c e n t r a t e d p r i m a r i l y on t h e inb r e d A188 a n d a few o t h e r i n b r e d s w h i c h w e r e f o u n d to be a m e n a b l e to t i s s u e c u l t u r e [ 12,14,15,18,22]. R e c e n t w o r k h a s d e m o n s t r a t e d t h a t o t h e r p u b l i c i n b r e d s a r e a l s o c a p a b l e of somatic embryogenesis, although the reported f r e q u e n c i e s of c u l t u r e i n d u c t i o n f r o m imma-

*Present address: School of Forestry, University of Montana, Missoula, MT 59812, U.S.A. **Present address: UCLA Medical Center, Division of General Surgery, 72-167 CHS, University of California, Los Angeles, CA 90024, U.S.A. Abbreviations: ABA, abscisic acid; 2,4-D, 2,4-dichlorophenoxyacetic acid; MCPA, 2-methyl-4-chlorophenoxyacetic acid; p-CPA, p-chlorophenoxyacetic acid; PGR, plant growth regulator; 2,4,5-T, 2,4,5-trichlorophenoxyacetic acid.

t u r e e m b r y o s a r e e x t r e m e l y l o w for m a n y comm e r c i a l l y i m p o r t a n t m a i z e i n b r e d s [9,10,12,27]. 2 , 4 - D i c h l o r o p h e n o x y a c e t i c (2,4-D) h a s b e e n the most commonly used plant growth regulat o r ( P G R ) in m a i z e t i s s u e c u l t u r e . O t h e r a u x i n l i k e PGRs, p a r t i c u l a r l y a n a l o g s of 2,4-D s u c h as p-chlorophenoxyacetic acid (p-CPA), 2-methyl-4-chlorophenoxyacetic acid (MCPA), a n d 2 , 4 , 5 - t r i c h l o r o p h e n o x y a c e t i c a c i d (2,4,5-T), h a v e a l s o b e e n u s e d w i t h l i m i t e d s u c c e s s [6]. O t h e r t y p e s of a u x i n - l i k e P G R s h a v e b e e n s h o w n to be e f f e c t i v e in t h e i n d u c t i o n of som a t i c e m b r y o g e n e s i s in s u g a r c a n e [17], a n d dicamba (2-methoxy-3,6-dichlorobenzoic acid) h a s b e e n u s e d e x t e n s i v e l y in t i s s u e a n d suspens i o n c u l t u r e of o r c h a r d g r a s s [5,16]. M o r e rec e n t l y , d i c a m b a h a s b e e n u s e d to i n d u c e r e g e n e r a b l e c a l l u s from a l a r g e n u m b e r of m a i z e i n b r e d s , a l t h o u g h t h e f r e q u e n c i e s of callus f o r m a t i o n by d i c a m b a w e r e n o t r e p o r t e d [91. T h e e x t r a c e l l u l a r o s m o t i c e n v i r o n m e n t is

0618-9452/87/$03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

82 also critical for many aspects of embryogenesis. High levels of exogenous sucrose have been found to increase the frequency of somatic embryogenesis in maize [21,26]. Both a high osmoticum and exogenous abscisic acid (ABA) have been shown to promote callus growth and root regeneration in maize tissue cultures [1], and ABA has been reported to promote the normal development of somatic embryos from cultured caraway cells [2]. This report describes the effects of auxinlike PGRs, as well as their interaction with osmotic regulation, on the frequency of somatic embryogenesis from immature embryos of several commercially important maize inbreds. The genotype specificity of response under a variety of culture induction conditions is also discussed.

Materials and methods Immature embryos from greenhouse-grown plants were used as the explant source in all experiments. Seed from maize (Zea mays L.) inbred lines A188, A619, A632, B14, B37, B73, B79, B84, C103, CM105, Mo17, MS71, Oh43, R168, and Va59 was obtained from Holden's Foundation Seeds. Donor plants were maintained under natural daylight supplemented with sodium vapor lamps and cool-white fluorescent lights a s necessary to maintain a minimum daylength of 14 h. Plants were fertilized weekly with a solution of 20-14-14 soluble fertilizer. Temperature was maintained at 25-30°C. All plants were self:pollinated. When the embryos had attained a lengih of 1.0-1.8 mm (approx. 9-12 days post-pollination), they were aseptically excised and placed in petri dishes with the embryo axis in contact with the medium. All media contained N6 salts [4] modified to include 0.025mg/1 CuSO4, 0.025mg/1 CoCl2, and 0.25 mg/1 Na2MoO4, and vitamins (0.5 mg/1 thiamine HC1, 0.1 mg/1 pyridoxine HC1, 0.5 mg/ 1 nicotinic acid, 2.0 mg/1 L-glycine, 100 rag/1 minositol, and 0.25 rag/1 calcium pantothenate). Media were solidified with 0.18% (w/v) Gelrite (Kelco Corp.). All PGRs were tested at a con-

centration of 10~M unless otherwise indicated, and the sucrose concentrations were as shown. When ABA was included in the medium, the concentration was 0.5 ~M unless otherwise indicated. Vitamin solutions were filter-sterilized and added to partially cooled autoclaved media before dispensing into 25 × 100 mm petri dishes. 2-Chlorophenoxyacetic acid and 2,5dichloro-, 2-amino-4-chloro-, 3-amino-4-chloro, 4-amino-2-chloro-, and 5-amino-2-chlorobenzoic acids were obtained from Aldrich Chemical Co.; 3-chlorophenoxyacetic acid was obtained from Alfa Products; 2,5-difluorobenzoic acid was obtained from Cooper Chemical Co.; 2,5dibromo- and 2,5-diiodobenzoic acids were obtained from ICN Pharmaceuticals; 3,5dichloro-4-aminobenzoic acid was obtained from Pfaltz and Bauer; 2,5-dichloro-3-nitro- and 2,3,5-trichlorobenzoic acids were obtained from Trans World Chemicals; 2,5-dichloro-6methoxy- and 2,3,5-trichloro-6-methoxybenzoic acids (dicamba and tricamba, respectively) were obtained from Velsicol Corp.; and 2,4-D, 3-amino-2,5-dichlorobenzoic acid (chloramben): 4-chlorophenoxyacetic acid, and ABA were obtained from Sigma Chemical Co. Cultures were incubated in the dark at 2627°C and scored for response (embryogenesis induction frequency) 3-4 weeks after isolation of immature embryos. The induction frequencies reported were calculated as follows: Induction Frequency = No. of explants which formed embryoids × 100 Total No. of explants Embryogenic tissue was distinguished from non-embryogenic growth of scutellar tissue by the presence of embryoids having a scutellarlike body and coleoptile-like structure. Each experiment was performed two or more times with a minimum of 20 embryos for each treatment in each experiment. All treatments in each experiment were performed in dt/plicate, and all data presented are mean values of all replicates in all experiments.

83

Results and discussion

Comparison of 2,4-D and chloramben B73, a BSSS (Iowa Stiff Stalk Synthetic) derivative, allowed little formation of embryogenic tissue when 2,4-D was used as the PGR, regardless of the sucrose c o n c e n t r a t i o n used (Fig. le). However, callus formation induced by chloramben showed a response p a t t e r n quite different from t hat produced by 2,4-D. There was no response at 3% sucrose. At 6% sucrose and above, culture induction was dependent on the sucrose concentration, with a maximum frequency of 70% at 12% sucrose (Fig. lf). The two Oh43 L a nc a s t er types, MS71 and

2,4-D

Chloramben

b 100 50 A619 o

~

L

~

~

d

c

o~ 100

A632 ~

o

~

Comparison of other 2,5-dichlorobenzoic acid analogs

e

g

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c:

A619, differed in their response to 2,4-D. MS71 showed a high frequency of somatic embryogenesis induction at all sucrose levels with a minor decline at 12% sucrose (Fig. lg), whereas A619 showed an improved response with increased sucrose levels and reached a maximum induction frequency of 52% at 9% sucrose (Fig. la). In contrast, chloramben caused an increase in induction frequency with increasing sucrose levels in the case of MS71, while A619 showed a high frequency of response at all sucrose levels tested with no decline at higher levels (9 or 12%). The maximum response of nearly 100% was at 12% sucrose for both inbreds (Fig. lb and h, respectively). A632, a B14 (BSSS) derivative, showed a low frequency of somatic embryogenesis induction at 3% sucrose and an increase in response frequency with increasing sucrose concent rat i ons in the presence of either PGR. The maximum response frequency was 82% at 9% sucrose with chloramben (Fig. ld), and 47% at 12% sucrose with 2,4-D (Fig. lc). A number of inbreds from diverse genetic backgrounds were also compared for their response to 2,4-D and chloramben at 9% sucrose. Chloramben induced a higher frequency of somatic embryogenesis t han 2,4-D for l0 of the 14 inbreds examined (Fig. 2).

so

B73 o

g

1°i 3

6

9

MS71

12

[Sucrose]

3

6

9

12

(%)

Fig. 1. Comparative frequencies of somatic embryogenesis induced by 10 gM 2,4-D or chloramben at 3, 6, 9, and 12% sucrose in cultures of maize lines A619 (a, b), A632 (c, d), B73 (e, f), and MS71 (g, h).

Six 2,5-dichlorobenzoic acid derivatives other t han chloramben were evaluated for their ability to induce embryogenesis in four maize genotypes (Table I). Chloramben and dicamba were each found to induce embryogenesis at relatively high frequencies in all four genotypes. 2,5-Dichlorobenzoic acid was effective only in B73. 2,3,5-Trichlorobenzoic acid, 3-nitro-2,5-dichlorobenzoic acid, and tricamba (2-methoxy-3,5,6-trichlorobenzoic acid) effectively induced embryogenesis only in MS71. Embryogenic tissue under these culture induction conditions typically consisted of a swollen scutellar-derived mass of tissue from which numerous embryoids arose. Embryoids were fused t oget her in many cases.

84

loo u e4) o" o "

50

C

.2 "o c

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A619

/I1

AS32

B14

B37

B73

B79

B84

C 1 0 3 C M 1 0 5 MS71

,o, 0h43

1

,o, R168

Va59

Genotype F i g . 2. C o m p a r a t i v e e m b r y o g e n e s i s induction frequencies in c u l t u r e s of 14 maize inbreds at 9% sucrose. PGRs used were 10 # M 2,4-D (~t) or c h l o r a m b e n ( I ) .

Comparison of benzoic acid derivatives containing other halogen substitutions in the 2 and 5 ring positions Benzoic acid derivatives containing either fluorine, chlorine, bromine, or iodine in the 2 and 5 ring positions were also evaluated under

similar conditions for PGR activity in vitro (Fig. 3). Although the C1 and Br derivatives slowed immature embryo germination and promoted expansion of the scutellum in both MS71 and B73, none of the derivatives were as effective as chloramben for induction of somatic

T a b l e I. R e s p o n s e frequency of e m b r y o g e n e s i s induction of c u l t u r e s of maize inbred lines B37, B73 and MS71 to 10 #M s u b s t i t u t e d 2,5-dichlorobenzoic acids.

COOH

R2~

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CI~__~R1 B37 R1

MS71

R2

na

freq. ( % )

n

freq. (%)

n

freq. (%)

Methoxy c -Methoxy

42 43 42 42 41 42

0.0 77.1 0.0 37.1 0.0 0.0

122 64 50 63 42 42

23.8 56.6 8.0 71.4 3.1 3.1

128 136 62 90 40 54

0.0 99.1 59.7 84.0 43.3 77.8

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a N u m b e r of i m m a t u r e e m b r y o s tested. bChloramben. CDicamba. dTricamba.

85

COOH

100

50 U B73

ml

mm mmm (0) I

o

COOH

m~ 100 "0 e-

5O

MS71 (o)

(o)

m

F

CI

Br

2,5-Disubstitution

{0)

I

Chlm (x)

Fig. 3. Inductionof somatic embryogenesisin cultures of maize lines B73 (a) and MS71 (b) by 10pM 2,5-dihalogenated benzoic acids at 9% sucrose. Responseto chloramben ('Chlm') is also shown.

embryogenesis in either genotype. In the case of the I derivative in B73 and the F, C1, and I derivatives in MS71, the scutellum expanded slightly but did not produce embryoids, and germination of the immature embryos occurred within a few days after being placed onto the culture induction medium. It is interesting to note that chloramben differs from 2,5dichlorobenzoic acid only by the addition of an amino group in the 3 position.

Structural analogs of chloramben Seven commercially available analogs of chloramben (2-amino-4-chloro-, 2-amino-5chloro-, 3-amino-4-chloro, 4-amino-2-chloro-, 5amino-2-chloro-, and 4-amino-3,5-dichlorobenzoid acids) were compared with chloramben for their effectiveness in induction of somatic embryogenesis. Of all the benzoic acid derivatives containing both amino and chlorine substitutions in the ring, only chloramben was able to induce somatic embryogenesis in B73 and MS71. Frequencies of culture induction by

chloramben were 55.7% and 99.1% for B73 and MS71, respectively. A number of workers have shown t h a t the molecular structure of a PGR is a major determinant in its growth-promoting activity in the intact plant [23,28]. Since only certain compounds were able to effectively induce embryogenesis in every genotype examined, it is likely t h a t the molecular structure of a PGR is an important factor in determining its ability to control the growth of plant tissues in vitro and to elicit a specific response such as embryogenesis. Charge distribution and separation in molecules of auxins and auxin-like PGRs may also influence their interaction with receptor sites in the cell and, thereby, determine biological activity [24]. Other work has demonstrated that the differential sensitivity of plant tissues to various auxin-like herbicides is due to different rates of metabolism and, hence, to differences in the levels of active forms of the PGRs in the tissues [3,11,19]. The observed differences in activity between the various forms of PGRs tested in this report are likely due to differences in molecular structure since, in some cases, a single change in structure caused a significant change in activity. Whether these structure- and genotypespecific responses are a result of differential metabolism, receptor recognition, or both, will require further investigation.

Osmotic considerations Consistent with previous reports [21,26], the sucrose concentration was often found to greatly influence the frequency of callus formation in each of the genotypes examined (Fig. 1). In most genotypes, the maximum frequency of embryogenesis was at either 9 or 12% sucrose. Addition of mannitol and sucrose to the medium in various combinations of concentrations altered the frequency of embryogenesis induced by chloramben from that observed with sucrose alone with inbreds B73 and A632 (Fig. 4a, b, respectively). In general, the addition of mannitol or an increase in the level of sucrose resulted in an increase in the frequency of embryogenesis. The induction

86

a

100

I J.Oo,:

(J -.t O" G) =.. U.

50

c 0 (3 .-i 10 C

(3)

(e)

(9)

[Sucrose],

molal

(%)

b

100 o~ c ® 0" U.

50

0.0

c

._o 0.09 "0 ¢,-

0.09

0.18

0,28

0.37

(3)

(6)

(9)

(12)

[Sucrose], mola I

(%) Fig. 4. Induction of somatic embryogenesis in cultures of maize lines B73 (a) and A632 (b) by combinations of sucrose and mannitol. All treatments included 10 ~M chloramben and 0.0 (m) or 0.5 ( [ ] ) p M ABA.

87 frequency in MS71 was not affected by the inclusion of mannitol (data not shown). Inclusion of ABA at 0.5 and 1.0 ~M (with 3% sucrose) in the medium was found to significantly alter the frequency of culture induction. This effect was genotype specific, concentration dependent and, for each inbred, was similar to t h a t seen with increased sucrose levels, both in terms of the concentration dependency of response (Fig. 5) and in the appearance of the cultures produced. The results indicate t h a t the role of ABA in the stimulation of embryogenesis might be due, at least in part, to its effects on cellular osmotic regulation. An increase in either osmoticum (sucrose or

sucrose + mannitol) or ABA produced cultures appearing more compact and opaque than those produced at a lower sucrose level (3%) without ABA or mannitol. These cultures were similar to 'Type I callus' previously described [13], and a gradual change from 'Type II' to 'Type I' tissue was noted with increasing sucrose or ABA levels in all genotypes examined. Iodine staining indicated t h a t the opacity of the tissue was due to increased starch accumulation. Increased levels of sucrose or mannitol also appeared to slow the rate of tissue growth (as judged by qualitative visual observation), and with further addition of 0.5 ~M ABA, this effect was more pronounced. When mannitol and sucrose were used in

a 100

A632 A

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.

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0.0

0.5

m 1.0

[ ABA] (pM)

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tl 6

m 9

c103

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[Sucrose ! (%)

F i g . 5. Effects of A B A a n d s u c r o s e c o n c e n t r a t i o n o n t h e f r e q u e n c y o f s o m a t i c e m b r y o g e n e s i s in c u l t u r e s of m a i z e lines A632 (a, b), B73 (c, d), a n d C103 (e, f). S u c r o s e c o n c e n t r a t i o n w a s 3% in all A B A t r e a t m e n t s (a, c, e); no A B A w a s p r e s e n t in t h e s u c r o s e t r e a t m e n t s (b, d, f).

88

combination with ABA in the culture medium, B73 showed a dramatic overall increase in induction frequency and a decreased sensitivity to a change in the osmoticum (Fig. 4a). The resulting frequencies often approached 100% where the response had been poor ( < 1 0 % ) without ABA. In contrast, the frequency of somatic embryogenesis in A632 was increased by ABA only at the lowest combined sucrose and mannitol levels (0.09 molal sucrose + 0.09 molal mannitol) (Fig. 4b). For these two treatments, the frequency of culture induction was higher th an any of the other t r eat m e nt s either with or without ABA. In fact, embryogenesis was inhibited by ABA with all other combinations of sucrose and mannitol. In the case of MS71, ABA did not cause a significant change (data not shown). In most treatments, MS71 exhibited a frequency of somatic embryogenesis n ear 100%, with or without ABA. Cultures induced on high osmotica were difficult to maintain and usually did not survive for more t h a n 6-8 weeks. However, those induced on low osmotica with ABA in the induction medium could be maintained for at least 3-4 months by lowering the level of ABA after culture induction. These results indicate that, in addition to the observed differences in the response to various auxin-like PGRs, there is also a genotypespecific requirement for ABA, a high osmoticum, or both, for a maximum frequency of somatic embryogenesis induction in some maize inbred lines. ABA has been shown to inhibit water uptake by seeds and to mimic the effect produced by a high ex~)genous osmoticum [25]. Both ABA and a high osmoticum promote the m a t u r a t i o n in vitro of immature rapeseed (Brassica napus L.) embryos [7,8], and ABA has been shown to control embryoid growth and to promote the normal development of somatic embryos from cultured cells of caraway (Carum carvi L.) when incorporated in the medium at a c o n c e n t r a t i o n of 0.1 1.0 pM [2]. These phenomena might explain the similarity in maize of the responses to ABA and to a high external osmoticum as observed in these experiments. The similarity of patterns of response to a range of

concentrations of sucrose, ABA and combinations of sucrose and mannitol suggests that, in addition to specific PGR-genotype interactions, there is also an osmotic component in the induction of somatic embryogenesis.

Conclusion The results demonstrate t hat somatic embryogenesis in maize can be stimulated by a variety of auxin-like PGRs and that, despite genotypic differences in response, several PGRs (chloramben, dicamba and tricamba) are capable of inducing this response at a high frequency in most inbred lines examined. In addition, other media components such as mannitol and ABA in combination with various sucrose levels are able to enhance PGR-induced embryogenesis, suggesting an additional osmotic requirement for embryogenesis in some genotypes. Regenerated plants from a number of inbred lines have been produced from cultures induced by the most effective of these callus induction treatments. Field observations of progeny up to the R5 generation are current l y being analyzed to determine if the use of different PGRs in vitro results in changes in the types or frequencies of genetic differences in regenerates within a given inbred line.

Acknowledgments The authors wish to express their thanks to Bob Erwin and Ernie Hubbard for constant support and encouragement.

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