Plant Cell Reports

Plant Cell Reports (1994) 14:145-150

.~ Springer-Verlag 1994

Somatic embryogenesis in peanut (Arachis hypogaea L.): stimulation of direct differentiation of somatic embryos by forchlorfenuron (CPPU) B. N. S. Murthy and Praveen K. Saxena Department of Horticultural Science, University of Guelph, Guelph, Ontario, Canada, N1G 2WI Received 29 April 1994,'Revised version received 4 July 1994 - Communicated by F. Constabcl

Summary. The ability of forchlorfenuron (CPPU), a substituted phenylurea compound, for inducing somatic embryogenesis in peanut (Arachis hypogaea L.) seedlings has been demonstrated. CPPU promoted somatic embryogenesis at a range of concentrations in all three peanut cultivars tested. Embryogenic response was dependent on applied CPPU concentrations. Exposure of seedlings for only two days to CPPU induced somatic embryogenesis, but the most effective treatment was to induce seed germination on media supplemented with either 2.5 or 4.0/aM CPPU and to maintain the seedlings on the same medium. Number of somatic embryos and the frequency of embryogenesis was higher for younger seedlings (up to 9 days), regardless of the CPPU concentrations and seedlings older than 21 days failed to produce somatic embryos. Removal of cotyledons from the seeds drastically reduced the embryogenic potential of the seedlings. Somatic embryos developed into whole plants following their separation and subculture on a medium lacking growth regulators. The induction of somatic embryos using CPPU as a sole growth regulator may provide a useful system to study the role of this compound in plant morphogenesis. Keywords: Forchlorfenuron, CPPU, Peanut, Somatic embryogenesis, Regeneration, Phenylurea Abbreviations: CPPU = N-(2-chloro-4-pyridyl)-N'phenylurea; DPU = N,N'-diphenylurea; IAA = Indole-3acetic acid.

INTRODUCTION Cytokinins play a major role in plant morphogenesis and are characterized by their ability to promote cell Correspondence to: P.K. Saxena

proliferation and tissue differentiation. There are two distinct classes of chemical compounds that elicit cytokinin-like activity : N6-substituted adenine derivatives and phenylureas. Some of the phenylureas exhibit strong cytokinin-like effects in a broad range of physiological responses. Forchlorfenuron (CPPU) is one such phenylurea that stimulates high frequency of cell division in tobacco callus cultures (Takahashi et al., 1978) and is 10-fold more active than N6-benzyl adenine (BA) in inducing shoot formation (Okamoto et al., 1978). Strong cytokinin-like activity of CPPU has been exploited to delay fruit maturky and for increasing fruit size and yield in several fruit crops including grapes, apple and kiwi fruit (Iwahori et al., 1988; Reynolds, 1992 and references cited therein). However, despite its potential in the regulation of cell division and growth, the use of CPPU in plant tissue culture has been limited. So far, CPPU has been used as a potent substitute for purine-based cytokinins in the induction of adventitious shoots (Mohamed et al., 1992; Fellman et al., 1987). Application of CPPU in combination with a strong auxin source, such as 2,4-D, induced embryogenic callus in Vitis vinifera (Matsuta and Hirabayashi, 1988). In this communication, we demonstrate the potential of CPPU in the induction of direct somatic embryogenesis in peanut, an important oil-seed legume, that has proven refractory to regeneration in vitro.

MATERIALS AND METHODS Seeds of peanut (Arachis hypogaea L. cv. Tango, Garroy, and Ruby) were provided by Dr. T. Smith, Crop Science Department, University of Guelph. All experiments reported in the present study w e r e conducted with the cv. Tango, except one experiment in which different cultivars were evaluated for their capacity to develop somatic embryos in response to CPPU. Seeds were surface sterilized by sequentially immersing them in 70% (v/v) ethanol tbr 3 min and in 1.5% (v/v) sodium hypoehlorite for 20 min, followed by rinsing 5 times in sterile distilled water. Four seeds were cultured in each Magenta culture box

146 (Magenta Corp., Chicago, IlL, USA) containing 50 ml of the culture medium. The culture medium consisted of MS salts (Murashige and Skoog, 1962), B5 vitamins (Gamborg etal., t968) and 30 g. L-~ sucrose. The medium was adjusted to pH 5.7 before autoclaving at 1.4 Kg. em "2 for 20 min. Gelrite (Scott Laboratories, Carson, USA) at 3 g.L-l was used as the gelling agent. Cultures were incubated at 250 C in the dark for the first seven days and then shifted to light (30 to 35 laE. m -2s-t; 16 h photoperiod) provided by cool white fluorescent tubes. Somatic embryos developed on the seedlings were counted after five weeks of culture initiation. In the first experiment, designed to evaluate different phenylnrea compounds and to identify the optimal concentrations for the induction of somatic embryogenesis, the seeds were cultured on MS medium amended with various concentrations (0.1, 0.5, 1.0, 2.0, and 4.0 gM) of CPPU [N-(2-chloro-4-pyridyl)-N'-phenylurea] or DPU (N,N'diphenylurca). Further experiments were conducted with only CPPU as DPU was tbund to be ineffective in inducing somatic embryogenesis at any of the above concentrations. To evaluate the efficacy of CPPU in inducing somatic embryos in different cultivars of peanut, an experiment was designed as a 3 x 5 factorial in a randomized complete block; the two factors tested were the genotype (Tango, Ruby, and Garroy) and CPPU concentration (0.1, 0.5, 1.0, 2,0 and 2.5 gM). The effect of the age of seedlings was tested with CPPU-induced embryogenie seedlings. In this experiment, the seeds were germinated and maintained on hormone-free MS basal medium for periods of 6,9,12,15 and 18 days and then transferred to various concentrations (0.1, 0.5, 1.0 and 2.0 pM) of CPPU. Another experiment was performed to determine the effects of the dose and duration of exposure to CPPU on the induction of somatic embryos Seeds were cultured on MS medium supplemented with 0,5, 1.0, 2.0 or 4.0 lxM CPPU for periods of 2,4,6,8 and 10 days and seedlings were transferred to hormone-free MS medium. As a control, one set of seedlings was subjected to continuous exposure to CPPU without transferring to MS basal medium. The final experiment was conducted to study the effect of cotyledon removal on the development of somatic embryos from CPPU-induced seedlings. The treatments consisted of removing one coty'ledon, both of them (leaving only embryonic axis), and a control with intac~ cotyledons. Tt~e explants prepared as above were cultured on MS medium amended with different concentrations of CPPU (0.1, 0.5, 1.0, 2 0 and 2.5 laM). In all experiments, each single treatment consisted of four replications and all experiments were repeated at least twice. Data were analyzed using analysis of variance (Proc GEM of PC SAS, version 6.0) and mean comparison made by protected Least Significant Differences (LSD) at the 5% level of probabili~,.

RESULTS

Seedling development P e a n u t s e e d s g e r m i n a t e d in all t r e a t m e n t s w i t h i n 48 to 72 h o f c u l t u r e i n i t i a t i o n . T h e p r e s e n c e o f C P P U or D P U i n t h e m e d i u m h a d n o a p p a r e n t effect o n e i t h e r the rate or t h e f r e q u e n c y o f g e r m i n a t i o n , b u t the g r o w t h o f t h e s e e d l i n g s w a s g r e a t l y affected. O v e r time, t h e c o t y l e d o n s o f t h e s e e d l i n g s c u l t u r e d in t h e p r e s e n c e o f C P P U o r D P U b e g a n to e x p a n d a n d a c q u i r e d d a r k - g r e e n colour, i r r e s p e c t i v e o f t h e c o n c e n t r a t i o n used. The c o t y l e d o n s a n d leaves o f p h e n y l u r e a i n d u c e d s e e d l i n g s w e r e c o m p a r a t i v e l y s m a l l b u t m u c h t h i c k e r (Fig. I A, r i g h t ) a n d g r e e n e r t h a n t h e control. A n o t h e r c o n s p i c u o u s effect o f C P P U a n d D P U c o n c e r n e d s h o o t a n d root

growth : the shoots remained stunted and root growth was i n h i b i t e d (Fig. 1A, arrow). A t h i g h e r c o n c e n t r a t i o n s (2.0 g M a n d a b o v e ) t h e r e w a s n o s e c o n d a r y r o o t formation.

147

Induction of somatic embryogenesis While CPPU was highly effective in inducing somatic embryogenesis at all concentrations in the range of 0.1 to 4.0/zM (Table 1), DPU was totally ineffective. An increase in the concentration of DPU to as high as 50 /~M did not induce somatic embryogenesis (data not presented). However, stunted growth of the seedlings in response to DPU indicated that applied concentrations (0.1 to 50/zM) were in the active physiological range. The first sign of the production of somatic embryos in CPPU-treated seedlings was the formation of pale-green or whitish nodular structures in the cotyledonary notch (2 to 3 mm hypocotyledonary region) of the seedling. These nodular structures became evident by the end of two weeks after culture initiation and later differentiated into somatic embryos (Fig. 1A,B; arrows). The development of somatic embryos on seedlings was asynchronous and it was possible to identify globular, heart-shaped and cotyledonary stages of embryo differentiation simultaneously after four to five weeks of culture. Of various CPPU concentrations tried, the highest number of somatic embryos (18.3) was produced at 4.0/~M; however, this was not significantly different from mean numbers of somatic embryos induced in the presence of 1.0 and 2.0 /zM CPPU (Table 1). At concentrations above 2.0 /~M, 93 to 100 % of the seedlings became embryogenic but the percentage of seedlings producing somatic embryos in response to 0.1 and 0.5/zM CPPU were relatively low (Table 1). The somatic embryos were well differentiated and could be easily separated from the mother tissue (Fig. 1C). Subculture of these embryos on a basal medium resulted in the development of whole plantlets (Fig. 1D). Occasionally, some of the seedlings developed somatic embryos with multiple cotyledons (crown-like structures) which upon extended periods of culture (8 to 10 weeks) produced flowers without shoot elongation.

Table 1. Effect of different CPPU concentrations on the average number of somatic embryos per seedling and frequency of somatic embryogenesis in peanut (Arachis hypogaea L.) cv. Tango. Number of seedlings (n)= 16.

Fig. 1. Somatic embryogenesis and plant regeneration in peanut (Arachis hypogaea L.). A. Six-weeks old seedling cultured on MS basal (left) and CPPUsupplemented medium (right; note the absence of secondary roots on CPPUtreated seedlings). B. Group of somatic embryos developed on a CPPU-induced seedling. C. An individual somatic embryo (note the presence of root pole; arrow). D. Various stages of plant regeneration from somatic embryos obtained from seedlings induced on CPPU-supplemented medium.

CPPU (#M)

Number of embryos/ seedling"

Embryo producing seedlings (%)

0.1

1.6"

43.8

0.5

5.9"

75.0

1.0

16.4 b

93.8

2.0

15.1 b

93.8

4.0

18.3 h

100.0

* Values with different superscripts are significantly different (P --< 0.005)

148

Effect of genotype All three cultivars produced somatic embryos on CPPU-supplemented medium. However, pronounced differences were observed among the cultivars with respect to their embryogenic potential for applied CPPU concentrations. At optimal concentration of CPPU for the induction of somatic embryogenesis, the cultivar Tango was the most responsive, and variation in the concentration of CPPU did not improve embryogenic response of low-responding cultivars (Fig. 2). The percentage of seedlings showing somatic embryos was comparatively lower (81.3%) in the cv. Garroy than in Tango (96.3%) or Ruby (90.0%).

25 0 2 DAYS Y7 4 D A Y S 9 6 DAYS

[] 8 D A Y S 9 tO D A Y S /k C O N T I N U O U S

r~

z 2o r.d r.d

30 I

9 GARROY V RUBY

25

9

20

a. co o

15

I:I:1 "~

Fig. 3. Effect of different concentrations and duration of exposure to CPPU on the frequency of somatic embryos produced per seedling in seed cultures of peanut (Arachis hypogaea L.).

jr

' /

The percentage of embryogenic seedlings also decreased following the removal of cotyledon; 86.3 %, 83.8% and 68.8%, respectively for the seedlings with intact cotyledons, one cotyledon and no cotyledons. The seedlings resulting from culture of only embryonic axes (both cotyledons removed) were pale, the stem was slender, and overall growth was inlaibited.

10

o

1

0.0

i

0.5

i

i

1.0

1.5

i

2.0

i

25

2.5

@ CONCENTRATION

4

T

r~

0

I

3

CONCENTRATION (NM)

,;it T

I

2

TANGO

z

r.-i o'/

I

1

(~M) Z

Fig. 2. Effect of different concentrations of CPPU and genotype on the frequency of somatic embryos produced per seedling in seedling cultures of peanut (Arachis hypogaea L.)

Dose response The experiment performed to study the effect of the duration of induction period revealed that the frequency of the production of somatic embryos per seedling increased with an increase in the duration of exposure as well as the concentration of CPPU (Fig. 3). Extended exposure to lower concentration or a brief exposure to a higher concentration of CPPU induced similar numbers of somatic embryos per seedling. A two-day treatment of germinated seeds to CPPU induced embryogenesis at a range of concentrations. However, the most effective treatment was to induce germination on a medium supplemented with 4.0 /~M CPPU and to maintain the seedlings on the same medium without subculturing on hormone-free medium (Fig. 3).

Role of cotyledons There was a drastic reduction in the frequency of somatic embryos produced per seedling when the cotyledons of the seeds were removed (Fig. 4).

INTACT

~7 O N E 9 NONE 20

r.1 r~a ~D

>. m

10

"~ o

5

I

I

I

I

I

I

0.0

0.5

1.0

1.5

2.0

2.5

CONCENTRATION (/zM)

Fig. 4. Effect of different concentrations of CPPU and cotyledon removal on the frequency of somatic embryos produced per seedling in seed cultures of peanut (Arachis hypogaea L.).

Age of seedlings The developmental stage of explant has been a crucial factor for the induction of somatic embryos in several species. Thus, it was of interest to test the effect of the maturity of seedlings on embryogenic response under the

149 influence of CPPU. The results of the present study (Table 2) clearly demonstrated that, at any particular concentration of CPPU, the frequency of somatic embryos per seedling and the percentage of embryogenic seedlings decreased with an increase in the age of the seedlings. Six and nine-day-old seedlings produced 13.90 and 12.75 somatic embryos per seedling, respectively; this was significantly greater than the number of somatic embryos produced by 12, 15 or 18 day-old-seedlings (Table 2). The percentage of embryo producing seedlings was higher (97.92 %) when seedlings were transferred to CPPU supplemented media from MS0 after 6 and 9 days of culture initiation; this progressively decreased for the day 12, 15 or 18 of subculture (Table 2). The influence of seedling age on somatic embryogenesis was attenuated to a certain degree by increasing the concentration of CPPU. For example, the response of 15-day-old seedlings receiving a dose of 2.0/~M of CPPU was the same as that of 6day-old seedlings developed on 0.1 /zM CPPU. However, seedlings older than 21 days did not produce somatic embryos, irrespective of the CPPU concentration used (data not shown).

Table 2. Effect of different concentrations of CPPU and age of the seedlings on the induction of somatic embryogenesis in peanut (Arachis hypogaea L.)

Age of the seedling

Number of somatic embryos/seedling 4- SE (Mean of 16 seedling) Mean~ Concentrationof(CPPU~M)

(days) 0.1

0.5

1.0

2.0

6

8.9+0.7 (100.0)

15.8_+1.8 (100.0)

13.2_+3.7 (9l .7)

17.6_+2.0 (100.0)

13.90" (97.92)

9

6.5_+1.1 (91.7)

13.7_+1.9 (100.0)

15.8-+1.7 (100.0)

15.0_+2.5 (100.0)

12.75 ~ (97.92)

12

3.8_+0.3 (83.3)

8.4_+3,0 (91.7)

9.9-+l.6 (91.6)

10.9-+2.8 (9l .7)

8.25 b (89.58)

15

2.0_+0.4 (50.0)

4.4_+0.6 (75.0)

8.3_+0.6 (91.7)

8.0_+0.6 (91.7)

5.68 b (77.08)

18

0.9_+0.3 (33.3)

3.2_+0.9 (75.0)

3.4+1.4 (58.3)

2.1_+0.7 (41.7)

2.39 ~ (52.08)

Mean ~

4.42 b (71.66)

9.08 a (88.33)

10.13 9 (86.66)

10.71 a (84.99)

a Values with different superscripts are significantly different (P_

Somatic embryogenesis in peanut (Arachis hypogaea L.): stimulation of direct differentiation of somatic embryos by forchlorfenuron (CPPU).

The ability of forchlorfenuron (CPPU), a substituted phenylurea compound, for inducing somatic embryogenesis in peanut (Arachis hypogaea L.) seedlings...
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