Plant Cell Reports

Plant Cell Reports (1996) 15:900-904

9 Springer-Verlag1996

Somatic embryogenesis and plant regeneration in Sorghum bicolor (L.) Moench C. Gendy, M. S6ne, Bui Van Le, J. Vidal, and K. Tran Thanh Van Institut de Biotechnologie des plantcs, URA CNRS D-t 128, Universit6 de Paris-Sud, F-91405 Orsay Cedex, France Received 20 August 1995/Revised version received 18 October 1995 - Communicated by A. M. Boudet

Summary. Callus induction, somatic embryogenesis and plant regeneration were obtained in two cultivars of Sorghum bicolor (L.) Moench. Transverse thin cell layers from roots/epicotyls of 15-day-old seedlings or of regenerated plantlets were used. Callus response depended on the genotype, the size of transverse thin cell layers, the level at which transverse thin cell layers were excised on the epicotyl, the composition of growth substances and the number of in vitro regeneration cycles undergone by the donor plant. Somatic embryos were differentiated under a defined dark/light sequence, from epidermised compact calluses (i.e having already differentiated an epidemlis), obtained directly with dicamba or from friable callus initiated with kinetin and 2,4 dichlorophenoxyacetic acid. The importance of kinetin and dicamba on the induction of embryogenic po'tential is reported. Abbreviations: 2,4-D: 2,4 dichlorophenoxyacetic acid, 2iP: N6(2-isopentyl)adenine, BAP: 6-benzylaminopurine, CaMV: cauliflower mosaic virus, CPPU: N-(2-chloro 4-pyridyl)-N'phenylurea, dicamba: 3,6-dichloro-o-anisic acid, IAA: indole-3acetic acid, K: kinetin, MS: Murashige and Skoog, NAA: c~naphthaleneacetic acid, PEPC: phosphoenolpyruvate carboxylase, SD: standard deviation, tTCL: transverse thin cell layer.

Introduction Sorghum is a tropical C4 plant with a high resistance to drought and widely used for food and feed. Our group is currently investigating the regulatory mechanisms that control the activity of the p h o t o s y n t h e t i c isoform of phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPC) from Sorghum, cv. Tanlaran (Lepiniec et al. 1994). Three members of the PEPC multigene family and their promoter sequences have been cloned and sequenced (Cr6tin et al. 1991); the corresponding full length cDNAs have been fused to the PEPC or 35S CaMV promoter in order to study gene regulation in planta, (in a homologous system) and the effect of over/ or underexpression of PEPC on the functioning of C4 photosynthesis of the Sorghum plant. As a first step in this direction, in vitro somatic embryogenesis and pl,'mt regeneration systems regenerating directly (or with a short callus phase to minimize somaclonal variation) are required to obtain transgenic plants. Most of the techniques reported in the literatuCorrespondence to: K. Tran Thanh Van

-re for S o r g h u m plant regeneration deal with the utilisation of immature embryos (Casas et al. 1993), the scutellum of immature (Dunstan et al. 1978) and mature embryos (Bhaskaran et al. 1987), inflorescences (Cai and Butler 1990), or leaf blade segments (Wernicke and Brettell 1980). Phenotype alterations such as growth delay, sterility or albinism were often observed. The methods of longitudinal (TCL) and transverse thin cell layer (tTCL) were developed in order to study, at the cellular, biochemical and molecular levels, the mechanisms which control in vitro morphogenesis of dicot plants (]?ran Thanh Van 1973, 1981; 1991; 1995; MeeksWagner et al. 1989; Trail Thanh Van et al. 1985a; Tran Thanh Van and Gendy 1993; Richard et al. 1992; Cousson and Tran Thanh Van 1993), and to overcome recalcitrance to regeneration in leguminous-, lignous- (Tran Thanh Van et al. 1985b) and monocotyledonous species (Jullien and Tran Thanh Van 1994). The thin cell layer system consists of a section of small size composed of one or a few (3 to 6) layers of differentiated cells for longitudinal TCL. For tTCL, thin transverse sections (0.1 to 3 mm thick) were made through different organ types. The concept, results, perspectives and applications of the methods of longitudinal and transverse thin cell layers have already been published (Tran Thanh Van et al. 1990; Tran Thanh Van and Gendy 1995). These methods were successfully adopted by different research groups for fundamental studies (Tiburcio et al. 1989; Neale et al. 1990; P61issier et al. 1990; Kaur-Sawhney et al. 1990; Mafia et al. 1991; Rajevan and Lang 1993) and were used as a rapid and powerful method for in vitro plant propagation (Becher et al. 1992; J6han et al. 1994; Kieffer et al. 1995; Lakshmanan et al. 1995) and for the obtention of transgenic plants (Pua et al. 1987; Trinh et al. 1987). We have extended this method to other dicot and monocot species and made it feasible in other research institutes (Van Den Endes et al. 1984; Klimaszewska and Keller 1985, Goh et al. 1994, 1995). In this paper, we report the induction of cell division, rapid somatic embryogenesis and plant regeneration using the tTCL method in Sorghum.

Materials and methods Plant material: The seeds of two commercial cultivars of Sorghum bieoIor, Tamaranand SquiroI, were surface sterilisedby soakingin a

901 detergent solution for 5 min and then in an aqueous solution of calcium hypochloride (6%) for 20 rain. After disinfection, seeds were rinsed three times in sterile water. They were allowed to germinate aseptically on a gelose (Difco) solidified medium containing Murashige and Skoog (1962) macro-/microelements. The culture conditions were 26+1~ and 16h photoperiod for the hght regime (100 gmol m -2 s- 1). Different types of inoculum: Segments of 5 mm size and transverse TCLs (tTCLs) of 2 mm, 1 mm or 0.3 mm thickness, were made by cutting transversely roots or epicotyls of 2-week-old seedhngs (in vivo tTCLs), or of in vitro regenerated plantlets (in vitro tTCLs). Since we found in a preliminary study that the orientation (apical or proximal side in contact with the N, medium) has no influence on the morphological responses of tTCLs, Zoner these were oriented randomly. ZoneiV Segments of 5 mm size were incubated Zone 111 horizontally. Epic~otyl For both genotypes, we analysed the influence of the size of the explants on Zone II cell proliferation i.e. callus induction. For epicotyls, different zones were tested: zone I, at the level of root ~ If'-'-, insertion; zone II, the median zone; Zone l zone III, the subapical zone; zone IV, at the level of the apical meristem and zone V, the base of the leaves above the apical zone (Fig. 1). Observations were made at 3 or 4 day intervals from day 0 to day 42. All experiments (5 replicates each) were repeated at least three times. The percentages were expressed as the Fig 1. Schematic representation average percentage • of a Sorghum plantlet

culture medium. For studies related to i), iii) and iv), only the cultivar Tamaran and the culture medium containing 2,4-D (3.10-6M) and kinetin (10-7M) (medium D) were used.

Influence ofexplant size: For the root as well as for zones I and I l l / I V of the epicotyl, the explant size (thickness) has an important influence on cell proliferation, i.e. callusing responses as shown on table 2. For example, for epicotyl zones IU/IV, 84% of 0.3 m m tTCLs formed callus; only 12% for 1 m m tTCLs and 0% for segments of 5 ram. The percentage of explants forming callus decreased when the explant size increased: the highest percentage was obtained with 0.3 m m for all organ types and zones of tTCLs. Thus, tTCLs of 0.3 m m thickness were used for further studies in both genotypes. Table 2 Average percentage +SD of Tamaran tTCLs forming callus in the dark in relation to explant size on a culture medium containing 2,4-D (3,10-6M) and kinetin (10-7M) after 10 days of culture.

Epicotyl Size (ram) 5 1

0.5 0.3

Root 0• 10• 20• 25•

Zone I

Zones IIIflV

2• 39•

0• 12•

63• 68•

72• 84•

Culture conditions: The explants were cultured on 20 ml of MS medium

Influence of the genotype: Of the two genotypes tested, the

containing 30 g 1"1 of sucrose, thiamine HC1 (0.4 mg l-1), myo-inositol 100 mg 1-1 The growth substances used were 2,4-D, NAA, dicamba, kinetin, BAP and CPPU. The concentrations tested ranged from 10-8M to 10-5M except for dicamba (from 10-5M to 10-2M). The analysis of the influence of the nature and concentration of growth substances was carried out after a preliminary study of the impact of explant size, genotype, nature of donor organ and level of excision. This preliminary study was conducted only on Tamaran tTCLs (0.3 mm size) using epicotyl zones I and IIUIV (results not shown). From this study, optimal concentrations (except for 5.10-6M of 2,4-D) were selected and used for further studies (Table 1). The pH of the culture medium was adjusted to 5.6 before autoclaving and the media were solidified with 10 g 1-1 of gelose and distributed into petri dishes (10x2 cm). The temperature of the incubation was 26+1~ In order to reduce oxidation of phenolic compounds, the cultures were conducted in the dark for the first two weeks after the inoculation. Friable or epidermised compact calluses obtained were then exposed to light, after being transferred onto fresh media which were either identical to (medium B, D or E) or different from (medium H or I) the previous ones.

percentage of callus response was higher for Tamaran than for SquirM, as regards to zones I and III/IV tTCLs (Tables 1 and 3). For Tamaran on medium B, 47% (zone I) and 58% (zones IIUIV) of tTCLs formed friable callus; for Squirol, the corresponding percentages were 38% and 15%. The largest difference between the two genotypes: 83% and 15% was recorded for zones m / I V on medium G.

Table 1 Nature and concentration (M) of different growth substances in ledia. Medium 2,4-D NAA Di K BAP CPPU A 10-6 B 3.10-6 C 5.10-6 D 3.10-6 10-7 E 3.10-6 3.10-4 3.10-4 10-7 F 3.10-6 10-8 3.10-4 G 3.10-6. 3.10-6 H 10-6 3,10-6 I 3.10-6

Influence o f the nature of donor-organs and o f the level o f excision on the epicotyh Callusing response of tTCLs varied according to the nature of the donor organ; in general, epicotyl tTCLs were more responsive (except for zone V) than root tTCLs (Table 3). For example, in Tamaran, on medium D and E, only 25% and 30% of root tTCLs formed callus instead of 84% and 82% respectively for epicotyl zones III/IV. For the epicotyl itself, the level where excision was made on the epicotyl was an important factor. In the case of Tamaran, on m e d i u m D: zones I (68%), IIIflV (84%) were more active in callus formation than zones 1I (16%) and V (2%). In contrast, for Squirt1, zone I was more responsive than the other zones: 56%, 29%, 20% and 0%, respectively, on the same medium.

Effect o f growth substances: Three concentrations (10-6M, 3.10-6M, 5.10-6M) of 2,4-D (medium A, B and C respectively) were used in order to test the responses of the genotype, the nature of the donor-organ and the level of tTCL excision. According to the results s h o w n on table 3, the optimal concentration for callus induction for all tTCL types tested was 3.10-6M (medium B, Table 1). All calluses obtained were

Results

Cell division induction. For monocot species, cell division is in many instances difficult to induce. The ability of tTCLs to form callus depends on i) the size of tTCLs, ii) the genotype, iii) the level on the epicotyl at which the explants were excised, iv) the number of regeneration cycles undergone by the donor plant, and v) the composition and concentration of growth substances in the

friable. A t 5.10-6M (medium C), the percentage of callusing tTCLs for zones III/IV dropped from 58% (medium B) to 3% in Tamaran and from 15% to 2% in SquirM. These results showed the high sensitivity of the TCL system to 2,4-D concentration. The addition of kinetin (medium D) at 10-7M (a concentration previously determined as optimal for callus induction) to medium B (2,4-D 3.10-6M) significantly increased the percenta-

902 -ge of callus formation: 84% (instead of 58%) for zones III/IV in Tamaran and 29% (instead of 15%) for Squirol. For both genotypes, in most cases, the following observations were made: i) the addition to meditma B of dicamba (medium E) increased the percentage of tTCLs forming callus;the addition of both kinetin and dicamba (medium F) increased the percentage of callusing but only for zones UI/IV tTCLs; ii) in contrast, the substitution in medium F (i.e. in presence of dicamba) of kinetin by a cytokinin-like substance, CCPU (10-8MI medium G), did not change significatively the percentage of callusing tTCLs of

Table 3 Average percentage --- SD of tTCLs forming callus in the dark in relation to the genotype, the nature of donor-organ, the level of excision on the epicotyl and the composition of growth substances, after 10 days of culture (T:Tamaran; S: Squirol).

Medium A B C D E F G

Root T S '7+3 3-+2' 9_+2 8+1 0-+0 0+0 25• 30-2_5 30-2-_3 25+3 3• 0-+0 19• 12-+5

Morphogenetic responses (friable/compact callus or somatic embryogenesis) of epicotyl tTCLs. In most types of tTCLs of the two genotypes, the culture media B, D and E were found to be optimal for induction of friable callus and medium E for epidermised compact embryogenic callus. In order to optimize somatic embryogenesis and/or organogenesis, different culture conditions were assayed using epicotyl zones I and IIIflV tTCLs of Tamaran and Squirol. Results obtained were qualitatively similar for both genotypes. In the following part, we report only results obtained on Tamaran (Table 6).

Zone I T S 47+3 11-+3 47• 38-+3 4+_2 0• 56_+7 68_+5 61• 55_+4 13• 7• 48• 62_+4

Epic, oWl ZoneII Zones IIUIV T S T S 9+__2 3+1 58+5 .2+1 17_+3 9_+3 58+4 15+_2 0_+0 2• 3+1 2+1 16• 20_+6 84+3 29+3 3+1 9_+3 82+4 21+4 0_+0 3+__2 83+6 25+4 0_+0 0_+0 83+7 15+1

Zone T 47_+ 4 45_+ 3 61_+ 4 49+ 4 55+ 4 58_+4

Zone II T S 17_+2 9_+3 17_+3 11_+3 9_+ 3 3+ 1 21-+ 3 6+ 2 18-+ 3 6+ 2 9_+ 3 1_+0.6

Zone V T S

3+_2

o+0

3+2 0_+0 2+1 2+1 5+2 1+1

1+1 0+0 0+0 1_+1 1+1 0+0

I

Dicamba Table 4 Average percentage + SD of tTCLs forming callus on medium B (2,4 D 3.10-6 M) in relation to the concentration of dicamba (T: Tamaran,S: Squirol).

(M) 0 10-5 3.10-4 10-4 10-3 10-2

Root T 9_+2 10_+2 30_+ 3 15_+ 3 22_+ 3 19_+3

S 8_+ 1 7+ 2 25_+ 3 10_+2 13_+4 10_+4

zones III/IV. However, it is important to note that the structm'e of calluses obtained in the presence of dicamba (medium E, F or G) was characteristic: these calluses were not friable but epidermised and compact, and thus more embryogenic (see next section). Table 4 shows the percentage of epidermised compact calluses obtained in relation to the concentrations of dicamba

I S 38_+ 3 40_+ 3 55+ 3 40_+ 3 48-+ 3 56_+ 3

Zones HI/IV T S 58_+ 4 15+2 56+5 17+ 3 82+ 4 21+ 3 60+ 4 20+ 3 75_+ 4 18+ 2 81_+5 17+ 3

Zone V S 3+2 1+0.3 2+ 1 2+ 1 2+ 1 1+0.7 1+0.6 2_+0.7 2+ 1 1+ 0.6 1__+0.7 0_+0

Thus, a dark/light sequence was applied to tTCLs cultured on B, D or E medium (Tables 1 and 6). Furthemore, other media were tested: H and I both containing BAP (3.10-6M) combined to a

6M. The optimal concentration (for zones I and III/IV) found,

reduced concentration of 2,4-D (10-6M) or to NAA (3,10-6M) respectively (Table 1). After the first two weeks in the dark, tTCLs were transferred onto fresh identical or different media (H or I) in order to test if the presence of 2,4-D after transfer inhibited somatic embryogenesis. They were then exposed to

3.10-4M, was used for further studies.

light (100 gmol m -2 s-l).

(ranging from 10-5M to 10-2M), in the presence of 2,4-D 3.10-

the number o f in vitro regeneration cycles undergone by the donor plant: In both genotypes, the

Effect o f

percentage of callusing tTCLs increased when in vitro regenerated plantlets, instead of seedlings (in vivo) were used as donor plant for tTCLs. In Squirol, on medium B, for zones III/IV epicotyl tTCLs, 56%, 73%, and 93% were obtained for the 1st, 2rid and.3rd cycle compared to 15% for in vivo plantlets; in Tamaran: 95%, 100% and 100%, compared to 55% for in vivo (Table 5). Table 5 Average percentage ---SD of tTCLs forming callus on medium B (2,4-D 3.10-6M) in relation to the number of in vitro regeneration cycles of the donor plant (T: Tamaran,S: Squirol). ~eectlmgs Regeneration cycle (in vitro) tTCLs (in vivo) 1st 2nd 3rd T S T S T S T S Zone I 45+5 40+8 78+6 57+9 93+5 82+9 100+_0 81+[ Zones 55+4 15+6 95+4 56+8 100+0 73+7 100_+0 93%' IIUIV

Transfer on identical medium: On medium B containing only 2,4-D (3.10-6M), friable calluses were obtained in the dark by day 10 from tTCLs of all zones tested. These calluses remained friable with no embryogenic potential whether they were transfeTable 6 Morphogeneticresponses of Tamaran tTCLs in relation to the composition of growth substancesof the culture medium and to the dark /light sequence. Twne course Irradiation Morphogenetic responses

Meditun (days) (gmol m-2 s-l) Zone I Zones HI/IV t3 0-14 0 Ic Ic 15-42 100 fc fc D 0-14 0 fc fc 15-42 100 fc fc. cc. se E 0 - 14 0 cc cc 15-42 100 cc se callus; cc: Compact callus; se: Somatic embryos

903 -rred or not to light (Table 6). On medium D containing 2,4 D (3.10-6M) plus kinetin (10-7M), friable calluses were obtained in the dark for all zones tested. Transferred on a fresh identical medium and exposed to light, 85% of friable calluses from zones HI/IV e v o l v e d progressively after 3 weeks into epidermised compact calluses from which somatic embryos differentiated after 5 to 6 weeks (Table 6, Figs 2 and 3). Fig 2 Plantlets (pl) and somatic embryos (se) differentiated on compact calluses (cc) issued from friable calluses (fc) of Sorghum epicotyl zones IIUIV after 7 weeks. Magnification:10. F i g 3: Epidermised compact nodules and, somatic embryos (se). Magnification: 15.

2,4-D. On medium E (2,4-D 3.10-6M and dicamba 3.10-4M), epidermised compact calluses were obtained directly from 100% of zones I, III/IV after day 10. Transferred to light, on zones IUJIV, somatic embryos were obtained from 90% of the compact calluses after 3 weeks (Table 6). The number of macroscopically developed embryos per tTCL on media D and E ranged from 4 to 6. In all cases, somatic embryos obtained evolved into plantlets on the same medium 1 or 2 weeks later. Eighty plants were separated and grown in the greenhouse. In 90% of the regenerated plants the morphological and physiological traits were similar to the plants germinated from seeds. Neither albinism nor chimerism was observed. However 10% of them flowered precociously (Figure 4). The different morphogenetic response, the corresponding culture media, light regime and time course are summarised in Fig. 5. Fig 5: Diagram of morphogenetic response of Sorghum tTCLs

Weeks

tTCLs

4-D~K~icamba

0

}Dark

9 2.

+ 3 -4

fc

~

+

4 N ~

*

Plantlet

F i g 4. Sorghum regenerated plant (left) showing precocious flowering. Control plant (right) after 3 months. Scale = 1/10

Plantlct

S

3Light

-6

7

- B-

Transfer on different media: It was shown in most cases, for example in Daucus carotta (Borkid et al. 1986), that somatic embryogenesis was induced in the presence of 2,4-D, but only expressed after the suppression of 2,4-D. In the variety G 522 of Sorghum (Wernicke and Brettell 1980), a transfer to a medium with a lower concentration of 2,4-D (2..2~tM instead of 9gM), was required in order to obtain globular structures from white tissue. Plantlets were only obtained after transfer to a medium without auxin. Our results in Sorghum indicated that the presence of 2,4-D did not inhibit somatic embryogenesis. However, we would like to test if a lower concentration of 2,4D or its suppression would increase the percentage of somatic embryogenesis (and presumably reduce the risk of somaclonal variation due to prolonged effect of 2,4-D). In the following experiments, friable calluses becoming compact calluses obtained from zones I]I/1V on medium D after day 15 (i.e. after their transfer to fight) were transferred onto a medium containing BAP (3.10-6M) combined either to N A A (3.10-6M) (medium I)

Its is importanf to point out that friable calluses obtained on tTCLs cultured on a 2,4-D plus kinetin medium were thus potentially embryogenic in contrast to those obtained with only

or to a lower concentration of 2,4-D (10-6M) (medium H) and maintained exposed to light. Four weeks after this transfer, 88% of the previously mixed friable-compact calluses formed somatic embryos on these two media. The percentage did not change signiflcatively when 2,4-D was suppressed or reduced after the transfer. Similar treatments were also conducted on friable calluses (obtained on medium B with only 2,4-D without kinetin). No embryos were obtained after transfer on medium H or I (results not shown).

904 Discussion Our contribution in this paper is to present a new and efficient method for a rapid and direct somatic embryogenesis. We have obtained somatic embryogenesis and in vitro plant regeneration of Sorghum bicolor, using epicotyl tTCLs and root tTCLs (results not shown). It was reported that somaclonal variation can affect the yield, the plant height or can induce albinism. In our work, the shorter time (3 to 6 weeks) required to obtain somatic embryogenesis, as well as the direct formation of epidermised compact calluses followed by embryogenesis on tTCLs might contribute to mininaize somaclonal variation. Different types of callus: friable with / without embryogenic potential, or epidermised compact embryogenic callus can be programmed, using the tTLC method. The size of the tTCLs, the level at which their excision was made on the epicotyl as well as the nature and quantity of growth substances are important factors. The positional effect was also demonstrated in Bambusa (Jullien and Tran Thanh Van 1994), Iris and Digitaria (unpublished results), where an horizontal gradient in the callusing and embryogenic responses of the tTCLs was juxtaposed to a vertical gradien L Somatic embr.yogenesis was only observed on callus with specific morphological traits: epidermised and compact. Friable calluses obtained on a 2,4-D medium remained friable, the ones obtained with the addition of kinetin (10-7M) evolved into compact calluses. With dicamba (3.10-4M), epidermised compact calluses were obtained directly and somatic embryos differentiated within 3 weeks on epicotyl and root tTCLs (results not shown). The influence of dicamba on somatic embryogenesis was previously reported on Zea mays leaf fragments (Conger et al. 1987) but the time required was longer (6 to 10 weeks instead of 3 weeks in S o r g h u m tTCL system). This is the first observation of such a short time for direct somatic embryogenesis in a m o n o c o t plant. For a dicot plant, Helianthus, a short time (13 days) was also reported for direct somatic embryogenesis (P61issier et al. 1990), using the longitudinal TCL method. For a given genotype, the embryogenetic potential increased with the number of regeneration cycles undergone by the donor plant. The adaptation of biochemical processes of the regenerated plantlets to the in vitro environmental conditions (supply of exogenous carbohydrates, growth substances and specific regime of temperature, light and humidity) can favor in vitro morphogenesis induction which occurs in identical environmental co/aditions. In conclusion, the following factors: the positional effect, the in vitro regeneration cycle effect as well as the high sensitivity of tTCLs to the quantity and quality of growth substances, especially of dicamba and kinetin, have permitted the programing of direct and rapid somatic embryogenesis in

Sorghum. The success in controlling direct and rapid somatic embryogenesis is a prerequisite condition for the next step in the investigation for producing of transgenic plants. The TCL m e t h o d was recognised as an appropriate experimental system which allows the control and analysis of the mechanisms of morphogenesis (Hicks 1981, Roberts 1985), and to "speed" the production of transgenic plants (Ammirato 1987).

Acknowledgements We thank Professor P. Gadal for his encouragement, Professor Henri Frisch for reading the manuscript, Y. Coudray and T. Do for their technical assistance and D. Froger for the photographs.

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