Plant Cell Reports

Plant Cell Reports (1996) 15:751-753

9 Springer-Verlag1996

High frequency plant regeneration via somatic embryogenesis in cell suspension cultures of coriander (Coriandrum sativum L.) Suk Weon Kim, Mi Kyung Park, and Jang R. Liu Genetic ResourcesCenter, Korea ResearchInstitute of Bioscienceand Biotechnology,KIST, P.O. Box 115, Yusung,Taejon, 305-600, Korea Received 29 September 1995/Revisedversion received28 December1995 - Communicatedby F'. Constabel

Abstract.

Hypocotyl segments and zygotic embryos of coriander formed embryogenic calli at frequencies of up to 75% when cultured on MS m e d i u m supplemented with 1 m g r 1 2,4-D. Calli were transferred to MS liquid medium with 1 m g P 2,4-D to initiate cell suspension cultures. Embryogenic cells became finely dispersible in the m e d i u m as the subculture proceeded. Cultures were transferred to a nitrogen compound enriched liquid MS m e d i u m containing 2% sucrose and 0.1 mgl "1 2,4-D, and cultured two weeks before plating on MS basal medium. Approximately 75% of cell aggregates (1 to two m m in diameter) underwent development into globular to cotyledonary somatic embryos after two weeks of plating. Most of the embryos were subsequently regenerated into plantlets. Regenerants were successfully transplanted to potting soil and grown to maturity in a phytotron.

Abbreviations: MS, Murashige and Skoog; MSID, MS m e d i u m + 1 mgl "1 2,4-D.

Introduction Coriander (Coriandrum sativum L.) is a herbaceous medicinal plant belonging to the family UmbelIfferae. It produces the unusual fatty acid petroselenic acid in the seed. Petroselenic acid can comprise as much as 85% (w/w) of the total seed fatty acid content. However, this fatty acid is virtually absent from other parts of the plant (Kleiman and Spencer 1982). Despite its Correspondence to: J. R. Liu

commercial importance only a few in vitro culture studies of coriander have been published. These studies include a report of successful micropropagation through shoot-tip cultures (Kataeva and Popowich 1993). So far, plant regeneration from cultured cells has not been achieved. This study

describes culture conditions

for high frequency plant regeneration via somatic embryogenesis coriander.

in

cell

suspension

cultures

of

Materials and methods Plant maten'al and callus induction. Seeds of coriander (Coriandrum sativum L.) were surface-sterilized with

70% ethanol for 1 rain, and immersed in a 0.4% sodium hypochlorite solution for 10 rain. They were then rinsed four times with sterile distilled water. The basal medium used throughout the experiments consisted of Murashige and Skoog's (1962) inorganic salts, 100 mgF1 myo-inositol, 0.4 mgl4 thiamine. HC1, 3% sucrose, and 0.4% Gelrite (MS medium). The pH of all media was adjusted to 5.8 before autoclavin~ Twenty-five ml of medium was dispensed into 87 x 15-ram plastic petri dishes. In each petri dish 10 seeds were placed onto MS basal medium and incubated at 25~ in the dark. Two-week-old seedlings and zygotic embryos excised from imbibed seeds were used as the source of explants. Transversely sliced cotyledon, hypocotyl, and root segments (approximately 2.5 mm in length) of seedlings and intact zygotic embryos (approximately 1 mm in length) were placed onto MS medium

752 supplemented with 0.1, 0.3, 1, or 3 mgl"~ 2,4-D. Three petri dishes per treatment with 10 explants each were cultured. After four weeks of culture, the number of explants with embryogenic calli were counted to determine the frequency of embryogenic callus formation. Embryogenic calli were distinguished from nonembryogenic calli by the presence of somatic embryos on the surface at early developmental stages. Embryogenic calli detached from the explants were subcultured on MS medium with 1 mgl"I 2,4-D (MSID) every four weeks. Unless mentioned otherwise, all cultures were maintained at 25"C in the dark.

weeks

Initiation and proliferation af embryogenic cell suspension

formed

2,4-D (SC-1) (Jeon et al. 1986) and cultured 2 weeks before plating on MS basal medium. The regenerants were transplanted to potting soil and maintained in a phytotron (27"(; day/22~ night at 16-h photoperiods).

Results and Discussion After two weeks of culture, calli

began to form

on the cut surface of all explants regardless of explant source and 2,4-D concentration. After four of

culture,

on

white

cotyledon,

compact

hypocotyl,

calli and

were zygotic

Fig. 1. Plant regeneration of coriander via somatic embryogenesis. A: Hypocotyl-derived embryogenic callus cultured on MS medium with 1 mgl"~ 2,4-D (bar = 2 ram); B: Shoot differentiation from somatic embryos (bar = 2 mm); C: Embryogenic cell suspension cultures of coriander (bar = 100 I~m); D: Somatic embryos developted from cell suspension cultures (bar = 100 I1 In); E: Flowering plant regenerated from somatic embryo (bar = 3 era).

culturea Subcultured hypocotyl-derived embryogenic calli (approximately 1 g) were placed in a 300 ml Erlenmeyer flask containing 50 ml of liquid MSID medium. Cell suspension cultures were maintained on a gyratory shaker (100 rpm) and subcultured every two weeks.

embryos, but not

Plant regeneratiara Cell aggregates (1 to 2 mm in

30%, and 77% respectively (Fig. 2). However, root

diameter) maintained for two weeks in liquid MSID medium were collected on stainless steel mesh (pore size: 1 mm) and rinsed with MS basal medium. Twenty cell aggregates were plated on MS basal medium in a petri dish. After four weeks of culture the regeneration frequency was determined from four petri dishes. To enhance the regeneration frequency, cell suspension cultures were transferred to MS medium supplemented with 4 gF1 KNO3, 0.29 gF~ NH4NO3, 3 mgF1 thiamine-HC1, 0.5 mgF1 pyridoxine 9 HC1, 5 mgl -~ nicotinic acid, 2% sucrose, and 0.1 mgl-~

explants were incompetent and did not produce

on

root

gave rise to n u m e r o u s culture

proceeded,

segments.

The calli

somatic embryos as the

indicating

that

they were

embryogenic (Fig. 1A, B). The highest frequencies of embryogenic callus formation

on hypocotyl,

cotyledon, and zygotic embryo explants were 75%,

embryogenic calli. The three different explants exhibited an o p t i m u m concentration of 2,4-D for embryogenic callus formation at 1 mg1-1 (Fig. 2). Hypocotyl-derived embryogenic calli were used

to

initiate

Embryogenic

cell

cell

suspension

suspension

cultures

primarily composed of small meristematic cells (approximately diameter)

containing

a

cultures.

prominent

were

isodiametric 10 pm in nucleus

and

753 dense cytoplasm with numerous vacuoles (Fig. 1C). Nineteen percent of cell aggregates maintained in MSID medium gave rise to numerous globular to cotyledonary embryos when transferred to MS basal medium (Fig. 1D).

80

,4 ~ o .O t-O

60.

O ~: O

40

~. X I.U

20

\

I ~ I 0 0.1 0.3

I 1

I 2

I 3

were reported in rice (Grimes and Hodges 1991; Jeong et al. 1991). It has also been reported that the inorganic NO3:NH4 § ratio alters cell sensitivity to auxin and regulation of hormone metabolism in rice (Grimes and Hodges 1991). Fifty plants were regenerated from somatic embryos. Ten of them were successfully transplanted to potting soil and grown to maturity (Fig. 1E). Recent increased interest in plant fatty acid metabolism has been stimulated by the potential to design new oilseed crops which produce higher value oils (T6pfer et al. 1995). Modification of fatty acid composion in seeds of transgenic oil crops has been accomplished in soybean, canola, and other plants by introduction of new genes or inhibition of existing enzyme activities using antisense RNA technology (Cahoon et al. 1992; Kinney et al. 1993). The plant regeneration system described in this study could be used for genetic transformation of coriander.

2,4-D (mgl -~)

Fig. 2. Effects of 2,4-D concentration on embryogenic callus formation on cotyledon ( - - . . ) , hypocotyl (--C)"), root (...111...), and zygotic embryo explants (--V-l--). Each treatment consisted of 10 explants with three replicates. Vertical bars represent SD.

Cell aggregates precultured in SC-1 medium gave rise to numerous somatic embryos at an approximate frequency of 75%. Approximately 70% of somatic embryos developed into plantlets. Thus, preculture in SC-1 medium resulted in a four-fold increase in regeneration frequency when compared with the treatment without preculture. Also, the precultured cell aggregates produced plantlets approximately two weeks earlier. The major difference between MS1D medium and SC-1 medium is nitrogen ion content. It is uncertain how nitrogen sources influence cell growth and plant regeneraton. However, similar effects of nitrogen source on plant regeneration

Acknowledgements. We thank Dr. Sang Soo Kwak for his critical reading of this manuscript, and Miss Chang Sook Kim for her assistance in manuscript preparation. References Cahoon EB, Shanklin J, Ohlrogge JB (1992) Proc Natl Acad Sci 89:11184-11188 Grimes HD, Hodges TK (1991) J Plant Physiol 136:362-367 Jeon JH, Liu JR, Yang SG, Lee HS, Joung H, Hart MH (1986) Korean J Plant Tissue Culture 13:119-128 Jeong WJ, Song NH, Min SR, Kim MK, Liu JR (1991) Korean J Plant Tissue Culture 18:209-214 Kataeva N, Popowich EA (1993) Plant Cell Tissue Organ Culture 34:141-148 Kinney AJ, Hitz WD, Yadav NS, Perez-Grau L (1993) Plant Physiol 102il Suppl 1 Kleiman R, Spencer GF (1982) J Am Oil Chem Soc 59:29-38 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 T6pfer R, Martini N, Schell J 0995) Science 268:681-686