Plant Cell Reports

Plant Cell Reports (1992) 10:621 623

9 Springer-Verlag1992

High frequency s6matic embryogenesis and plant regeneration in tissue cultures of Codonopsis lanceolata Sung R. Min 1, Seung G. Yang ~, Jang R. Liu a, Pil S. Choi z, and Woong Y. Soh 2 1 Plant Cell Biology Laboratory, Genetic Engineering Research Institute, Korea Institute of Science and Technology, P.O. Box 17, Taedok Science Town, Taejon, 305-606, Korea 2 Department of Biology, Chonbuk National University, Chonju, 560-756, Korea Received July 19, 1991/Revised version received October 23, 1991 - Communicated by I. K. Vasil

ABSTRACT Culture c o n d i t i o n s f o r high frequency somatic embryogenesis and plant regeneration from c o t y l e d o n a r y e x p l a n t s of Codonopsis lanceolata a r e described. The maximum induction frequency of somatic embryos from c o t y l e d o n a r y e x p l a n t s was 80~ on Murashige and Skoog (MS) medium containing 6~ sucrose with 1 mg/1 2,4d i c h l o r o p h e n o x y a c e t i c acid and 10~ coconut water. Upon t r a n s f e r onto MS basal medium c o n t a i n i n g 324 sucrose, most somatic embryos developed i n t o p l a n t l e t s . Key words : regeneration embryogenesis

Codonopsis

Plant

tissue

Plant c u l t u r e - Somatic

lanceolata -

Abbreviations : 2,4-D, 2,4-dichlorophenoxyacetic acid ; GA3, g i b b e r e l l i n As ; MS, Murashige and Skoog INTROI?iJCTION

Codonopsiz lanceolata is a perennial herb belonging to the family Campanulaceae. I t s taproot is used in medicineor as a wild vegetable in Korea, Japan and China. Recently, i t has b e e n adopted as an ornamental crop. Hence, the demand for such a herb is increasing considerably. In the Japanese bellflower (Platycodon grandiflorum), which belongs to the same family, somatic embryos can be obtained from c u l t u r e d a n t h e r s (Ham and Lee, 1976). However, no merphogenesis could be induced in c a l l u s o b t a i n e d from a n t h e r s of C. l a n c e o l a t a (Lee e t a l . , 1980). Likewise, protoplasts Offprint requests to. J. R. Liu

i s o l a t e d from stun segment-derived c a l l u s form c e l l c o l o n i e s without producing any organs (Ahn et al., 1986). In t h i s communication we r e p o r t the culture c o n d i t i o n s f o r high frequency somatic embryogenesis and p l a n t r e g e n e r a t i o n from c o t y l e d o n a r y e x p l a n t s of C. l a n c e o l a t a . MATERIALS AND METHODS Seeds of C. l a n c e o l a t a (S. e t Z.) Trautv. were disinfested with 70~ ethanol f o r 5 min and subsequently, in 25~ Clorox s o l u t i o n for 20 min. They were r i n s e d t h r e e times with s t e r i l e double-distilled water and placed onto a n u t r i e n t agar medium supplemented with 1 mg/1 GA3 to promote germination. A f t e r 4 weeks of incubation, 50~ of the seeds germinated (without GA3, 25~4 germinated). Unless mentioned otherwise, incubation was at 25~ in the dark. Two x 3-mmcotyledonary explants were excised and placed o n t o media to induce somatic embryos. The basal culture mediumconsisted of Murashige and Skoog's (1962) inorganic salts, i00 mg /l myo-inositol, 0.4 mg/l thiamine. HCl, 3~ sucrose, and 0 . 8 ~ Bacto-agar (MS basal medium). The medium was supplemented with factorial combinations of either 0, 0. i, i, or 3 mg/l 2,4-D and either 0 or I0~ coconut water (GIBC0). In addition, a medium containing 6 ~ sucrose was also supplemented with the same factorial combinations. All media were adjusted to pH 5.8 before autoclaving 150-ml-aliquots for 15 min at 121~ Twenty-five-ml of medium was dispensed into 87 x 15-mm plastic Petri dishes. Ten explants were placed in each Petri dish and sealed with Parafilm. Three replicates were prepared for each treatment. Periodically,

622 cultures

were

observed

microscope. For shaped were

somatic fixed

dehydrated embedded at 1~

under

histological

in

formalin-acetic a

heart-

acid-alcohol,

tertiary-butanol

in p a r a f f i n .

Serial

10 /2m and stained with

plantlets,

dissecting

embryos induced from the explant in

Safranin

a

studies,

(Sass,

0.5~

1971).

cotyledonary

s e r i e s and

sections were cut

explants

on media

structures

were

explants or from

transferred

onto

MS basal

with

cool-white fluorescent tubes

medium at a 16-hr

intervening

media

without

an

intervening

the

photoperiod (5 W/m2).

e i t h e r d i r e c t l y from

on

mg/1 2,4-D).

embryos were

induced

explants

To

somatic

2,4-D. The organized compact callus. Up

to 20~ of the organized s t r u c t u r e s obtained

Hematoxylin and regenerate

with

However,

organized

intervening were

callus.

The

on

structures. identified

During

morphology

The as

developed

organized

explants callus

without

RESULTS AND DISCUSSION

c a l l u s (only 10~ at 1

at 3 mg/1 2,4-D, most of

No

friable

divisions

0.1 mg/1 2,4-D arose

structures

observed

2,4-D.

with

and of

second week of culture, both compact

friable the

c a l l i developed from the cut edges

explants

cultured

with or without coconut without two

2,4-D.

types

structures

of

After

on media with 2,4-D,

water. 4

calli

proliferated

weeks

and

Few c a l l i formed of culture, the

numerous on

the

organized surface

of

structures

maintained

producing

organized

somatic

from

on media without mitotic organized

structures

embryos on the basis

t h e i r globular, heart-shaped, and the

from

were of

torpedo-shaped

(Fig. 1A). A h i s t o l o g i c a l examination

of one of the heart-shaped

structures

exhibited

a bipolar organization with future shoot and root apices

(Fig.

1B).

Hence,

friable

c a l l i were recognized as embryogenic and

nonembryogenic c a l l i , concentration

of

the

compact

respectively.

2,4-D

for

the

The optimum induction

Fig. 1. Somatic embryogenesis and plant regeneration of

C. lanceolata.

A: Globular to

torpedo-shaped

embryos

derived

explants

(scale

Longitudinal

from bar

somatic

cotyl edonary =

and

900 /Am); B:

section of a heart-shaped

somatic embryo (scale bar = 50 /~m). C: Cotyledonary somatic embryos (scale bar = 900 ~m) ; D: Plantlet regenerated from somatic embryo (scale bar = I cm).

of

623 somatic embryos from

the explants was 1 mg/1

a l l treatments except 3~

sucrose

highest

for

without

frequency

the medium containing

coconut of

in

water, wherein the

embryogenic c a l l us

was

obtained at 0.1 m g / 1 2,4-D. In general, the inclusion of 10~ coconut water in the medium promoted the induction of embryogenic callus in the range of 0.1 to 3 mg/1 2,4-D. However, a double level (6~) of sucrose in the medium enhanced the induction of the cal l us at only 1 mg/1 2,4-D. The maximum induction frequency of somatic embryos from explants reached as high as 80~ on the medium containing 6~ sucrose with 1 mg/1 2,4-D and 10~ coconut water (Fig. 2). Many of the somatic embryos had more than two cotyledons (Fig. 1C). Cotyledonary somatic embryos grew into p l a n t l e t s (Fig. 1D) when t r a n s f e r r e d onto MS basal medium. Fi nal l y 30 plants were prepared for t r ans pl a nt a t ion to p o t t i n g s o il.

Pri or to

this

communication, the description of

somatic

embryogenesis

in

(Ham and Lee, 1976) has been

the

Campanulaceae

reported

for only

one species, the Japanese bellflower, Most species in t hi s family are widely used as medicinal herbs in Korea, Japan and China, and due to the recent increase in demand for these species, propagation by somatic embryogenesis can be an a t t r a c t i v e a l t e r n a t i v e to the current practice of propagation by root cuttings. Somatic embryogenesis described in this communication will cont ri but e to the practice of micropropagation. ACXNOWLEDGES~VFS

We thank Drs. Sang S. Kwak and Nam H. Song for t h e i r c r i t i c a l reading of t hi s manuscript. REFERENCES

t

i00

0--0 9O 80

~

o-o [~--Z3 3s + lo cw u--mm

6s + io cw

T/

~l

7o

-~ 60 ~- 50

)

N

N

N 30 e 20

~-~ 10 0,1

1

3

Concentrationof 2,4-D(~/1) Fig. 2. E f f e c t s o f various concentrations of 2,4D, sucrose, and coconut water on the formation of somatic embryos from cotyledonary explants of C. lanceolata. 3S: MS medium containing 3~ sucrose ; 6S: MS medium containing 6~ sucrose ; I0 CW: I0~ coconut water. Data were c o l l e c t e d a f t e r 4 weeks of culture. Vertical bars represent •

Ahn CS, Chung SJ, Koh OC, Park SH (1986) J Korean Soc Hort Sci 27:205-212 Harn C. Lee, YI (1976) Korean J Plant Tissue Culture 4:1-3 Lee MS, Lee JH, Kim DY (1980) Korean J Plant Tissue Culture 7:9-12 Murashige T, Skoog F (1962) Physiol Plant 15: 473-497 Sass JE (1971) Botanical microtechnique. 3rd ed., The Iowa State Univ. Press, Ames, Iowa, pp 55-77