Plant Cell Reports

Plant Cell Reports (1992) 11:155-158

9 Springer-Verlag1992

Direct somatic embryogenesis and plant regeneration from protoplasts of Bupleurum scorzonerifolium Willd Xia Guang-min, Li Zhongyi, Guo Guang-qin, and Chen Hui-min Department of Biology, Shandong University, Jinan, Shandong, 250100, P.R. China Received July 12, 1991/Revised version received January 9, 1992 - Communicated by I.K. Vasil

Abstract Protolasts of Bupleururn scorzonerifolium were prepared from stem node-derived an enzyeme

embryogenic

calli with

mixture, in which snailase was a necessary

After

subculture on the same

medium

with 1 mg1-1

2,4-I) every 20 days for half a year,another two types of ealli

appeared. One was

yellow and compact (type

component. Follolwing cell wall regeneration protoplasts

B) and the

other was yellow, loose and granular (type

divided

C). On the

medium

and directly

developed direct

into

embryo

formed

somatic

embryos which

plantlets. The conditions formation were

favorable to

investigated,

and the

0.1

'without

mgV 1 zeatin, only

type B and C

ferentiate plantlets, type B via

nature of the callus used for protoplast preparation was

C via

found to be a critical factor. The osmotic concentration

the isolation of protop]tasts.

and the composition of the culture the phytohormone Key words:

combinations

Bupleurum

medium

including

were also important.

scorzonerifolium, somatic e m -

bryogenesis, plant regeneration, protoplast.

phytohormone

ealli could dif-

organogenesis and type

embryogenesis. Type C calli were

selected for

Among the different combinations of enzyme mixture tested, the one containing 1.5% cellulose Onozuka R-10, 0.3%

Maeerozym

R-10, 0.5% snailase, 0.6 M

mannitol, 5 mM CaCL2, and 0.3%

Introduction

or with

potassium dextran

sulfate at pH5.8 gave the best results.

Protoplast yield

Protoplasts are being used increasingly in both ba-

was significantly decreased without snailase. The ealli

sic and applied research, particularly in genetic manipu-

were dispersed in the enzyme mixture at 25"Cfor about

lation. There are several reports of protoplast culture of

4 h with

medicinal herbs belonging to the Umbelliferae ( Li and

filtered through a 50 #m stainless steel Falter, collected

Chen 1986; 1987; Jia et a1.1989; Sheng and Chen 1990;

by eentrifugation

Wang and Chert 1991; Zhang et al. 1985 ). We report

with washing

plant regeneration from protoplasts of Bupleurum scor-

5 mM CaCL 2 at pHS.8, and once more with protoplast

zonerifolium

culture medium.

Willd, an

important

medicinal

herb in

China via direct embryogenesis.

and

protoplast

shaking.

at 100xg

solution

The

protoplasts

for 5 min,

washed

were

twice

containing 0.6 M mannitol and

Protoplast culture : The purified protoplasts were soft

Materials and methods Callus induction

occasional

embedded

isolation : The stem

in

modified

5 • 105ml-l,and cultured

MS in

medium at a density of small petri

dishes with a

nodes of Bupleurum scorzonerifolium Willd were s t e -

diameter of 3.5 em. "['he culture medium contained MS

rilized with 0.1% HgCL 2 and cultured on MS medium

inorganic salts and vitamins supplemented with 90 gl-t

(Murashige and Skoog 1962) containing 2 mgV t 2,4-D.

glucose,

White and soft calli (type A) were formed in 2 weeks.

glutamine,

Correspondence to: X. Guang-min

10 g1-1 sucrose, 250 mgl -t D-ribose 40 mgl -l

aspartie

acid,

2 mg -l

100mgl -I eysteine,

156 2 mgl -~ ascorbie acid, 500mgl -~ casein mgl-aNAA, 0.25mgl -~ K T and

hydrolysate, 1.5

0.3% agarose at pH5.8.

hies grew only slightly although the number of dividing protoplasts

increasd

continuously to about

40%

(on

Fresh liquid culture medium was added every 15 days

the 45th day after culture).Many proembryos were seen

after initial culture. After the formation of multicelinlar

at this stage. After about

(about 100 cells)

proembryos (about 70 days

division in protocolonies accelerated and many multieel

after culture),the glucose and phytohormones in the ad-

luar globular proembryos composed of dozens to h u n -

ded medium were omitted.

dred of cells without an

spherical

This led to further

devlop.

two months, the rate of cell

evident

suspensor

merit of the proembryos into globular embryos and em-

(Fig.4).

Very few of the

bryo aggregates. The embryos ( l . 5 - 3 m m ) were transfer-

(Fig.5).

The frequency of proembryo

red onto solid MS medium with

number of

0.1 mgl -a zeatin

or

appeared

proembryos had a suspensor

proembryos

divided

formation

by

the

(the

number of

without phytohormones for plant regeneration.

protoplasts originally plated)was 0.2--0.5% on the 70th

Results and discussion

day after culture. At this time,it was necessary to omit

Protoplast culture : Isolated protoplasts were small and

the glucose and phytohormones in the added fresh me-

densely cytoplasmic (Fig.l). First

dium in order to promote further

toplasts was asymmetrical

division of the p r o -

and was generally seen on

proembryos into globular embryos which became more

the 4th day (Fig.2). Three or four-celled colonies were

compact

formed in seven days (Fig.3). The

without significant

division

frequency

development of the

because of the cell

sustained

internal

enlargement

divisions

(Fig.6).

Careful

was 10-15%( five duplicates ) on the 15th day. There

examination revealed that secondary and tertiary em-

was a rather long lag phase (about 60 days) after this

bryos often arose on

early division stage,

young embryos. A primary embryo associated with the

during which the small protocolo-

the

surface

of the

developing

Fig. 1. Freshly isolated protoplasts X 100 Fig. 2. First asymmetrcal division of a protoplast-derived cell Fig. 3. A small protocolony with polarity of eel1 arrangement X200 Fig. 4. A globular proembryo X200 Fig. 5. A proembryo with a suspensor X 150 Fig. 6. Globular embryos with compact inner cells X 100 Fig. 7. Embryo aggregates X 50 Fig. 8. Further development of the embryos on the solid medium X 10 Fig. 9. Plantlets regenerated from protoplasts X0.6.

157 The

secondary and tertiary e m b r y o s derived from it com-

frequency

0.2--0.5%

prised an embryo-aggregate (Fig.7). At this time,most of the culture consisted of em-

of

early

embryo

formtion

was

on the 70th day of culture in medium

con-

taining 0.5M o f glucose. I f the a d d e d medium con-

bryo-aggregates together with some single globular em-

tained only 0.2M

bryos and developing early proembryos.Embryo-aggre-

ture,the frequency was twenty-fold

gates, l . 5 - 3 m m in diameter,were placed on solid MS me-

quency of embryogenic callus

dium

without

about 2% .Maintenance at high

Some

embryos in

phytohormones or with 0.1mgl -~ zeatin. the

plantlets, and the others

of

time

glucose

was

from the 15th day of cullower but the

formation

fre-

increased to

osmotieum for a long

aggregates

grew directly into

period

grew and

produced more se-

embryogenesis. Direct embryogenesis from cultured pro-

direct

toplasts has

quently, many cotyledonary embryos and plantlets were

most

formed on the calli. The

into embryos in a medium with decreasing osmotic con-

of crowding, but they could grow normally calli were

cut

into

pieces

or

because

when

when plantlets

the were

described

beneficial to

condary embryos and embryogenic callus (Fig.8/. Subse-

plantlets were weak

heen

therefore

in only a few species. In

instances the protoplast-derived cells developed

centration, such as in White spruce (Attree et al. 1987)

Pennisetum americanum (Vasil and Vasil 19801. In

and

separated from each other (Fig.9).

Citrus sinensis( Kobayashi 1985), the optimum osmotic

Factors affecting direct embryogenesis : Three types of

concentration

calli were used (Table 1).Direct somatic embryogenesis

sity of protoplasts.

could be obtained only when protoplasts were

isolated

was

variable

to be

potential of the source

medium in respect to the

protoplasts

is

a

critical

factor for direct embryogenesis.

to

the

superior to K M 8 p

(Kao and Michayluk 1975) division of

protoplasts

Time for maximum yield of protoplast

Protoplast appearence

(11) A

C

and

proembryo initiation (Table 2). MS is most suitable for

Table 1. The effects of source of calli on protoplast divison and direct embryo formation

Callus type

den-

MS and N~( Chu et al. 1975 ) medium were found

from type C callus.This indicates that the embryogenic of

according

Protoplast yield (x 10~g-l

Division frequency on the 15th day

Results

fresh weight) Large, vaeuolated, with few inclusions Small, with some inclusions Small, densely eyto plasmic

1

0

2.5

2

1.25

10

Formation of cell clusters and small caUi Direct formation of embryos and plantlet regeneration

158 embryo development

although KM8p

medium is

richer in organic chemicals. Media with higher nitrogen

(such as NAA) were suitable for somatic embryo formation (Table 2).

content (especially reduced nitrogen) and weaker auxins

Table 2. The effects of different media and phytohormones on direct embryogenesis.

Culture medium

N~)

KM8p

MS a)

MS I)

MS ~)

phytohormones

2,4-D 1+

2,4-D 1+

2,4-D 1+

NAA 1.5+

2,4-D 1+

(mgl -i)

KT 0.25

KT 0.25

KT 0.25

KT 0.25

ZT 0.5

5-10

2-3

5-10

10-15

5

50-100

10-20

100-200

200-500

20-50

none

none

30-50

100-150

5-9

Division frequency on the 15th day(%) Number of proembryos / ml on the 70th day Number of globular embryos and embryo aggregates / ml after 3 months

1) For added chemicals see Materials and Methods. 2) Supplemented chemicals same as in MS medium.

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