Biotechnology in Agricultural Chemistry - American Chemical Society

Chapter 6

U s e of S o m a t i c E m b r y o g e n e s i s for t h e of

Regeneration

Plants

David A. Stuart and M. Keith Redenbaugh

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on June 9, 2016 | http://pubs.acs.org Publication Date: March 18, 1987 | doi: 10.1021/bk-1987-0334.ch006

Plant Genetics, Inc., 1930 Fifth Street, Davis, CA 95616

Somatic embryos of many crop species can develop in plant cell cultures as the result of discrete manipulations of growth regulators and nitrogen levels in the culture medium. Somatic embryos express some of the same morphological and biochemical attributes in vitro as do zygotic embryos, their counterpart in vivo. Somatic embryos represent an opportunity to explore the manufacture of embryo specific storage products as well as plant propagules. Techniques for coating naked somatic embryos which will permit survival and subsequent germination are discussed.

In p l a n t s , a s e x u a l embryo development can be t r i g g e r e d from somatic t i s s u e s t o o c c u r under a v a r i e t y of c o n d i t i o n s both in v i v o and i n v i t r o . T h i s p r o c e s s i s r e f e r r e d t o as somatic embryogenesis ( 1 ) . Somatic embryos a r e m o r p h o l o g i c a l l y (2) and b i o c h e m i c a l l y (3,4) s i m i l a r t o t h e i r s e x u a l l y d e r i v e d c o u n t e r p a r t , the z y g o t i c embryo. However, somatic embryos a r i s e by a p r o c e s s of c l o n i n g which does not i n v o l v e m e i o t i c r e c o m b i n a t i o n events a s s o c i a t e d w i t h f e r t i l i z a t i o n and t r u e seed f o r m a t i o n . A somatic embryo can be germinated d i r e c t l y i n t o a p l a n t by a one step p r o c e s s s i m i l a r t o seed g e r m i n a t i o n , s i n c e i t c o n t a i n s both the embryonic shoot and r o o t axes. However, somatic embryos l a c k s t r u c t u r e s a s s o c i a t e d w i t h z y g o t i c embryos such as the seed coat o r endosperm. Thus, a somatic embryo develops as a naked embryo. Somatic embryos can be used f o r a number of purposes such as f o r regeneration of genetically modified c e l l s , f o r production of secondary p r o d u c t s and mass c l o n i n g f o r a r t i f i c i a l seeds. F i g u r e 1 i s a schematic o f the p r o c e s s o f somatic embryogenesis from e x p i a n t to r e g e n e r a t i o n o f a c l o n e d p l a n t . Because somatic embryos can be produced from c a l l u s c u l t u r e s ( u n d i f f e r e n t i a t e d c e l l s ) , c a l l u s can f i r s t be s u b j e c t e d t o g e n e t i c m a n i p u l a t i o n and somatic c e l l s e l e c t i o n b e f o r e embryogenesis and r e c o v e r y o f complete plants. Somatic embryogenesis i s a l s o an e f f i c i e n t method f o r r e c o v e r y o f p l a n t s 0097-6156/87/0334-0087$06.00/0 © 1987 American Chemical Society

LeBaron et al.; Biotechnology in Agricultural Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

B I O T E C H N O L O G Y IN A G R I C U L T U R A L C H E M I S T R Y


88


6.

following lacking

experiments cell

walls)

liposome/cell such

as

corn,

regenerated Control these

of

rice,

this


of

somatic permit

a

ability

economical

parents

on

there

would

be

since

Another

example

delivery

of

the

next

traits be

could

now

be

using

oil

control

with

components (8)

are

develop

production

seeds.

found

in

produced

by

this of

(3,4),

production

associated

palm

and

embryos

vitro

process

certain

may

plant

use

and

hybrid

for

work

cloned

of

somatic

such

soybean

or

tested, that

a

the

direct

field

production

one

biology plant

suitable

of

these year.

of

using

If

to

pollination

with

cloned

for

hybrid

outcrossing

delivery

example, seed.

year

the

of

is may

sterility,

systems

is

system

plant

and

field

male

from

embryos no

For

hybrid

for

restorer

for

If

of

embryos embryos

production.

lettuce,

allow

pollinating.

somatic somatic

repeatedly

where

currently

produced,

of

of

production

out

be

conceivable

Use

incompatibility

hybrids

as

application

to

agronomic

somatic

embryo-

such hybrids

would

possible. Problems

Delivery least

of

solved

major

(Figure

frequency. (11), have

Some

or

second allow

non-sterile for

for

problem to

naked

or

of

assess

envisioned

analoguous

covering

and

embryos

somatic

current requires

celery

embryos. that

control;

embryos

at

a

(10),

existing,

of

the

autonomously

field

to

high

alfalfa

high-frequency

However, produce

embryo into

environment.

field

somatic

they

covering

of

at be

most

species

embryos

either

the

stage

that

a vigorous

to

plant

in

While

procedures

development,

tests

have

readiness

these

embryos,

of

a

exist

not

been either

(14,15).

since

that

(9),

have

that

embryogenesis

culture

somatic

systems

embryo

the

reliable

of

carrot

maturation

or

encapsulated

Finally,

The

is

is

requires

somatic

frequencies.

germinate

greenhouse to

lower

situation

with

(13)

production

much

normalization

conducted

as

orange

Seeds

field

problem

embryogenesis

at

it

a

populations such

and

somatic

to

Somatic

associated

first

species

(12),

sporadically A

The

available

poor

will

2).

with

embryos

problems

synchronous

coffee

protocols

Associated

somatic

three

differentiate

3).

and

cloned

to

could

hand

be is

be

need

not

without

Research

it

can

experiments

in

aroma

production.

crossed

does

it

and crops

embryogenesis.

zygotic

for

are

and

reliable

suitable

for

hand

to

breeding

could

no

crops

genesis,

alfalfa

many

to

which

immediate

plant

they

similar

(6,7),

plant

contain

parents,

crop

for

lines

these

In

important

somatic

makes

cells

level.

more

tool

impact

of

plant

macromolecules

and

of

opportunity

flavor,

and

industrial and

an

products

The

exists.

cotton,

pathway

oil,

embryos.

inbred

the

plant cocoa

on an

an

(naked of

agronomically

process

biochemically

(5),

valuable

parent

the

present

essential

cloning

have

are

also

secondary

more

A

Many

soybeans,

using

celery

products as

they

example,

seeds

wheat,

developmental

embryos

numerous

For

protoplasts

electroporation

possible.

Because of

as

procedures.

primarily

species

somatic

employing such

fusion

89

Use of Somatic Embryogenesis

STUART AND REDENBAUGH

will

to

the

embryo

techniques that

this

embryos need

seed is for

to coat

are be of

referred

zygotic to

production

coating

be

naked, coated

a

as and

a

it with

seeds. somatic

is a

The seed

maturation

hydrated

gel

presently protective

of

combined (Figure somatic

matrix,


but


BIOTECHNOLOGY IN AGRICULTURAL CHEMISTRY

Establish Somatic

Increase Mature

Package in

Quality Somatic

Somatic

2.

Critical

research

of and Embryos

Embryos

G e l Capsules

Germinate

Figure

a n d Improve Embryogenesis

Somatic

problems

Seeds

for production

of

somatic

seeds·



STUART A N D R E D E N B A U G H


Figure 3. Somatic seeds. i n calcium a l g i n a t e beads. in diameter.

A l f a l f a s o m a t i c embryos e n c a p s u l a t e d E a c h c a p s u l e i s a p p r o x i m a t e l y 4 mm


92


there

is

no

reason

compatible

with

desiccating

somatic

useful. have

Attempts

not

that

the

a

dry

embryos

As

high

could

not

techniques

develop,

to use d r i e d

given

coating

embryo.

dry coatings

coatings

frequency

with

be

for

would

somatic

germination

used

if

it

dehydrating be

especially

embryos

of

is and

of

embryos,

carrot however

(16,17). Reliable

Culture

Embryogenesis


In

alfalfa,

can

be In

and o t h e r

which

a

as

to

increase

and

amino

acid The

frequency

of

hormone

genesis

by

the

and

(2,4-D)

four and

day

5

bred

for

varieties

Maturation

of

Embryos

Alfalfa

In

addition

amino

to

acids

Amino

Embryos

containing

no amino

highest

proline,

of

frequency

Further lower

levels

alfalfa in

were

the

size

a

more

give

high U S

a

clear

of

embryos

of by

that

a

acid

embryos

(20).

selected

Quality

and

be

more

was

high

used

to

of

Embryos zygotic

embryos

altered.

in protein

a

balance by

By

using

treating

in this t h a n were

manner those

the biochemistry

When

compared

do n o t Perhaps

a 50% i n c r e a s e

treated

Furthermore,

was

(18).

achieved

50 uM)

give

arginine,

frequency

formation.

to

medium

embryos

achieve

are

embryo

easily

alanine,

performance

quality

(4). to

give

(18). formed

culture

Glutamine-treated

which

formation,

and best

dilute by

Somatic

embryos

convert

on

of

embryos

largest

also

the best

protein

difference

the

embryo-

shown

resulting

followed

2,4-D treatment. storage

in

effected

has

somatic

the

will

embryo

similar

the

the

10 uM 2 , 4 - D ( i n s t e a d

improved-quality

embryo-specific 2,4-D-induced,

of

of

should

c o n v e r s i o n was a c h i e v e d after

in

aseptically

with

of

including

(20).

in

with

work

of

induction of

2,4-D for

also

induction

been

improvements

morphologically

produced of

acids quality.

is

the

have

treatments

and

degree

NH^

yield

or hormones.

amino

such yields

the c o n t r o l

alfalfa

frequency

Thus,

of

highest

embryos

i n embryos

the acids

of

of

cultures

embryo

.

result

combinations between

acids

to

the

arginine

placed

conversion

a n d NH,

necessarily

are

acids

embryo

amino

acid which

understood.

Earlier

the frequency the

amino

presence

nitrogen

L-proline

may a f f e c t

Improvements

acid-treated

plantlets. the

and

the

the

50 uM 2 , 4 - d i c h l o r o p h e n o x y a c e t i c

to regenerate

a lanine,

but

certain

during

used.

gives

increase

Other

in

10 a n d 25

The amino

is

not

of

ion

reduced

(13).

t h e most

ammonium

and l i n e s

increasing

other

embryogenesis

to

Embryo:

also

Glutamine, embryos.

the

of

way a s y e t n o t

genotype

exposure

the a b i l i t y

in

Somatic

embryogenesis

between

levels

exposure

uM k i n e t i n

Additionally,

lies

improve

on

Thus,

somatic

of

to

of

ammonium

L-arginine

depends

i n some

optimization three

and

somatic

tested

formation

NH,

(19).

metabolism

been

the effect

L-arginine

range

i n embryogenesis.

optimized

medium

of of

the e f f e c t s have

L-alanine,

the

regeneration

the Frequency

level

optimal

embryo

Interestingly,

L-proline

the

The

acids

somatic

2.5-fold

compared

(13).

amino

L-glutamine,

L-proline.

in

the frequency

adjusting

investigations,

such

stimulates

causes as

species,

by

medium

further

sources

Improvement

Alfalfa

optimized

regeneration mM.

Control:

in

in

deposition

the 10

level and

was


of

50 uM

evident.

6.

Low

2,4-D

found less of

treated

in than

20 n g

storage

10%

the

there


Gel In

is

each

(Figure

the 4).

represent

level

level

expected

for

Encapsulation for

room

of

system,

11S

to

that

assess

however,

in

the

somatic

level

of

embryo

somatic

embryo

accumulation

seed,

protein

accumulated

is

less

than

suggesting

that

embryo

quality.

Embryos

embryos a

the

normalization

assay

alfalfa

of

embryos

indicates

to

protein

improvement

Somatic

somatic

propagation

for

of

each

result

related

useful

maxiumum

ug

uM-treated

This

a

2.4

50

is

The

additional

order

accumulated

while

accumulation

a n d may

(4).

of

seed,

protein

development quality

embryos

alfalfa

93



to

delivery

be

useful

method

as

is

a

low-cost,

required

with

high-volume

the

following

parameters : 1.

The

2.

A provision

use

of

low-cost

3.

The

for

delivery

materials

singulation

system

should

that

are

non-toxic.

of

the

somatic

be

compatible

embryos. with

existing

planting

equipment. A

hydrogel

encapsulation

satisfies fluid

these

drilling

delivery

because

using

quality

of

somatic

artificial

embryos

and

the

singulates or

cup

embryos

is

a

one

is

the

vacuum

seeds.

gel

particularly

Encapsulation

it

existing

the

producing

system,

requirements.

planters.

further

on

and

As

both

technology

than

allows

for

discussed,

the

parameter

for

critical

in

alginate,

advantageous

embryos

Improvement

encapsulation

based

more

the

quality

need

to

of

progress

simultaneously. Somatic

embryos

lettuce 3).

were

The

lack

most

of

useful

damage

alginate, bean

In

uncomplexed reagent. range does

hardness

not

impede from

somatic

embryos.

due

to

when

of

firm

gels

into to

100

mM

(2-3%

components

majority

of

our

cost.

range).

Other

Complexation calcium

encapsulating

or

time

chloride

somatic

of

This but

somatic

non-encapsulated equivalent

in

was

chosen

alginate

forms

0.5

ferrous

alginate.

for

capsules,

to

5.0%.

calcium

complexing or

w/v)

and

the

Sodium

chloride

chloride,

capsule.

alginate

from

push-pull

per

essentially

ranging

calcium

Chatillon

to

the

complexing

Germination

sodium

locust

with

agents

(w/v)

nitrate such

as

chloride

are

is

minutes.

20-30

(100

embryos

mM) in

also

are the

the great

experiments.

alginate

softer

a

cobaltous

complexing

standard

lower

concentrations either

chloride,

alginate

and

are

with

appropriate

equal

and

potassium

mixed

handling

is

alginates

at

were

germination.

these

or

carrageenan

a

and

Figure

encapsulation

pressure

for

Consequently,

availability

sodium

with

breaking

capsules.

(30

produces

embryo

of

are

an

measured

kg

(21,23;

ease

and

into

Brassica,

hydrogels

embryos

potassium

lanthanum

Sodium

as

sufficiently

capsules

capsules

for

gelatin,

dropped 1.2

somatic

solution suitable

to

of

embryos

and

dropped

Sodium

terms

somatic

celery,

of

form

its

suitable

0.2 is

the

Sodium

with

and

alfalfa,

variety

in

the

hardness,

of

a

somatic

alginate

from

embryos

in

the

general,

Capsule

to

species,

hydrogels

hydrogel

range

ability

four

to

sodium

gum.

gauge,

of

encapsulated

(2%

capsules

(w/v)

that

when

still

mixed

retained

with

gelatin

sufficient

(5%

integrity


w/v) for


94


Somatic Embryos pH7; I M NaCI; I 0 mM DTT ; 200^M PMSF 60

50

Seed IIS Embryos Induced i With o-o

50μΜ

2,4-D

·—·

10/aM

2,4-D

40

\ α» 3

Ο

oc CL

30

1*1

20

10

40

Top

30

20

FRACTION NUMBER

10

1

Bot

co

-

-° CO

CO

?

Ο

CM

CSJ

Ο

Ο

ΙΩ

F i g u r e 4 . A l f a l f a s o m a t i c embryo p r o t e i n s s e p a r a t e d a f t e r e x t r a c t i o n by sucrose d e n s i t y g r a d i e n t s e p a r a t i o n ( l e f t s i d e o f chromatogram) and S D S - p o l y a c r y l a m i d e e l e c t r o p h o r e s i s ( r i g h t s i d e of chromatogram). The 2 , 4 - D - t r e a t e d embryos e x p r e s s more U S p r o t e i n as shown i n t h e s u c r o s e g r a d i e n t . The 11S p r o t e i n h a s p o l y p e p t i d e s t h a t c o - m i g r a t e w i t h U S s e e d p r o t e i n a s shown b y SDS-PAGE. (Reproduced w i t h p e r m i s s i o n from r e f e r e n c e 4 . C o p y r i g h t 1985, Plenum P u b l i s h i n g . )


6.

handling. to

The sodium

complexation

temperature

does

encapsulating and

will

n o t emerge

potassium

similar

in firm


geenan

is

somatic geenan

as

to

the

do

not

ammonium

i t

bean

(23).

either

carbohydrate

appears

endosperm

for

vigorously

In

in

are

order

for

encapsulating of

embryos)

t o 30 C .

research

has been

carra

conducted

Initial

results

a prominent

endosperm

storage

reserves

are required

f o r ex-albuminous are

the

itself

such

species

not

in with

necessarily

may s e r v e

requirements

to

kappa-carra-

temperature

or celery.

of

capsules

capsules.

the capsule to

These

was r i c h

carbohydrates

that

gum ( 0 . 4 t o 1.0%

with

whereas

tailored

This

5 0 0 mM s o l u t i o n

essential

alfalfa

species

additional

It

a

the gelling

to somatic

f o r germination,

endosperm

that

encapsulation

of

prior

may b e u s e f u l

bean

into

gum i s

lowers

embryo

at 40^C chloride.

germinate

chloride.

carrageenan

f o r albuminous

required

dropped

the alginate-gelatin

Locust

embryos

reduced

artificial

or

a

since

additional

capsule

i s held calcium

capsule.

when

50 C ( d e t r i m e n t a l

that

celery,

the

capsules

required.

of

of

The mixture

that

a harder

capsules,

somatic

indicate

embryos

chloride

embryos

Much using

t h e embryos.

from

hardness

from

mixture

mM s o l u t i o n

( 0 . 2 t o 0 . 8 % w/v) a n d l o c u s t

firm

produce

50

somatic

produce

either

a

damage

Carrageenan w/v)

alginate-gelatin

in

95



of

as an

species

of

and conversion

to

concern. Finally, complete, on

in vitro

crops, that

plants

conversion

the conversion for

alfalfa

naked

naked

to

of

somatic

experiments

is

40-50%.

s h o w t h e same

research

celery

seeds

For celery,

this

the

has

and a l f a l f a .

f o r somatic

Although

within

seeds

To date,

with

embryos.

experiment,

embryos

the

is essential.

frequencies

somatic

conversion

experiment and

germination

vigorous

is is

F o r both

identical 60-90%

frequencies

any one e x p e r i m e n t ,

focused

vary

somatic

to

while from seeds

response.

Acknowledgments The and

contributions maturation

studies, studies

of

Steve

studies,

and David are greatly

Slade

Strickland of

Janet

and Peter

and Carol

Nelsen Viss

to

McCall the

to the y i e l d

storage

t o t h e embryo

protein

encapsulation

appreciated.

Literature Cited 1. 2. 3. 4. K.W.; 5. 6. 7. 8.

Tisserat, B.; Esan, E.B.; Murashige, T. Horticultural Reviews. 1979, 1; 1-78. Halperin, W.; Jensen, W.A. J . Ultrastruct. Res. 1967, 18, 428-43. Crouch, M.L. Planta. 1982, 156, 520-4. Stuart, D.A.; Nelsen, J.; Strickland, S.G.; Nichol, J.W. In Tissue Culture in Forestry and Agriculture Henke, R.R.; Hughes, Constantin, M.P.; Hollaender, Α . , Eds. Plenum Publ. Corp., New York, 1985, pg. 59-73. Al-Abta, S.; Galpin, I . J . ; Collin, H.A. Plant Sci. Lett. 1979, 16, 129-134. Jalal, M.A.; Collina, H.A. New Phytol. 1979, 83, 343-349. Janick, J.; Wright, D.C.; Hasegawa, P.M. J. Amer. Soc. Hort. Sci. 1982, 107, 919-22. Turnham, E . ; Northcote, D.H. Phytochem. 1984, 23, 35-9.


96 9. 10. 11. 12.


13. 14. 15.

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