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A M e thod f o r in Vitrd- Storage* atloJfum multiflorum

Lam.

,

P.

J. DA LE '

P/a/tf Breeding Station, Aberystwyth,

Dyfed,

SY23 3EB

Accepted: 25 October 1979

A B S T R A C T

A means of storing

Lolium multiflorum

Lam. stock plants

in vitro

has been developed over a period of

three years. Various explants and culture conditions were examined. Shoot tips 0-3-0-9 mm long were found

to be best for establishing storage cultures because they gave a high yield of plantlets in culture and

important pathogens are eliminated. F or sub-culturing, after each annual cycle of storage at 2- 4 °C,

shoot tips, tiller buds, tiller bases and nodes can be used. Tiller buds were most abundant and best for

increasing the number of stored plantlets when necessary. T here was no advantage of including an auxin

in the culture medium for shoot tips and Murashige and Skoog's medium w ith 0-2 mg 1

- 1

kinetin has

been ado pted for initiating, sub-culturing and storing cultures. T he optimum temperature range for

regenerating plantlets was 20-25 °C. Light was necessary for a high rate of plantlet regeneration and

light quality and intensity affected their growth.

Key words: Lolium multiflorum Lam., ryegrass, storage in vitro, tissue cultu re.

I N T R O D U C T I O N

In forage grass breeding it is necessary to maintain particular plant genotypes for many

years . During progeny testing of a potential variety the parents m ust be maintained and

later when a variety is in commercial production the basic plants, from which seed

stocks are multiplied over several sexual generations, must be kept alive and healthy.

Shoot tip culture is an accepted method for removing pathogens and for propagating

valuable genotypes in a wide range of species (Murashige, 1978). Cultured plants

established from shoot tips have also been stored at tem peratu res from 1-9 °C where

they remain for several mo nth s (Morel, 1975; T omes, 1979) or even years (Mu llin and

Schlegel, 1976) in the same culture vessel. A method of regenerating several forage

grass species from shoot tips and for virus elimination has been reported earlier (Dale,

1977a, b) . This paper describes the application of shoot tip culture for storing plant

stocks of the biennial forage grass species, Lolium multiflorum Lam.

MATERIALS AND METHODS

T o initiate the storage cultures,

Lolium multiflorum

Lam. plants were taken from the

glasshouse and split into individual tillers. Shoots were trimm ed t o 5 cm and roots to

1 5 cm. T illers were washed thorou ghly in tap w ater, surface sterilized in 50 per cen t

sodium hypoch lorite (5-7 per c ent available chlorine) for 7 min and washed six times in

sterile water. S hoot tips 0-1-15 mm long were dissected asep tically in a lamina flow

cabine t with a stereo-microscope at a m agnification of x 10. Excised sh oot tips were

placed on to a cu lture m edium solidified with 4-4- 5 g I"

1

of Sigma (type IV) agar in

Sterilin Universal plastic tube s. T he culture m edium, when no t specified, was M S

(Mu rashige and Skoog , 1962) with kine tin at 0-2 mg I"

1

. T he medium was autoclaved for

15 m in at 121 °C after adjusting the pH to 5-6.

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498 Dale—In vitro Storage of L. multiflorum Lam.

T A B L E 1.

The effect of culturing shoot tips of different sizes on contamination and plant-

let regenera tion in culture

Shoot tip size (mm)

Number of shoot tips

cultured

Number contaminated

Number giving plantlets

Number of plantlets with

only one shoot

01 -0 -3

10

2 (20% )

3 (30%)

0 (0%)

0-3-0-9

21

0 ( 0 % )

16

(76%)

0 ( 0 % )

0-9-3

11

1 (9%)

6 ( 5 5 % )

3 ( 2 7 % )

3-15

56

16(29%)

36 (64%)

2 1 ( 3 8 % )

Contingency

_

< 5 %

< 10%

< 1%

Unless otherwise stated plantlet regeneration occurred in cultures maintained at

25 + 1 °C unde r con tinu ou s white fluorescent light at an intensity outside the cu lture

vessel of approxim ately 6000 lx. After 1-2 m on ths of cultu re, plantlets with sho ots

3- 6 cm long were placed directly into the cold stora ge con dition s of 2-4 °C with a n

8 h daily photoperiod of white fluorescent light at approximately 300 lx.

After one year from culturing (10-11 months at low temperature) the plantlets were

returned to the high temperature regenerative conditions for about one week before

sub-culturing. Various explants were used for sub-culturing; shoot t ips, t i l ler buds

(successive leaves were stripped downwards from the stem to reveal buds in their axes),

tiller bases (a whole tiller was separated, the roots trimmed to 1-2 mm long and the

shoot cut off 2-5 mm from the base of the stem) and nodes (in some cases internode

extension occurred in culture and nodes could b e excised). T he culture medium and

conditions used for sub-culturing were the same as for initial culturing. Explants were

measured (base to tip) with a micrometer eyepiece in the stereo-microscope used for

dissection.

Data were analysed where possible by an analysis of variance. Otherwise Contingency

X

2

was used to determ ine the probability tha t the observed frequencies were cha nce

variations from the overall mean frequency for the experiment.

R E S U L T S

T he first s torage cultures of L. multiflorum were established in 1976 and have undergone

three annual sub-cultures . During this t ime various observations have been made on

how to establish and maintain storage cultures in this species.

Production of pathogen-free p lantlets* for storage

Optimal shoot tip size.

Four size categories of excised shoot tips were examined for

obtaining pa thog en free plantlets for storage. T he smallest (01 mm long) consisted of

the meristem dom e and one leaf primord ium. T he largest (3-15 mm ) had several leaf

prim ordia enveloping the meristem dom e. T he level of contam ination, the number of

shoot t ips that gave plantlets and the number of shoots produced by each plantlet

varied from one size category to the next (T able 1). Sh oo t tips of the second catego ry

(0-3-0-9 mm ) were the least infected and gave the highest yield of plantlets. P lantlets

from this category also had more than one shoot which is a decided advantage at sub-

culturing.

Each stock plant genotype needs to be represented in storage by about ten cloned

plan tlets in individual culture vessels. T his enables stocks to be taken from storage w hen

required for seed multiplication and insures against the occasional death of individual

* Plantlets free of certain important pathogens.

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Dale

—In vitro

Storage ofL.

multiflorum

Lam.

499

T A B L E 2. The effect of various concentrations of

IAA*

and NAA ^ on the frequency of

shoot tips of L.

multiflorum

regenerating plantlets in culture. The shoot tip size range was

0-1-0-3 mm and survival was scored five weeks after culturing

Auxin concentration 0

(mgl -

1

) (Control) 0 0 2 0-2 2 5 10

IA A

Plantlets regene rated/ 14/19 10/20 14/19 14/19 10/19 3/19

shoot tips cultured (74% ) (50%) (74%) (74%) (53%) (16% )

NAA

Plantlets regenerated/ — 10/18 11/18 14/19 5/18 —

shoot tips cultured (56%) (61%) (74% ) (28% )

* IAA, indol-3yl acetic acid.

t NA A, naphthalene acetic acid.

An analysis of variance was significant at 5 per cent.

plantlets. T he mo re explan ts available at sub-culturing the easier it is to increase the

number of plantlets to the original level.

Ryegrass mosaic virus is known to be eliminated from

L. multiflorum

by culturing

shoot tips up to 11 mm long and there is evidence that other viruses can be eliminated

by the same method in this species (Dale, 1977a and u npub lished). Sho ot t ips 0-3-0-9 m m

long were therefore found to be the best, in several respects, for initiating storage

cultures.

Culture medium.

M S m edium supplemented with 0-2 mg I"

1

kinetin was previously

found to be suitable for regenerating plantlets from shoot tips of

L. multiflorum

(Dale,

1975). T he use of auxins in the med ium ha s sometimes increased survival and gro wth of

excised shoot tips

in vitro

(Murashige, 1974) but the addit ion of IAA or NAA to the

kinetin m edium in concentrations ranging from 0-02 to 10 m g l"

1

did not improve

plantlet production in

L. multiflorum

(T able 2). At con centration s of 5 mg 1

- 1

and

above, the auxins were inhibitory.

Light and temperature requirements.

Preliminary experiments suggested that light was

required for the development of the excised shoot tips. On average only 34 per cent of

cultures kept in darkness gave plantlets compared with 59 per cent of cultures given

light (probability < 2 per cent). Similar results were obtained with excised tiller buds.

More detailed tests on shoot tips and tiller buds indicated that plantlet production was

influenced by light intensity and quality. Cool white light at an intensity of 1000 lx gave

a higher number of plantlets than G ro-lux at the same intensity (T able 3). However, the

num ber of shoots produced by plantlets was on average doubled in Gro-lux l ight . H igher

intensities of white light also increased the number of shoots per plantlet while main-

taining a high number of successful cultures.

Shoot tips of

L. multiflorum

can give plantlets when incubated at temperatures from

15-30 °C but 2 0-2 5 °C prov ed to be m ost effective. Co mb ining da ta from several

experiments w here bo th 20 and 25 °C were used 24 per cent (27 /113) of sh oot tips

gave plantlets at 20 °C compared with 19 per cent (21/112) at 25 °C, a difference which

is not statistically significant.

Survival and sub-culture of cold stored plantlets

If plantlets were well established in culture, ideally with sho ots longer than 3 cm,

there was apparently no special requirement for survival during the 10-11 month

periods at low tem perature . F rom 88 to 100 per cent of plantlets survived du ring each

period and most deaths resulted from the culture vessels becoming infected.

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T A B L E

3.

The effect of light quantity and quality on the frequency of shoot tips size

0-25-0-8

mm) and tiller buds 0-4-3 mm) giving

plantlets in culture and on the mean tiller number, shoot height and root length. Data scored six weeks after culturing

Light quality . . .

Light intensity* . . .

Number of explants cultured

Number giving plantlets

Mean tiller number with standard error

Mean shoot height (mm)

Mean root length (mm)t

Dark

0

15

. 4 ( 2 7 % )

1 3 + 0-25

25

3

Gro-lux

(continuous)

1000

15

7 ( 4 7 % )

2-6 + 0-37

> 50

19

Cool white fluorescent (continuous)

1000

15

10(67%)

1 2 + 013

> 50

34

10000

18

11(61%)

2-7 + 0-43

> 50

32

20000

15

9 ( 6 0 % )

2-4 + 0-38

> 50

36

Contingency

X

3

NS

(10-20%)

o

Co

s

o

f

L

m

u

• Measured outside the culture vessel.

t The mean length of the longest root for each plant.

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Dale—In vitro Storage ofh. multiflorum Lam. 501

T A B L E 4. T he efficiency of various explants upon sub-culturing. D ata scored 6 weeks

after sub-culturing

Explant . . .

Size range (mm) ...

Plantlets regenerated/

explants cultured

Mean tiller number six

weeks after culturing, with

standard error

Approximate mean number

of explants available for

sub-culturing, per plantlet

Shoot tips

0-1-3

43 /80(54%)

2-8 + 0-34

2

T iller buds

0 2^ 5

115/173(67%)

1-7 + 0-11

7

T iller bases

2-5

18 /30(60%)

2 9 ±0-44

2

Contingency

X

2

NS

(10-20%)

T he requireme nt at sub-cu lturing is to provide new culture m edium, to confirm the

stocks are alive, to replace plantlets that have been used for seed multiplication or have

died and to produce multi-tiller plantlets with several explants for the next sub-culture.

T here w as little difference between shoo t tips, tiller buds a nd tiller bases in their yield of

plantlets (T able 4). More tiller buds were available for sub- culturing bu t they gave

plantlets with the lowest average tiller number.

Nodes can also be used for sub-culturing. Internode extension regularly occurs in

culture to give up to nine or more easily detectable nodes. Tiller buds (and sometimes

roots) are usually present at the nodes and the apical meristem at the highest node. In

cultured nodes w here the apical meristem is absent, the tiller bud gives a plantlet. T he

frequency of plantlet production from nodes is around 60 per cent.

In practice all four explants have been used for sub-culturing. Where stocks need to

be multiplied, tiller buds are the best because there are m ore of them . T he lower tiller

number of plantlets from tiller buds can be overcome to some extent by culturing small

tiller buds (0-2 -1 mm ) because, as with small sho ot tips (T able 1), they tend to give

plantlets with more tillers.

DISCUSSION

Conventionally, stock plants are maintained in the field or glasshouse where they have

to be weeded, watered and cared for generally. L. multiflorum is not a strict biennial

and can be ma intained by careful man agem ent for many years. T o do this plants need

to be transplanted once or twice per year after splitting them into individual or small

clumps of tillers and removing dead leaf material. Even when this is done, pathogens

accumulate and frequently plants are lost. Plantlets stored in culture are in aseptic

conditions and over the three year period have shown no obvious signs of deterioration.

When moved to soil, they are healthy and grow vigorously.

It is important that stock plants are genetically and to some extent physiologically

stable duri ng st orag e. T his can only be tested over a period of several years and will be

reported on later. Supplementation of the culture medium with an auxin was found to

be unnecessary and hence the risk of callus formation and attendant genetic instability

problem s is reduced (Hold gate, 1977; Hussey, 1978). T he add ition of cytokinin was

found to be beneficial in earlier work so Murashige and Skoog's basal medium with

0-2 mg I-

1

kinetin has been adopted routinely for all stages of the storage system which

is summarised in Fig. 1.

In vitro

stora ge is being evalua ted for storing several othe r forage grass species,

Lolium perenne, Festuca pratensis, F. arundinacea, F. rubra, Phleum pratense and Dactylis

glomerata, some of which have been maintained for over 2 years.


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502 ale

—In

vitro Storage

of

L. multiflorum

Lam.

fnttlation of storage cultures

shoot tips 0-3-0-9 mm

eliminoiion of pathogens

Plantlet regeneration

1-2 months, 2O-25*C

IOOOO Ix continuous

white fluorescent light

Plontlet storage

10-11 months. 2 -1 C

30 0 l i , 8 h photoperiod

white fluorescent light

Seed multiplication

nnual subculture

generally using tiller

buds but also shoot

tips, tiller bases and

nodes

FIG.

1 . T he

in vitro

storage system adopted for

Lolium multiflorum.

A C K N O W L E D G E M E N T S

I thank Ms S. J. Dalton for technical assistance and Dr E. L. Breese for helpful

discussion.

L I T E R A T U R E C I T E D

DALE, P . J., 1975. Meristem tip culture in Lolium multiflorum. J. exp. Bot. 26, 731-6.

1977a. T he elimination of ryegrass mosaic virus from

Lolium

multiflorum by meristem tip culture.

Ann. appl. Biol. 85, 93-6.

1977A. Meristem tip culture in Lolium, Festuca,

Phleum

an d Dactylis. PI. Sci. Lett. 9, 333-8.

HOLDGATE, D . P., 1977. Propagation of ornamentals by tissue culture. In Applied and

Fundamental

Aspects of Plant Cell, Tissue and Organ Culture,

eds J. Reinert and Y. P. S.

Bajaj,

pp .

18-43.

Springer Verlag, Berlin.

HUSSEY, G., 1978. T he application of tissue culture to vegetative propagation of plants. Sci. Prog. Oxf.

65, 185-208.

MOREL, G., 1975. Meristem culture techniques for the long-term storage of cultivated plants. In Crop

Genetic

Resources

for Today and Tomorrow, eds. O. H. Frankel and J. G. Hawkes, pp. 327-32.

International Biological Programme 2, Cambridge University Press, Cambridge.

MULLIN, R. H. and SCHLEGEL, D . E. , 1976. Cold storage maintenance of strawberry m eristem plantlets.

Hort. Science 11, 100-1.

MURASHIGE, T., 1974. Plant propagation through tissue cultures. A. Rev. PI. Physiol. 25, 135-66.

1978. T he impact of plant tissue culture on agriculture. In

Frontiers

of

Plant

Tissue Culture 1978,

ed T .A .T ho rpe , pp . 15-26. T he International Association for Plant T issue Culture, Calgary,

Alberta, Canada.

an d SKOOG, F . , 1962. A revised medium for rapid growth and bioassay with tobac co tissue cultures.

Physiologia. PI.

15, 473- 97.

TOMES, D. T ., 1979. A tissue culture procedure for propagation and maintenance of Lotus

corniculatus

genotypes. Can. J. Bot. 57, 137-40.

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