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A Method for in Vitrd-Storage*'atloJfum

Lam.

multiflorum
,

P. J. DALE

'

P/a/tf Breeding Station, Aberystwyth, Dyfed, SY23 3EB

Accepted: 25 October 1979

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. For 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. There was no advantage of including an auxin
in the culture medium for shoot tips and Murashige and Skoog's medium with 0-2 mg 1 -1 kinetin has
been adopted for initiating, sub-culturing and storing cultures. The 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 culture.
INTRODUCTION

In forage grass breeding it is necessary to maintain particular plant genotypes for many
years. During progeny testing of a potential variety the parents must 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 temperatures from 1-9 C where
they remain for several months (Morel, 1975; Tomes, 1979) or even years (Mullin 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

To initiate the storage cultures, Lolium multiflorum Lam. plants were taken from the
glasshouse and split into individual tillers. Shoots were trimmed to 5 cm and roots to
1 -5 cm. Tillers were washed thoroughly in tap water, surface sterilized in 50 per cent
sodium hypochlorite (5-7 per cent available chlorine) for 7 min and washed six times in
sterile water. Shoot tips 0-1-15 mm long were dissected aseptically in a lamina flow
cabinet with a stereo-microscope at a magnification of x 10. Excised shoot tips were
placed on to a culture medium solidified with 4-4-5 g I"1 of Sigma (type IV) agar in
Sterilin Universal plastic tubes. The culture medium, when not specified, was MS
(Murashige and Skoog, 1962) with kinetin at 0-2 mg I"1. The medium was autoclaved for
15 min at 121 C after adjusting the pH to 5-6.
O3O5-7364/8O/05O497+O6 $02.00/0
17

1980 Annals of Botany Company

BOT45

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ABSTRACT

498

DaleIn vitro Storage of L. multiflorum Lam.

TABLE

1. The effect of culturing shoot tips of different sizes on contamination and plantlet regeneration in culture
Contingency

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(55%)
3(27%)

3-15
56
16(29%)
36 (64%)
21(38%)

_
<5%
< 10%
< 1%

RESULTS
The first storage cultures of L. multiflorum were established in 1976 and have undergone
three annual sub-cultures. During this time various observations have been made on
how to establish and maintain storage cultures in this species.
Production of pathogen-free plantlets* for storage
Optimal shoot tip size. Four size categories of excised shoot tips were examined for
obtaining pathogen free plantlets for storage. The smallest (01 mm long) consisted of
the meristem dome and one leaf primordium. The largest (3-15 mm) had several leaf
primordia enveloping the meristem dome. The level of contamination, the number of
shoot tips that gave plantlets and the number of shoots produced by each plantlet
varied from one size category to the next (Table 1). Shoot tips of the second category
(0-3-0-9 mm) were the least infected and gave the highest yield of plantlets. Plantlets
from this category also had more than one shoot which is a decided advantage at subculturing.
Each stock plant genotype needs to be represented in storage by about ten cloned
plantlets in individual culture vessels. This enables stocks to be taken from storage when
required for seed multiplication and insures against the occasional death of individual
* Plantlets free of certain important pathogens.

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Unless otherwise stated plantlet regeneration occurred in cultures maintained at

25 +1 C under continuous white fluorescent light at an intensity outside the culture
vessel of approximately 6000 lx. After 1-2 months of culture, plantlets with shoots
3-6 cm long were placed directly into the cold storage conditions of 2-4 C with an
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 tips, tiller 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 be excised). The 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
X2 was used to determine the probability that the observed frequencies were chance
variations from the overall mean frequency for the experiment.

DaleIn 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

0
(Control)

002

0-2

10

IAA
Plantlets regenerated/
shoot tips cultured

14/19
(74%)

10/20
(50%)

14/19
(74%)

14/19
(74%)

10/19
(53%)

3/19
(16%)

10/18
(56%)

11/18
(61%)

14/19
(74%)

5/18
(28%)

NAA
Plantlets regenerated/
shoot tips cultured

* IAA, indol-3yl acetic acid.

t NAA, naphthalene acetic acid.
An analysis of variance was significant at 5 per cent.

plantlets. The more explants 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 unpublished). Shoot tips 0-3-0-9 mm
long were therefore found to be the best, in several respects, for initiating storage
cultures.
Culture medium. MS medium 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). The use of auxins in the medium has sometimes increased survival and growth of
excised shoot tips in vitro (Murashige, 1974) but the addition of IAA or NAA to the
kinetin medium in concentrations ranging from 0-02 to 10 mgl" 1 did not improve
plantlet production in L. multiflorum (Table 2). At concentrations 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 Gro-lux at the same intensity (Table 3). However, the
number of shoots produced by plantlets was on average doubled in Gro-lux light. Higher
intensities of white light also increased the number of shoots per plantlet while maintaining a high number of successful cultures.
Shoot tips of L. multiflorum can give plantlets when incubated at temperatures from
15-30 C but 20-25 C proved to be most effective. Combining data from several
experiments where both 20 and 25 C were used 24 per cent (27/113) of shoot 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 shoots longer than 3 cm,
there was apparently no special requirement for survival during the 10-11 month
periods at low temperature. From 88 to 100 per cent of plantlets survived during each
period and most deaths resulted from the culture vessels becoming infected.
17-2

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Auxin concentration
(mgl- 1 )

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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* . . .

Dark
0
15
.4(27%)

Mean tiller number with standard error

Mean shoot height (mm)
Mean root length (mm)t

1-3 + 0-25
25
3

Measured outside the culture vessel.

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

Cool white fluorescent (continuous)

1000

10000

15
7(47%)

15
10(67%)

18
11(61%)

15
9(60%)

2-6 + 0-37
> 50
19

1-2 + 013
> 50
34

2-7 + 0-43
> 50
32

2-4 + 0-38
> 50
36

20000

Contingency
X3
NS
(10-20%)

Co

ge of L. multif

Number of explants cultured

Number giving plantlets

Gro-lux
(continuous)
1000

DaleIn vitro Storage ofh. multiflorum Lam.

TABLE

501

4. The efficiency of various explants upon sub-culturing. Data scored 6 weeks

after sub-culturing
Explant . . .
Size range (mm) . . .

Tiller buds
0-2^-5

Tiller bases
2-5

Contingency
X2

43/80(54%)

115/173(67%)

18/30(60%)

NS
(10-20%)

2-8 + 0-34

1-7 + 0-11

2-9 0-44

The requirement at sub-culturing is to provide new culture medium, 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.
There was little difference between shoot tips, tiller buds and tiller bases in their yield of
plantlets (Table 4). More tiller buds were available for sub-culturing but 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 where the apical meristem is absent, the tiller bud gives a plantlet. The
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 more of them. The 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 shoot tips (Table 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 maintained by careful management for many years. To 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 during storage. This 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
problems is reduced (Holdgate, 1977; Hussey, 1978). The addition 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 storage is being evaluated for storing several other 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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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

502

DaleIn 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
300 l i , 8 h photoperiod
white fluorescent light

FIG. 1. The in vitro storage system adopted for Lolium multiflorum.

ACKNOWLEDGEMENTS

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 CITED
DALE, P. J., 1975. Meristem tip culture in Lolium multiflorum. J. exp. Bot. 26, 731-6.
1977a. The 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 and 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. The 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 meristem plantlets.
Hort. Science 11, 100-1.
MURASHIGE, T., 1974. Plant propagation through tissue cultures. A. Rev. PI. Physiol. 25, 135-66.
1978. The impact of plant tissue culture on agriculture. In Frontiers of Plant Tissue Culture 1978,
ed T.A.Thorpe, pp. 15-26. The International Association for Plant Tissue Culture, Calgary,
Alberta, Canada.
and SKOOG, F., 1962. A revised medium for rapid growth and bioassay with tobacco 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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Seed multiplication
Annual subculture
generally using tiller
buds but also shoot
tips, tiller bases and
nodes