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IN VITRO PROPAGATION OF THE OSTRICH FERN (Matteuccia struthiopteris)

BRIAN W, DYKEMAN and BRUCE G. CUMMING Plant Industry Branch, New Brunswick Department of Agriculture, Fredericton, N.B. E3B 5HI; and Department of Biology, Univeristy of New Brunswick, Fredericton, N.B. E3B 6EI. Received Il Jan. 1985, accepted )6 Apr. 1985

DyreveN, B. W. eNo CurravrNc, B.

G. 1985.

In vitro propagation of the ostrich

fern(Matteuccia struthiopteris). Can. J. Plant Sci. 65: 1025-1032.


Methods were developed for the successful in vitro propagation of ostrich fern (Matteuccia struthiopteris (L.) Todaro) clones utilizing shoot tips derived by forcing

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lateral buds on the rhizome. Maximum shoot proliferation was attained with 6furfurylaminopurine (kinetin) at 1.0 mg/L with half-strength Murashige and Skoog (MS) inorganic salts and sucrose, agar, NaH,PO., adenine sulphate, i-inositol and thiamine.HCl at 30 000, 4000, 85, 40, 100, 0.4 mglL, respectively. Excellent frond and root development was achieved with half-strength MS salts and sucrose, agar, i-inositol and thiamine.HCl at 7500, 4000, 100 and 0.4 mg/L, respectively. The methods developed were satisfactory for a cross section of clones. Morphogenesis in vitro was dependent on medium osmotic potential.

Key words: Matteuccia struthiopteris, in vitro propagation, tissue culture, morphogenesis, fern (ostrich) [Propagation in vitro de la matteuccie fougdre-ir-l'autruche (Matteuccia stuthiopteris) .l Titre abr6g6: Propagation in vitro de la fougdre-d-l'autruche. Nous avons 6labor6 des m6thodes pour la propagation in vitro de clones de la matteuccie fougdre-d-l'auftvche (Matteuccin struthiopteris (L.) Todaro) d I'aide d'extr6mit6s de tiges obtenues par forgage des bourgeons lat6raux des rhizomes. Nous avons obtenu une prolif6ration maximale avec 1,0 mg/L de furfurylamino six purine (kin6tine), une dilution de 50Vo de sels inorganiques de Murashige et Skoog (MS) et un m6lange de sucrose, d'agar, de NaH,PO., de sulfate d'addnine, de i-inositol et de thiamine.HCl ir raison de 30 000, 4000, 85, 40, 100 et 0,4 mg/L respectivement. Par ailleurs, nous avons obtenu un excellent diieveloppement des frondes et des racines avec des sels MS dilu6s d 50o/c et un melange de sucrose, d'agar, de i-inositol et de thiamine.HCl i raison de 7500, 4000, 100 et0,4 mglL respectivement. Les mdthodes mises au point donnaient des r6sultats satisfaisants avec toute une gamme de clones. La morphog6ndse in vitro d6pendait du potentiel osmotique du milieu utilis6.

Mots cl6s: Matteuccia struthiopteris,, propagation


morpnogenese

in vitro, culture de tissus,

The croziers of the ostrich fern. Matteuccia

struthiopteris (L.) Todaro, have been harvested as a spring food delicacy by the inhabitants of eastern North America for sev-

eral centuries. The largest harvest of


croziers or fiddleheads takes place in New Brunswick and Maine where over 400 000
Can. J. Plant Sci.65: 1025-1032 (Oct. 1985)

kg are picked annually from the wild. Present demand for fiddlehead greens is far greater than available supply. Therefore, clonal selection and crop management research have been initiated with the objective of developing a cultivated production system for the species. This also necessitates the development of methods for asexual propagation of superior clones.

t025

t026

CANADIAN JOURNAL OF PLANT SCIENCE

The natural asexual reDroductive svstem

in the species is through the producrion of rhizomes. New rhizomes arise from detached meristems (Wardlaw 1943) which
are laid down at regular intervals along the

here utilized shoots that were obtained by in vitro proliferation on the above medium.

In Vitro Shoot Multiplication


Ten shoots, each ca. 8 rng in weight, were placed on 50 mL of medium in a 300-mL ointment iar. Jars were covered with a double layer of siran film and maintained in continuous light (1000 lx from cool white lamps) at 25-r loC. Four rep-

rhizome and erect shoot (crown). This


normally results in the production of one to

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four new rhizomes per crown per year. If rhizomes are detached from the crown and are sectioned into lengths of 5 cm, for example, the detached meristems are activated, resulting in the development of new lateral shoots. The number of new olants
which can be derived from this o.o..du... however. is inadequate for commercial
needs. For this reason coupled with the verv

lications per treatment of four jars (40 shoots)


each were used. Data were recorded after 8 wk in culture and included fresh weight, dry weight,

number

of

shoots, average shoot weight, the

number of fronds and roots per culture. Analysis of variance and an LSD test were carried out on

following study.

successful in vitro systems developed ior other ferns (Hughes l98l), effort was directed towards developing an in vitro propagation system. This is the subject of the

all data. Three cytokinins: kinetin, N6-benzylamino purine (BA) and N6 isopentenylamino purine
(2iP) and three auxins NAA, 3 indoleacetic acid (IAA) and indolebutyric acid (IBA) were initially assayed, for their effectiveness. Based on these assays, kinetin and NAA were utilized exclusively for all studies. NAA and kinetin were evaluated at concentrations of l 0. 2.0. 4.0 and 8.0 mg/L each in a 4 x 4 factorial study. This was followed by a 5 x 4 factorial study which included NAA at concentrations of 0.0, 0.1, 0.5, 1.0 and 2.0 mglL and kinetin at concentrations

MATERIALS AND METHODS


Explant Establishment in Vitro Various parts of the sporophytic plant were screened as sources of explant material for culture. Shoots produced from detached meristems on the rhizome were the only successful explants
and these were used as the initial source of explants for all studies. Rhizomes were sectioned

into 2-cm pieces, placed in plastic bags

and

Skoog 1962) at 0.5 x strength and the following in milligrams per litre: thiamine.HCl, 0.4; monobasic sodium phosphate, 85; adenine sulphate, 40; i-inositol, 100; sucrose, 30 000; tissue culture (TC) agar (K.C. Biologicals), 8000; 6-furfurylamino purine (kinetin), 1.0; and naphthalenaecetic acid (NAA), 1.0. All studies reported

maintained at 25'C in low light intensiry (100500 lx). When the lateral shoots were 5-10 mm long (6-8 wk), 5-mm shoot tips were removed, surface sterilized tn 10o/a commercial bleach (0.67o NaOCI) and0.l%o C5-28 1-3 wetting agent (Aerosol 22, American Cyanamid) for 10 min, and then rinsed twice in sterile deionized water. Shoots were then recut to l-2 mm and one explant was placed in each 30-mL ointment jar containing l5 mL of medium. From preliminary studies a basal medium for establishment was developed which contained Murashige and Skoog (MS) salts (Murashige and

of 0. l, 0.5, 1.0 and 2.0 mglL. MS inorganic macronutrient salts at half (0.5 x ), three quarters (0.75 x ) and full strength (1.0 X ), and sucrose at concentrations of 15, 30, and 45 glL were evaluated in a 3 x 3 factorial study. Concentrations of TC agar of 0 (using a

filter bridge), 4, 8 and 12 mglL were also evaluated. Five selected cones of the ostrich fern
were evaluated for growth and multiplication in vitro using the final medium described in Table

In vitro Plantlet Development


The basal medium for plantlet development
studies unless altered for study purposes, included: MS macronutrient salts, 0.5 x : MS micronutrient salts; and, in milligrams per litre iinositol, 100; thiamine.HCl, 0.4; sucrose, 7500; and TC agar, 8000. Forty shoots per treatment were used for all studies. These were placed, 10 shoots to a 300mL ointment jar with 50 mL of medium and covered by a double layer saran film. Cultures were

maintained in continuous light at 3000 lx at 25 + l'C for 4 or 6 wk. Plantlets were then evaluated for fresh weisht. the number of fronds with

DYKEMAN AND

CUMMING IN VITRO PROPAGATION

OF THE OSTRICH FERN

r02'7

three or more pinna, and the number of roots greater than 5 mm in length. Sucrose levels of 0.0, 7.5, 15.0, 30.0 and 45.0 g/L; MS macronutrient levels of 0.125 x , 0.25 x , 0.5 x and 1.0 x the orininal concentrations and TC agar levels of 0 (using a filter bridge), 4, 6, 8, 10, and 12 glL werc evaluated sepaiately in randomized complete block design for their influence on plantlet development Five clones were evaluated for their response to the selected plantlet development medium indicated in Table 5.

LSD..

6 d ::: g :::
soo 100

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Post Culture Establishment The move from in vitro culture to soil was best achieved by moving the culture vessels to a shaded greenhouse for 1 wk prior to transplanting. Plants were then transferred to a soiiiess mix in 4-cm cell packs and kept in a high-humidity chamber for 2 wk. They were then moved to a regular shaded greenhouse bench. After 2 mo they were transplanted to 7.5-cm peat pots.

f l.^ o"'

P o

RESULTS
Shoot Multiplication PHvroHonvroNE LEVELS. In the initial study, NAA had no effect on shoot multiplication, and increasing levels of NAA significantly inhibited culture growth (Fig. 1) as expressed by fresh weight. Rates of kinetin higher than 2 mg/L also inhibited growth and reduced shoot proliferation.

o r! I or0

',,

Fig. l. Influence of growth regulators on in vitro growth and shoot multiplication of the ostrich
fern (clone NB44).

There was no significant difference between 1 and 2 mglL of kinetin. There was
a significant interaction between auxin and

levels. There was no interaction between NAA and kinetin at the low levels tested in
this second study.

cytokinin in culture growth. At the high levels of kinetin, NAA had no effect on fresh weight, but at low kinetin concentrations, growth was depressed by increasing NAA rates (Fig. l). In the second study, in which relatively low concentrations of NAA and kinetin were tested, NAA had no stimulatory effect on culture growth or shoot multiplication (Table 1), regardless of kinetin concentrations. As in the initial experiments, multiplication was greatest at kinetin rates of I
and 2 mglL. Culture fresh weight decreased

NurntrtoN AND MEDIUM


ruRE. Half strength MS

srRUC-

macronutrient salts produced the greatest fresh and dry weights (Table 2). Shoot multiplication was greatest with 0.5 x salts while individual shoot weights were greatest at0.15 x. Su-

crose levels did not affect multiplication


rates but strongly influenced culture fresh weight (Table 2). The 30-glL sucrose treatment yielded the greatest fresh weight, but dry weight did not differ between 30 and 45 g/L of sucrose. There was no interaction between inorganic salt and sucrose levels in the medium. Increasing agar levels did not significantly reduce either fresh weight or

with increasing kinetin concentrations;


therefore individual shoot weight dramatically decreased with increasing kinetin

10213
Table

cANADIAN JouRNAL oF pLANT sctENCE

l.

Response of ostrich fern (clone NB44) tissue to levels of


Fresh

NAA and kinetin in culture

NAA
(mg/L) 0.0
0.1 0.5
1.0

Kinetin (mg/L)

weight (mg)
L)l

Shoots per culture 33.9 30.2 32.9 29.0

Calculated weight per shoot (mg)


7.0
8.1

D.y
weight
(mg)

3l
31

245

2t6
225

6.6 7.8

2.0
0.1
t,. -)

216
211

28.6
1

/.o
18.7

26 21 28 30 30 29 25

4.-5

255

30.2
41 .9

Can. J. Plant Sci. Downloaded from pubs.aic.ca by 109.125.141.1 on 05/25/11 For personal use only.

1.0

239
190

2.0 LSD

37.0
8.3
NS NS

8.4 5.7 5.1

(P-0

05)

2I
Signi.ficance (ANOVA)
NS

6.8
NS NS NS

NAA
Kinetin

NAA x kinetin *xSignificant at

l%c

level, NS, not significant.

Table 2. lnfluence of inorganic salts and sucrose on in vitro shoot proliferation and growth of ostrich fern (clone

NB44) trssue MS macronutrient

salts Sucrose (concentration) (mg/L)


0.50 x 0.75 x
1.00 x

Culture
fresh

weight
516

Shoots per c ul ture

Culture dry weight


56.1 49.4

Fresh shoot

weight
(mg)

2t.3
I 1.3

304
48.6
31 .0

)l

216

8.8

l5,000
30,000

45,000

439 543 396


102

l2.7
15 .7

38.4 40.8 52.0


51.1 12.1

36.5
31 .6

13.0 8.9

35.9

LSD

(P:0

0s)

t2.9

SigniJit'ance (ANOVA)

MS salts
Sucrose

Salts x Sucrose

NS

NS NS

NS

NS NS

x+Significant at rhe l7a level: NS, not significant.

the number of shoots per culture. The rate of 4 glL of TC agar produced good growth and shoot multiplication. When the optimum medium presented in Table 5 was evaluated using five different clones, there were large differences in re-

face ofthe developing rachis. Occasionally

sponse between clones,

but growth

ap-

peared to be healthy for all clones (Table

Shoot Morphogenesis Observations indicated that shoots arose adventitiously, largely on the adaxial sur-

shoots would arise from leaf apical and marginal meristems. When phytohormones were present in the medium, leaves underwent little or no lamina development and frond extension but remained as tight croziers in compact, multiple shoots resembling miniature dormant crowns of the species or leaf buds of higher plants (Fig. 2a). When placed on phytohormone-free medium. croziers unfurled without notable delay followed by rapid frond growth and root development (Fig. 2b). On a medium con-

DYKEMAN AND CUMMINC

IN VITRO PROPAGATION OF THE OSTRICH

FERN

IO29

Table 3. Clonal responses to in vitro shoot multiplication and plantlet development media
Shoot multiplicaton medium Clone Shoots per cul ture
12.5 + Fresh

Fronds per

Roots per

weight (mg)
369 + 47 225 +24
194

culture

culture
2.0 + 0.4

NB5
NB I2 NB I4

2.1*
4 .6

ll0+2.1
ll.2:x1.2
'7.0+0.2
6.7 0.8 t2.6 + 3.5 Roots per

22.3 +

0.0
0.7 + 0.1
1.7 +0.'7

118+l
16

I
I

+ l5

NB35 NB39
P

4.8+1.4

t+3

334 + 60 351 +64

4.1

+2.5

lant

Le

dev

Io pme

nt medi um
Fresh

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Clone

weight (mgl
366 + 30 214 + 25 t36 + 2l

Fronds per

No. shoot aplces per


plantlet
2.1 + 0.9 2.0 + 1.0 | .6 + 0.'7

plantlet
8.2 + 0.5

plantlet

NB5
NB I2 NB I4 NB35

3.2+0.4
3.1 + 3.0

+3.0 3.6+2.7
9.',l

NB39

188 + l9 440 + 30

4 8+3
7

31+3.0
7.9 + 0.5

13.2 + 5.0

.8):2

5 8

1.6+0

3.0 + 0.9

*Mean and standard error of 30 cultures per clone.

taining kinetin, each newly initiated leaf


developed a bud on the adaxial surface of its rachis. Thus, when macroscopically sin-

mones, a plantlet development stage was


required. Plantlet development was very responsive to inorganic nutrients, carbohydrate, and agar levels in the medium (Table 4). NAA had no positive effect on plantlet

gle shoots from kinetin-treated

cultures were placed on kinetin free medium, all initiated buds developed resulting in a plantlet with several shoot apices.

development. MS macronutrient salts were finally used at half-strength because root development was greatly inhibited at full
strength. Sucrose levels of 7.5 glL resulted in the greatest fresh weight and the highest num-

Plantlet Development Because of the lack of frond and root development in the presence of phytohor-

Fig. 2. In vitro culture of the ostrich fern on (a) shoot multiplication medium (3 x ), and (b) plantlet
development medium (2 x ).

1030

CANADIAN JOURNAL OF PLANT SCIENCE

Table 4. lnfluence of addenda levels on plantlet development and growth of ostrich fern (clone NB44) shoots

in a pretransplant medlum
Fresh weight Addendum
(mgJ

No. developed
fronds

No. roots

)5mm
4.4
11.2

MS macronutrient salt concentration


1.0

353

4.6

0.5

x
x

0.25 x
0. 125

406 251
133

13.9

I1.5
8.1

9.0

24
3.0
0.1

Significance LSD (P:0.05)

69
24

28
/1

Can. J. Plant Sci. Downloaded from pubs.aic.ca by 109.125.141.1 on 05/25/11 For personal use only.

Sucrose (glL)

0.0

1s
15.0

329
2'75

l3.l

129

t2 l
88
3.0

tt.4
2.'7

30.0

134

450
Significance (ANOVA) LSD (P:0.05) TC agar (glL)
0 4
6
8

5l
44
148 189

0.3

8.4
3.1

t.2
3.4

2.8

40
3.5

120
87 78 53 42

2.2
1.8
I -:)

3l
NS

l0
t2 Significance (ANOVA) LSD (P:0.05) NAA (metL)

J.Z

0.9 0.8 9.0


8.3 NS

2-6

00
0.1

208

'7.4

l15
45

2.9 1.0

Significance (ANOVA) LSD (P:0.01)

**,*Significant

at

P=0.01 and P:0.05, respectively; NS, not significant

ber

of roots per plantlet when evaluated


It

DISCUSSION ventitiously on the adaxial surface of the developing crozier rachis. Hoffmeister
857) reported the existence of buds on the adaxial surface of the rachis which were laid
(I

after 4 wk (Table 4). The largest number of developed fronds was attained with 7.5 and 15 .0 g/L of sucrose. If plantlets were left in

was observed that new shoots arose ad-

culture for 6 wk, the l5-g/L treatment yielded larger plantlets which were darker
green.

Agar concentrations of 0 and 4 g/L in the medium facilitated the greatest growth and frond development. Each level above 4 gl L progressively depressed fresh weight, frond height and the number of developed fronds. Agar concentration had no effect on rootlet development.

down early in the ontogeny of the leaf of the ostrich fern. He found that this bud did not develop beyond a few initials and was soon overgrown by marginal tissue. Hoffmeister's work suggested that the morphogenic potential of these initials was lost very early in the development of the leaf. In the
presence of kinetin in vitro, these initials appear to continue developing to produce

Various clones responded differently to


the plantlet development medium but useable plantlets were produced for all clones tested (Table 3).

new shoots.
In vitro propagation studies on other fern species have consistently reported optimum

DYKEMAN AND CUMMING

IN VITRO PROPAGATION

OF THE OSTRICH FERN

1031

Table 5. Proposed media for in vitro propagation of the ostrich fern (per litre) Explant establishnrent and shoot multiplication MS macronutrient salts 0.5 x MS micronutrient salts 0.5 x
Sodrum monobasic phosphate

Adenine sulphate

i-inositol
T'hiamine.HCl

Kinetin
Sucrose

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TC agar pH to 5.8 Plantlet development MS macronutrient salts MS micronutrient salts

R5 mo 40 mg 100 mg 0.4 mg 1.0 mg 30 000 mg 4000 mg

significantly less at higher sucrose levels. During plantlet development (a 4-wk passage) maximum growth was attained with only 7.5 g/L sucrose ( : - 0. 17 mPa). When plantlet developmeni cultures were

suggests that a fine line exists between adequate carbohydrate nutrition and the inhibitory effect of high sucrose concentra-

left for 6 wk, 15.0 gil of sucrose (rfn': -0.23 mPa) was superior to 7.5 g/L. This

tions (and low osmotic potentials of the


medium) on plant growth and morphogenic development. There have been many references in the literature to the effects ofcarbohydrate levels on callus growth and organogenesis (Thorpe 1982), but relatively few referring to morphogenic development. Most reports have concluded that high carbohydrate levels and/or low medium osmotic potentials are most conducive to organogenesis (Brown and Thorpe 1980), the optimum level being about 37o (wtivol) for many species (Upper et al. 1970; Barg and

0.5 0.5

x x

i-inositol
Thiamine.HCl
Sucrose

100 mg

Agar pH to 5.8

0.4 mg 7500 mg 4000 mg

kinetin levels of l-2 mglL (Harper 1976; Beck 1980) but optimum levels of auxins have varied from 0.1 to 10.0 mg/L of NAA. In the studies reported here, greatest shoot multiplication was also obtained with 1-2 mgil of kinetin. Nevertheless, the fresh weight of cultures increased with decreasing kinetin levels and much larger individual shoots were produced at kinetin levels below I mg/L. No advantage was found in the addition of NAA to the medium at any concentration in these studies.
The levels of inorganic salts, sucrose and agar all significantly affected growth, multiplication and morphogenesis. 'fhe normal

Umiel 1976; Aitken et al. 1981).

Con-

versely, plantlet development of some species has been depressed at high carbohydrate levels with optimum levels ranging

from 0.5 to

2Vo

(Al Talib and Torry

1959;

Barg and Umiel 1976; Aitken et al. 1981; Cheng and Vogui 1911).The differences in response obtained in the present studies could be explained in part by the differ-

multiplication and plantlet development passages. It is likely however, that osmotic potential of the
ences in duration of the shoot

MS inorganic nutrient levels were too high for both the multiplication and plant development stages for this species. Shoot mul-

tiplication was most sensitive to inorganic


salt levels. The response to sucrose levels differed markedly with the developmental process. While shoot multiplication rate was not significantly affected by sucrose concentration. culture growth was very sensitive to

medium also plays a role with the proof shoot organogenesls requiring or tolerating lower medium osmotic potential than the process of morphogenic developcess ment. Plantlets obtained by utilizing the media

described here have been successfully


transplanted to soil and grown to a size suf-

concentration. During shoot multplication (an 8-wk passage) maximum growth was achieved at 30 glL sucrose (osmotic potential (rfn) : - 0.33 mPa) with growth being

ficient for field establishment. To date. l0 000 in vitro cultured plants have been successfully transplanted to soil. Tissueculture-derived plants are currently being evaluated for clonal integrity and field performance.

1032

CANADIAN ]OURNAL OF PLANT SCIENCE

ACKNOWLEDGMENTS
Appreciation is extended to Ms. Terri Brodie for her valuable technical assistance. This work was supported by the New Brunswick Department of Agriculture and Rural Development; also a grant in aid of research from the Natural Sciences and Engineering Research Council of Canada to B. G. Cumming is gratefully acknowledged.

Harper, K. L. 1976. Asexual multipiication of


leptosporangiate ferns through tissue culture. M.S. thesis. University of California, Riverside, Calif. Cheng, T. Y. and Vogui, T. H' 1977. Regeneration of Douglas Fir plantlets through tissue culture. Science 198: 306-307. Hoffmeister, W. 1857. Beitrage zur kenntnis der Geffaskryptogamen. Abh. Kon. Sachs Ges. Wiss. 3: 603-682. Hughes, K. W. 1981. Ornamental species. Pages 5-50 in B. V. Conger, ed. Cloning agricultural plants. CRC Press, Boca Raton, Fla. Murashige, T. and Skoog, T. 1962, A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15: 47349'7
.

Aitken, J., Hargan, J. and Thorpe, T. A. Can. J. Plant Sci. Downloaded from pubs.aic.ca by 109.125.141.1 on 05/25/11 For personal use only.

1981. Influence of explant selection on shootforming capacity of juvenile tissue of Pinus radiara. Can. J. For. Res. ll: 112-ll7.

Al-Talib, K. H. and Torrey, J. H. 1959. The


aseptic culture of isolated buds of Pseudotsuga taxifoLia. Plant Physiol. 34: 630-637. Barg, R. and Umiel, N. 1977. Effects of sugar concentration on growth, greening and shoot formation in callus cultures from four genetic lines of tobacco. Z. pflanzenphysiol. 81: 153160.

Thorpe, T. A. 1982. Carbohydrate utilization and metabolism. Pages 325-268 in J. M. Bonga


and D. J. Durzan, eds. Tissue culture in forestry. Nijhoff and Junk, The Hague, The Netherlands.

Beck, M.

The effects of kinetin and naphthaleneacetic acid on in vitro shoot multiplication and rooting in the fishtail fern. M.Sc. thesis. University of Tennessee, Knoxville, Ten.
31 pp.

J. f980.

Upper, C. D., Helgeson, J. P. and Haberlach' G. T. 1970. Limitations of tobacco callus, tissue growth by carbohydrate availability. Plant Physiol. 46: ll8-122.

Brown, D. C. W. and Thorpe, T. A. 1980.


Changes in water potential and its components during shoot formation in tobacco callus. Physiol. Plant 49: 83-87.

Wardlaw, C. W. 1943. Experimental and

an-

alytical studies of pteridophytes. I. Preliminary observations in the development of buds on rhizomes of the ostrich fern (Matteuccia struthiop-

terls Tod.). Ann. Bot. N.S. 7: l'71-184.