Plant Physiol. (1975) 56, 390-393
The Effect of Sucrose on the Differentiation of Excised Fern
Leaf Tissue into Either Gametophytes or Sporophytes1
Received for publication September 20, 1974 and in revised form May 6, 1975
ANN M. HIRSCH2
Department of Botany, University of California, Berkeley, California 94720
ABSTRACT
Excised juvenile leaves of Microgramma vacciniifolia
(Polypodiaceae) develop sporophytic regenerants when
grown on mineral agar with sucrose. The ratio of sporophytes to gametophytes produced from the leaf tissue increases with higher percentages of sucrose such that at
4%V sucrose, the induction of aposporous gametophytes is
a rare occurrence. Experiments varying the osmotic potential with sorbitol and those holding the osmotic potential
of the culture medium constant while varying the sucrose
level indicate that the effect of sucrose on the differentiation of fern leaf tissue into either gametophyte or sporophyte is nutritional rather than osmotic. A significant
effect of sucrose in altering the differentialion of fern leaf
tissue is the increased rate of senescence promoted by high
sucrose concentrations.
sporophytic differentiation can be examined. Besides serving as
a metabolite, sucrose, like any solute, exerts an osmotic effect.
It is therefore necessary to determine whether sucrose is acting
as a nutrient or an osmoticum when investigating any sucrosemediated phenomenon.
MATERIALS AND METHODS
Cultures of juvenile plants were established from callus tissue
of rhizomes of Microgramma vacciniifolia (Langsd. and Fisch.)
Copeland (Polypodiaceae) grown on modified Prague's (14)
medium (NH4N03, 400 mg/l; Machlis and Torrey [9] micronutrients; 2% [w/v] sucrose). Under the greenhouse and culture
conditions employed, M. vacciniifolia was completely infertile.
To test the effects of sucrose on the differentiation of either
sporophytic or gametophytic outgrowths, juvenile leaves 2 to 5
mm in length were excised from young plants growing in vitro
and transferred to 125-ml Erlenmeyer flasks containing 50 ml of
Prague's nutrient medium, pH 5.7, 0.8% agar, supplemented
with various carbon sources or osmotic agents, and autoclaved
at 15 lb/in2 pressure for 15 min. Three juvenile leaves were
transferred to each flask. The leaves were grown with 16-hr light,
8-hr dark, at 23 C.
After 6 weeks, the leaves were harvested and scored for production of either aposporous gametophytes, regenerated sporophytes, or intermediates. Intermediates were classed as those
structures possessing features of both sporophytes and gametophytes and in general were found to have stomates and vascular
tissue on a crenulated but slightly thickened prothallus. The
number of the growths produced on the excised leaves were
totaled with regard to each treatment.
The number of leaves with sporophytes produced divided by
the number of leaves showing gametophytes was used as a convenient method for comparison of the various sucrose culture
media. This number is designated as the s/g ratio. In scoring the
percentage of growths produced, only viable leaves were counted
since an increase in mortality was evident with increased concentrations of sucrose.
Both apogamy, the formation of a haploid sporophyte directly
from a gametophyte without syngamy, and apospory, the production of a diploid gametophyte directly from sporophytic
tissue, circumventing meiosis, can be induced in vitro in ferns
and have been shown to be mediated by the concentration of
sucrose in the culture medium. High levels of sucrose (>1 %)
bring about the formation of apogamous sporophytes (16),
whereas the lack of sucrose or low levels of sucrose ( < 1 %) in
the culture medium promote apospory (3, 13). In addition to
producing aposporous gametophytes, sporophytic fern tissue
can also give rise to regenerated sporophytic plantlets (4, 10).
This has also been found to be dependent on the percentage of
sucrose in the culture medium.
In the epiphytic fern species Microgramma vacciniifolia, either
aposporous gametophytes or sporophytic plantlets can be induced
to form in vitro on the laminar surface of the juvenile leaves.
Significantly, regenerated sporophytes as well as gametophytes
RESULTS
are readily induced on leaves growing in culture medium lacking
sucrose. Because both generations of the life cycle can be induced
Effects of Sucrose. When leaves of M. vacciniifolia were exon excised M. vacciniifolia leaves without added sucrose, the cised and transferred to culture medium lacking sucrose, both
effects of increasing sucrose concentrations on gametophytic and gametophytes and sporophytes developed on the leaf surfaces
(Fig. 1). The addition of sucrose to the culture medium was
found
to have a profound effect on the promotion of sporophytic
I This work was
supported in part by a University of California
development on excised Microgramma leaves.
Chancellor's Patent Fund grant and by a Grant-in-Aid from Sigma Xi, over gametophytic
2
illustrates
a leaf which had been cultured on medium
Figure
the Scientific Research Society of North America. Submitted in partial
fulfillment of the Ph.D. degree, to the Department of Botany, Univer- with 2% sucrose and which after 6 weeks was covered with new
sporophytic plantlets. Increasing the sucrose concentration from
sity of California, Berkeley.
2 Present address: Department of Botany, University of Minnesota, 0 to 4% (w/v) greatly favored this increase in sporophyte deSt. Paul, Minn. 55108.
velopment (Fig. 3). At 0% sucrose, 46.5%- of the viable juvenile
390
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Plant Physiol. Vol. 56, 1975
391
DIFFERENTIATION OF EXCISED FERN TISSUE
FiGs. 1 and 2. Excised leaves of Microgramma vacciniifolia. 1: Leaf grown in medium lacking sucrose. Both gametophytes (g) and sporophytes
(s) have developed on the laminar surface. X 15. 2: Leaf grown in medium with 3% sucrose. Only sporophytes (s) are present on the leaf surface. X 12.
leaves produced sporophytic regenerants and 35.5 % developed
aposporous gametophytes (s/g = 1.31). With a sucrose concentration of 4%, the number of leaves having aposporous gametophytes decreased such that 5.2% of the excised leaves show
bc
Sporophytic
Gametophytic
Intermediate
90
U
a- 8.92
v
G- -1687
F
gametophyte development and 88.1% show sporophytic growth
(s/g = 16.87). Concomitant with an increase in the sucrose
concentration of the culture medium, there was a decrease not
only in the percentage of viable leaves showing gametophyte development but also intermediate growths. The percentage of
intermediates formed decreased from 18 to 6.7% when the level
of sucrose was raised from 0 to 4%. Although intermediates
shared some of the morphological features of sporophytes, they
initially appeared gametophytic and later assumed more sporophytic characteristics (Hirsch, unpublished results).
Viability of the excised juvenile leaves decreased from 62.5%
in no sucrose to 29.9% in 4% sucrose. This increase in mortality
does not seem to be caused by the higher I7r of 3 and 4% sucrose
because equiosmotic concentrations of sorbitol did not lower the
viability of the leaves (Table I). This toxic effect of sucrose
II.
Table I. Effect of Inicreasing Osmotic Potential of the Culture
Medium from 0 to -3 Bars Using Sorbitol and of Holding Osmotic
Potential Conistant at -3 Bars while Inzcreasing Sucrose
Conicentration from 0 to 4%
80
S
7z5.23
-c0
°-
w
)F
70
3 60 I0
-
70
I.
.5
0
'0
-o>40
S.6
-S5 / 3/
3
50
-0
0
cJ
4-,
o
0 30
>0)
40
10
F F FI
0%
1%
2%
Medium
Bars
0
4%
-0.75
Viability:
ioc
62.5X
55
42.3%
45
37
49.5%
36.6%
32
29.9 %
-1.50
3
FIG. 3. The effect of increasing sucrose concentrations (0-4%) on
the development of sporophytes, gametophytes, and intermediates
from excised fern leaf tissue. The s/g ratio refers to the number of viable leaves that produced sporophytes divided by the number of leaves
thatdeveloped gametophytes.
-2.25
-3
-3
-3
-3
-3
1v
0
0
'0
0
1
2
3
4
Viability
s/g
Sorbitol
%o
3%
Percentage of sucrose in culture medium
Sample size:
1 Sample Size
Sucrose
Mf
0
0.03
0.06
0.09
0.12
0.09
0.06
0.03
0
160
39
39
41
67
54
49
57
107
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Copyright © 1975 American Society of Plant Biologists. All rights reserved.
% sample size
ratio
62.5
1.31
1.37
1.32
1.63
1.89
89.7
82.1
68.3
80.6
46.3
57.1
59.6
29.9
3.90
5.11
5.40
16.87
392
HIRSCH
which is not related to 'I7r has also been observed in other plants
(11).
Increase in Osmotic Potential. A range of 0 to 4% sucrose in
the culture medium has a resulting I'r ranging from about 0 to
-3 bars. Since the nutrient medium was dilute, it was omitted
from calculations of I7r.
Experiments employing various osmotic agents were used to
determine whether the promotion of the sporophytic mode of
development is a consequence of the nutritional or the osmotic
effects of sucrose. Mannitol, polyethylene glycol 6000, and sorbitol were used to increase the J7r of the culture medium. Leafy
explants grown in the dark on 0.06 M mannitol ('7r equivalent to
2% sucrose) showed a 1% increase in dry weight as compared
to cultures grown in the dark on no sucrose. In the light, leafy
explants grown in 0.06 M mannitol increased in dry weight by
81% as compared to 115% for no sucrose. Mannitol resulted in
a high mortality of the isolated leaves; most explants were brown
at the end of the experimental period. Polyethylene glycol 6000
also did not prove to be a satisfactory osmoticum because the
pH of the culture medium dropped significantly (pH 5.7 to 3)
during the course of the experiment. A high level of mortality
was also evident for leaves grown in polyethylene glycol medium.
Sorbitol was found to be more satisfactory. Although 0.03 M
sorbitol (I7r equivalent to 1% sucrose) increased the dry weight
of leafy explants grown in the dark by 19% as compared to those
grown without sucrose, viability of the cultures was not adversely
affected as in experiments using mannitol. (Cultures grown for 6
weeks in the dark on 1 % sucrose increased their dry weight
1140% over that for no sucrose). Viability of the isolated leaves
grown in sorbitol concentrations of the equivalent I7r of 0 to
4% sucrose did not show a large decrease as did sucrose alone
(Fig. 3) or sucrose plus sorbitol (Table I).
Increasing the I7r from 0 to -3 bars by using higher concentrations of sorbitol resulted in s/g ratios comparable to the s/g
ratios exhibited by leaves grown without sucrose (Table I). At
I7r equal to -3 bars, the s/g ratio was slightly higher than the
no sucrose ratio of 1.31. Of the viable leaves grown on 0.12 M
sorbitol, 60.2% developed sporophytes (46.5% on no sucrose)
and 31.9 % had gametophytes (35.5 % on no sucrose). Doubtless,
this is attributable to the slight action of sorbitol as a carbohydrate source (19% dry weight increase in the dark). The s/g
ratio shown by leaves grown in 0.12 M sorbitol was still considerably lower than the ratio for 1% sucrose.
Holding the I7r equivalent to -3 bars by decreasing the
amount of sorbitol and increasing the percentage of sucrose in
the culture medium (Table I) gives further evidence that the production of new sporophyte plants is correlated with the increase
in available carbohydrate supply. As in Figure 1, increasing the
percentage of sucrose in the culture medium resulted in the production of more sporophytic outgrowths and a concomitant
decrease in the number of gametophytes produced by viable
leaves. The data show that sucrose promotes the appearance of
sporophytic regenerants regardless of the increase in I7r, an
effect which can be duplicated by using glucose or glucose plus
fructose.
DISCUSSION
The initiation of aposporous gametophytes has been linked to
starvation conditions (13), whereas increasing the level of available carbohydrate supply to fern cells has been implicated both
in the development of apogamous sporophytes (16) and in the
proliferation of sporophytic plantlets from excised fern leaf
tissue (4). The nutritional effect of sucrose on differentiation
into either sporophyte or gametophyte has long been suspected
because the vast morphological differences between the two plants
imply different nutritional levels. The gametophyte is a small,
Plant Physiol. Vol. 56, 1975
flattened thallus limited to moist environments, whereas the
sporophyte is a highly complex plant with elaborated leaves.
Increased osmotic potential has also been assumed responsible
for the differences between growth forms, particularly with
respect to aquatic and terrestial leaves of Marsilea drummondii
(1). Although the fern sporophyte is adapted to a terrestial environment and thus is probably under greater osmotic stress
than the gametophyte which inhabits moist areas, the present
investigation has shown that increasing I'r is an unlikely cause
for the increasing numbers of sporophyte growths formed on
excised fern leaves grown in high sucrose medium. In apogamy,
sucrose has also been shown to have a nutritional, as opposed to
an osmotic, effect (16). Apogamous sporophytes were induced
to develop in higher concentrations of sucrose but were not induced if equiosmotic concentrations of mannitol or polyethylene
glycol were used.
How then does sucrose exert its effect on sporophytic development? Whittier and Steeves (16) suggested that increased carbohydrate supply allows the transition from gametophytic to sporophytic morphology by increasing the energy available to the
cells. Other sucrose-mediated effects such as sporogenesis and
increase in pinnae number have been attributed to the higher
energy level afforded to the explant (5, 15), although Sussex and
Clutter (15) have stated that additional factors may also play a
role. Another effect of increased sucrose levels is a greater mortality of excised fern leaves; these adverse effects do not seem to
be osmotic. Other workers (7, 12) have found that sucrose increases the rate of aging in plant tissue. The plant organs accumulate starch and reducing sugars, and show marked Chl degradation. Excised M. vacciniifolia leaves exhibited symptoms of
senescence within one week after transfer to culture medium
containing 3 and 4% sucrose. The majority of leaves died after
6 weeks; those that survived developed sporophytic growths
more often than gametophytic growths. Leaves grown in medium
with 0 or 1% sucrose do not show the excessive yellowing and
necrosis of leaves grown on medium with higher sucrose concentrations. On the lower sucrose levels, aposporous gametophytes developed on a large number of excised leaves.
Munroe and Sussex (13) suggested that several developmental
processes including chloroplast differentiation are involved in
the redifferentiation of sporophyte cells as gametophyte cells.
They found that kinetin, by retarding senescence, had a stimulatory effect on bracken root cells which subsequently gave rise
to aposporous gametophytes. Although exogenously applied
kinetin does not seem to have an effect on M. vacciniifolia leaves
(unpublished results), cells which redifferentiate into aposporous
gametophytes go through the same processes as do root cells of
bracken, i.e., Chl synthesis and cell division. It seems that for
gametophytes to form from cells of sporophytic tissue, senescence
must be retarded in those cells either by low sucrose levels or by
additional kinetin. The addition of sucrose to the culture medium
would then result in a decrease in the number of gametophytes
produced with the net effect of more sporophytes being observed
on excised leaf tissue.
The mechanism of sporophytic induction from excised leaf
tissue by sucrose seems to involve both nutrition and senescence
by increasing the energy available to the leaves, thereby promoting sporophyte development, and by inhibiting aposporous
gametophyte development by accelerating senescence. The possibility that these two effects work together might be examined by
hastening senescence of leaves grown on 0% sucrose through
ethylene or ABA treatment. In this way, the nutritional effect of
sucrose can be separated from the senescence effect.
It seems that not only apogamy but also apospory and the
proliferation of sporophytic regenerants from excised fern leaf
tissue are controlled by the nonosmotic effects of sucrose. The
influence of sucrose on in vitro apospory and apogamy has direct
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Plant Physiol. Vol. 56, 1975
DIFFERENTIATION OF EXCISED FERN TISSUE
application to the study of alternation of sporophyte and gametophyte generations. It has been suggested that the difference
between the two generations of the fern life cycle can be traced
back to the different environmental conditions under which the
spore and egg develop (8). Nutrition has been implicated as one
of the causal agents here (6), but other factors come into play
which control the differentiation of these two specialized initial
cells (2). With regard to apogamy and apospory, the effects of
nutrition can be more direct, because in these instances the initial
cells are somatic cells which differentiate to form either gametophytes or sporophytes under the conditions of either starvation
or high carbohydrate supply.
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Am
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