CryoLetters 23, 375-384 (2002)

CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK

SUCROSE PRECULTURE TO SIMPLIFY CRYOPRESERVATION OF

BANANA MERISTEM CULTURES

Bart Panis*, Hannelore Strosse, Sofie Van Den Hende and Rony Swennen

Laboratory of Tropical Crop Improvement, Catholic University of Leuven, Kasteelpark

Arenberg 13, 3001 Leuven, Belgium. *Email: bart.panis@agr.kuleuven.ac.be

Abstract
A simple cryopreservation method is described for proliferating meristem cultures of
banana (Musa spp.). It relies on a 2-week preculture on media containing 0.4 M sucrose
followed by rapid cooling in liquid nitrogen. Different preculture media were screened for
efficient protection of banana meristems during cryopreservation. Sucrose can be replaced by
both fructose and glucose without significantly affecting post-thaw survival. A high BA
concentration (100 M) in the preculture medium results in less material available for
cryopreservation, but does not affect cryoprotection. Culture in liquid media significantly
improved post-thaw regeneration. The optimized cryopreservation protocol was applied on 36
banana accessions belonging to 8 different genomic groups. It is shown that post-thaw
regeneration frequencies (ranging between 0 and 66 %) are highly dependent on the genomic
constitution of the banana cultivar.
Keywords: banana, Musa spp., cryopreservation, cryoprotection, benzyladenine, fructose,
glucose, meristem culture, sugar, sucrose

INTRODUCTION
Banana plants (Musa spp.) are monocotyledoneous giant herbs and provide a staple food
source for millions of people in the tropics (13). Being a highly sterile, vegetatively
propagated crop, seed conservation is not an option for the conservation of cultivated banana
germplasm, also called cultivars. Field conservation is very labour and space demanding and,
moreover, subject to pests, diseases and adverse weather conditions. In vitro collections
provide a welcome alternative but their maintenance is also labour intensive and during
storage, true-to-type germplasm can be lost due to contamination and somaclonal variation
(35, 37). Thus, the ultimate long term storage strategy for banana germplasm relies on
efficient cryopreservation protocols.
Since the deve lopment of vitrification-based protocols, the list of plant species that can be
cryopreserved has continuously grown. So far, cryopreservation procedures have been
established for in vitro and recalcitrant seed tissues of about 100 and 40 species, respectively.
For each species and tissue type, the cryopreservation protocol needs to be adapted in relation
to its natural freezing resistance, explant size and water content. It is believed that
cryopreservation of tropical plant species is more problematic tha n of temperate species,
since the latter are intrinsically tolerant to low temperatures. Indeed exposure of explants to
low temperatures prior to cryopreservation often proved essential for a high post-
thaw regeneration frequency (15, 23, 25, 26). Abscisic acid (ABA) (4) and sugar (10, 29) have
been shown to have similar effects in many plant species.
For banana, cryopreservation methods have been established for embryogenic cell
suspensions (20), seed and zygotic embryos of wild, non-edible banana relative s (1, 5)
(also
1
called varieties) and meristematic tissues. For meristematic tissues, three cryopreservation
methods are available; a simple freezing protocol relying on sucrose preculture of highly
proliferating meristem clumps (21), vitrification of sucrose-precultured meristem clumps (19)
and vitrification of apical meristems (32). Each method has its advantages and disadvantages.
For example, the first two protocols depend on the time consuming production of
cauliflower- like meristem clumps while for the latter all leaf primordia need to be
carefully removed from the meristematic dome. Hence the challenge lies in the development of
plant cryopreservation protocols that demand minimal labour and consist of a minimum
number of preparatory steps.
In this paper we present the different steps leading to an improved simple freezing
protocol (21) and its application to 36 different cultivars belonging to different genomic
Musa
groups.

MATERIALS AND METHODS

Starting material
All cultivars were obtained from the international Musa germplasm collection at the
INIBAP Transit Centre situated at K.U.Leuven, Belgium (35). Multiplying meristems were
grown in 25 x 150 mm test tubes on 25 ml Murashige and Skoog (18) semi-solid medium
supplemented with 10 M BA and 1 M IAA (p5 medium)(2). They were cultured at 25
-2 -1
2C under continuous 50 E m s illumination provided by 36 W Osram cool-white
fluorescent tubes. The pH was adjusted to 5.8 prior to autoclaving. 'Cauliflower-
like' meristem clusters (Figure 2) were produced on p4 medium, i.e. a p5 medium with a 10
times higher BA concentration (100 M). Every 1 to 2 months, small clumps of
'cauliflower- like' meristems were selected and transferred to fresh medium.

Preculture
As soon as cauliflower- like clumps were obtained, white meristematic clumps of about
4 mm diameter and containing at least 4 apical domes were excised 6 weeks after the last
subculture. They were then transferred onto a preculture medium that contained all p5
medium elements but the final sucrose concentration was increased to 0.4 M. In two series of
experiments, modified preculture media were applied; (i) p5 medium supplemented with
either 0.4 or 0.5 M sucrose, fructose or glucose or a combination of these components and (ii)
0.4 M sucrose combined with p5 medium (containing 10 M BA) or p4 medium (containing
100 M BA). These cultures were kept for 2 weeks under conditions identical to those of
normal meristem growth (see above). At the end of the pregrowth phase, meristem survival
and growth were visually determined.

Cryopreservation
Small clumps of 2-3 mm diameter, containing at least 5 meristematic domes, were
excised from the precultured clumps. Brown tissues were removed and only white-yellowish
tissues retained. These clumps were transferred to 2 ml sterile Cryo Vials, directly plunged
into liquid nitrogen and stored for at least 1 h. Each cryotube contained 7 to 10 clumps.

Post-thaw recovery
After storage, the frozen cryotube was rapidly thawed by stirring in a water bath at 40C
for 1.5 min. Control (precultured but non-frozen) and frozen meristems were transferred to 9
cm Petri dishes containing semi-solid regeneration medium which was identical to the p5
medium but with a 10 times lower BA concentration (1 M) and sealed with parafilm.
Alternatively, regeneration was executed in liquid medium whereby thawed meristems were
transferred to 100 ml Erlenmeyer flasks containing 30 ml liquid regeneration medium and
placed on a rotary shaker at 70 rpm. After 1 week of culture in the dark, Petri dishes and
-2 -1
flasks were transferred to continuous light at 50 E m s and kept at 25 2C. Four weeks
after freezing, regrowth was determined under a binocular microscope. Two types of
surviving tissues were distinguished, i.e. shoots and callus.

Statistical evaluation
Means and standard deviations were calculated and the statistical difference between
mean values of post-thaw regeneration was assessed using Analysis of Variance (ANOVA)
using Duncans multiple range test (P < 0.05). Prior to the analysis, the original
percentage data were arcsin transformed. Arcsin transformation (y = arcsin y1/2 ) is
necessary to stabilise the variance of data that are proportions (binomial distribution).

RESULTS AND DISCUSSION

Production of starting material
The morphological aspect of proliferating banana cultures is very variable and cultivar-
dependent. For example, on p5 multiplication medium, shoot cultures of bananas belonging to
the ABB group gave rise to 'cauliflower- like' meristem clumps whereas AAA highland
bana nas yielded single rooted plantlets. Bananas belonging to other groups exhibited an
intermediate behaviour. Previous experiments revealed that regrowth after cryopreservation
could only succeed with 'cauliflower- like' meristem clusters (Fig. 2). To produce this kind of
material in all Musa accessions, shoot cultures need to be transferred to p4 medium. The
presence of 100 M BA in this medium suppresses the outgrowth of meristems, thereby
favouring the formation of numerous white apical domes.

Optimisation of sucrose preculture medium

In earlier experiments, sucrose at different concentrations (21) and mannitol (22) was
applied in the cryoprotective preculture medium. From these experiments it was concluded
that the cryoprotective effect of sucrose was more than purely osmotic and that adaptive
metabolism was involved. To investigate whether the disaccharide sucrose is replaceable by
other metabolically active sugars, its cryoprotective activity was compared with equimolar
concentrations of the monosaccharides glucose and fructose. Figure 1 shows that other sugars
than sucrose can cryoprotect banana meristems; cryoprotection with 0.5 M glucose or 0.4 and
0.5 M fructose resulted in post-thaw regeneration frequencies that were not
significantly
different from 0.4 M sucrose. Surprisingly, the combination of sucrose and glucose with a
total molarity of 0.5 M resulted in significantly lower survival rates. These results deviate
from those obtained with oil palm polyembryonic cultures, where sucrose preculture could not
be replaced by a wide range of other sugars and polyols (9). Other sugars were only effective
when a preculture was followed by dehydration. Here sugar preculture probably improves
resistance towards dehydration rather than to cryopreservation itself. However, when such
dehydration was applied to banana meristematic clumps no increased survival rate after
freezing was noted (21). Also Asparagus shoot-tips and carrot somatic embryos could be
efficiently dehydration-protected with a wide range of sugars and polyols, prior to the
application of the classical slow freezing protocol (28, 30). Resistance towards dehydration
with plant vitrification solutions (PVS) prior to cryopreservation could also be induced in
Anigozanthos viridis shoot apices using different sugars and polyalcohols (33). The protective
role of sugars is far from understood. Besides its osmotic and colligative effects, thus
reducing the amount of intracellular freezable water, it is known that sugars are competitive
protein and membrane stabilisers (6). In addition to physical changes, sugar treatments can
also result in different physiological and metabolical changes leading to cryoprotection. These
include alterations in proteins (31, 16), membrane fatty acids (36) and amino acids (7).
 Next, it was determined whether the same high amount of BA (100 M), necessary to
induce cauliflower- like meristem clumps was required in the sucrose preculture medium.
Two weeks culture on media containing 0.4 M sucrose did not result in increased proliferating
tissue masses (Fig. 2). On the contrary, a high amount of tissue death was often obtained.
Different reactions were observed (Fig. 3); the tissue became (i) grey, indicating complete
tissue death, (ii) completely black, indicating production and oxidation of polyphenols, (iii)
partially black (75 % (Fig. 2D), 25-50 % (Fig. 2B)) and (iv) completely healthy. This negative
effect of sucrose on tissue growth is clearly cultivar-dependent (Fig. 3) and can be attributed
to the high osmotic value of the medium. We observed that high BA concentrations had a
negative effect on the viability of meristem clumps during preculture. On average, 1.6
precultured meristem clumps per inoculated clump could be excised from the 10 M BA
containing medium, compared to only 1.2 meristem clumps for the 100 M BA containing
medium. Post-thaw viability rates, however, were not significantly affected by the different
preculture media (Fig. 4). Therefore, it was decided for practical reasons to use 10 M BA in
the preculture medium since more viable material becomes available after preculture for
cryopreservation.
When preculture was executed at 15C, no post-thaw survival could be obtained (results
not shown). Growth of non-frozen meristems however, was not influenced by this treatment.
Hence unlike temperate crops, cold hardening is not effective for the tropical banana plant.
Moreover, it proves that a temperature of 15C which proved to be optimal for the medium
term in vitro storage of banana germplasm (2) is too low fo r adaptive metabolism, needed
to survive cryopreservation.

100
% Cal
80
S a % Reg
ur 60 ab
vi
ab
va 40 bc abc
l
( cd cd cd
20

0 0. 0. 0. 0. 0.
0. 5 4 5 4 5 0. 0.
4 M M M M M 3 3
M S G G F F S S
S +0 +0
.2 .3
Sugar in preculture mediu m

Figure 1. Effect of different preculture media on post thaw survival of proliferating clumps of
the cultivar Bluggoe (ABB group). Error bars represent the standard deviation of regenerable
regrowth. Bars marked by the same letter are not significantly different for regenerable
regrowth according to Duncans test after arcsin transformation (P < 0.05) (S = Sucrose; G =
Glucose; F = Fructose; Cal = callus; Reg = regeneration of shoots).
Optimisation of the regeneration medium
After rapid thawing, clumps were initially transferred to semi-solid regeneration medium.
Blackening, due to polyphenol oxidation, was often observed. This can cause cytotoxic effects
but mainly causes an impermeable layer on the recovering clumps thereby preventing nutrient
uptake for further outgrowth. Different strategies could be developed to overcome this
problem, e.g. addition of antioxidants to prevent oxidation, active charcoal to adsorb toxic
substances and regeneration in liquid culture to dilute the released polyphenols.

A B

C D

Figure 2. Proliferating cauliflower-like excised clumps of Kluai Hom Khom (A and B) and
Kluai Roi Wi (C and D), before (A and C) and after (B and D) a 2 week culture period on p5
medium supplemented with 0.4 M sucrose (bar = 500 m).

100 0% black
90
Ex 25% black
80
pl
70 50% black
an
t 60 75% black
re 50
ac 100% black
40
tio 30 greyish
n
20
(%
10
0 Kl Na Ng Ng Pl
Ca Ca Kl Kl Na Pl
ua m a a a
ch ch uai ua m a
Kl i wa m m m
ac ac Ho i wa m
uai Ro Kh u u p5
o o m Ro Kh p4
Ho i o p5 p4
p5 p4 Kh i o
m Wi m
o Wi m
Kh p4 p4
m p5 p5
o
m p4
p5

Cultivar/medium
Figure 3. Effect of BA concentration in the 0.4 M sucrose preculture medium on the
appearance of proliferating meristem clumps of 6 banana cultivars after a 2-week culture
period (p5: medium containing 10 M BA or p4 medium containing 100 M BA).

Neither an increased level of ascorbic acid to 100 mg/l nor the addition of active charcoal at
0.05 % in the regeneration medium resulted in reduced blackening and any higher post-
thaw
survival (results not shown). In general, the use of liquid media with a relative low osmotic
value immediately after cryopreservation is not preferred because of the osmotic shock it can
cause. In banana however, meristem clumps of 8 cultivars out of 10 responded favorably to
such treatment. In Bluggoe, Cachaco, PrataJD and Nakitengwa, regenerable regrowth was
even significantly higher in liquid medium (P < 0.05) (Fig. 5). Only the cultivar Mbwazirume
failed to recover after cryopreservation. The post-cryopreservation application of liquid
regeneration medium for organized tissues is rather unique. Its positive effect for banana
meristems can be attributed to the fact that banana tissues produce massive amounts of
polyphenols that oxidise rapidly under stress conditions (14). Moreover, the final
concentration of the used cryoprotectants (0.4 M) is relatively low in comparison to, for
example, protocols based on the addition of vitrification solutions.

100
90 a Cal

80 ab R eg
ab
70
S
ur 60
c bc bc
vi 50
va c c c c
40
l c c
(% 30
20
10
0 Kl Kl Na
Kl Kl Na Ng Ng Pl Pl
Ca Ca ua ua ua ua m m a a a a
ch ch i i i i wa wa m m m m
ac ac Ho Ho Ro Ro Kh Kh u u p5 p4
o o m m i i o o p5 p4
p5 p4 Kh Kh Wi Wi m m
o o p5 p4 p5 p4
m m

Cultivar/medium

Figure 4. Effect of BA concentration in the 0.4 M sucrose preculture medium on post-thaw

survival of proliferating meristem clumps of six banana cultivars. Error bars represent
standard deviation of regenerable regrowth. Bars marked by the same letter are not
significantly different for regenerable regrowth according to the Duncans test after arcsin
transformation (P < 0.05)(p5 medium containing 10 M BA; p4 medium containing 100 M
BA; Cal = regrowth of non-regenerable callus, Reg = regeneration of shoots).

Application of the optimised cryopreservation protocol to 36 cultivars

The response after cryopreservation is very cultivar dependent, ranging from 0 to 66 %
regeneration (Table 1). Bananas are classified according to their genomic constitution
depending on the relative contribution of Musa acuminata which provides the A genome and
Musa balbisiana which provides the B genome (27). The following combinations exist: AA,
AB, BB, AAA, AAB, ABB, BBB, AAAA, AAAB, AABB and ABBB whereby two, three
and four letters stands for diploids, triploids and tetraploids respectively. Three significantly
different groups were distinguished according to their post-thaw regeneration frequency: (i)
ABB types with an average regeneration frequency of 53 %, (ii) AAA highland bananas with
an average regeneration frequency of 3.9 % and (iii) the other genomic groups with
regeneration frequencies around 20 % (Figure 6). Such differential behaviour was already
observed in other species and can be caused by many factors. In Diospyros, temperate species
responded better to cryopreservation procedures compared to subtropical species (17). Also
dormant apple buds excised from accessions with a low cold hardiness were more responsive
(12).
 100

90

80 Cal

70
Reg
Su 60
rvi
va 50
l
(% 40
)
30

20

10

0
so so so so so so so li so li so so
Bluggo Saba Cacha Prata Prata Grande Wi Wi Na Na Mbwaz Kisubi
e co JD Naine llia llia kit kit irume
li li m m en en li
li li li li gw li
s s gw
Cach Prata Grand a Mbwa
a
e zirum

Regeneration medium/cultivar

Figure 5. Effect of solid (so) versus liquid (li) regeneration medium on post-thaw survival of
sucrose-precultured proliferating meristem clumps of 10 banana cultivars. Error bars
represent the standard deviation of regenerable regrowth.
(Cal = regrowth of non-regenerable callus; Reg = regeneration of shoots).

100
90 Cal
a
80 Reg
70
S
ur 60 b b
vi 50 b b
b
va b
l 40
(% 30 c
20
10
0 A B A
A A
A A A B A B A A
B A A A B Ah
B B Bp

Genomic group

Figure 6. Effect of the genomic group on post-thaw survival of proliferating meristem clumps.
Values are averages of different experiments on cultivars belonging to specific genomic
groups. Error bars represent the standard deviation of regenerable regrowth. Bars marked by
the same letter are not significantly different for regenerable regrowth according to the
Duncans test after arcsin transformation (P < 0.05)
(Cal = callus; Reg = regeneration of shoots).
Table 1. Post-thaw regeneration and survival of 36 banana cultivars belonging to 8 genomic
groups after undergoing the optimised simple freezing protocol.
c
Accession Genomic Regeneration Survival n
group (% SD) (% SD)
Guyod AA 17 21 20 19 416
Lep Mu Nang AA 69 10 4 21
Kluai Khai AA 45 68 43

Plam AAA 24 12 27 13 149

Williams BF AAA 24 17 29 20 451
Williams JD AAA 17 11 25 13 102
Williams BSJ AAA 17 9 23 14 50
GN JD AAA 17 12 22 15 296
Kluai Hom Khom AAA 16 7 24 7 131
Highgate AAA 12 17 24 22 34
Kluai Hom Thong AAA 76 12 1 52
a
Nakitengwa AAAh 8 13 20 22 228
Igitsiri AAAh 00 13 173
Mbwazirume AAAh 00 00 47

Prata AAB 35 23 45 24 301

Prata JD AAB 20 13 25 17 174
Kluai Roi Wi AAB 15 5 20 5 149
Lady finger AAB 00 13 13 45
b
Ngamu AABpl 26 18 32 20 170
Cemsa AABpl 17 13 22 18 244
Three Hand Planty AABpl 14 20 18 23 235
Obino L'Ewai AABpl 10 14 16 6 19
Bise Egome AABpl 6 12 6 12 99
Agbagba AABpl 23 17 6 37

Kamaramasenge AB 32 19 37 21 415
Kisubi AB 10 14 20 19 317

Burro Cemsa ABB 66 18 73 21 133

Cachaco ABB 65 18 71 17 729
Monthan ABB 62 30 65 33 146
Bluggoe ABB 60 20 61 19 326
Cardaba ABB 47 25 47 25 58
Namwa Khom ABB 32 8 37 8 115
Fougamou ABB 30 19 39 30 69
Kluai Namwa ABB 12 11 18 12 73
Kluai Namwa Khom ABB 96 17 11 44

Lep Chang Kut BBB 18 20 25 30 40

TOTAL 18 13 27 8 6131
a
AAAh : East African highland banana
AABplb : AAB plantain
nc : number of cryopreserved meristem clumps
In cassava, the best responding varieties were those that also showed a high drought tolerance
(11). The same tendency can be observed in banana. Cultivars with a higher contribution from
the Musa balbisiana (B) genome, such as ABB bananas tend to be more drought tolerant
compared to cultivars of the Musa acuminata (A) type, like AAA highland bananas. The
differential response in banana, however, cannot be explained completely by the relative
presence of A and B genomes. Other factors that play an important role are proliferation rate
and amount of blackening (35). Bananas belonging to the AAAh group for example produce a
limited number of meristem tips per surface unit and a lot of polyphenols.

CONCLUSIONS

For tropical crops like banana, where cold hardening is not effective, sucrose treatment is
often essential to achieve survival after cryopreservation. For some temperate plant species,
such a sucrose treatment can substitute for cold hardening (29, 15, 23). The originality of the
cryopreservation protocol described in this paper lies in its simplicity. Post- thaw survival is
obtained following preculture of meristems on media with a high sucrose concentration and
not by combining different treatments as used with most other plant species. Previously, post-
thaw survival following a sucrose treatment was only reported for Asparagus buds (34),
embryogenic tissues of sweet potato (3) and oil palm somatic embryos (8). For these three
species, and in contrast to banana meristems (21), post-thaw recovery could be increased
using an extra air dehydration phase.
Despite substantial improvement in survival, regeneration frequency in banana still
remains rather low. Nevertheless, it can be used efficiently for most cultivars belonging to the
ABB group. We recently observed that the application of plant vitrification solutions
following the sucrose pretreatment is very promising for banana meristem cultures belonging
to the other genomic groups (19). Besides its role for banana germplasm conservation, this
simple cryopreservation system provides an excellent model for investigating the
cryoprotective effect of sucrose. As such, we were recently able to correlate changes in
polyamines and membrane fatty acids with survival of sucrose precultured meristem clumps
(24).

Acknowledgements: Financial support of DGIC (Directorate General of International

Collaboration), Belgium and INIBAP (International Network for the Improvement of Banana
and Plantain) within the framework of the Genetic Improvement Group of the Global
Programme for Musa Improvement - PROMUSA is gratefully acknowledged.

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Accepted for publication 17/11/02