SUMMARY: The effect of sucrose concentration during the pregrowth treatment and of the freezing procedure on the survival of encapsulated apices of 6 sugarcane varieties was investigated. The optimal sucrose concentration was 0.75M. Survival was noted with both rapid freezing by direct immersion in liquid nitrogen and slow freezing using a programmable freezer but it was generally higher &amp;er rapid cooling. Important genotypic variation was observed for both parameters studied. Histological examination revealed that cells were slightly harmed during pregrowth and freezing. Apices could withstand freezing as a whole. Direct regrowth of cryopreserved apices occurred within 3 days after thawing

Engelmann, Florent

Gonzalez Arnao, M.T.

Huet, Christine

Urra, C.

M T Gomlez Amaol

F Engelmk2

C Huet2

C Urra&apos

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CRYOPRESERVATION OF ENCAPSULATED APICES OF SUGARCANE: EFFECT OF FREEZING PROCEDURF: AND HISTOLOGY

Horizon / Pleins textes

Cryo-Le t t e  r s  14, 303-308( 1993). 
Published by Cryo-Letters,  7, Wootton Way, Cambridge C E 3  9 L X ,  U K  
CRYOPRESERVATION OF ENCAPSULATED APICES OF SUGARCANE: EFFECT 
OF FREEZING PROCEDURF: AND HISTOLOGY 
M.T. Gomlez Amaol, F. Engelmk2*, C. Huet2 and C. Urra' 
1: CNIC, Ave. 25 y 158, Apartado 6880, Cubanacan, Playa, La Habana, Cuba. 
2: ORSTOM, BP 5045, 34032 Montpellier Cédex 1, France. 
*: to whom correspondence should be addressed. 
SUMMARY: The effect of sucrose concentration during the pregrowth treatment and of the 
freezing procedure on the survival of encapsulated apices of 6 sugarcane varieties was 
investigated. The optimal sucrose concentration was 0.75M. Survival was noted with both 
rapid freezing by direct immersion in liquid nitrogen and slow freezing using a programmable 
freezer but it was generally higher &er rapid cooling. Important genotypic variation was 
observed for both parameters studied. Histological examination revealed that cells were slightly 
harmed during pregrowth and freezing. Apices could withstand freezing as a whole. Direct 
regrowth of cryopreserved apices occurred within 3 days after thawing. 
KEY-WORDS: sugarcane, cryopreservation, apex, encapsulatioddehydration, freezing 
procedure, histology. 
INTRODUCTION * 
Apices oftenly represent the material of choice for germplasm conservation of species 
with recalcitrant seeds and of those which reproduce asexually. Until recently, ipices were 
cryopreserved using cl;ssical procedures including pretreatment with cryoprotectants in liquid 
medium and controlled slow freezing (1). However, survival rates were generally low, growth 
recovery was slow, often including a transitory callusing phase and results were not always 
reproducible. A new cryopreservation method termed encapsulatioddehydration was 
developed for apices. It appears to offer high survival rates, rapid growth recovery and 
reproducible results. It was applied firstly to species fiom temperate climates (2, 3, 4, 5) and 
experimented more recently with plants from tropical origin (6, 7, 8,9). 
In the encapsulatioddehydration process, fieezing is generally rapid but slow 
controlled cooling proved beneficial in some cases (2,4). With sugarcane apices, this technique 
has so far been tested with a total of 8 varieties but using rapid freezing only (7, 8). 
In this paper, the effect of freezing procedure on the survival rate of 6 commercial 
sugarcane vaneties was investigated and histological study of apices was performed during the 
whole cryopreservation process. 
MATERIALS AND METHODS 
Plant material 
Plants of 6 commercial sugarcane varieties were used: 3 coming from the in virro 
collection of CIRAD-CA, Montpellier (B69566, My55 14, IAC54480), 3 fiom that of CNIC, 
La Habana ((28751, C26670, B34104). 
303 
In vitro culture conditions of mother-plants r 
Plants originating from CIRQD were cultivated on semi-solid medium devoid of 
growth regulators and containing 50 g.1-I sucrose (7). At CNIC, mother-plants were 
maintained on semi-solid medium supplemented with 0.1 mg.1-l 6-be laminopurine (BAP), 
were placed at 25&l"C, with a photoperiod of 16 hrs light/8 hrs dark with a photon dose of 36 
Cryopreservation protocol 
The cryopreservation protocol employed consisted of a combination of the conditions 
used by Paulet et al. (7) and G o d e z  Arnao et al. (8).  M e r  dissection, apices were placed 
overnight on CNIC's standard medium (8), encapsulated in the same medium supplemented 
with 3% alginate and pregrown for 24 hrs in liquid medium with various sucrose 
concentrations. Beads were dehydrated for 6 hours in the air current of the laminar flow 
cabinet down to 20% water content (fresh weight basis), then placed in 2 ml polypropylene 
sterile cryotubes and submitted to one of the three following freezing procedures: rapid 
freezing by direct immersion in liquid nitrogen, slow fieezing either at 0.5°C.min-1 fiom 2OoC 
to -40°C (Minicool, L'Air Liquide, France) or at 0.5"C.min-l from 20°C to -4OOC and 
1"C.min-1 from -40°C to -80°C (Bio-Cool, FTS Systems, USA), followed by immersion in 
liquid nitrogen. After one hour of storage at -196"C, beads were thawed by placing the 
cryotubes for 10-15 min at room temperature. Recovery took place on semi-solid medium 
containing 0.5 mg.1-1 BAP, 0.1 mg.1-l KIN, 2.5 g.1-l activated charcoal and 20 g.1-l sucrose 
(8). After one week in the dark, apices were transferred to standard conditions of illumination. 
Survival was noted after 3 weeks by counting the number of apic-s which had developed into 
shoots. Four to 30 apices were used per condition. Pregrown and desiccated apices were used 
as controls. 
Histology 
Samples were fixed for 48 hours at room temperature in a phosphate buffer (pH 7.2) 
containing 10% pardomaldehyde, 1% glutaraldehyde and 1% caffeine. They were then 
dehydrated by successive transfers in alcohol baths with progressively increasing alcohol 
concentrations. Inclusion was camed out in Kulser 7100 resin. Three pm thick sections were 
cut using an automatic microtome (Historange 2218, LKl3). Samples were treated with 
periodic acid-Schiff reaction and naphtol blue black. This double staining technique allows 
specific characterizatiov of polysaccharide compounds which stain red and the soluble and 
non-soluble proteins which stain blue black (1 1). Five apices were prepared and examined after 
each of the following steps: dissection, overnight culture on standard medium, 24-hr 
pregrowth with 0.75M sucrose, 6-hr desiccation, rapid freezing and thawing, 1, 3 and 7 days 
of recovery. 
1 mg.1-1 kinetin 0, 0.5 mg.1-1 indole acetic acid (AIA) and 20 g.1' 7 sucrose (10). Plants 
pmo1.m' 2 1  s- . 
RESULTS 
Survival of pregrown non desiccated control apices was close to 100% in all conditions 
(data not shown). With two varieties (B34104 and B69566) control apices pregrown with the 
lowest sucrose concentration (0.3 M) did not withstand the 6-hour desiccation period. In 
general, survival after fieezing in liquid nitrogen increased in line with increasing sucrose 
concentrations and was optimal for 0.75M (Table 1). Higher concentrations led to a drastic 
decrease in survival (data not shown).There was no apparent effect of the culture medium of 
the mother-plants on survival of apices after the various treatments. Direct immersion in liquid 
nitrogen (RF) generally gave better results than controlled freezing (SF1 and SF2). However, a 
high genotypic effect was noted regarding. the resistance to pregrowth and fieezing with the 
different varieties used. -. 
Figure 1A corresponds to a longitudinal section of an untreated control apex. It 
consisted of the apical dome, several foliar primordia and of a basal part. Cells of the apical 
304 
>L ~ -I 
J 
\ 
region displayed characteristics of meristematic cells: they had a high nucleocfloplasmic ratio, 
a dense cytoplasm, small vacuoles, a large nucleus with one or two apparent nucleoli. B a d  
cells contained a high protein concentration and numerous lipoproteic bodies (Fig. 1B). 
Table 1: Effect of sucrose concentration during pregrowth and of freeing procedure on the 
survival rate ("A) of control (Ctrl) and cryopreserved (RF, SF1, SF2) apices of 6 varieties of 
sugarcane. 
RF: rapid freezing; SF 1 : controlled freezing, 0.5"C.min-1 to -40°C; SF2: controlled freezing 
0 . 5 " c . ~ n - l  to -4OoC+ 1"C.min-1 from -40°C to -80°C. -: not tested. 
1: mother-plants cultivated on CNIC medium; 2: mother-plants cultivated on C W  medium. 
Y 
10 O O 
I l  
n 
63 
44 33 67 
75 25 O 
OJ 68 
Fig. 1A: Control apex immediately after dissection. Bar: 50 pm. 
Fig. 1B: Basal cells of the same apex containing numerous Iipoproteic bodies (lp) and 
proteins (p). Bar: 50 pm. 
Fig. 2: Apex after overnight culture on standard medium. Note the accumulation of starch 
grains in cells of the meristematic region. Bar: 50 pm. 
Fig. 3: Apex after pregrowth and desiccation. Note retracted and intensely stained nuclei and 
cytoplasm retracted from the cell 'wall in some cells (arrows). Bar: 50 pm. 
Fig. 4: Apex 24 hrs after the freeze-thaw cycle. Bar: 50 pm. 
Fig. 5A: Apex 3 days after the freeze-thaw cycle. Note damaged and destroyed cells in the 
basal part (BP) and in the larger primordia. Bar: 100 pm. 
Fig. 5B: Magnification of boxed area on Fig. 5A: detail of the same apex showing recovery of 
cell divisions (arrow) in a young foliar primordium. Bar: 10 pm. 
306 
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After overnight culture on standard medium, accumulation of starch was observed, 
mostly in cells of the meristematic region (Fig. 2). Vacuolization seemed to increase in cells of 
the underlying zone. An important decrease in the quantity of proteins was noted. After the 
24-hr pregrowth in medium with 0.75M sucrose, synthesis of starch increased in intensity. 
After desiccation, the structure of cells was drastically modified pig. 3): nuclei were retracted 
and displayed a particular aspect; being intensely and uniformely stained. Nucleoli were no 
longer visible. The cytoplasm was oftenly retracted, detached fiom the cell wall. Cells of 
samples observed immediately after the freeze-thaw cycle displayed a similar structure. 
However, recovery of apices was very rapid, as shown by examining samples 24 hrs &er 
thawing Fig. 4). At this stage, almost all cells of the apical region had recovered their initial 
appearance, as noted in comparison with Fig. 1. The cytoplasm had -reexpended and was 
densely stained and nuclei, some of which were lobed, showing several well defined nucleoli. 
Three days &er thawing (Fig. SA), most cells of the whole apical zone were alive whereas 
those of the basal part were severely damaged or destroyed. Some cells of the foliar primordia 
were also destroyed. Cell divisions could be observed at the extremity of one young foliar 
primordium (Fig. 5B). 
DISCUSSION/CONCLUSION 
During this work, we showed that encapsulated apices of sugarcane could withstand 
freezing in liquid nitrogen using various freezing procedures after a suitable pregrowth 
treatment with sucrose. Growth recovery of apices after thawing was very rapid and direct, 
due to the fact that most cells of the apical region had been only slightly harmed, as revealed by 
histocytological examination. 
Cryoprotection with sucrose at high concentrations was employed in some cases during ’ 
classical fieezing processes (12, 13, 14) and is used during all encapsulatioddehydration 
procedures (15). In optimal pregrowth conditions, the decrease in survival rate between 
desiccated and cryopreserved sugarcane apices was low, thus indicating that desiccation wai 
the key step in reducing viability and not cryopreservation. A large increase in starch content 
was observed during pregrowth of sugarcane apices, as noted in the case of date palm (16). 
This allows us to assume that intracellular concentration of free sugars was. drastically 
increased, as measured in Aqaragus apices (17) and oil palm somatic embryps (18). In 
contrast to the osmotic dehydration phenomenon occumng during a classical fipezing process 
where only freezable wCer is extracted during pretreatment and prefreezing (19), the extensive 
dehydration performed during desiccation in air allows to remove both freezable and non 
freezable water (20). This was confirmed by microcalorimetric studies which showed that a 
glass transition only was recorded in dehydrated samples (1 5,22,23). Sucrose may protect the 
structural iiitegrity of cells by stabiliig proteins and phospholipid bilayers (21). . 
Histological studies performed on apices of various species fiozen using a classical 
procedure revealed that a considerable number of cells are damaged and only groups of cells, 
oftenly localized in primordial leaf tissues and the meristematic dome area, remained alive (16, 
23,24,25,26). Intense callusing (16,27,28,29) and sometimes production of multiple shoots 
from a single meristem (23) were oftenly reported. On the contrary, the present study showed 
that almost all cells of the meristematic area and of young leaf primordia survived &er freezing 
in liquid nitrogen, thus allowing rapid and direct growth recovery of the whole structure. 
Contrarily to what was observed with potato and grape (2, 4) controlled freezing did 
not improve the survival rate of sugarcane apices. The present study also confirmed the high 
genotypic variability of respoqse to cryopreservation which has already been observed in the 
first report on cryopreservation of sugarcane apices (7). However, fiom a practical point of 
view, it is essential to note that even though the present experiments were performed on a 
limited number of apices, satisfactory survival rates could be obtained with 6 new sugarcane 
varieties using the same freezing procedure. 
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In conclusion, encapsulatioddehydration, which has already been successíùlly applied 
to several plant species from different origins appears as a very promising process for the 
cryopreservation of sugarcane apices and should be developed for a larger range of plant 
species. 
i 
. .  
.. . ... . 
ACKNOWLEDGEMENTS 
This work was performed in the framework of an FAO project (TCP/CUB/OO56). M.T. 
Gonzalez Arnao greatefblly acknowledges the hancial support provided to carry out this 
reaearch. The authors wish to thank F. Paulet and J.C. Glaszmann (CIRAD-CA, Montpellier) 
for kindly providing some of the plant material used in this work and N. Michaux-Ferrière 
(CIRAD-BIOTROP, Montpellier) for her helpfbl comments on the histological study. 
REFERENCES 
F.A. Hammerschlag and RE. Litz eds., CAB Intl., Walliigford, UK, 1992, pp. 169-200. 
1 L.A. Withers, in "Biotechnology of Perennial Fruit Crops", Biotechnology in Agriculture nos, 
2 J. Fabre and J. Dereuddre, Cryo-Letters 11,413-426 (1990). 
3 J. Dereuddre, C. Scottez, Y. Arnaud and M. Duron, C.R Acad. Sci. Paris 310, Sér. III, 265- 
4 P. Plessis, C. Leddet and J. Dereuddre, C.R. Acad. Sci. Paris 313, Sér. III, 373-380 (1991). 
5 C. Scottez, E. Chevreau, N. Godard, Y. Arnaud, M. Duron and J. Dereuddre, Cryobiology 29, 
6 E.E. Benson, N. Chabrillange and F. Engelmann, in Proc. Soc. for Low Temp. Biol. Autumn 
7 F. Paulet, F. Engelmann and J.C. Glaszmann, Plant Cell Rep. in press (1993). 
8 M.T. Gonzalez Arnao, F. Engelmann and C. Urra, Biotecnologia Aplicada (submitted). 
9 S. Mari, F. Engelmann, N. Chabrillange and A. Chamer, in Proc. 15th Intl. Coll. on Coffee, 
10 A. Komeva, (personal communication). 
11 D.B. Fisher, Histochemie 16,92-96 (1968). 
12 J. Fabre and J. Dereuddre, C.R. Acad. Sci. Pans 304, Sér. III, 485-487 (1987). 
13 H. Bouman and G.J. de Klerk, Vakblad voor Bloemsterij 12, 52-53 (1990). 
14 S. Bagniol and F. Engelmann, Cryo-Letters, 12,279-286 (1991). 
15 J. Dereuddre, in "Reproductive Biology and Plant Breeding", Y. Dattée, C. Dumas and A. 
16 S. Bagniol, F. Engelmann and N. Michaux-Femère, Cryo-Letters 13,405-412 (1992). 
17 A. Uragami, PhD Thesis, Kyushu Univ., Japan, 121 p. (1991). 
18 D. Dumet, (personal communication). 
19 P. Mazur, Am. J. Physiol. 247, 125-142 (1984). 
20 J.H. Crowe, J.F. Carpenter, L.M. Crowe and T.J. Anchordoguy, Cryobiology 27, 219-231 
21 J. Dereuddre, N. Hassen, S. Blandin and M. Kaminski, Cryo-Letters 12, 135-148 (1991). 
22 D. Dumet, F. Engelmann, N. Chabrillange, Y. Duval and J. Dereuddre, Cryo-Letters 14 (in 
23 R.H. Haskins and K.K. Kartha, Can. J. Bot. 58,833-840 (1980). 
24 B.W.W. Grout and G.G. Henshaw, Ann. Bot. 46,243-248 (1 990). 
25 K. Taniguchi, R. Tanaka, N. Ashitani and H. Miyagawa, Jpn. J. Genet. 63,267-272 (1988). 
26 S. Fukai and M. Oe, J. Jpn. Soc. Hort. Sci. 59,383-387 (1990). 
27 K.K. Kartha, N.L. Leung and L.A. Mroginski, Zeitschrift Planzenphysiol. 107, 133-140 
28 L.E. Towill, Cryobiology 20, 567-573 (1983). 
29 S. Fukai, Scientia Hort. 45, 167-174 (1990). 
272 (1 990). 
67 1-700 (1992). 
Meeting, Stirling, UK, 8-9/09/92, 1992 (in press). 
Montpellier, France, 6-1 1/06/93, in press (1993). 
Gallais eds.,'Springer Verlag, Berlin, 1992, pp. 291-300. 
(1 990). 
press). 
(1 982). 
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Cryopreservation of encapsulated apices of sugarcane : effect of freezing procedure and histology

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Cryopreservation of encapsulated apices of sugarcane : effect of freezing procedure and histology

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