Temperate Fruit Plant Propagation through Temporary Immersion

C. Damiano, S. Monticelli, S.R. La Starza, A. Gentile and A. Frattarelli

Fruit Tree Research Institute, Propagation Department
00040 Ciampino Aeroporto – Rome – Italy
E-mail: isf.propag@mclink.it

Keywords: Carotenoids, chlorophylls, fructose, hyperhydricity, in vitro multiplication,

liquid medium

Abstract
So far many research activities have been reported on automation in
micropropagation; many liquid media systems for tissue culture have been proposed
in this regard. In this work the temporary immersion system (TIS) has been tested
for in vitro propagation of strawberry, pear, apple, peach, cherry, plum, raspberry
and papaya. Because the objectives of the experiments were to assay the media
composition, to establish the immersion-period length and to define environmental
parameters, a simple double bottle device, manually operated, was used. The
temporary immersion of the explants was achieved filling the bottle containing the
explants with the medium transferred through a silicon tube from the second bottle,
in which an overpressure was created by a manual air pump. The effects on growth
were compared to those of the stationary liquid and agarized cultures (control). The
shoot multiplication rate was very high in strawberry, showing the typical juvenile
unilobate morphology, raspberry and papaya. In pear the multiplication efficiency
was strictly genotype dependent; TIS revealed to be highly efficient to prevent the
apices necrosis. For the other species the TIS strongly affected the quality of the
explants preventing the necrosis and the hyperhydricity. All the species maintained
the juvenile habit during the TIS culture. Data on photosynthetic pigments and
sugar contents seem to suggest a positive influence of TIS on the autotrophic
activity.

INTRODUCTION
The high cost of in vitro plant propagation, mostly due to the dependence on the
manual work, was a limiting factor for the application with commercial purposes to many
species. The automation of the entire process engaged several researchers bringing to the
development of large-scale culture systems exploiting the liquid media, as in bioreactors.
In fact, gelling agents do not make easy the automation and are not inert (Berthouly and
Etienne, 2002), while the use of liquid media ensures more uniform growth conditions,
permits the aeration and it is easy to control exudates and contaminants in the medium
(Ziv, 2002). The use of the bioreactors was firstly directed to the cell culture and to the
secondary metabolite production, then to the plant propagation by somatic embryo-
genesis, organogenic regeneration (Ziv, 1995), bulbs, microtubers and corms propagation
(Takayama, 2002). However, when the explants are continuously submerged, they exhibit
clear oxidative stress symptoms, physiological disorders, hyperhydricity and morpho-
genic malformations. These disadvantages have induced the development of modified
alternative methods of liquid media culture.
Among these, the Temporary Immersion Systems (TIS) furnishes good results in
terms of proliferation and vigour of the plants and frequency of morphologically normal
explants. The optimisation of parameters, as the immersion duration and/or frequency,
volume of the containers and volume of the nutrient medium is decisive to improve the
efficiency of the system. Proliferation, microtuberization, somatic embryogenesis are
positively affected by TIS (Berthouly and Etienne, 2002) as well as the production of
secondary metabolites from officinal plants as Hypericum and Lavandula (Hohe et al.,
2002). Several TIS designs exist providing rocking, tilting, renewal of the nutrient
medium, misting, thin film system, with variable level of automation and efficacy.

Proc. XXVI IHC – Biotechnology in Hort. Crop Improvement

Eds. F.A. Hammerschlag and P. Saxena
Acta Hort. 625, ISHS 2003
193
Publication supported by Can. Int. Dev. Agency (CIDA)
 So far TIS has been successfully applied for tropical crops using a system, easily
automatized, called RITA (Teisson and Alvard, 1995). Coffee, Musa spp., Hevea
brasiliensis, sugarcane, Citrus, pineapple have been propagated with this system (Alvard
et al., 1993; Etienne et al., 1997; Cabasson et al., 1997; Lorenzo et al., 1998; Escalona et
al., 1999). However for commercial scale-up the twin flask TIS has been preferred
(Jiménez-Gonzáles, 2002). Propagation by TIS has been extended to forestry,
horticultural and ornamental plants. More recently the interest has been focused on
temperate fruit tree species (Damiano et al., 2000; Damiano et al., 2002; Zhu et al., 2002).
In this work we resume the results over last years of the application of twin flasks
TIS to in vitro propagation of strawberry, pear, apple, peach, cherry, papaya, plum,
raspberry, compared to stationary liquid and solid media. The influence of the system on
multiplication response, shoots quality and physiological state of the treated plants is
reported.

MATERIALS AND METHODS

Twin Flasks TIS

The device was composed of two glass bottles connected with a silicone tube (Fig.
1A-B); one of the bottles was used to contain explants, the other one to contain culture
medium. The medium was periodically transferred from one bottle to the other through an
overpressure created by an air pump; the air insufflated to the bottle was sterilised with
0.2 µm sterile filter (Acro50 - Pall Gelman) with hydrophobic (PTFE) membrane. Glass
beads (2 mm diameter) on the bottom of the bottle containing plants were used to isolate
the plants from the liquid medium thin layer remaining in the bottle during the not
immersion period and for a better regulation of gravitropism.

Plant Material and Cultural Conditions

Micropropagated shoots of Malus domestica ‘Jork 9’, Prunus cerasus ‘Bigarreau
Burlat’, P. persica ‘Yumyeong’, Pyrus communis ‘Conference’, ‘Harvest Queen’,
‘Precoce di Fiorano’, ‘William’, wild pear (Pyrus communis var. pyraster L.), Papaia
spp., P. cerasifera ‘Adara’, Rubus idaeus ‘M. Exploit’ and Fragaria x ananassa
‘Teodora’ were used. Plants were cultured for 60 days on: 1) solidified medium (0.6%
agar); 2) TIS with an immersion period of 30 min or 60 min per day; 3) stationary liquid
condition, using the media reported in Table 1. For all the treatments the medium was
renewed every 20 days. The culture conditions were: 24°C ±1, photoperiod of 16 h, light
intensity of 25 µmol m-2s-1, provided by Osram fluora L58 W/77 lamps.

Experimental Plan
For each treatment 10 single shoots were used and three replications were carried
out. After 60 days of culture, multiplication rate (calculated as number of new shoots
produced per cluster), fresh (FW) and dry weight (DW, obtained after dehydration in the
oven at 110°C for 24 h), were recorded. Water content (WC) percentage was calculated as
WC = [(FW – DW)/FW] x 100. The SE was calculated after the normalization of the data
according to the formula by Bliss (arcsin√%). The content of chlorophylls and carotenoids
(Jensen, 1973), fructose (Davis et al., 1967) and water were measured for all the species
except than pear and wild pear.

RESULTS
After 60 days of TIS the influence of the immersion time in terms of
multiplication rate was detectable, in particular for papaya, raspberry (Fig. 1C-E) and
apple (Table 2). The time corresponding to the highest proliferation performance from
now onwards will be indicated as “optimum time”. This time was 30 min for cherry, wild
pear, Conference and 60 min for all the other genotypes. As regards the control (solid
culture) the multiplication rate was significantly higher at the optimum time in pear
(‘Conference’, ‘Harvest Queen’ and ‘William’), raspberry, strawberry and wild pear. On

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the contrary, the shoot formation resulted highly inhibited by liquid stationary medium
(Table 2).
In TIS, after 60 days, vitrification and necrosis of the explants were never
detected, whereas they were present in solid culture (Fig. 1D). The water content of
temporarily immersed plants in apple decreased at 60 min and increased at 30 min; it
decreased at both time for cherry and peach and at 60 min for plum and increased in
raspberry at 60 min (Table 3).
In stationary liquid condition plants appeared always vitrified: in apple, cherry and
peach (Fig. 1F) a reduced water content, probably due to the necrosis of the plants,
occurred, while in plum and raspberry, where plants appeared hyperhydric but not necrotic,
the water content increased. In strawberry and papaya the WC did not vary (Table 3).
In TIS chlorophylls and carotenoids content was significantly higher than the
control at the optimum time, except in papaya (Table 3).
On the contrary in liquid stationary condition the content of those photosynthetic
pigments was notably lower.
The total amount of fructose was significantly higher in plant grown under TIS at
the optimum time and in apple and raspberry at 30 min (Table 3). In liquid medium the
total fructose decreased significantly. The total fructose content is the sum of the free
fructose and the fructose compounding the sucrose molecule. The fraction of these two
components could vary. In fact, the percentage of free fructose decreased significantly
(therefore the sucrose content increased) in the most part of the genotypes tested, when
cultured by TIS. Only in plum free fructose decreased in stationary liquid medium,
whereas in the other cases it did not change or increased (Table 3)

DISCUSSION AND CONCLUSION

The more clear and immediate effect of the TIS on the generality of the plants
tested so far, was the drastic reduction or the total absence of the hyperhydricity of the
tissues and the necrosis of the apices. This effect, widely reported in the literature, is due
to the high humidity of the containers, that prevents the dessiccation of the explants, the
thin film of medium standing on the explant surfaces, that allows gaseous exchanges but
ensures a constant nutrient supply, and the renewal of the air inside the bottles, that avoids
the ethylene accumulation (Alvard et al., 1993; Cabasson et al., 1997; Etienne et al.,
1997). Stationary liquid condition induced always the decay of plants, due to
hyperhydricity and necrosis, whereas in the solid condition physiological alterations
occurred with different frequency among the genotypes. In cherry and peach the reduction
of hyperhydricity is accompanied to a perceptible reduction of water content in TIS.
The duration of immersion is of great importance in the TIS: it must ensure a
supply of nutrient sufficient to stimulate the development and the growth of plants but
avoid toxic effects occurring in the constant contact of the medium with the plants. So
varying the specific requirements of the genotypes, the optimal immersion time varied
accordingly. The optimal immersion time could affect in the same way both the quality of
the plants and the multiplication rate.
In plants grown in TIS the total amount of fructose significantly increased. Since
the total fructose includes the sucrose fraction, actually in the plants cultured in TIS is
mainly the sucrose to increase, while in plants grown in solid and stationary liquid
conditions the free fructose prevailed (Table 3). These results seem to support the
hypothesis that the plants cultured in TIS, being in contact with the sugars in the medium
only for very short period during the culture, could partially restore the autotrophic ability
and the capability to accumulate sugars. In fact, low or not supply of sucrose to herbaceous
plants has been already shown to increase shoot growth, promoting autotrophic behaviour
(Aitken-Christie and Davies, 1988). Furthermore, the content of chlorophylls and
carotenoids also appeared higher in plants cultured in TIS at the specific optimal
immersion time. In conclusion, in our experiments the TIS was shown to improve mainly
quality of the plant material. Further experiments are needed to determine the response of
plants cultured in TIS to the rooting and the stress of acclimation.

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ACKNOWLEDGEMENTS
Mr. Frattarelli has provided technical implementation of experimental plan and
collection of data.

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Tables

Table 1. Multiplication media.

Apple Cherry Papaya Peach Pear Plum Raspberry Strawberry

Macrosalts MS MS MS LP LP LP LP MS
Microsalts MS MS MS MS LP LP MS MS
Chelates MS MS MS MS MS MS MS MS
Vitamins MS* MS MS MS MS** MS MS MS
-1
Calcium pantothenate mg L - - - - 1.0 1 - -
-1
Biotin mg L - - - - 0.1 0.1 - -
-1
Riboflavin mg L - - - - 0.5 0.5 - -
-1
Sucrose gL 30 30 20 30 20 30 30 20
BA mg L-1 0.5 0.5 0.15 0.4 0.4 0.25 0.4 0.3
IBA mg L-1 0.1 0.1 0.015 0.06 0.05 0.1 - 0.1
-1
GA3 mg L - - - 0.03 - - - 0.1
-1
Adenine sulphate. mg L - - - 3 - - - -
-1 -1
*Thiamine ten-fold concentrated (1 mg L ); **150 mg L myo-inositol
MS: Murashige & Skoog, 1962; LP: Quoirin et al., 1977

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Table 2. Multiplication rate in plants grown in different cultural conditions (Data are the
mean of three values ± S.E.). L = liquid, S = solid.

Species cv Cultural conditions

L S 30 min 60 min
Apple Jork 9 3.00±0.28 10.00±1.97 2.00±0.41 8.00±1.63
Cherry Bigarreau Burlat 2.00±0.31 5.00±1.05 6.00±1.17 4.00±0.77
Papaya spp. 3.00±0.65 22.00±2.36 9.00±0.75 24.00±3.25
Peach Yumyeong 3.00±0.28 20.00±2.06 8.00±1.57 14.00±2.79
Pear Conference 2.66±0.33 4.00±0.57 6.33±0.66 4.33±0.33
Pear Harvest Queen 2.33±0.33 2.66±0.33 4.00±0.57 5.66±0.33
Pear Precoce di Fiorano 1.33±0.33 7.33±0.66 3.66±0.33 4.66±0.33
Pear William 2.66±0.88 3.00±0.57 4.33±0.33 6.33±0.66
Plum Adara 3.00±0.32 30.00±2.89 n.r. 28.00±2.54
Raspberry M. Exploit 4.00±0.56 9.00±0.71 12.00±1.05 45.00±3.27
Strawberry Teodora 5.00±0.37 8.00±0.66 10.00±0.67 15.00±0.67
Wild pear spp. 4.00±0.33 4.00±0.33 13.00±0.88 11.00±0.66

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Table 3. Water (WC), chlorophills, carotenoids, total and free fructose contents in plants grown in different cultural conditions (Data are
the means of three values ± s.e.). L = liquid, S = solid, 30′ e 60′ = TI period of 0 or 60 min.
Cultural Apple Cherry Papaya Peach Plum Raspberry Strawberry
conditions
W.C. (%)* L 82.21±1.04 80.82±2.56 94.13±0.24 87.86±0.75 86.36±0.22 91.30±0.71 92.36±1.82
S 85.93±0.32 92.46±0.36 94.81±0.37 89.89±0.96 83.08±0.92 86.34±2.00 92.73±2.01
30’ 97.56±2.99 79.79±1.26 93.62±0.55 77.76±1.50 n.r. 86.55±0.62 93.15±1.52
60’ 78.53±0.43 76.66±0.76 94.33±0.27 77.27±0.90 80.72±0.74 90.73±0.77 91.27±1.19

Chlorophylls L 1.35±0.13 0.17±0.02 1.08±0.06 0.90±0.05 0.44±0.01 0.57±0.02 0.49±0.06

mg/g D.W. S 3.36±0.10 3.06±0.11 2.97±0.23 2.26±0.09 1.25±0.05 2.88±0.24 7.34±0.25
30’ 3.81±0.17 4.78±0.18 2.95±0.13 9.87±0.13 n.r. 5.85±0.14 8.15±0.44
60’ 5.96±0.09 2.99±0.10 3.77±0.28 4.02±0.51 4.38±0.19 3.87±0.09 9.19±0.31

Carotenoids L 0.54±0.01 0.13±0.01 0.58±0.03 0.43±0.04 0.39±0.05 0.17±0.01 0.39±0.02

mg/g D.W. S 2.11±0.25 1.04±0.07 1.96±0.19 1.48±0.19 0.72±0.06 1.32±0.05 2.64±0.33
30′ 2.05±0.26 1.82±0.08 1.63±0.06 2.69±0.05 n.r. 1.98±0.06 3.37±0.23
60′ 2.59±0.07 1.67±0.02 2.01±0.08 2.19±0.02 1.72±0.04 1.70±0.13 4.21±0.31

Total fructose L 21.54±0.72 39.51±0.32 33.24±1.91 49.02±2.51 56.30±2.10 53.66±2.35 58,06±0,60

mg/g D.W. S 40.62±1.83 53.91±2.43 58.52±2.03 75.64±1.22 67.31±1.80 66.02±3.01 66.01±1.38
30’ 43.23±0.61 67.03±0.71 65.25±3.11 71.43±2.23 n.r. 69.14±3.15 75.44±1.97
60’ 36.71±2.04 52.54±2.41 72.03±3.28 84.21±2.43 77.11±1.51 57.31±2.98 81.39±1.48

Free fructose L 19.96±2.10 49.53±4.46 34.71±1.58 53.85±4.85 2.42±0.22 30.32±1.23 77.23±1.91

** S 19.83±1.78 12.19±1.28 31.31±1.26 33.55±3.52 24.94±2.12 16.92±1.01 67.97±0.98
% 30′ 11.85±1.01 10.46±0.89 26.03±1.02 2.82±0.27 n.r. 8.20±0.95 28.25±1.02
60′ 4.73±0.43 4.30±0.47 19.39±0.98 8.41±0.88 7.49±0.71 14.57±0.99 27.93±0.94

* Calculated as WC = [(FW – DW)/FW] x 100. FW: fresh weight; DW: dry weight
** Calculated as = (Free fructose/Total fructose) x 100.

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Figurese

E F

Fig. 1. A-B: devices used for temporary immersion. C: Papaya in multiplication by TIS after
60 days. D: hyperhydricity of peach cultured in solid medium. E: raspberry in multi-
plication by TIS after 60 days. F: stunted growth of peach in liquid stationary culture.

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