African Journal of Biotechnology Vol. 10(19), pp.

3762-3765, 9 May, 2011

Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2060
ISSN 1684–5315 © 2011 Academic Journals

Full Length Research Paper

Adaptation of regenerants of Vaccinium corymbosum

L. and Vaccinium vitis-idaea L. to ex vitro conditions
Elena Kutas
Central Botanical Garden of the NAS of Belarus, 220072 Minsk, Surganova, 2v, Republic of Belarus. E-mail:
[email protected]. Tel: (+375 17) 284-15-89. Fax: (+375 17) 284-14-84.
Accepted 22 February, 2011

The benchmark analysis of the structured-functional particularities of regeneration has introduced

varieties of Vaccinium corymbosum L. and Vaccinium vitis-idaea L. under in vitro and ex vitro
conditions. The anatomical structures of the leaves of introduced varieties of V. corymbosum and V.
vitis-idaea, cultivated in aseptical culture, greenhouse and open ground were studied. First, it is shown
that the condition of cultivation superimposes the imprint on the structure and function of regeneration;
secondly, the structured-functional organization regeneration (a mobile system) can be reformed in
accordance with the changed condition surrounding ambiences. The differences in construction and
functions of the sheet plants growing in an aseptic culture, in hothouses or open ground conditions,
are indicative of the plastic sheet, which is an organ capable of reconstructing its structure and
function adequately to the condition of cultivation that is theoretically, a guarantor to successful
adaption of the plants when carrying them from in vitro (the cultural container) to ex vitro (the
greenhouse and open ground) conditions.

Key words: Aseptic culture, greenhouse, open ground, anatomical structure, blueberry, cowberry.

INTRODUCTION

In the foundation of clonal micropropagation of plants, times faster with plants obtained via in vitro culture than
there are two completely different stages (in vitro and ex plants obtained from the greenhouse. The thickness of
vitro). During the first stage (in vitro), vital functions of the palisade cells was much lower with regenerants raised in
material that is being propagated occur in a closed sterile aseptical conditions than that of regenerants from the
space, on the nutrient medium under strictly controlled greenhouse and open ground. According to researches
conditions. After the regenerants are transferred from in by Grout (1975) and Sutter and Langhans (1979), the
vitro conditions, the second stage begins ex vitro system, leaves are deprived of wax bloom with plants cultivated in
quite different from in vitro conditions. In ex vitro vitro, and stoma function is imperfect as a result of the
conditions, the plants have to pass from a heterotrophic open-closed mechanism’s failure. Similar conclusions
to autotrophic nutrition, conjugated with structural and about stoma functioning were obtained by Lee et al.
functional transformation of the organism in new (1988), Brainerd et al. (1982) and Wardle and Short
conditions. They must adjust themselves to changeable (1983).
environmental factors inherent to them. According to the data given by Bunning and Sagromsky
The transition of plants from in vitro to ex vitro (1948), O’Leary and Knecht (1981) and Penfound (1931),
conditions is critical in most cases and entails death of the stoma development is influenced by such factors as
plants. From this study’s point of view, the comparative CO2 concentration in the retort, water regime and
analysis of the ex vitro and in vitro conditions of the hormone level. The stomata of plants in vitro conditions
structural and functional peculiarities of regenerants will are usually open which is not true in respect of the
help to understand and prevent the cause of death of stomata of plants ex vitro conditions. In the study’s
plants during the adaptation period. opinion, such behaviour of stomata ex vitro conditions is
Researches conducted by Brainerd et al. (1981) on leaf quite justified because in cultural retorts, a very high
anatomy and water stress with plump plants, in vitro and constant relative humidity rate is kept (over 90%), and the
ex vitro, showed that the loss of water occurred three temperature and illumination degrees are not expected to
 Kutas 3763

fall over the limit because they are being controlled. cultivated in vitro, had no clear differentiation of
However, if any condition should occur in the cultural mesophyll into palisade and spongy tissues. Thus, they
container, the stomata reaction will follow in response to had a thin leaf plate, weakly developed cuticular
the changes of the given conditions. integument and under developed stomata apparatus
From this point of view, the failure clearly overtakes entailing continuous opening of stomata and over
some researchers seeking to interfere with the efficient transpiration.
performance of stomata responding to conditions in The leaves developed in greenhouse, had a clear
which they are found. For instance, the use of mesophyll differentiation in palisade and spongy
antitranspirants during transfer of plants from in vitro to mesophyll. Also, it had cuticular integument and well-
ex vitro conditions promoted a decrease of developed stoma apparatus enabling normal
photosynthesis caused by a deterioration of plant growth transpiration.
(Danies and Kozlowski, 1974). The leaves of plants transplanted into open ground did
According to researches by Fabbri and Sutter (1986), not differ from greenhouse leaves in a general structure.
the leaf structure of wild strawberry, which formed in vitro They had a leaf structure that is clearly differentiated into
culture, was characterized by a relatively thin leaf plate, palisade and spongy mesophyll, a well-developed
under developed palisade cells, big air cavities, and cuticular integument and a stoma apparatus. However, it
weakly developed cuticular integument. At the same time, should be pointed out that the difference was observed in
the leaf of wild strawberry, which formed ex vitro the change of the quantitative indices of the leaf
conditions, was differentiated into palisade and spongy structure. Thus, leaves from open ground had a thicker
tissues with a well-developed cuticular integument. leaf plate, more layers of palisade tissue, longer cells and
Similar results were obtained by Donnelly and Vidaver reduced volume of ductus intercellularis when compared
(1984) when studying raspberry leaves regenerated in with the greenhouse leaves in vitro (Table 1).
vitro. Waldenmeier and Schmidt (1990) observed It should be pointed out that the differences in the leaf
histological differences in rhododendron leaves in vitro structure are conjugated with their functional differences.
and ex vitro when tempering on them. The differences An example is a thorough research on the comparative
included absence of breathing pores and weakly- anatomy and physiology of Asian birch (Betula
structured mesophyll with leaves in vitro. With the leaves platyphylla) cultivated in greenhouse on aseptic culture,
found ex vitro, the anatomical structure of leaves conducted by Smith et al. (1986). The author came to a
changed. As a result, their thickness grew, the number of conclusion that a weak development of the vascular
layers of epidermis and palisade tissue increased, and system was seen in vitro, followed by increased
the cuticle appeared. The acclimatization by low humidity sensitivity of such plants to water stress inherent to ex
rate led to a clear differentiation of the tissue into vitro conditions.
palisade and spongy mesophyll. A low intensity of photosynthesis was discovered by
The objective of this research is to study the adaptation them through a very low illumination degree conjugated
of the regenerants of the introduced varieties of with the absence of clear differentiation of the leaf into
Vaccinium corymbosum L. (Dixi, Bluecrop) and palisade and spongy tissues in an in vitro culture.
Vaccinium vitis-idaea L. (Koralle). After transfer of plants into ex vitro conditions
(greenhouse), the researchers observed the increase in
photosynthesis intensity and changes in leaf anatomy. In
MATERIALS AND METHODS their opinion, the plants grown in aseptic conditions
considerably change their anatomical and physiological
The leaves of V. corymbosum L. (Dixi, Bluecrop) and V.vitis-idaea
features when compared to their double cultivated ex
L. (Koralle) were preserved in alcohol-acetic acid (3:1). The cross
sections were made in the middle part of the leaf, at microtome, by vitro conditions. The changes are accounted for by the
histological technique and razor. The sections were cleared with influence of a specific environment in aseptic culture and
chloral hydrate and then stained with Genevez and Sudan III a disappearance of the transfer of plants into ex vitro
reagents. The thickness of the leaf plate and other indices of conditions due to a quick recovery of metabolism
anatomical leaves structure were measured by micrometer. resulting from normal development of plants.
The analysis of the anatomical structure was realized according
to the previous method described by Brainerd et al. (1981), Grout According to researches done by Donnely et al. (1984)
(1975), Sutter et al. (1979) and Lee et al. (1988). and Grout and Millam (1985), the photosynthetical activity
is lower with in vitro shoots compared to that of ex vitro
shoots. The minimum photosynthetical activity until 14
RESULTS AND DISCUSSION days after transfer of leaves from in vitro culture was
observed for plants that survived during acclimatization
The researches conducted by the author on dependence using the stock of metabolites. The normal recovery of
of the internal leaf structure on cultivating conditions the structure and function occurs with the regenerants
showed that regenerants of introduced species of V. within a month after placing them in ex vitro conditions.
corymbosum (Dixi, Bluecrop) and V. vitis-idaea (Koralle), To increase the survival rate of plants during adaptation,
 3764 Afr. J. Biotechnol.

Table 1. Quantitative indices of anatomical leaves structure of V. corymbosum and V. vitis-idaea cultivated in the aseptical culture, greenhouse and open ground*.

Aseptic culture (in vitro) 4000

Greenhouse >15000 Lx Open Ground > 50000 Lx
Lx

Leaf thickness (μm)

Leaf thickness (μm)

Leaf thickness (μm)

Stoma size length x

Stoma size length x

Stoma size length x

Palisade coefficient

Palisade coefficient
stomata per 1 mm2

stomata per 1 mm2

stomata per 1 mm2

Length : width of

Length : width of
cells of palisade

cells of palisade
The number of

The number of

The number of
Grade

tissue ratio

tissue ratio
width (μm)

width (μm)

width (μm)
V. corymbosum
Bluecrop 76±2 16±1 15x11 154±16 0.75 1.8:1 251±11 25x17 210±11 0.87 2.5:1 260±12 23x16
Dixi 85±3 16±1 15x12 173±13 0.71 1.9:1 250±9 26x16 221±12 0.9 2.7:1 265±10 24x15
V. vitis-idaea
Koralle 91±4 19±1 16x10 286±9 0.63 2.61:1 410±20 24x15 450±19 0.86 3.31:1 430±23 21x14
* No indices are shown for palisade coefficient and palisade tissue cells with the leaves of plants from aseptic culture, since the mesophill of the leaf was not differentiated into palisade and spongy
mesophills.

it is necessary to gradually decrease the relative CO2 concentration is one of the low leaves’ growth in the aseptic culture, in
air humidity and increase irradiation. This photosynthetical intensity observed with greenhouse or open ground, testify to the
promotes increase of space occupied by palisade regenerants plants in in vitro culture. The CO2 flexibility of the leaf, that is, the organ has the
cells which, in turn, causes increase in intensity of concentration increases by transfer of plants into ability to transform its structure and function
photosynthesis. ex vitro conditions causing an increase in intensity according to the cultivating conditions. This is
Interesting researches were conducted by of photosynthesis followed by the growth theoretically the guarantor of a successful
Solarova (1989) on the study of round-o’clock acceleration. adaptation of plants when transferring them from
variability of CO2 concentration in cultivating On the foundation of the comparative analysis in vitro to ex vitro conditions.
retorts, where the cultivated regenerants plants of the structural and functional features of the In practice, we managed to avoid losses of
were obtained from leaf pieces. It turned out that regenerants in in vitro and ex vitro conditions, material at the critical point due to the use of
CO2 concentration in retorts increased in a dark based on the written sources and results of the techniques based on conclusions confirmed by
period and was connected to the regenerant size author’s own researches, this study came to a the results of experimental researches. This was
and sucrose content in the medium. The conclusion that: (1) the in vitro and ex vitro proven by the study’s observations over the
concentration in retorts decreased in light period cultivating conditions leave imprint on the adaptation process of the introduced species of V.
and the illumination reached the compensation structure and functions of regenerants, and (2) corymbosum (Dixi, Bluecrop, Herbert, Rancocas
point in 3 to 4 h, despite the low illumination structural and functional organization on and Covill, Early blue) and V. vitis-idaea (Koralle,
degree (100 µmol.m-2.s-1). A conclusion was made regenerants is a mobile system that has the ability Masovia, Erntedank, Erntecrone and Erntezegen)
by the author that the low CO2 concentration in to be transformed in accordance with the changed when transferring them from in vitro to ex vitro
closed retorts for cultivation of regenerant plants environmental conditions. This means that the conditions.
is different in growth. Therefore, the decreased differences in the structure and function of plant To prevent death of the material from over
 Kutas 3765

transpiration (which refers not only to V. corymbosum and V. vitis-idaea not only in greenhouse conditions, but
and V. vitis-idaea) caused by reasons known to the also in open ground conditions.
study: (1) the humidity drops ex vitro conditions, and (2)
the imperfect structural and functional organization of the
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