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DOI: 10.

1515/agr-2016-0013

THE ANNALS OF “VALAHIA” UNIVERSITY OF TARGOVISTE


2016

EXPLORATION OF REGENERATION POTENTIAL OF EXTINCT PLANTS


STARTING FROM OLD SEEDS, BY IN VITRO TECHNOLOGY
Aurelia Corina Cosac1, Gabriela Teodorescu1, Stephane Buord2,Valentina Isac3, Claudia Nicola3
1
Valahia University of Targoviste, Romania
2
Conservatoire Botanique de Brest, France
3
Research Institut for Pomology Pitesti-Maracineni (Roumanie)
e-mail: aureliacorinacosac@yahoo.com

Abstract
The aim of the study is to try a systematic and rational exploration of regeneration potential of extinct taxons, starting from
old seeds found in the soil or in the herbarium, to study their germination potential with the purpose of biodiversity
conservation. The species involved in this study are: Achillea spinosa, Chenopodium wolfii. Several protocols for the in
vitro propagation of the species, starting from the seed are described in this study. The presence of callus during
micropropagation of plants has also been the object of research for different compositions of nutritive medium.

Keywords: extinct species, regeneration, seeds, in vitro culture

1. INTRODUCTION

The IUCN Red List of Threatened Species provides of 5 years. The age of seed is over 100 years old. So,
taxonomic, conservation status and distribution we try to see if the germination capacity is
information on plants, fungi and animals that have conserved, despite the age and the conservation
been globally evaluated using the IUCN Red List method.
Categories and Criteria.
This system is designed to determine the relative
risk of extinction, and the main purpose of the IUCN
Red List is to catalogue and highlight those plants
and animals that are facing a higher risk of global
extinction (i.e. those listed as Critically
Endangered, Endangered and Vulnerable). The
IUCN Red List also includes information on plants,
fungi and animals that are categorized
as Extinct or Extinct in the Wild.
The IUCN Red List Index measures overall trends
in extinction risk for groups of species based on
genuine changes in their Red List status over time.
The aims of this study is to find Balkan endemic
extinct seed species on their old natural stations or
in ancient herbals and study their germination
potential.

2. MATERIAL AND METHOD

The species were selected on longevity criteria,


taxonomic validity and on the advice of field
botanists.
The 2 species were selected by the Red Liste and
they are extinct in Romania. Figure 1. Chenopodium wolfii in the Cluj Herbarium
The Achillea spinosa is an endemic and extint
specie, founded last time 50 years ago, in Cluj The seed of Achillea spinose were prelevated from
county. the soil bank, from different depths, in Valea Morii
The Chenopodium wolfii was seen for the last time region, in Cluj county (fig. 2,3).
in the Cluj county also, 100 years ago.
The seeds of the Chenopodium wolfii were taken
from the conservated plants from the Cluj
Herbarium (fig.1). The method of conservation is to
put a thin film of oil, to protect against insects, once

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THE ANNALS OF “VALAHIA” UNIVERSITY OF TARGOVISTE
2016

5 seeds of Achillea spinose were found in the soil


samples and they were scanning by electronic
microscopy to see the integumentary area. We
observed the structure of external wall of seeds and
it gas a perfect preserved structure (fig.5,6).

Figure 6. The surface of Achillea spinosa seed, at


electronic microscopy
Figure 2. Achillea spinosa in the Cluj Herbarium

Figure 6. The surface of Achillea spinosa seed, at


electronic microscopy
Figure 3. Soil sample from Valea Morii region Parallel to this research, similar species of the 2
species selected will allow to perform culture tests
After the prelevation, the soil sample were analised in vitro. The objectif is to germinate the seeds
to find the seeds of the Achillea spinosa (fig.4). collected.
The in vitro technology was realized at Research
Institut for Pomology Pitesti-Maracineni, starting
from the seeds, but with related species, such as
Achillea millefolium and Chenopodium glaucum to
establish the in vitro technology.
The working methodology for biotechnology in
vitro : 2 nutritive mediums Murashige-Skoog and
Gamborg, in support solide - agar Difco (6 g/l), pH
= 5,6 – 5,8 and sucre = 10 g/l.
Based on bibliographic searches, we establish 2
variants of seeds sterilization:
1. short immersion in ethanol 700,
sterilization in Ca hypochlorite 6% for 15
minutes and rinse 3 times with sterile
double distilled water and maintain in
water until prelevment.
Figure 4. Soil sample analysis

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2016

2. wash in double distilled sterile water and In vitro technology of Achillea millefolium in the
maintain in water for 20 minutes; images (fig.7-10).
sterilization in ethanol 700 for 5 minutes
sterilization in Ca hypochlorite 6% for 30
minutes and rinse 3 times with sterile
double distilled water and maintain in
water until prelevment.
The best results were obtained on the first variant.

3. RESULTS AND DISCUSSIONS

The establish methodology for biotechnology in


vitro of Achillea millefolium and Chenopodium
glaucum :
1. In vitro germination stage: nutritive
medium Murashige - Skoog + complex de
5 vitamins + phytohormons (AIA = 1,5
Figure 7. Sterilisation seeds Figure 8. Germination
mg/l, AG3 = 0,1 mg/l, BAP = 1 mg/l). seeds of Achillea millefolium
2. In vitro multiplication stage:
- pour Achillea millefolium: nutritive
medium Murashige - Skoog + complex of
5 vitamins + phytohormons (AIA = 0,1 g/l,
BAP = 0,5 mg/l. Sans AG3).
- pour Chenopodium glaucum: nutritive
medium Murashige - Skoog + complex of
5 vitamins + phytohormons (AIA = 0,1 g/l,
BAP = 0,5 mg/l. Sans AG3 ).
3. In vitro rooting stage :
- pour Achillea millefolium: nutritive
medium Murashige - Skoog + complex of
5 vitamins + phytohormons (AIA = 0,5 g/l,
AG3 = 0,1 g/l, BAP = 1 mg/l).
Fig. 9. Multiplication stage Fig.10. Rooting stage
pour Chenopodium glaucum: nutritive
medium Murashige - Skoog + complex of
Concerning the 2 extinct species involved in this
5 vitamins + phytohormons (AIA = 0,5 g/l,
study - Achillea spinosa and Chenopodium wolfii-
BAP = 1 mg/l, AG3 = 0,1 g/l).
based on establish technology, we started the study
4. Aclimatisation stage:
the regeneration capacity of seeds.
- nutritive mixture = perlit.
For the seeds of Chenopodium wolfii, prelevated
from the herbarium, aged of 100 years, we followed
The presence of callus during micropropagation
the established technology for seeds sterilization and
stage of plants has also been the object of research
initiation stage. During 4 months, the seeds
for different compositions of nutritive medium. We
remained on the nutritive medium, but the seed
established that the best nutritive medium
didn’t germinate. No seed has not germinated
composition, in the multiplication stage, should
despite increased its volume.
contain 0,5 mg/l BAP. At a concentration of 2 mg/l
For Achillea spinosa, we found 5 seeds on the soil
BAP, the callus was frequent and dense. In the same
bank (aged probably of 50 years) and put on the
time, on the same variant, the hyperhydricity of
nutritive medium, following the establish
plants was produced.
technology with related specie.
Table 1. The presence of callus (%), in the During 3 months, the seeds didn’t germinate.
multiplication stage, at different concentration of BAP
4. CONCLUSIONS
Species % of callus
BAP concentration For Chenopodium wolfii, after four months of
0,5 mg/l 1 mg/l 2 mg/l culture, the seeds did not germinate. One of the most
Chenopodium 0 10 25 probably explanation is the fact that petroleum is an
glaucum organic solvent that destroys the plant organic
Achillea 0 10 20 matter and in the end the plant tissus. For loss of
millefolium
regenerative capacity, it could be an explanation,
besides the advanced age of seeds.

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2016

For Achillea spinosa, we continue the study of [14] Saeidnia S, Gohari A, Mokhber-Dezfuli N, Kiuchi
regeneration capacity by in vitro culture, despite of F. A review on phytochemistry and medicinal
advanced age. The seeds found in the soil showed a properties of the genus Achillea. Daru 2011;19:173-
86
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