THE ANNALS OF “VALAHIA” UNIVERSITY OF TARGOVISTE2016

EXPLORATION OF REGENERATION POTENTIAL OF EXTINCT PLANTSSTARTING FROM OLD SEEDS, BY IN VITRO TECHNOLOGY

Aurelia Corina Cosac1, Gabriela Teodorescu1, Stephane Buord2,Valentina Isac3, Claudia Nicola3 1 Valahia University of Targoviste, Romania2 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 fromold seeds found in the soil or in the herbarium, to study their germination potential with the purpose of biodiversityconservation. The species involved in this study are: Achillea spinosa, Chenopodium wolfii. Several protocols for the in vitropropagation of the species, starting from the seed are described in this study. The presence of callus during micropropagationof 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 providestaxonomic, conservation status and distributioninformation on plants, fungi and animals that havebeen globally evaluated using the IUCN Red ListCategories and Criteria. This system is designed to determine the relative riskof extinction, and the main purpose of the IUCN RedList is to catalogue and highlight those plants andanimals that are facing a higher risk of globalextinction (i.e. those listed as CriticallyEndangered, Endangered and Vulnerable). The IUCNRed List also includes information on plants, fungiand animals that are categorized as Extinct or Extinctin the Wild.The IUCN Red List Index measures overall trends inextinction risk for groups of species based on genuinechanges in their Red List status over time.The aims of this study is to find Balkan endemicextinct seed species on their old natural stations or inancient herbals and study their germination potential.

2. MATERIAL AND METHOD

The species were selected on longevity criteria,taxonomic validity and on the advice of fieldbotanists.The 2 species were selected by the Red Liste andthey are extinct in Romania. The Achillea spinosa is an endemic and extint specie,founded last time 50 years ago, in Cluj county.The Chenopodium wolfii was seen for the last time inthe Cluj county also, 100 years ago.

The seeds of the Chenopodium wolfii were takenfrom the conservated plants from the Cluj Herbarium(fig.1). The method of conservation is to put a thinfilm of oil, to protect against insects, once of 5 years.The age of seed is over 100 years old. So, we try tosee if the germination capacity is conserved, despitethe age and the conservation method.

Figure 1. Chenopodium wolfii in the Cluj Herbarium

THE ANNALS OF “VALAHIA” UNIVERSITY OF TARGOVISTE2016

The seed of Achillea spinose were prelevated fromthe soil bank, from different depths, in Valea Moriiregion, in Cluj county (fig. 2,3).

Figure 2. Achillea spinosa in the Cluj Herbarium

Figure 3. Soil sample from Valea Morii region

After the prelevation, the soil sample were analised tofind the seeds of the Achillea spinosa (fig.4).

Figure 4. Soil sample analysis

5 seeds of Achillea spinose were found in the soilsamples and they were scanning by electronicmicroscopy to see the integumentary area. Weobserved the structure of external wall of seeds and itgas a perfect preserved structure (fig.5,6).

Figure 6. The surface of Achillea spinosa seed, atelectronic microscopy

Figure 6. The surface of Achillea spinosa seed, atelectronic microscopy

THE ANNALS OF “VALAHIA” UNIVERSITY OF TARGOVISTE2016

Parallel to this research, similar species of the 2species selected will allow to perform culture tests invitro. The objectif is to germinate the seeds collected.The in vitro technology was realized at ResearchInstitut for Pomology Pitesti-Maracineni, startingfrom the seeds, but with related species, such asAchillea millefolium and Chenopodium glaucum toestablish the in vitro technology.The working methodology for biotechnology invitro : 2 nutritive mediums Murashige-Skoog andGamborg, in support solide - agar Difco (6 g/l), pH =5,6 – 5,8 and sucre = 10 g/l.Based on bibliographic searches, we establish 2variants of seeds sterilization:

1. short immersion in ethanol 700, sterilizationin Ca hypochlorite 6% for 15 minutes andrinse 3 times with sterile double distilledwater and maintain in water untilprelevment.

2. wash in double distilled sterile water andmaintain in water for 20 minutes;sterilization in ethanol 700 for 5 minutessterilization in Ca hypochlorite 6% for 30minutes and rinse 3 times with sterile doubledistilled water and maintain in water untilprelevment.

The best results were obtained on the first variant.

3.RESULTS AND DISCUSSIONS

The establish methodology for biotechnology in vitroof Achillea millefolium and Chenopodium glaucum :

1. In vitro germination stage: nutritive mediumMurashige - Skoog + complex de 5 vitamins+ phytohormons (AIA = 1,5 mg/l, AG3 = 0,1mg/l, BAP = 1 mg/l).

2. In vitro multiplication stage:- pour Achillea millefolium: nutritivemedium Murashige - Skoog + complex of 5vitamins + phytohormons (AIA = 0,1 g/l,BAP = 0,5 mg/l. Sans AG3). - pour Chenopodium glaucum: nutritivemedium Murashige - Skoog + complex of 5vitamins + phytohormons (AIA = 0,1 g/l,BAP = 0,5 mg/l. Sans AG3 ).

3. In vitro rooting stage :- pour Achillea millefolium: nutritivemedium Murashige - Skoog + complex of 5vitamins + phytohormons (AIA = 0,5 g/l,AG3 = 0,1 g/l, BAP = 1 mg/l). pour Chenopodium glaucum: nutritivemedium Murashige - Skoog + complex of 5vitamins + phytohormons (AIA = 0,5 g/l,BAP = 1 mg/l, AG3 = 0,1 g/l).

4. Aclimatisation stage:- nutritive mixture = perlit.

The presence of callus during micropropagation stageof plants has also been the object of research fordifferent compositions of nutritive medium. Weestablished that the best nutritive mediumcomposition, in the multiplication stage, shouldcontain 0,5 mg/l BAP. At a concentration of 2 mg/lBAP, the callus was frequent and dense. In the sametime, on the same variant, the hyperhydricity ofplants was produced.

Table 1. The presence of callus (%), in themultiplication stage, at different concentration of BAP

Species % of callusBAP concentration

0,5 mg/l 1 mg/l 2 mg/lChenopodiumglaucum

0 10 25

Achilleamillefolium

0 10 20

In vitro technology of Achillea millefolium in theimages (fig.7-10).

Figure 7. Sterilisation seeds Figure 8. Germination seeds of Achillea millefolium

Fig. 9. Multiplication stage Fig.10. Rooting stage

THE ANNALS OF “VALAHIA” UNIVERSITY OF TARGOVISTE2016

Concerning the 2 extinct species involved in thisstudy - Achillea spinosa and Chenopodium wolfii-based on establish technology, we started the studythe regeneration capacity of seeds.For the seeds of Chenopodium wolfii, prelevatedfrom the herbarium, aged of 100 years, we followedthe established technology for seeds sterilization andinitiation stage. During 4 months, the seeds remainedon the nutritive medium, but the seed didn’tgerminate. No seed has not germinated despiteincreased its volume. For Achillea spinosa, we found 5 seeds on the soilbank (aged probably of 50 years) and put on thenutritive medium, following the establish technologywith related specie.During 3 months, the seeds didn’t germinate.

4. CONCLUSIONS

For Chenopodium wolfii, after four months of culture,the seeds did not germinate. One of the mostprobably explanation is the fact that petroleum is anorganic solvent that destroys the plant organic matterand in the end the plant tissus. For loss ofregenerative capacity, it could be an explanation,besides the advanced age of seeds.For Achillea spinosa, we continue the study ofregeneration capacity by in vitro culture, despite ofadvanced age. The seeds found in the soil showed agood conservation status.

5. ACKNOWLEDGEMENTS

This research was realized with the Research Institutfor Pomology Pitesti-Maracineni (Roumanie)support.

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