Somaclonal variance in Deschampsia antarctica Proposal for MS Thesis Mihai Ionita, BS

1.

Overview

Poaceae family includes along Deschampsia antarctica from the Descampsia genus important crops like that from Oryza L., Triticum L., Zea L. genera containing an overall of 338 genera and 3014 accepted taxa1. The extreme environment of Antarctic region where D. antarctica thrives consists of low temperature, limited moisture availability, frequent and often rapid freeze-thaw and hydration-dehydration cycles, exposure to high photosynthetic photon flux density and ultraviolet radiations, seasonal snow cover, high winds, cryoturbation and considerable daylight in summer as described by Block W et al, 20092 This proposal project will try to increase the scientific background of micropopragation experiments in D. antarctica Cuba M. et al. 20053,4 documenting somatic variability in various micropropagation and environmental stress conditions. 2. Reason

Understanding the stability of D. antarctica is of paramount importance for future transformation work. In the same time allows the construction of a proper morphological, biochemical and genetic workflow of experimental procedures for characterisation, selection and propagation of plants with desire characteristics. 3. Your research project

The project consist in the study of somaclonal variations in D. antarctica by callogenesis and organogenesis with the induction of root differentiation fallowed by morphological, biochemical and genetic experiments on in-vitro seedling and acclimatized plants. 4. Research Aims

(I) Callogenesis: asses the response for various types of tissue explants (1) finding the optimal phytohormonal concentration (2) determine the environment condition for: circadian rhythms (3), environmental temperature (4); Image analysis of plantules (6), statistical studies on the morphological / biochemical and genetic studies (7), (II) Organogenesis: (2), (1), (5), (7), (3), (7) determine the regeneration response and the number of shoots and there length (10)

(III) Root differentiation: (2), (4), (5), (7), (8), (6) (IV) Acclimatisation of seedlings: (4), (6), (7)

5.

Questions for research may include the following : Assessing the EA for common enzymes, AFLP, RAPD, SSR studies;

6.

Methodology

Practical experiments / studies performed in USAMV, Faculty of Biotechnology, Bucharest, Romania with the possibility of extending the investigation in other laboratories in collaboration with Institute of molecular biology and genetics NAS of Ukraine (under discussion). I.1. The protocol described by Cuba M. et al. 2005 presents the fallowing stages in surface sterilization in D. antarctica: Stage Solution 1 2 undisclosed Time Observation

undisclosed the plants are washed to eliminate substrate In an Erlenmeyer flask under constant steering

300 ml solution of 1.5 g l-1 Captan 80W 20 min (BASF Chile-SA)5 + 0.5 gl-1 Benomyl 500WP (CYANAMID Chile-SA)6 sterile distilled water sterile distilled water sterile distilled water undisclosed undisclosed undisclosed

3 4 5

Tissue explants taken into the study

I.2. Determine the optimal concentration for callogenesis induction According with Cuba M. et al. 2005 paper callogenesis response with > 98% shooting response was obtained in low 2,4-D concentration (2.2 uM) and low (0.2 uM) to 0 BAP concentration on MS medium (0.2) generation 100% callus formation.
CODE Hormone concentration 2,4-D A_1 0* A_2 A_3 B_1 0.44 B_2 B_3 C_1 0.88 C_2 C_3 D_1 1.32 D_2 D_3 E_1 1.76 E_2 E_3 F_1 2.2* F_2 F_3 -

BAP 0 0.1 0.2* 0.04 0.1 0.16 0.08 0.1 0.12 0.12 0.1 0.08 0.16 0.1 0.04 0.2* 0.1 0

Table 1: Effect of 2,4-D / BAP in MS medium on callogenesis (*100% response callogenesis form Cuba M. et al. 2005 studies ) I.3./4. Dark/light circadian rhythms / Environmental temperature

According with Xiong et all, 1999 the 20 degree Celsius day temperature / 7 degree Celsius at night + 16 h / 8 h created an improvement on relative growth rate, biomass, leaves number / area, leaf area ratio, leaf mass area 1-4 folds versus plants grow 12 degree Celsius in daytime / 7 degree Celsius at night on the same day / night cycle, The whole-canopy net photosynthesis was optimal at 10 degree Celsius,

Code A-F*_1-3*_D0_T0 A-F*_1-3*_D0_T10 A-F*_1-3*_D0_T20 A-F*_1-3*_D10_T0 A-F*_1-3*_D10_T10 A-F*_1-3*_D10_T20 A-F*_1-3*_D25_T20 A-F*_1-3*_D25_T20 A-F*_1-3*_D25_T20

Dark incubation (days) 0

Temperature 0 10 20

14

0 10 20

30

0 10 20

1. USDA, NRCS. 2012. The PLANTS Database (http://plants.usda.gov, 3 February 2012). National Plant Data Team, Greensboro, NC 27401-4901 USA. 2. Block W, Smith RIL, Kennedy AD (2009) Strategies of survival and resource exploitation in the Antarctic fellfield ecosystem. Biol Rev Camb Philos Soc 84:449484. 3. Cuba M., Gutirrez-Morgan A., Buten-dieck B. , Gidekel M. 2005 Micropropagation of Deschampsia antarctica a frostresistant Antarctic plant. Ant. Sci. 17: 6970. 4. Katarzyna J. Chwedorzewska, Piotr T. Bednarek. (2011) Genetic and epigenetic studies on populations of Deschampsia Antarctica Desv. from contrasting environments on King George Island. Polish Polar Research 32:1, 15-26, 1-Jan-2011. 5. http://aesop.rutgers.edu/~plantbiopath/links/bbcpestweb/BRAMBLELABELS/CAPTAN-80W.PDF on 18.02.2012, 09:00 6. http://www.villacrop.co.za/products/docs/Benomyl%20500%20WP%20E_UCP.pdf on 18.02.2012, 09:00 7. Xiong FS, Mueller EC, Day TA., Photosynthetic temperature response of the Antarctic vascular plants Colobanthus quitensis and Deschampsia antarctica, PHYSIOLOGIA PLANTARUM 106: 276286. 1999 8. Price, C.A., O. Symonova, Y. Mileyko, T. Hilley and J.S. Weitz Leaf Extraction and Analysis Framework Graphical User Interface: Segmenting and Analyzing the Structure of Leaf Veins and Areoles. Plant Physiology

9. , 155 (236-245).