Somatic embryogenesis from in vitro anther culture of apomictic buffel grass genotypes and analysis of regenerated plants using flow cytometry Edgardo Carloni ; Andrea Ribotta ; Eliana López Colomba ; Sabrina Griffa ; Mariana Quiroga ; Exequiel Tommasino ; Karina Grunberg | Ana Rafaela Teixeira | Daniela Sousa | João Rodrigues |

Pennisetum ciliare (L.) Link syn. Cenchrus ciliaris L. Introduction

ApomicticDrought resistantEasy to establishHigh crop eficiency

Initiation and Progression of Apomictic Mechanisms Relative to Events in the Sexual Life Cycle of Angiosperms.The normally dominant vegetative phase of the life cycle is curtailed in this figure to emphasize the events of gametophyte formation, particularly... ©2004 by American Society of Plant Biologists Bicknell R A, and Koltunow A M Plant Cell 2004;16:S228- S245

Introduction For apomictic plants For buffel grass Conventional breeding programs are difficult to set up In vitro techniques provide the possibility to transform these plants Successful in vitro regeneration using a variety of explants Regeneration mostly by somatic embryogenesis But efficiency depends largely on the explant and genotype

Introduction  Increase regeneration efficiency  Establish genotypes that do not respond to in vitro culture New explants Anthers For buffel grass Successful in vitro regeneration using a variety of explants Regeneration mostly by somatic embryogenesis But efficiency depends largely on the explant and genotype

Introduction Genetic Stability DNA content DNA Structure Flow Cytometry (FCM)

Aims To establish a protocol for in vitro regeneration in apomictic genotypes of buffel grass using anther as explants and to evaluate the genetic stability of regenerated plants using FCM

1 - 3 cm 3 genotypes: RN51 RN147 RN158 (Registados no Instituto de Fisiología y Recursos Genéticos Vegetales of the Instituto Nacional de Tecnología Agropecuaria (INTA), Córdoba, Argentina) Methodology

Tillers No treatmentDistilled water 5 days 7 days 0,3 M mannitol 5 days 7 days

3 anthers = 1 explant Methodology 70% ethanol 1 min 10% sodium hipocloride + HCl + Tween min Distilled water X 3

Methodology 2,4-D: 2,4-dichlorophenoxyacetic acid; NAA: napthaleneacetic acid ; BAP: 6-benzylaminopurin Induction MS medium + 3% sucrose + 2,4-D (6 mg/L) 90 days (subculture d45) Dark 25°C Germination MS medium + 3% sucrose + NAA (0,5 mg/L) + BAP (1 mg/L) 12 months (subculture every 60 days) 16h/8h light/dark 25°C Rooting MS medium + 1,5% sucrose + NAA (0,5 mg/mL) 16h/8h light/dark 25°C

Methodology  Histology :  Samples were fixed in a 5:3,5:1:0,5 solution of ethanol:water:formalin:glacial acetic acid, dehydred in ethanol and embedded in parafilm.  Dyes used were saffranin and fast green  FCM  Both donor plants and regenerated plants’ nuclei were analised against a standard of Z. mays, using propidium iodide and following these equations: DI – DNA Index

Methodology  Statistical Analysis  Proportion of Embryogenic Calli (PEC) and Number of Regenerated Seedling (NRS)  Induction/Regeneration vs PEC/NRS  PEC & NRS vs date collection/treatment  Genetic stability  donor plants vs regenerated plants  genotype, days in vitro, treatment

Results RN 51 RN 147 RN 158 All genotypes were able to induce calli in MS medium suplemented with 2,4-D. Day 7 Day 7 Start of callus development Day 15 Day 15 Day 30 Day 30 Non-embriogenic calliEmbriogenic calli Proembryos

Results Day 20 Day 20 First shoots Germination of embryogenic calli in regeneration medium under light conditions. Week 4 Subculture performed at 60 days stimulated the development of new seedlings. Well-developed seedlings

Results The number of regenerated seedlings (NRS) was variable (1-55 seedlings ) Plant in greenhouse after the hardening period

Results  Embryogenic calli, and consequently regenerated plants, are of somatic origin. Cellular growth Connective tissue Wall Anther filament Anther Day 15 Day 15

Results Absence of embryogenic cells Somatic embryos with bipolar organization (root and caulinar apices), leaf primordia and vascular system Nonembriogenic callus Embriogenic callus  Seedling regeneration occurred only from embryogenic calli. Day 20 Day 20

Results RN 51 RN 147 RN 158 Highly significant differences (P < 0,0001) both for the proportion of embryogenic calli (PEC) and the number of regenerated seedlings (NRS) Best response

Results Maximum embryogenic potential on earlier dates

Results Maximum embryogenic potential on earlier datesMaximum embryogenic potential on later dates At second and third collection date, the plants have already reached full flowering This behavior may be related to physiological conditions of the donor plant

Results Two dominant peaks - nuclei in G0/G1 Two lower peaks - nuclei in G2 No peak overlap and low coefficients of variation in theG0/G1 phase. The FCM analysis yielded histograms with four peaks.

Results GenotypeMean DNA index RN 510,7103 RN 1470,7083 RN 1580,8504 Genotype2C / pg RN 513,8568 RN 1473,8462 RN 1584,6527 Genotypes with different ploidy levels

Results Five plants with lower nuclear DNA content 1,6 - 3,3% of variation Two plants with higher nuclear DNA content 2C are approximately the double of the original cytotypes

Results Three plants with lower nuclear DNA content 2,62 – 3,16% of variation Plants from genotype RN 147 did not show an increase in nuclear DNA content

Results Lower nuclear DNA content Higher nuclear DNA content Aneuploidy Complete or partial loss of a chromosome N early twice the nuclear DNA content In vitro polyploidization FCM revealed variations in nuclear DNA content. Genetic instability

Results Only RN 51 showed an increase in nuclear DNA content Mannitol 0,3 M 5 days No significant effect in the modification of nuclear DNA content 4,7% of polyploidization Consistent with values found for other species The stresses applied to tillers might not be responsible for polyploidization Not detected in plants of the remaining pretreatments

Results The changes observed in nuclear DNA content might be caused by the effect of growth regulators used in the culture media. Need for additional assays to confirm the possible role of growth regulators as promoting factors for the variations observed 2,4-D ?! 2,4-D NAABAP

Conclusion  The three apomictic genotypes of buffel grass responded to in vitro anther culture, but differed in their efficiency both for embryogenic callus induction and plant regeneration.  Somaclonal variation would provide an opportunity to obtain new genotypes that might be included as sources of genetic variability in breeding programs

References  Rowson HM, Macpherson HG. (2000). Irrigated wheat - managing your crop. Rome: Food and Agriculture Organization of tha United Nations. Available at:  Heuzé V., Tran G., Baumont R., Buffel grass (Cenchrus ciliaris). Feedipedia.org. A programme by INRA, CIRAD, AFZ and FAO. Last updated on August 23, 2013, 8:25http://  Triticum Aestivum (wheat)." Triticum Aestivum, Bread Wheat at GeoChemBio: Taxonomy, Brief Facts, Developmental Stages, Flower Anatomy. Nemose, 19 May Web. 13 Nov  Bicknell RA, Koltunow AM. (2004). Understanding Apomixis: Recent Advances and Remaining Conundrums. The Plant Cell. 16 (Suppl): S doi: /tpc

Somatic embryogenesis from in vitro anther culture of apomictic buffel grass genotypes and analysis of regenerated plants using flow cytometry Edgardo Carloni ; Andrea Ribotta ; Eliana López Colomba ; Sabrina Griffa ; Mariana Quiroga ; Exequiel Tommasino ; Karina Grunberg | Ana Rafaela Teixeira | Daniela Sousa | João Rodrigues |