Garlic and Health, P9 report 23/06/2018 Garlic and Health, P9 report WP2 Breeding systems Munich, 29-3 October 2003 INRA, Avignon, France V. Chovelon, research scientist JP Leroux, research technician E. Clini, temporary agent P nine -assistant contractor of P eight- three persons had worked for this program at INRA Avignon in 2003: V. Chovelon, research scientist JP Leroux, research technician E. Clini, temporary agent (1.5 month)

Objectives Somatic embryogenesis, genetic characterisation 23/06/2018 Objectives Somatic embryogenesis, genetic characterisation plant regeneration acclimatisation and field evaluation sanitary aspects meristem culture on selected clones production of virus-free bulbs Objectives: Somatic embryogenesis: P9 is involved in the somatic embryogenesis program to assure plant regeneration, acclimatisation, field cultivation and evaluation to control plant conformity, in comparison with the original material. Sanitary aspects: P9 bring competencies for regeneration of selected garlic clones by meristem culture and for production of virus-free bulbs for other partners.

Milestones Embryogenesis 23/06/2018 Milestones: Four varieties are experimented in embryogenesis program: Rouge de la Réunion, Messidrôme, Morasol and Printanor. Plants issued from callus and plants issued from cell suspension were regenerated, acclimatised and field cultured for evaluation. Particular attention should be paid on the genetic integrity of the regenerated plants from somatic embryos. Analytical methods are: - morphological notations of regenerated plants during 2 years in field (P9) - flow cytometry to determine the ploidy level (by P8). - AFLP analyses, (by P1). - biochemistry (dry-matter and sulphur contents, by P5). All the tasks were realised at time during the four years of the project

Milestones • Sanitary aspects 23/06/2018 Milestones: Sanitary aspects 1) virus elimination on 4 clones selected by P7 and P9: - meristem culture, - acclimatisation, - virological control, - and field culture. 2) production of virus-free bulbs for other partners: three hundred plants of each variety (Rouge Reunion, Messidrôme, Morasol and Printanor). All the tasks were realised at time during the four years of the project.

Deliverables 23/06/2018 Deliverables: DP one: virus-free bulbs materiel for introduction in vitro DP five: Evidence of a somatic embryogenesis process for plant regeneration and acclimatisation in garlic (Allium sativum L.). L. FEREOL, V. CHOVELON, S. CAUSSE, N. MICHAUX-FERRIERE and R. KAHANE- “Plant Cell Report”, 2002 DP thirteen: Establishment of embryogenic cell suspensions cultures of garlic (Allium sativum L.) plant regeneration and biochemical analyses. L. FEREOL, V. CHOVELON, S. CAUSSE, D. TRIAIRE, N. MICHAUX-FERRIERE and R. KAHANE (done submitted to Plant Sciences in 2003) DP twenty one: paper on “ embryogenic cell suspension cultures of garlic as method for mass propagation and convenient material for genetic improvment” L. FEREOL, V. CHOVELON, S. CAUSSE, R. KAHANE (done, on the web-page in 2003).

Rouge Reunion plants from callus and cell suspensions 23/06/2018 Rouge Reunion plants from callus and cell suspensions In vitro culture (1) acclimatisation (2) field evaluation (3) (4) AFLP analyses flow cytometry phenotypic notations Rouge Reunion: plants issued from embryogenic calli and cell suspensions - in vitro culture was realised during the first year of the project. - plants acclimatisation was realised at different seasons during the second year, in greenhouse, and then in field under an insect-proof tunnel. - AFLP analyses were realised by P1 on plants issued from calli and cell suspensions and plant multiplied by cloves, as reference. The autoradiogram shows that the patterns are almost equal with one exception in plant multiplied by cloves in which there is an extra fragment present. - the ploidy level was checked by flow cytometry on one hundred and twenty plants from callus and from cell suspensions: all the plants were diploids (P8). - Normal phenotypic notations were observed during the culture; with however less vigorous plants and bulbs obtained from cell suspensions. - the first field evaluation was realised during year 3 (2002) under tropical conditions by P8. After 2 weeks at 5°C the material has been planted under tunnel (June and July 02). Different notations were given from plantation to harvest (weight clove mean, height plants mean, leaves number mean, vigour and colour of plants) showing a good and homogeneous development of plants from embryogenesis, however less developed and less vigorous than plants from vegetative multiplication. -Alliine and allicine contents were analysed by P5 on bulbs after harvest. - a second field evaluation after plantation of calibrated cloves was realised during the fourth year of the project (2003).

plants from callus or cell suspensions 23/06/2018 Messidrôme, Morasol, Printanor plants from callus or cell suspensions • In vitro culture (2) • Acclimatisation (2) • 1st field culture (3) (4) • 2nd field culture (4) - AFLP analyses - flow cytometry - phenotypic notations - 143 Mes-cal; 157 Mes-ecs - 106 Mol-cal; 114 Mol-ecs - 205 Pri-cal; 102 Pri-ecs Morasol and Messidrôme plants issued from embryogenic callus or cell suspensions: in vitro plants were obtained and acclimatised in greenhouse during year 2 (2001) and transferred in field under an insect-proof tunnel in year 3 for a first field evaluation (2002) and in year 4 for a second field evaluation (2003). Notations were realised and bulbs were harvested in June 03.

Ploïdy level 23/06/2018 First field evaluation: - Ploïdy level was tested by flow cytometry on one hundred plants of each variety : - all the plants RReunion from callus or cell suspensions are diploids; - Messidrôme plants: ten % plants from callus and 1% plants from cell suspensions were tetraploïds. - Morasol plants: three % from callus and four % Morasol plants from cell suspensions are tetraploïds. Printanor plants: ten % from callus and three % plants from cell suspensions are tetraploïds. In all the case, a low% of tertraploïd plants (less than 4%) was obtained on plants issued from cell suspensions.

Morasol notations plants bulbs 23/06/2018 Second field evaluation : all the bulbs obtained from diploïd and tetraploïd plants were planted under tunnel in November 02, after clove homogeneous calibration (1-2g; 2-3g; 3-4g), for a second field evaluation. Notations of plants in vegetation (plant development, vigour, height, leaves colour) were realised until harvest and notations of bulbs (size and shape, cloves number and weight, cloves and external scale colours, multiplication rate, bulb diameter and weight, storage capacity) were also realised in July 03. 106 Morasol-cal and 114 Morasol-ECS bulbs were harvested (20 June 2003). Notations of plants (table 1 and table 2): for an homogeneous cloves class at the plantation, plants issued from callus or from cell suspension have similar height; but plants issued from callus present a more important vigour and an higher leaves number (April to June 03). After harvest, bulbs weight and multiplication rate are quasi similar from callus or from ecs issued plants.During the second year of field evaluation, tetraploïd plants are still irregular, less high and less vigorous than diploïd plants. No floral scape development was observed on tetraploïd plants; and inferior bulb weight were generally obtained at maturity (MOL-cal).

Messidrôme notations plants bulbs 23/06/2018 Messidrôme plants issued from callus (MES-cal), second field evaluation : all the bulbs obtained from diploïd and tetraploïd plants were planted under tunnel in November 02, after clove homogeneous calibration, for a second field evaluation. Notations of plants in vegetation and notations of bulbs after harvest were realised (February to August 03). 143 Messidrôme-cal bulbs were harvested (20 June 2003). Notations of plants: During the first field culture (MES-ecs), very heterogeneous plants were observed but no particular difference was observed between diploïd and tetraploïd plants. The second field evaluation showed that plants issued from callus (MES-cal) are more vigorous (leaves number more important), but less high than plants vegetatively multiplied (table 3). The bulbs obtained on plants issued from callus are bigger than those vegetatively multiplied, for a same cloves weight at plantation (table 4). During the second field culture, tetraploïd plants were less vigorous and less high than diploïd plants; with inferior bulbs weight and multiplication rate.

Printanor notations plants bulbs 23/06/2018 Printanor plants issued from cell suspensions (PRI-ecs), first field evaluation : plants in vitro obtained were acclimatised in pot under greenhouse (September 03) and transferred in field under an insect-proof tunnel (February 03). Notations of plants in vegetation and notations of bulbs after harvest were realised (February to August 03). 102 Printanor-ecs bulbs were harvested (July 2003). Printanor plants issued from callus (PRI-cal), second field evaluation : all the bulbs obtained were planted under tunnel in November 02, after clove homogeneous calibration, for a second field evaluation. Notations of plants in vegetation and notations of bulbs after harvest were realised (February to August 03). 205 Printanor-cal bulbs were harvested (July 2003). Notations of plants (table 5 and table 6): - During the first field culture (PRI-ecs), the cloves were not homogenised before plantation, and very heterogeneous plants were observed. - The second field evaluation showed that plants issued from callus (PRI-cal) are still heterogeneous, with irregular height (table 5). More vigorous plants (leaves number more important) and bigger bulbs were obtained from planted cloves up to 2 g (table 6). During the second field culture, tetraploïd plants were less vigorous and less high than diploïd plants; with inferior bulbs weight and multiplication rate.

Sulphur analyses (HPLC) 23/06/2018 Sulphur analyses (HPLC) concentrations Morasol and Messidrôme Sulphur content tests on bulbs after harvest (P5): five bulbs of each origin were sent to P5 and tested by HPLC method in comparison with five bulbs of Morasol and Messidrôme vegetatively multiplied. The results showed that: 1- for all the varieties, alliin concentration is quasi similar on the different bulbs produced by embryogenesis or vegetatively multiplied. (the little superiority on Messidrôme-cal (57) and Morasol vegetatively multiplied (61) and RReunion-ECS (44) are not significatively different) 2- for all the varieties, there is not difference in alliin or dipepetid concentrations between embryogenic plants issued from callus or from cell suspensions. 3- but, for all the varieties, there is a great difference of dipeptides concentration between garlic from embryogenesis (cal and ECS) and garlic vegetatively multiplied: dipeptides concentrations are highest on plants issued from embryogenesis. Rouge Reunion

sulphur compounds proportion (HPLC) 23/06/2018 sulphur compounds proportion (HPLC) Morasol and Messidrôme The allin and dipeptids proportions amount is stable in all the bulbs, but the distribution is different: - the alliin proportion is higher on bulbs vegetatively multiplied (more than fifty %), whereas it is only at thirty to thirty five % in bulbs from embryogenesis. - the proportions of the two dipeptids are higher on bulbs from embryogenesis, and they are reversed comparatively with Messidrôme and Morasol vegetatively multiplied. - It is important to note the great weight difference of the analysed bulbs: twenty to forty grams for bulbs from embryogenesis and seventy grams for bulbs vegetatively multiplied. In conclusion, for all the varieties, the garlic sulphur compounds are different on bulbs issued from embryogenesis and on bulbs vegetatively multiplied. These results must be confirmed next year, after the second field culture, on bulbs with similar weight. Rouge Reunion

Sanitary aspects production of virus-free plants virological control 23/06/2018 Sanitary aspects production of virus-free plants Messidrôme, Morasol, Printanor 300 plants / var virological control potyvirus-free latent viruses: Messidrôme: 100% GCLV Printanor: 100% MbFV Sanitary aspects: Production of virus-free plants - For each variety (Messidrôme, Morasol and Printanor) three hundred cloves were planted in November and December 02 in field, under insect-proof conditions to assure a new production for June 03. - ELISA tests were realised about pathogen viruses and latent viruses: - all the varieties were free of potyviruses, but some one contained latent viruses: - Messidrôme: all the plants contained GCLV - Printanor: all the plants contained Allexivirus.

Sanitary aspects virus elimination by meristem culture 23/06/2018 Virus elimination by meristem culture on 4 garlic clones - Four garlic clones were selected by P7 and P9 for their fertility , and regenerated by meristem cutlure, after thermotherapy treatment to eliminate more easily pathogen and latent viruses. - plants well developed with roots and bulblets were obtained, acclimatised in, cultured in pots under greenhouse and planted in field under an insect-proof tunnel. - Virological control were realised on plants in vegetation against pathogen (OYDV, LYSV) and latent viruses (GCLV, MbFV): all the plants are free of viruses. - matured bulbs were harvested , and the bulbs were sent to P7 for sexual seed production.