1. Garlic and Health, P9 report
Liverpool, February 2003
INRA, Avignon, France
V. Chovelon, research scientist
JP Leroux, research technician
F. Mirrione, temporary agent (1.5 month)
P nine -assistant contractor of P eight-
three persons had worked for this program at INRA Avignon in 2002:
V. Chovelon, research scientist
JP Leroux, research technician
F. Mirrione, temporary agent (1.5 month)

2. 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.

3. Milestones Embryogenesis 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 three first years of the project.

4. Milestones • Sanitary aspects Milestones: Sanitary aspects
1) virus elimination on two clones selected by P7 (Rina):
- 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 three first years of the project.

5. Deliverables 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.) and plant regeneration. L. FEREOL, V. CHOVELON, S. CAUSSE, D. TRIAIRE, N. MICHAUX-FERRIERE and R. KAHANE (in progress, to be published in 2003)
DP twenty one: paper on the use of somatic embryogenesis for rapid multiplication of garlic in vitro (in progress, to be published in 2003).

6. plants from callus and cell suspensions
Rouge Reunion
plants from callus and cell suspensions
In vitro culture (1)
acclimatisation (2)
AFLP analyses
flow cytometry
phenotypic notations
- 1st field evaluation at Reunion Island by P8 (3)
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 will be realised during the fourth year of the project (2003).

7. plants from callus or cell suspensions
Messidrôme and Morasol
plants from callus or cell suspensions
• In vitro culture (2)
- regeneration
- bulb maturation
• Acclimatisation (2)
• 1st field culture (3) (4)
166 Mes cal; 145 Mol cal; 125 Mol ecs; 153 Mes ecs
• 2nd field culture (4)
- 282 Messidrôme cal
- 143 Morasol cal
- 145 Morasol 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 (2002) for the first field evaluation.
Notations were realised and bulbs were harvested in June 02.
Calibrated cloves were planted in November 02 for a second field evaluation.

8. 1st field evaluation
notations (plant development, height, leaves colour, bulbs shape and weight)
ploïdy level
First field evaluation:
- notations of plants in vegetation (plant development, vigour, height, leaves colour) and after harvest (bulbs shape and weight) were realised: very heterogeneous plants were observed during the first field culture, but no particular difference was observed between diploïd and tetraploïd plants.
- 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;
- ten % Messidrôme plants from callus, three % Morasol plants from callus and four % Morasol plants from cell suspensions are tetraploïds.

9. 1st field evaluation • sulphur compounds concentration (HPLC)
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

10. 1st field evaluation • 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 vegetaively multiplied.
These results must be confirmed next year, after the second field culture, on bulbs with similar weight.
Rouge Reunion

11. plants from callus Printanor
Printanor plants issued from embryogenic calli:
- three hundred and forty height plants were in vitro cultured and acclimatised in greenhouse in April 02: one hundred and seventeen plants in vegetation and one hundred and twenty six bulbs were obtained in October 02.
One clove per bulb was planted under an insect-proof tunnel for a first field evaluation, and two hundred and twenty two plants are now in vegetation.
Flow cytometry will be realised on plants in April/May 03,
and biochemistry analyses will be realised on bulbs after harvest in July 03.

12. Sanitary aspects production of virus-free plants virological control
Messidrôme, Morasol, Printanor
300 plants / var. (Nov-Dec 02)
virological control
all the plants were potyvirus-free
some one contained latent viruses
Messidrôme: 100% GCLV
Morasol: 83% MbFV
Printanor: 100% MbFV
Sanitary aspects: Production of virus-free plants
- For each variety (Messidrôme, Morasol and Printanor) three hundred bulbs were harvested in June-July 02 and dried at room temperature before sending to the different partners in September-October 2002.
- 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
- Morasol: eighty three % contained Allexivirus
- Printanor: all the plants contained Allexivirus.

13. Sanitary aspects virus elimination by meristem culture
Virus elimination by meristem culture on garlic clones (1) and (2)
- Two garlic clones were selected by P7 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 September 01 and cultured in pots under greenhouse (Octob. 01 to February 02).
- All the plants were planted in field under an insect-proof tunnel in March 02.
- Virological control were realised in April 02 on plants in vegetation against pathogen (OYDV, LYSV) and latent viruses (GCLV, MbFV): all the plants are free of viruses.
- matured bulbs were harvested in June 02:
eight. one. ninety six (VIII.1.96): thirty seven bulbs (twenty g < weight bulb < seventy five g)
seven. eighty one. ninety seven (VII.81.97): ten bulbs (twelve g < weight bulb < eighty g)
- for each clone one bulb was kept by P9 and planted under tunnel, and the other bulbs were sent to P7 for sexual seed production.
Virus elimination by meristem culture on garlic clones (3) and (4)
two clones were selected by P9 for their fertility, regenerated and acclimatised in greenhouse in September 02: nineteen plants of Clone (3), and thirty plants of Clone (4) will be transferred in field in March 03 and tested by ELISA.

14. Workplan for 2003 Embryogenesis, genetic characterisation
Rouge Reunion
2nd field evaluation (P8), biochemistry (P5)
Messidrôme, Morasol and Printanor from callus
2nd field culture and evaluation (P9)
flow cytometry (P8), AFLP (P1), biochemistry (P5)
Messidrôme, Morasol and Printanor from cell suspension
1st field evaluation
Sanitary aspects
production of virus-free bulbs (P9)
1) embryogenesis, genetic characterisation:
- Rouge reunion:
- bulbs will be cultured at Reunion Island for a 2nd field evaluation under tropical climate.
- biochemistry analyses (dry mater and sulphur content) will be realised a second time by P5 on bulbs after harvest.
- Messidrôme, Morasol and Printanor plants issued from embryogenic callus:
- plants will be cultured in field under tunnel for a second field evaluation, flow cytometry and AFLP analyses will be realised on Printanor plants in May 03, alliine content will be revaluated in July 03.
- Messidrôme, Morasol and Printanor plants issued from cell suspension :
- bulbs plantation and first field evaluation, flow cytometry, AFLP and biochemistry
2) sanitary aspects
- production of virus free material: new cloves were planted and will be cultured in 2003