Participant No: P7 & P10 Participant number, names and address of the participating organizations




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Participant No: P7 & P10

Participant number, names and address of the participating organizations

P7 R. Kamenetsky. The Volcani Center, ARO, P.O.Box 6, Bet Dagan, Israel; Tel. 972-3-968-3511; Fax 972-3-966-0589; E-mail: rmgold@agri.huji.ac.il

P10: H.D. Rabinowitch. Hebrew University of Jerusalem, Faculty of Agriculture, food and Environmental Quality Sciences, P.O. Box 12, Rehovot 76100; Tel 972 8481244; Fax: 972 89468265; E-mail: rabin@agri.huji.ac.il
Scientific team

Research leader (P7):

Dr. R. Kamenetsky

(6 person months)

Duties: supervision of research, collection missions, flowering physiology and fertility restoration

Research assistant

H. Zemah

(7 person months)

Duties: flowering physiology, fertility restoration

Research technician

M. Shafran

(8 person months)

Duties: evaluation and maintenance of collected plant material
Research leader (P10)

Prof. Dr. H.D. Rabinowitch

(1 person month)

Duties: supervision of the research, collection missions

Research assistants

Agr. A. Harazy

(2 person months)

Duties: maintenance of the quarantine and collection. Systematic evaluation.

Agr. I. London

3 person months

Duties: evaluation of plant material, flowering physiology

Contractual links to other participants

P1, P6, P7, P8, P9 and P10 are involved.



Objectives

Study of the sexual hybridisation system and fertility restoration in garlic.



Workplan

Environmental requirements (storage conditions, temperature and day length during plant growth) for flower transition, scape elongation, flower development and restoration of fertility will be investigated. Light and scanning electron microscopy will be employed for the developmental work.


Milestones & Deliverables
M&D 2000

Milestones:

Morphological analysis of flower development

in the selected clones done

Initial developmental, florogenesis and fertility studies done

Plant physiological age and environmental effects of development

and flowering in selected clones partly done

Deliverables:

DP4: Paper on morphological and physiological aspects of floral/

topsets initiation and development partly done


M&D 2001

Milestones:

Plant physiological age and environmental effects on floral

initiation and development done

Mechanical and chemical treatments to initiate normal flowering,

restore fertility and produce seeds partly done1

Deliverables:

DP4: Paper on morphological and physiological aspects of floral/

topsets initiation and development done
M&D 2002

Milestones:

Forcing selected garlic clones for flowering done

Establishing mechanical or chemical treatments to obtain normal

flowering done1

Pollination and study of seed production, seed viability and germination

ability done

Producing self-pollinated and cross-pollinated populations within

and between selected clones partly done

Deliverables:

DP 11: Paper on environmental regulation of flower differentiation

and flowering in process



Research Activities During The Third Reporting Period




Introduction
Garlic (Allium sativum L.), a completely sterile plant, is asexually propagated by cloves, or by topsets of inflorescences. A number of reasons for the plant inability to produce fertile inflorescence were suggested, including competition for nutrients supply between the developing floral buds and topsets (Koul and Gohil, 1970; Kononkov, 1953; Novak and Havranek, 1975; Etoh et al., 1988, Konvicka, 1984); degeneration of the tapetum (Novak, 1972), degenerative diseases (Konvicka, 1973), hormonal balance (Pooler and Simon, 1994), and more. None of the above, however, provided a comprehensive clarification for this phenomenon, and/or tools for a complete fertility restoration (Kamemetsky and Rabinowitch, 2001, 2002; Etoh and Simon, 2002).

The discovery of fertile garlic in the 1980s of the 20th century (Etoh and Simon, 2002) brought the study on the flowering of garlic to the fore, and opened new research avenues on garlic genetics and physiology, as well as on breeding.

A recent review on florogenesis indicated that in all studied Alliums, environment serves as a major causal agent in flowering (Kamenetsky and Rabinowitch, 2002), but little was done to elucidate the environmental effects on induction, initiation, and floral differentiation in garlic. The availability of flowering fertile genotypes, however, made a research on the effect of environment on flowering in garlic both possible and important for better and efficient exploitation of this trait.

The transition of apical meristem from vegetative to reproductive state occurs in the field, during the active growing stage, after the formation of six to eight leaves, including leaf primordia (Kamenetsky and Rabinowitch, 2001). Low field temperatures may promote inflorescence induction, but cold requirements vary with genotype. Little, however, is known about the physiological regulation of florogenesis by temperature and light, as well as on the phases of the garlic’s annual cycle most responsive to various environmental treatments.

Garlic genotypes vary markedly in their ability to produce scape (flower stalk), and were classified by Takagi (1990) as follows: (1) Nonbolters do not normally form inflorescence and in many cases produce cloves inside the false stem. Occasionally, however, scapes may develop. (2) Incomplete bolters – produce thin, short scape, and bearing only a few large topsets, and usually forming no flowers; (3) Complete bolters – produce a long, thick scape, with many flowers and topsets. Our observations showed a great variation between clones with bolting capacity, including: minimum leaf number prior to bolting, final stem length, earliness, flower to topset ratio in the umbel, and pollen viability (Kamenetsky et al., in press).

In addition to the genetic traits, scape elongation in garlic is strongly affected by environment. In non-bolters or incomplete bolters, temperature and light may promote scape elongation. In contrast, adverse conditions may inhibit scape elongation of bolting plants (London, pers. obs.). In bolting plants, day length in the field plays a dominant role in controlling scape elongation. Long days are essential for both the actual elongation of the scape and its normal development (Takagi, 1990).

Garlic inflorescence is an umbel-like flower arrangement, the branches (flower clusters) of which arise from a common meristem (Kamenetsky and Rabinowitch, 2001). The flowers have a distinct morphology typical of the genus Allium (Etoh, 1985). Differentiation of topsets begins in the peripheral part of the apical surface following that of the flowers. The immediate contact between the growing topsets and the young flowers, results in physical pressure on the young developing floral buds, thus causing their degeneration (Kamenetsky and Rabinowitch, 2001). Therefore, in some bolting garlic clones, a continuous removal of topsets at the early stages of their development resulted in normal flowering. Thereafter, when viable pollen was available, pollination and fertilization was followed by seed production (Pooler and Simon, 1994; Konvicka, 1984; Etoh et al., 1988; Koul and Gohil, 1970, Jenderek, 1998)

We hereby report on the effect of environmental conditions on sequential morphological processes during the vegetative and reproductive development of garlic.


Materials and Methods
Plant material: Bulbs of accession #2091, introduced to Israel from Russia in 1990-s, were received from the Field Gene Bank for Vegetatively Propagated Short-Day Allium spp., in Rehovot, Israel. Medium sized cloves were randomly sampled for growth experiments. They were treated with 0.2% Benlet (Du Pont De Nemours, France) and 1% Marpan 50 wp (Machteshim, Israel) and planted in 1.8 L plastic pots containing 0.8 mm cinder and peat (RHP; Dega Potground Delft Company, Holland), at 4:1 v/v.

Mature bulbs were harvested in July 2001, and treated as above until September 5th 2001, when randomly selected medium size bulbs were stored at 40C, RH 65-70%, for 60 days. Following planting, the plants were exposed to a number of thermo- and photoperiod treatments (Tab. 1).

Phenological observations were performed during all growth period. The number of leaves and leaf primordia, number and developmental stages of cloves, floral scape length, degree of floral development, bulb weight, and bulb diameter, were determined during growth and after harvest. During the period of 9-12 weeks after planting, 3-10 plants were sacrificed weekly for detailed morphological analysis. At the end of the experiment, the morphology and physiological status of the remaining plants were also studied; altogether 3-14 plants were dissected at each of the sampling dates.
Effect of thermo- and photoperiod on CSO accumulation during bulb maturation: Phytotron thermo- and photoperiod treatments are presented in Fig. 1 After bulb maturation and harvest, 5 bulbs per treatment were transferred from Israel (P7) to France (P5) for CSO analysis.

CSO analysis : Lichtwer's method


The method used is a technique without derivatization. Chromatographic separations were performed using Waters instrument with Hypurity Elite C18 column (150 mm X 3 mm). The detection is an UV detector with a wavelength fixed at 208 nm. The separation requires an elution gradient with two solvents A and B. The solvent A constitution is 20 mM sodium dihydrogen phosphate + 10 mM heptane sulfonic acid at pH of 2,1. The solvent B constitution is 50% A + 50% acetonitrile. The flow-rate of the eluant is 0,4 ml/mn.

Gradient:




Time (min)

% A

% B

0

100

0

5

70

30

25

46

54

26

0

100

28

0

100

30

100

0

40

100

0


Results (Tables and Figures are in Annex)
Transition from vegetative to reproductive state
Following storage at 4°C, garlic plants were exposed to a variety of phytotron conditions, for developmental studies of reproductive organs. Garlic inflorescence is terminal, and new leaf production ceases with the transition of the apex to the reproductive state. Under all growth conditions, the apical meristem underwent floral transition, and floral differentiation occurred in all plants. Further development, however, was temperature and day-length dependent (Tab. 1).

The earliest meristem transition from vegetative to reproductive state occurred 40 days after planting, under long photoperiod, in plants at the physiological age of 7-8 leaves (Tab. 1). Under SD and 20/120C, this transition was first observed in the plants with 10-12 leaves (including leaf primordia), while higher temperatures of 23/150C further delayed this process (Tab. 1).


Scape elongation
Morpho-developmental studies revealed the strong environmental effect on scape elongation. Following floral initiation and transfer from SD to LD, final scape length was markedly affected by temperature, and reached 80 and 55 cm under cooler and warmer phytotron conditions, respectively (Tab. 1, Fig. 2).

Under LD, scape elongation immediately followed the initiation of floral meristem, continued at a fast rate in tandem with flower differentiation, and spathe break was reached 99 days after planting. However, scapes of more than 80% of the plants failed to lengthen (Tab. 1, Fig. 2).

Under uninterrupted short photoperiod, scape elongation was slow and temperature played a significant role (Tab. 1, Fig. 2). Abortion of the differentiated flowers occurred in the the inflorescence occurred, when the scapes were still enveloped by the leaf sheaths inside the false stem. Under lower temperatures, scapes were 18-20 cm long, while at higher temperatures elongation was completely inhibited (Tab. 1).

When LD interrupted SD regime for one week, scape elongation was slightly inhibited, and at higher temperatures, inflorescence was aborted (Tab. 1, Fig. 2). Two to four weeks of LD interruption were sufficient for normal scape elongation both at lower and higher temperatures.

Higher temperature reduced percentage of scape elongation under all photoperiod conditions (Tab. 1)
Inflorescence differentiation and completion of floral development
At the initial stages of the inflorescence development, the apical meristem subdivides to form several swellings, each of which gives rise to a number of individual floral primordia (Fig. 3 a). This is followed by an increase in both the size of the developing inflorescence and the number of floral primordia. The differentiation of individual flowers begins when the developing inflorescence reaches a diameter of about 2-3 mm. This process occurs in the oldest floral buds, while younger ones still emerge as undifferentiated meristematic domes (Fig 3 b).

Following flower differentiation, new undifferentiated domes become visible at the periphery of the inflorescence, these quickly differentiate and grow to form vegetative buds, i.e., topsets (Fig. 3 c). Under our experimental conditions, the photoperiod regime prevailing during scape elongation significantly affected the development of the topsets in the inflorescence.

When SD was followed by LD, and under LD conditions, both flowers and topsets developed in the inflorescence, and final inflorescence diameter reached 4 cm. After spathe break, the fully differentiated flowers were physically squeezed by the growing topsets, and thus degenerated and dried out before anthesis (Fig. 4a).

At 20/120C and SD, an interruption of one week LD resulted in normal flower differentiation with the formation of only a few vegetative buds. Full anthesis of the inflorescence of 1- 1.2 cm in diameter was reached after spathe break (Fig. 4 b). At 23/150C a one week interruption of SD by LD resulted in a abortion of young floral buds at the early stages of differentiation. The inflorescence dried out before spathe break. Two, three or four weeks interruption of SD by LD, both at low and high temperatures, had no such an adverse effect. The longer the LD period, the bigger was the diameter of the inflorescences, thus reaching 1.5 cm, 2.7 cm and 2.9 cm, respectively. The number of topsets increased progressively, concurrently with inflorescence size with the consequent squeezing of the flowers, which never reached anthesis. In plants exposed to only two or three weeks of LD, the topsets formed mainly slender leaves (Fig. 4 c), whereas four weeks of LD interruption resulted in a mixed population of dormant and non-dormant topsets.


Effect of thermo- and photoperiod on CSO accumulation during bulb maturation
The effect of various thermo- and photoperiod on concentrations of S-compounds AlCSO, GLUAlCS, isoGLUAlCS and allicin in matured bulbs is presented in Table 2 and Figures 5-6. Higher growth temperatures seems to have an effect compared to lower temperatures (20/12°C). Higher temperatures (23/15°C) and long days, with incandescent light, seem to induce more GLUAlCS, isoGLUAlCS and allicin but no similar effect is observed on alliin content in bulbs.
Discussion

The recent discovery of fertile garlic in Central Asia (Etoh et al, 1988; Kotlinska et al., 1991; Etoh and Simon, 2002), and the consequent knowledge acquired on garlic florogenesis (Kamenetsky and Rabinowitch, 2000), improved our understanding of the environmental regulation of the development of vegetative and reproductive organs.

Under our experimental conditions, all plants underwent a complete transition of the apical meristem from vegetative to reproductive state during the active growth phase. Low temperatures were suggested to promote floral induction in many cultivated Allium crops (Kamenetsky and Rabinowitch, 2002). Similar effect was recorded in garlic (Table 1). Yet, our results with clone #2091 show clearly that daylength might promote floral transition in garlic. Early exposure to LD resulted in reduction of the number of leaves and leaf primordia prior to floral transition, in comparison with SD (Table 1). Based on our results we conclude that in garlic accession # 2091, meristem transition from vegetative to reproductive state occurs under a vast variety of storage and growth conditions, but that environment has only a quantitative effect. Similar mechanism was described for Arabidopsis, were "autonomous" pathway was suggested as an alternative to the major effects of low temperatures or LD on floral initiation (Reeves and Coupland, 2000).

Interactions between storage and growth temperatures play the most important role in scape elongation, and photoperiod markedly affects this process in Allium species (Kamenetsky and Rabinowitch, 2002). We clearly show that in garlic LD is obligatory and quantitative for scape elongation (Table 1, Fig. 2). Hence, under SD, scape remained enveloped by the leaf sheathes, and without a LD interruption, had never become visible. Under exclusive LD conditions, scape elongation was fast, yet only a few plants reached maximum scape length and spathe break (Table 1). In Japanese bunching onion, LD promotes scape elongation only when the inflorescence reached a certain stage of development. Otherwise, it may even inhibit flowering (Yamasaki et al, 2000). In garlic, even a short, one to four weeks' interruption of SD regime by LD was sufficient for scape elongation (Fig. 2). Growth temperature affected scape elongation only under margin daylength conditions. Hence, low temperatures enhanced scape elongation under SD, or when LD interrupted SD for one week. Longer LD treatment of more than two weeks had only a minor effect and scape elongation was similar at low and high temperatures.

In the inflorescence, differentiation of newly formed flowers and topsets proceeds concomitantly with scape elongation. The development of topsets, however, is dominated by photoperiod. While long days promote topset growth with the consequent maturation and dormancy, short photoperiod (with one week interruption of LD which promotes scape elongation) was found to inhibit their development. As a result, only flowers appeared in the inflorescence. These flowers reached full anthesis after spathe break (Fig. 4b). Exposure to LD for two or three weeks was too short for dormancy induction, and thus resulted in non-dormant vegetative buds in the inflorescence (Fig. 4c).

We conclude that in bolting genotypes, sophisticated manipulation of environment prior to- and after-planting can lead to the development of flowers in a topsets’ free umbel, thus regaining fertility without the continuous removal of the developing floral sets.


References

Etoh T (1985) Studies on the sterility in garlic, Allium sativum L. Memoirs of the Faculty of Agriculture, Kagoshima University 21:7-132.

Etoh, T. and P.W. Simon (2002) Diversity, fertility and seed production of garlic. In: Rabinowitch, H.D. and L. Currah, eds. Allium Crop Science: Recent Advances. CABI, Wellingford, U.K. pp. 101-117

Etoh T, Noma Y, Nishitarumizu Y, Wakomoto T (1988) Seed productivity and germinability of various garlic clones collected in Soviet Central Asia. Memoirs of the Faculty of Agriculture, Kagoshima University 24:29-139.

Jenderek, M.M. (1998). Generative reproduction of garlic (Allium sativum). Sesja Naukowa 57, 141-145. (in Polish).

Kamenetsky, R., London Shafir, I., Baizerman, M., Khassanov, F., Kik, C. and H.D. Rabinowitch (2003)

Garlic (Allium sativum L.) and its wild relatives from Central Asia: evaluation for fertility potential. Acta Horticulturae, in press

Kamenetsky, R. and H. D. Rabinowitch (2001). Floral development in bolting garlic. Sexual Plant Reproduction 4, 235-241.

Kamenetsky, R. and Rabinowitch, H. (2002) Florogenesis. In: Allium Crop Science: Recent Advances (H. D.Rabinowitch and L. Currah, eds) CAB International, pp. 31-57

Kononkov PE (1953) The question of obtaining garlic seeds. Sad i Ogorod 8:38-40 (in Russian).

Konvicka O (1973) The causes of sterility in Allium sativum L. Biologia Plantarum (Praha) 15(2):144-149 (in Czech).

Konvicka O (1984) Generative reproduction of garlic (Allium sativum). Allium Newsletter 1: 28-37.

Kotlinska, T., Havranek, P., Navratill, M., Gerasimova,I., Primakov, A and Neikov, S. (1991) Collecting onion, garlic and wild species of Allium in central Asia, USSR. FAO/IBPGR Plant Genetic resources Newsletter 83/84,31-32

Koul, A.K., Gohil, R.N. (1970) Causes averting sexual reproduction in Allium sativum. Linn. Cytologia 35: 197-202.

Novak, F.J. (1972) Tapetal development in the anthesis of Allium sativum L. and Allium longicuspis Regel. Experientia 28(1): 1380-1381.

Novak, F.J., Havranek, P. (1975) Attempts to overcome the sterility of common garlic (Allium sativum L.) Biologia Plantarum (Praha) 17 (5): 376-379.

Pooler, M.R, Simon, P.W (1994) True seed production in garlic. Sexual Plant Reproduction 7:282-286

Reeves, P.H. and Coupland, G. (2000) Response of plant development to environment: control of flowering by daylength and temperatures. Current Opinion in Plant Biology, 3:37-42

Takagi, H. (1990) Garlic Allium sativum L. In: Rabinowitch HD, Brewster JL (eds) Onions and allied crops (vol III) Biochemistry, Food science, and minor crops. CRC Press, Boca Raton, Florida, pp 109-146.

Yamasaki, A., Tanaka, K., Yoshida, M. and Miura, H. (2000) Effects of day and night temperatures on flower-bud formation and bolting of Japanese bunching onion (Allium fistulosum L.) Journal of the Japanese Society for Horticultural Science 69, 40-46.



Significant difficulties or delays experienced during the reporting period

No bottlenecks



Sub-contracted work during the reporting period

No subcontractors


Future actions




  1. Producing self-pollinated and cross-pollinated populations within and

between selected clones

  1. Developmental analysis of seedling populations

  2. Vegetative development of plants and environmental regulation of

Floral initiation, differentiation and fertility restoration

ANNEX

Tables 1-2

Figures 1-6



Table 1. Effect of phytotron conditions on florogenesis in garlic in 2001-2002 (details in Fig. 1). Following storage at 40C X 60 days, medium size cloves were planted in pots and placed on November 5, 2001, in the phytotron. Records were taken when >50% of the population within a treatment reached the developmental stage in question. All time measurements refer to the planting date

Treatment

Growth conditions

Leaf number prior to scape formation

Time to apex floral transition, days

Final scape length,

cm

Plants with visible scape, %


Time to spathe break, days



Temperature

Weeks at LD


1

20/120C

12

12.5±0.5

60

80.8±4.0

84.6

136

2

20/120C

-

11.0±0.5

60

18.8±5.2*

-

-

3

20/12 -23/150C

11

11.5±0.5

60

54.2±3.0

66.7

122

4

20/12 -23/150C

-

13.0±0.6

70

0.97±0.3*

-

-

5

20/12 -23/150C

16

7.7±0.7

40

57.4±4.1

19.2

99

6

20/120C

1

11.7±0.3

60

49.2±2.4

95.0

160

7

20/120C

2

11.8±0.3

60

58.8±2.3

85.7

160

8

20/120C

3

11.5±0.5

60

56.9±1.9

68.4

160

9

20/120C

4

12.5±0.4

60

64.8±2.9

79.8

154

10

20/12 -23/150C

1

11.4±0.3

60

50.5±5.5

18.7

aborted

11

20/12 -23/150C

2

12.7±0.3

60

54.7±2.5

66.7

134

12

20/12 -23/150C

3

12.3±0.5

60

60.6±2.1

56.2

134

13

20/12 -23/150C

4

12.0±0.9

60

56.0±3.3

80.0

134


Table 2. Concentrations and standard deviations in nmole/mg of fresh bulb


Treatment

Alliin

Sd

alliin

GLUAlCS

sd

GLUALCS

Iso

GLUALCS


sd iso

GLUALCS

allicin

sd

allicin

1

30,7

10,7

13,6

4,4

45,6

6,9

5,5

1,6

2

34,7

1,6

6,6

1,1

27,5

7,6

5,1

2,7

3

31,1

2,2

20,6

3,4

47,9

3,4

6,9

2,9

4

41,1

10,4

17,0

4,8

39,8

7,6

7,2

1,3

5

32,5

3,1

26,2

3,3

56,3

1,6

3,7

0,3

6

35,5

2,2

4,7

0,5

25,4

5,8

3,3

2,0

7

42,1

6,8

8,0

0,4

31,3

2,5

3,5

1,1

8

32,5

5,1

7,2

1,8

31,9

5,8

5,9

1,7

9

38,7

4,1

10,0

2,4

34,1

7,3

4,2

1,4

10

33,1

2,2

12,9

0,8

42,4

3,0

7,0

2,0

11

37,6

2,4

15,4

1,5

37,3

2,6

10,1

3,3

12

32,0

4,4

15,8

3,2

35,7

4,2

7,7

0,4

13

26,0

5,7

18,5

1,6

40,3

2,5

7,5

1,7






Fig. 2. Effect of growth conditions on mean weekly rate of scape elongation. Intact bulbs were stored at 40C for 60 days prior to planting in the phytotron on November 5, 2001. (N=14). White and black columns represent growth temperatures of 20/120C and 23/150C, day and night, respectively.



Fig. 5. Total amount of Aliin in mature garlic bulbs, as affected by growth temperatures and photoperiod. For details, see Fig. 1 (in M&M)





1 Garlic clones from our core collection were able to produce seeds without any mechanical or chemical intervention. Thus, the performance of this part of the program was terminated





1

Fig. 6. Quantification of S-compounds (AlCSO, GLUAlCS, isoGLUAlCS, and allicin) in mature garlic bulbs, as affected by growth temperatures and photoperiod. For details, see Fig. 1.



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