Seed Storage and Dormancy in Chatham Island forget-me not (Myosotidium hortensia (Decne.) Baillon)

C.R. McGill1, M.J. Park1,2, J. Nadarajan3, W.M. Williams4, B.R. MacKay1 & H.A. Outred5

1Institute of Natural Resources, College of Sciences, Massey University, New Zealand 2Korea Seed & Variety Service, Anyang-Si Gyeonggi-do, Republic of Korea 3Seed Conservation Department, Royal Botanic Gardens Kew, Wakehurst Place, UK4Margot Forde Germplasm Centre, AgResearch Grasslands, Palmerston North, New Zealand5Institute of Molecular BioSciences, College of Sciences, Massey University, New Zealand

Myosotidium hortensia - the plant

• herbaceous perennial plant

http://www.bestgardening.com/bgc/plant/native02.htm

• can grow up to 1 metre across

• dense pyramidal flower clusters appear in spring

• each flower contains a four-lobed ovary that develops into four large nutlets

• each nutlet contains a single seed. http://www.bestgardening.com/bgc/plant/native02.htm

Myosotidium hortensia - the interest

• a monotypic genus endemic to the Chatham Islands of New Zealand

Myosotidium hortensia - the interest

Source: http://www.chathams.com/framesets/mapset.html

Myosotidium hortensia - the interest

• a monotypic genus endemic to the Chatham Islands of New Zealand

• trampled by cattle, sheep and pigs

• palatable to sheep and pigs

• threatened by introduced weeds (Ammophila arenaria)

• classified as nationally vulnerable in the wild

• an important ornamental propagated mainly by seed.

Myosotidium hortensia

Presentation Outline

Seed storage potential

• storage reserves

Seed dormancy

• seed coat structure

• oxygen uptake

How long can seed of M. hortensia be stored?

• seed of M. hortensia can be desiccated to 7.5% moisture without loss of viability and stored for at least 21 months at this moisture at 5°C

• at 9.5% moisture and 5°C seed showed a significant loss in viability after 9 months.

McGill et al. (2002)

Does the rapid loss of viability at 9.5% moisture occur at a range of storage temperatures?

Can M. hortensia be desiccated to moisture contents in equilibrium with 15% relative humidity and stored without loss of viability?

How long can seed of M. hortensia be stored?

• seed of M. hortensia has been stored in water-impermeable 12/20/50 micron laminated polyester/aluminium foil/polythene packets at 4.5% (equilibrium moisture content at 15% RH), 5.9% and 9.2% moisture at 20°C, 5°C, 0°C or -20°C. Seed will remain in storage for ten years

• seed has been sampled after three, six, twelve, eighteen and twenty-four months storage.

How long can seed of M. hortensia be stored?Viability after storage at three moisture contents and four temperatures

Storage Time (Months)0 5 10 15 20 25

Radi

cle

Emer

genc

e (%

)

0

20

40

60

80

100

4.5% moisture

Storage Time (Months)0 5 10 15 20 25

20oC 5oC 0oC-20oC

Storage Time (Months)0 5 10 15 20

Radi

cle

Emer

genc

e (%

)

0

20

40

60

80

100

9.2% moisture5.9% moisture

• at 9.2% there was a loss of germination after 18 months storage at all temperatures

• there was no decline in germination after 12 months storage at 4.5% or 5.9% moisture• at both 4.5% and 5.9% moisture there was a decline in germination at 24 months at all

storage temperatures.

Seed Storage Reserves in M. hortensiaQuantification of Seed Storage Reserves

5µM

McGill et al. (2002)

TEM section of a M. hortensia embryo (Magnification: 2300x)

Seed Storage Reserves in M. hortensia

Seed Storage Reserves (% seed dry weight)

Seed Lot Oil Content (± SE)

(%)

Protein (± SE)

(%)

Chatham Island 38.5 (±0.46) 21.6 (±0.06)

Egmont 41.0 (±0.45) 23.5 (±0.98)

Seed Storage Reserves in M. hortensiaDifferential Scanning Calorimetry (DSC) identified two lipid melting peaks and an ice

melting peak at 100%RH.

30% relative humidity warming cycle 100% relative humidity warming cycle

DSC indicates that lipid 2 starts melting at -30oC and finishes at around 10oC. It is in half fluid-half crystalline phase at -20 to 5oC. The crystalline phase of lipid may be

responsible for the decline in germination

Ice melting

-100 -80 -60 -40 -20 0 20 40 50

Temperature (oC)

210

205

200

195

190

185

180

-100 -80 -60 -40 -20 0 20 40 50

Temperature (oC)

Heat

Flo

w E

ndo

Up (m

W)

Heat

Flo

w E

ndo

Up (m

W) 400

300

250

Lipid 1 melting

Lipid melting

Lipid 2 melting-20.29°C

-60.56°C

Dormancy in M. hortensia seed

McGill et al. (2000) reported that M. hortensia seed has coat-imposed dormancy. It is not clear if this dormancy is a function of:

• restriction of gas exchange across the seed coat

• physical constraint of embryo growth by the seed coat or

• a combination of both

Dormancy in M. hortensia seedSeed Coat Structure

100µM 40µM

Scanning electron microscopy images of the outer seed coat surface showing the ridging on the coat surface and the lack spaces or fissures in the coat.

Dormancy in M. hortensia seedSeed Coat Structure

10µM

40µM

Scanning electron microscopy images of the seed coat showing the vascular-like elements with secondary thickening.

Dormancy in M. hortensia seedSeed Coat Structure

40µM

10µM

EN

Seed Coat

Light microscopy transverse section through the seed coat stained with safranin and fast green showing cherry-red staining characteristic of lignin (magnification 1000x).

Dormancy in M. hortensia seed

Oxygen uptake was measured in intact seed and seed that had beenmanipulated in one of three ways

• coat removed by hand after an incision was made in the seed coat (cotyledon area) with a scalpel - decoated seed

• coat pierced with a 0.1-0.2 mm needle in the centre (cotyledon area) of the seed

• coat pierced with a 0.1-0.2 mm needle and then Vaseline® applied to the pierced area.

Dormancy in M. hortensia seedThe rate of oxygen uptake in seeds after application of dormancy-breaking techniques (bars indicate the least significant difference between mean oxygen uptake at each time (P ≤ 0.05))

Time (hours)2 24 48 72

Oxy

gen

upta

ke (µ

l see

d-1 h

our-1

)

0

5

10

15

20

25

Intact (Control)Decoated PiercedPierced with Vaseline

Dormancy in M. hortensia seed

Time (days)0 7 14 21 28 35 42 49 56 63

Radi

cle

Gro

wth

(%)

0

20

40

60

80

100

Intact (Control)Decoated Decoated with seed coat replaced Pierced Pierced with Vaseline

Cumulative radicle growth (%) after application of dormancy-breaking techniques (bars indicate the least significant difference between mean radicle growth for each day (P ≤ 0.05))

Myosotidium hortensia

Summary

Storage potential

• appears to store poorly, even at -20°C and 4.5% moisture

• lipids with a melting temperature above 0°C are not the cause of the short storage life

Where to next?

• is instability of lipid 2 the cause of the short storage life?

• would storage at -196°C increase storage life?

Myosotidium hortensiaSummarySeed dormancy

• seed coat structure suggests that physical constraint of embryo growth may be a cause of dormancy

• seed coat does not appear to be limiting oxygen for germination

• physical constraint of embryo growth is a likely a cause of dormancy

Where to next?

• why is there a higher rate of oxygen uptake in decoated seed?

• role of phenolic compounds?

AcknowledgementsIn closing we wish to acknowledge and thank:

• Massey University Agricultural Research Foundation• The Nutrition Laboratory, Institute Food, Nutrition and Human Health,

Massey University• Doug Hopcroft, Manawatu Microscopy and Imaging Centre, Massey

University • Institute of Natural Resources, Massey University• Massey University Doctoral Scholarships• New Zealand International Doctoral Research Scholarships• Millennium Seed Bank, Wakehurst Place, Royal Botanic Gardens, Kew• Korea Seed & Variety Service