Hong, ToO., Ellis, R.H., Astley, D., Pinnegar. A.E.. Groot, S.P.e. and Kraak. H.L. (2005), Sud Sci. & Technol., 33, 449-460

Survival and vigour of ultra-dry seeds after ten years of hermetic storage

T.D. HONG!, R.H. ELLIS\*, D. ASTLEY2. A.E. PINNEGAR2, S.P.e. GROOT3 ANO H.L. KRAAK l

IDepartment of Agriculture, The Universily of Reading, Earley Gate, P.O. Box 237, Reading RG6 6AR, UK (E-mail: r.h.cllis@reading.ac.uk)

2Genetic Re~ources Unit. Horticulture Research Intemational, Wellesbourne, Warwick. eV35 9EF, UK lPlant Research Intemational, Droevendaalsesteeg 1, P.O. Box 16,6700 AA Wageningen. TIte Netherlands

(Accepred June 2004)

Surnrnary

Seeds of carrot, groundnut, IetlUce, oilseed rape and onion were stored hermelically in laminated aluminium foil packets in four environments (dry or ultra-dry moisture contents combined factoriaJJy wiLh temperaturcs of 200 e or _20°C), replicaled at scveral sites. Afler ten years' hermetic storage, seed moisLUre content, equilibrium relative humidity, viability (assessed by ability to germinare nonnaJly in standard gennination tests) and vigour were determined. Afier a decade, rhe change in seed moisture content of samples stored at _20°C was small or ni!. Exeept for groundnut and lelluce (where loss in viability was about 8 and 3%, respeetively), no loss in viability was deleeted after lO years' hennetic storage al _20°e. In all cases, there was no difference in seed survival between moisture conteuts at this temperature (P> 0.25). Comparison of seed vigour (root lengLh aud rate of germination) al so confirmed that drying (O moisture contents ,in equilibriurn with 10-12% r.h. had no detrimental effect to longevity when stored al _20°C; rhe only significant (P< 0.05) differences detected were slightly greater roollengths for uHra-dry storage of four of Ihe six. seed Jots. Seed moisture content had inereased afrer a decade at 200e (general1y lo the level in equilibrium wiLh ambienL reJative humidity). Hence, sub-zero temperature storage helped m<lintain the long-rerrn integrity of the laminated aiuminium foil packets. as well as rhat of ¡he seeds wilhin.

Introduction

There ls considerable practical and lheoretical value in determining as precisely as possible the extent to which seed moisture content can be reduced by desiccation yet still ¡ncrease subsequent longevlty in alr-dry storage. Different approaches to this subject have been developed in lhe UK (Ellis, Hong and Roberts, 1988) and lhe USA (Vertucci and Roos, 1990).

The tenn "ultra-dry" seed storage was inlroduced by the International Board for Plant Genetic Resources (IBPGR, 1992), now lhe International Plant Genetic Resources Institute (IPGRI), for seed banks conserving plant genetic resources, in response to results which quantified the response of seed longevity to a very wide range of seed storage

"" Author for correspondence

449

T,D, HONG. R.H. ElUS, D. ASlLEY, A.E. PINNEGAR, S.P.e. GROOT ANO H-L. KRAAK

moisture contents. The results of four years of hennetic storage of sesame (Sesamum indicum L.) seeds al 50°C wilh 2.0% moisture content showed lhat a surprisingly large proportion of seeds (38%) remained able lo germinale nonnany al lhe end of lhis period while reduction in seed storage moisture content from 5 lo 2% increased longevity 40-fold (Ellis el al., 1986). The considerable survival of seeds at very low moisture content from that study led to further investigations in more than 25 other crop species of survival in hermetic storage at low and very low moisture conlents. as low as 1.0% in several species, at 65°C (Enis el al., 1988, 1989, 1992). Those investigations showed that there was a low-moisture-content limit (mc) to the negative logarithmic relation between seed storage moisture content and longevity within the seed viability equation (Ellis and Roberts, 1980), such lhat below m, seed longevity no longer increased wilh further reduction in moisture content (Enis el al., 1992 and references lherein). Despite considerable variation in the estimates of mc among contrasting species (from 2.0 to 6.0% moislure conlent). mc was in equilibrium with about 10- t 20/(, relative humidity at 20°C (EHis el al.. 1992 and references lherein), where 20°C was the temperalure al which seeds were first dried before subsequent hermetic storage. From that early research. it was therefore proposed that in order to maximize the longevity of orthodox seeds in genebanks in those circumstances where refrigeration lo -] goC couId not be provided, seeds should tirst be dried al 20°C 10 moislure conlents in equilibrium with about 10-12% Lh. and lhen be stored hermetical1y at ambient, or where possible, eooler temperatures (Ellis el al., 1989). Apart from lhese results in experimental hennetic storage over 9 monlhs to 4 years at 50-65°C, results at eooler temperatures over longer periods have supported this approach (Ellis el al., 1991, 1993, 1995, 1996; Steiner and Ruckenbauer, 1995).

These suggestions have, however, been eriticised. Investigations based on a combination of thermodynamic principIes and measurements of seed vigour following short-term storage (several months) at 35°C led to reports that (i) moisture contents in equilibrium with 19-27% r.h. are optimal for seed longevily and (ii) drying seeds to moisture eontents in equilibrium with 10-12% r.h. would be detrimental to longevity, particularly if slored at cool lemperalures (Vertucci and Roos, 1990, 1991, 1993, 1994). Subsequently, fonowing results of seed viabitity and vigour during 1000 days' hennetic slorage, lhe optimum storage relative humidily suggesled was reduced from 19-27% lo aboul 15-20% depending upon slorage lemperature (Walters-Vertucci el al., 1996; Walters, 1998; Walters el al., 1998).

While lhe difference belween Ihe Iwo groups' suggestions have reduced, see Walters (1998) and Ellis (1998) for recenI summaries, the dilernma faced by genebank managers as lo whal lhey should do is considerable (Smilh, 1992). Moreover, the real world would appreciate results over longer periods of experimental storage than is possibIe from the comparatively short periods that are possible with short-tenn research granls. In this paper we report the resuIts of a ten-year programme of collaborative research in which "ultra­dry" seed slorage has been evalualed. This updates an eartier report (Enis el al.. 1996) on results after five years' storage.

450

ULTRA-ORY SEED S1DRAGE

Materials and methods

Seeds of six 101s of five crops - carrot (Daucus carota L.), groundnut (Arachis hypogaea L.), lettuce (Lactuca saliva L.), oilseed rape (Brassica napus L.) and oníon (Allium cepa L.) - were stored hermetically at each of Reading (Seed Science Laboratory, University of Reading), Wellesbouroe (HRI, Horticullure Research Inleroational) and Wageningen (onion and letluce only. Planl Research Interoational) beginning in March 1990. The four hermetic storage environments for each seed lot comprised a factorial combination of two moisture contents (table 1) and two temperatures, 20°C and _20°C. The two moisture contents comprised ''ultra-dry'' (2.0 - 3.7% moisture content, dried to equilibrium wilh 9.8 - 10.6% r.h. al 20°C) and a more conventional low moislure conlenl trealmenl (5.5 - 6.8% moisture conlent, dried lo equilibrium with 19.1 - 54.6% Lh. at 20°C). The wanner temperature of storage provided a value close lo laboratory ambient values, the cooler is that recommended for seed storage in genebanks. These four storage environments were duplicated (carrot, groundnut, oilseed rape) at Reading and Wellesbouroe or lriplicaled (lettuce and onion) at Reading, Wageningen and Wellesboume. Drying, packaging, storing, sampling and germinalion testing procedures and results after 5 years of hermetic slerage were published by Ellis el al. (1996).

Table 1. Change in seed moislure conlent (%. w.b.) alld equilibrium relative humidity (%. in brackets, detennined al 20°C) rOT seed~ stored over a decade in sealed laminared aluminium foil packets at 2WC and -20"C.

Crop Initial moislllre conlent Final moisture contenl

200C -20"C

3.6 (10.6) 6.7 (42.5) 4.2 (\8.0) Carrot

5.6 (2J.2) 6.8 (42.7) 5.9 (24.8)

2.0 (10.5) 2.9 (20.7) 2.0 (10.5) GroundnUl

5.5 (42.5) 46 (4S.t) 48 (46.t)

2.6 (9.8) 5.6 (42.8) J.6 (22.4) Letruce

5.8 (J4.3) 6.0 (43.2) 6.3 (45.3)

2.8 (10.5) 51 (41.6) JO IIJ.O)Oílseed rape

6.1 (54.6) 5.4 (43.1) 5.9 (45.5)

J.6 (9.8) 7.3 (406) J.7 (12.6) Onion A

6.0 (19.1) 7.6 (44.2) 6.3 (26.5)

3.7 19.8) 7.7 (43.0) J.6(114)Onion B

6.8 (27.5) 8.0 (44.8) 6.3 (24.5)

In March 2000, i.e. after len years of hermetic slerage, lwo sample packets fram each treatment combinatíon were removed from storage at each of Wageningen and Welles­boume. Qne sample was tested for gennination at eíLher Wageningen or Wellesboume and lbe duplicate sample returned by post for testing al Reading. Receipl al Reading was no laler lban three days after dispatch. The samples stered al Reading were lested only al Reading. Excepl for graundnut (where 5 g sample was used), four packels of seeds (about 1-2 g totally) stored in the same environment were used foc Lhe determination of moisture conlent and equilibrium relative humidily as described previously (Ellis el al.. 1996).

451

T.D. HONG. RoH. ELLlS. D. AS1l...EY, A.E PtNNEGAR, S.P.e. GROar AND A.L. KRAAK

Immediately before the gennination tests, aH seeds were humidified aboye water at 20°C for 24 h for carrol, lelluce, oilseed rape and onion, or 48 h for groundnut in order to avoid imbibition damage (Ellis el al., 1985). Three hundred seeds (six replicates of 50 seeds) were tested for gennination on top of moistened Whatman filter papers (Grade 181) in 90 mm Petri dishes (Reading and Wellesbourne), or in plastic boxes 15 x 20.5 x 2.5 cm (onion, Wageningen), or on a Copenhagen Table (lettuce, Wageningen), except for groundnut seeds which were tested between moist roUed paper towels with five or six replicares of 40 seeds. Seeds of carrot, groundnut and oilseed rape were lesled at 20/30°C (16/8 h), while those of lettuce and onion at 15°C and 20°C, respectively, for 14 d, except 10 d for oilseed rape. Germination was evaluated for normal seedling development according to the criterion of normal germination as defined by the [STA (2004). Given that seedling evaluation is subjective, total germination (i.e. normal plus abnormal seedlings) was also used to compare among storage environments. storage sites and germination sites.

Two hundred seeds (four replicales of 50) for carrot, lettuce, onion and oilseed rape, or 120 seeds (six replicates of 20) for groundnut were used to determine rate of germination (only at Reading). After humidification as described previously, seeds were tested for germination at 20°C, except 15°C for lettuce, in 90 mm Petri dishes for carrot, lettuce, onion and oilseed rape, or in moistened rolled paper towels for groundnut. In each case, lhe progress of germination was monitored at frequent intervals (e.g. every 2-3 hours between 16 and 48 h in test and every 6 hours after 48 h) until they had gerrninated fully. In this test. germination was defined as radicIe protrusion (>0.5mm for carrot, lettuce, onion and oiIseed rape and > l mm for groundnut). These short lengths were used to ensure the time of first visible germination was recorded. The rate of germination (R, do') was estimaled as R = I,n/I,(d*n) where n is number of seeds germinating on day d, where day d is calculated to two decimal places.

One hundred seeds (four replicates of 25) from each storage environment for onion, lettuce, carrot and oilseed rape, or 120 seeds (six replica tes of 20) for each storage environment for groundnut, were subjected to a slant growth test to determine root length (only at Reading). After humidification as described previously, 20 (groundnut) or 25 seeds were placed between moist paper towels and then gently roUed (i.e. as in the rolled towel test for viability). Four rolls were placed within an unsealed polyethylene bago These were supported at an angle of 70° from the horizontal in an incubator maintained al 15°C (lettuce) or 20°C (olher species). The roots of seedlings developing norrnally were measured after 96 hours for lettuce and oilseed rape, 144 hours for groundnut, or 240 hours for carrot and onion.

Resulls

Seed moülure conlent and equilibrium relative humidity Except for groundnut where seed moisture content increased from 2.0% (in equilibrium with 10.5% r.h.) in 1990 to 2.9% (in equilibrium wilh 20.7%) in 2001, seed moisture content of samples stored ultra-dry (especially) at 20°C increased considerably after ten

452

ULTRA-DRY SEED SroRAGE

year, hermetic slorage, sueh lhat lhe dry and (originally) ullra-dry seeds attained almosl the same moísture contents within each species and almost the same equílibrium relative humidity, between 40 and 46% (table 1). In contrast, seed moisture content in storage at -20oe remalned unchanged for groundnut and both seed 10ts of onion, although small increases occurred in carrol and lettuce (0.6 and 1.0%, respectively). Consequently, except in carrot and lettuce where the equilibrium relalive humidity of ultra-dry seeds increased from about 10% lo about 18.0 and 22.0%, respeelively, the equilibrium relative humidity 01' ullra-dry seeds remained unehanged (10-13%) over a deeade's hermetie slorage al -20°C (table 1).

Seed sun'ival No differenee (P> 0.50) in either normal or total germination was deteeled (analysis 01' variance after angular transformatíon) between dry and ultra-dry storage after ten years at -20°C (figures Id. h, 1; 21', 1, r). Comparison among lols, siles and storage trealment (figures I and 2) revealed differences (P< 0.001) among the six seed lols for both normal and lolal germinalion, nol surprisingly, but no significant effeel (P> 0.10) 01' lest sile and no significant interactions among test site, seed lot and moísture contenl (P> 0.50).

Given that analysis of results after ten years' slorage had revealed no dífferences belween results for dry and ullra-dry storage, ehanges in viabilily lhroughoul lhe ten years were lnvestigated by combining current results (figures 1 and 2) with those for the firsl fi ve years 01' storage (ElIis el al., 1996). Observations were subjected lo probit analysis using GENSTAT (Genstat 5, 1997) in aeeordanee with the equation v = K,- plcr where vis probil pereentage viability (assessed by abilily lo germinale normally) after p days in storage, K, is the seed lot constant and (J is the standard deviation of the frequency distribution 01' seed dealhs in time (ElIis and Roberts, 1980) and the effeel 01' the lwo moisture contents thereon determined. No 10ss in viability was detected (P> 0.25) during len year, storage al -20°C 01' oilseed rape (figure 3 a), carral (figure 3 e) and either onion seed 10t (figure 3 e, f) al either moisture content. In groundnut and lettuce the declines in viability were slightly greater, sorne 3-8% over the ten-year perlod and significant (P< 0.05). No differences (P> 0.25) in cr at -20"C belween dry and ultra-dry storage were deteeted, however (figure 3 b, d; lable 2).

Survival afler a deeade at 20°C was grealer (P< 0.001) for seeds slored inilially ultra­dry rather lhan dry (dala not shown) in groundnut, oilseed rape, lettuee and onian A and slightly greater (but nol signifieant. P> 0.10) in carral and onion B. This eontinued lhe lrends over time reported after five years' storage (ElIis el al., 1996). Hawever, given the increase in seed moisture content towards the end of the slorage períod, the results at 20°C are not discussed further.

Seed vigour Rate of germination after ten years' hermetic storage at -20oe did not differ between the two moisture eontenls in any 01' lhe seed lots (P> 0.25) (figure 4). Following hermetie storage at _20°C for len years, ultra-dry seeds of oilseed rape, lettuce and onion produced longer roots than dry seeds (P< 0.05) (figure 5 e-f), whereas in the other two seed lots no signifieant differenee was deteeled (P> 0.25).

453

T.D. HONG, R.H. ELLlS. D. ASTI...EY. A.E. PINNEGAR. S.P.e. GROar AND H.L. KRAAK

RJR VGB/R VGBNGB Mean

d 1

aHJO b e 1 11

95 90

"" 70

Carral50 "" 4il 30 20

10 nno

5.6 3.6 5.6 3.6 5.6 3.6 5.6 3.6 5.9 4.2 5.9 4.2 5.9 4.2 5.9 4.2

~

~ " .~ Groundnut

.~ " tJ

o 5.5 2.0 5.5 2.0 5.5 2.0 5.5 2.0 4.8 2.0 4.8 2.0 4.8 2.0 4.8 2.0

HJO

95 90 80 70 Oilseed60 50 rape40 30 20 10

o

6.1 2.8 6.1 2.8 6.1 2.8 6.1 2.8 5.9 3.0 5.9 3.0 5.9 3.0 5.9 3.0

Moisture content (%, w.b.)

Figure l. Comparison of pereentage (angular-transfonned seale) nonnal gennination (solid eolumns) and total germinalioll (eombined height of solid and open eolumns) of earrot (a, b, e, d), groundnut (e, f, g, h) and oil seed rape (i, j, l, 1) seeds stored hermetieally fOr ten years at -20°C at two moisture eonteOls (upper values detennined when storage began in 1990, lower values after storage). Seeds were slored at Reading (R) (a, e, i) or Wellesboume (VGB) (b, e, f, g, j, l) from Mareh 1990, until tested for gennination at Reading (IR) (a, b, e, f, i, j) or Wellesboume (NGB) (e, g, l) in Mareh 2000. Mean germination for eaeh storage moisture eontent eombining storage and gennination sites is shown for earrol (d), groundnut (h) and oilseed rape (1). The vertical bars represent the least signifieant differenee (l.s.d) (P= 0.05) for eomparison of total gennination between moisture eontents.

454

ULTRA-DRY SEED STORAGE

RIR VGB/R VGBNGB NUR NUNL Mean

so 70 60 Onion A 50 40 30 20

10

o

6.0 3.6 ,O 3.6 6.0 3.6 6.0 3.6 6.0 3.6 6.0 3.6 6J 3.7 6.3 37 6.3 3.7 6.3 3.7 6.3 3.7 6.3 3.7

6.~ 3.7 '.8 3.7 6.8 3.7 6.8 3.7 6.8 3.7 6.8 3.7 6.3 3.6 6.3 3.6 6.3 3.6 6.3 3.6 6.3 3.6 6,3 3.6

100~ h k

~ 1 1 1 1

95 90

e soo 70 .~ 60 OnionB e 60

.~ 40 30

<U 20

" 10

o

Lettuce

"'" 95 90 so 70 60 60 40 30 20 10

5.8 2.6 5.8 :2.6 5.8 2.6 5.8 2.6 5.8 2.6 5.8 2.6 6.3 3.6 6.3 3.6 6.3 3.6 6..1 3.6 6.3 3.6 6.3 ].6

Moisture content (% . w.b.)

Figure 2. Comparison of percentage (angular-transformed sca\e) normal germination (solid columns) and total germination \combined height of solid and open columns) of onion lot A (a - f). onion 10t B (g - 1) and lettnce (m - r) seeds stored hermetically for ten years at -20°C at two moisrure eontents (upper values determincd when swrage began in 1990, lowee values aftee storage). Seeds weee stored al Reading (R) (a. g, m) oc Wellesbourne (VGB) (b, c. h, i, n, o) or Wageningen (NL) (d, e, j, l, p, q) feom March 1990, until tested for germinatíon al Reading (IR) (a, b, d, g, h, j, m, n, p) or Wellesboume (IVGB) (e, l, o) or Wageningen (INL) (e, l, q) in March 2000. Mean gennination for eaeh storage moistuee content eombining storage and germination sites is shown for onion lol A (f), onion lar B (1) and letlllee (e). The verlical bars represent the least signiticant differenee (I.s.d) (P= 0.05) for eOlllpanson of total gennination between moisture contenls.

455

T.D. HONG, R.H ELLIS, D. ASTLEY, A.E. PINNEGAR, S.Pe. GROOT ANO R.L. KRAAK

90

80

70

e Carrat100

~ e 90 o .~

e .~ 80 <.:) O

70

e Dnjon (A)100

a Oilseed rape

80 L

70

o

d Lettuce

Dnjon (B)

~~~ CJ·=·=·=·=·=·~·~·~·9

o 1 2 3 4 5 6 7 8 9 10 O 2 3 4 5 6 7 8 9 10

Duration of storage (years) Figure 3. The effecl of hermetic slOrage al -20°C on lhe survival (a!>ses!>ed by abilily lo germinate nonnally) of seeds of oil!>ccd rapc (a), groundnut (b), carrol (c), leuuce (d) and onion (e, f) al Iwo moi~ture contents (Iable 1); dry storage, i.e. as reeommended by IBPGR (1976) and FAO/IPGRl (1994) (0), or ultra-dry storage, Lc. dried lo equilibrium with aboul 10-12% Lh. al 20°C beforc hermelic !>lorage (O). Eaeh observalion shown is the mean of test results al aH eombinalions of storage and test sites determined for a total of 600 groundnut, 900 carrol or oilseed rape, 1,200 onion, or 1,500 lertuce seeds. The survival curves for seeds slored dry and ultra­dry al _20°C (-) were filted lo these observations by probit analysis; Ihe fined values of K, and (J are shown in table 2. Observations for up lo five years' slorage were published previously (Ellis el 01., 1996). Predicted survival curves for seeds stored dry (- - -) and ultra dry (....) al _20°C lar groundnut (b). letluce (d) and onion (e. f) are estimated from the viability eonstants of these species provided by Usberti and Gomes (1998), Kraak and Vos (1987) and ElIis and Roberts (1981), respeclively.

456

ULTRA·DRY SEED STQRAGE

Carrol Groundnut Oilseed rape Onioo A OnionB Lettuce

1.0 a b e d e f

"':l 0.8~

B ~ '" 0.6e o .~

0.4e.§ ~ 0.2... " 0.0

5.6 3.6 5.5 2.0 6.1 2.8 6.0 3.6 5.8 2.6 5.9 4.2 4.8 2.0 5.9 3.0 6.3 3.7 6.3 3.6

Moisture content (%, w.b.)

Figure 4. Rate of seed gennination (d· l ) when tested after len years of hermelic slorage at -20°C with two moisture eonlents (shown) for earrot (a), groundnut (b), oilseed rape (e), onion lol A (d), onion lot B (e) and lelluee (f). The upper values of moisture conlent were detennined in 1990 when storage llegan, lower values at the end of storage. The vertical bars represenl means ± s.e.

Table 2. Estimates of K, and cr provided by probit analysis from observations of seed survival (percentage normal germination) of s;x lots stored hermeliealIy at two moisture eontent.~ for ten years al -20"'e (see figure 3).

6.8 3.7 6.3 3.6

Equilibrium relalive Moislure humidity (%) content (%)

Species at 200 e delermined delermined K, (s.e.)* l/o o, d initial1y initially (s.e.) x 104

(final) (final)

Carrol 23.2 (24.8) 5.6 (5.9) 0.791 (0.013) .f 10.6 (18.0) 3.6 (4.2) P'> 0.25

GroundnUl 42.5 (46.1) 5.5 (4.8) 1.674 (0.027) 1.219 (0.164) 8.203 10.5 (10.5) 2.0 (2.0) P'> 0.10 1.219 (0.164) 8,203

Lettuce 34.3 (45.3) 5.8 (6.3) 1.906 (0.019) 1.271 (0.116) 7,868 9.8 (22.4) 2.6 (3.6) P'> 0.25 1.271 (0.116) 7,868

Oilseed rape 54.6 (45.5) 6.1 (5.9) 2.399 (0.068) .f 10.5 (13.0) 2.8 (3.0) P> 0.25

Onion A 19.1 (26.5) 6.0 (6.3) 1.291 (0.016) .f 9.8 (12.6) 36 (3.7) P'> 0.25

B 27.5 (24.5) 6.8 (6.3) 0.678 (0.0231 .f 9.8 (11.4) 3.7 (3.6) P'> 0.25

... Probabilities shown are for (he effect of constraining both seed survival curves for Ihat lot to a common origino ;; No estimate for 1/0' and O' provided as no significant loss in viabillly was detected (h 0.25) al either moisture conlenl al -20°e.

457

T.D. HONG, RH ELLlS, D. ASTLEY, A.E. PINNEGAR, S.P.e. GRoar AND H.L. KRAAK

Carrot Groundnut Oilseed rape Onion A Onion B Lettuee 100

E S .,s 00 c

.2

8 ¡>::

a bl­ e d e f 80 ¡­ ¡­ ¡­ 1­

1­

60 1­ 1­ 1­ 1­1­40 1­ 1­ 1­ 1­¡­

20 1­ ¡­ ¡­ 1­ 1-. lLLO~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~

5.6 3.6 5.5 2.0 6.1 2.8 6.0 3.6 6.8 3.7 5.8 2.6 5.9 4.2 4.8 2.0 5.9 3.0 6.3 3.7 6.3 3.6 6.3 3.6

Moisture eontent (%, w.b.)

Figure 5. Seedling root length (mm) when tested l'ollowing ten years ol' hermetic seed sloragc at -20°C with two moisture contents for carrol (a), groundnut (b), oilseed rape (e), onion lot A (d), onion 1m B te) and lettuce (f). The upper values of moisture conten( were determined in J 990 when srorage began, the lower value.~ at the end ol' storage. The vertical bars represenl means ± s.e.

Discussion

Hermetic storage at -20°C with dry and ultra-dry seeds for ten years resulted in no detectable loss in viability for oilseed rape, earrot and both seed lots of onion (figure 3a. e, e, f), but about 3-8% loss in viability was deteeted in lenuee and groundnut (figure 3b, d). In the latter cases, there was no difference in loss in viability between dry and ultra-dry seed slorage at -20°C. Neither were lhe vigour tests 'able to distinguish consistently between moisture contents in seed deterioration after 10 years at -20°C: while the results for root length showed a significant advantage of ultra-dry storage in oilseed rape, 1ettuce and onion the differences were small (figure 5) and rate of gennination showed no differences between moisture contents (figure 4). The question we posed at the beginning of the ] 990' s was whether or not reduction in seed storage rnoisture content from lhe value (5 ± 1%) reeommended by the IBPGR (now lPORI) for genebanks to values in equilibrium with about 10% r.h. al 20Q C ("ullra dry") was daruaging (Vertueei and Roos, 1990) or advanlageous (Ellis el al., 1986, 1989, 1992, 1995, 1996) to seed surviva1 at ambient and coo)er ternperatures.

The current results for 10 years' storage, together with those reported elsewhere for survival after storage for 5 years (Ellis el al., 1996),16 years (Bass and Stanwood, 1978), 24-25 years (Ellis el al., 1993) and 110 years (Steiner and Ruekenbauer, 1995) show that ultra-dry storage cornpared with conventional dry storage at ambient temperatures is advantageous, while for storage at -20°C there is evidence of neither an advantage nor a disadvantage to seed survival of uLtra-dry storage compared with conventional dry storage. The results confirm that survival at -20°C is considerable across a wide range of (ultra dry lo dry) moisture eontenlS. This should be most reassuring lo managers of genebanks operating long-tenn seed stores at -20°C, although in groundnut and lettuce

458

ULTRA·DRY SEED STORAGE

(bUI oot ooioo) viabilily afler 10 years was sligbtly lower lhao would have beeo predicted (figure 3). Less reassuriog is lhe failure of lamioated alumioium foil packels after a decade at ambient temperatures: as with the seeds, the integrity of packets stored at sub­zero temperatures was much beHer.

Acknowledgements

The work at Wageoiogeo was supported io pan by lhe Dulch Mioistry of Agricultore. Nature Maoagemeot aod Fisheries (DWK388).

References

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