United States

Department of

Agriculture

Forest Service

Southern Forest

Experiment Station

New Orleans,

Louisiana

General Technical Report

so-107

September 1994

Tree Seed Technology

Training Course

F. T. Bonner, J. A. Vozzo, W. W. Elam, and S. B. Land, Jr.
SUMMARY

Th is m anual is intended prim arily to train seed colle ctors, seed-plant m anage rs, seed

analysts, and nurse ry m anage rs, but it can se rve as a resource for any training course in forest

re gene ration. It includes both te m pe rate and tropical tree spe cies of all intended uses. Th e

m anual cove rs th e follow ing topics: seed b iology, seed colle ction, seed h andling, seed-quality

evaluation, seed prote ction, seed basics for nurseries, and seed program s. It also includes

practical e xe rcises.
‘Ik e e Se e d Te ch nology W aining Course

Stude nt Outline

F. T. Banne r, J. A. Voz z o, W . W . Elam , and S. B. Land, Jr.

F.T. Bonne r and J.A. Voz z o are supervisory plant ph ysiologist and research plant ph ysiologist, respective ly, U.S. De partmen t of Agriculture,

Forest Se rvice, South e rn Forest Expe rimen t Station, Stark ville, MS 39 759 ; W .W . Elam and S.B. Land, Jr., are professors of forestry, Mississippi

State Unive rsity, Mississippi State, MS 39 762. In coope ration w ith : Forest Tree Seed Program , De partmen t of Forestry, Mississippi Agricultural

and Forest Expe rimen t Station, and Mississippi State Unive rsity.

. . . 111

CONTENTS

Introduction .................................................................. .

Goalsofth e Course .................................................. .

Scope ............................................................... .

Sources of Inform ation and Te ch nical H e lp. .............................

Biology ....................................................................... .

I. Flow e ring, Pollination, and Seed Maturation. ...... : .....................

II. SeedDorm ancy ...................................................... .

III. Ge rm ination ......................................................... .

Colle ction .................................................................... .

I. Genetics and Seed Source. .............................................

II. Production .. ........................................................ .

III. Colle ction Operations. .................................................

. M atu rity ............................................................ .

. Posth arvest Care .................................................... .

H andling..................................................................... .

I. Drying and Extracting ............................................... .

II. Cle aningandupgrading .............................................. .

III. Storage Principles .................................................... .

IV Storage Applications ................................................. .

E va lu a ti n g Qua li ty............................................................ .

I.

II.

III.

IV

V.

VI.

VII.

Sam pling ............................................................ .

M oisture Content.................................................... .

Purity and W eigh t ................................................... .

Ge rm ination Tests.. ................................................. .

Rapid Tests: Cutting, Vital Stains,

Excised Em b ryo, and H ydrogen Pe roxide. ...............................

Rapid Tests: X Rays and Le ach ate Conductivity. .........................

Vigor Tests .......................................................... .

Prote ction .................................................................... .

I. Inse cts .............................................................. .

II. Path ogens ........................................................... .

Basics for Nurse ries ........................................................... .

I. Production Syste m s ...................................................

Seed Program s............................................................... .

I . NationalProgram s ................................................... .

II. Seed Cente rs ........................................................ .

III. Labeling and Ce rtification .............................................

. Ge rm plasm Conse rvation ..............................................

. Applied Research .................................................... .

Laboratory Exe rcises .......................................................... .

Lite rature Cited .............................................................. .

1

2

2

2

5

6

11

13

17

18

20

21

23

24

27

28

29

30

32

37

38

39

40

41

43

45

46

49

50

51

53

54

57

58

59

60

62

62

65

77

Introduction
GOALS OF TH E COUR SE

Th e obje ctive of th is course is to provide a basic

understanding of th e follow ing topics:

A.

B.

C.

D.

E.

F.

G.

H .

I.

J.

K.

L.

M .

N.

0.

;.

R.

Reproductive cycles, from flow e ring th rough seed

ge rm ination

Seed origin

Seed colle ction

Seed m aturity

Colle ction and posth arvest care

Seed extracting, cle aning, and conditioning

Insect and disease proble m s

Seed storage

Seedlot sam pling

Tests for m oisture, purity, w e igh t, ge rm ination,

and vigor

Rapid viab ility estim ates

Seed test results

Seed h andling in nurse ries

Seed program s

Seed labeling and ce rtification

Ge rm plasm conse rvation

Seed cente r design and staffing

Applied seed research

SCOPE

Th e e m ph asis in th is course w ill be on indigenous,

m ultipurpose tree spe cies suited for forestry and rural

agroforestry. H ow e ve r, e xotics are included, because

fast-grow ing e xotics h ave a de finite place in forestry

program s.

SOUR CES OF INFORM ATION

AND TECH NICAL H ELP

Sources of inform ation and te ch nical h e lp include

journals, re fe rence book s, inte rnational organiz ations,

re gional organiz ations, and research institutions.

Journals

Seed Science and Te ch nology

Inte rnational Seed Testing Association (ISTA)

Reckenh olz, P.O. Box 412, CH -8046

Z urich

Sw itz e rland

Journal of Seed Te ch nology

Association of Official Seed Analysts (AOSA)

Seed Abstracts

CAB Inte rnational Inform ation Se rvices

W alling-ton, Oxon OX 10 8 DE

UK

Agrofore stry Abstracts

(sam e as above )

New Fore sts

Dr. M ary Durye a, Editor-in-Ch ie f

Departm ent of Forestry

118 N-Z H all

Unive rsity of Florida

Gainesville, FL 32611

U.S.A.

Indian Fore ste r

(includes seed te ch nology articles1

Indian Journal of Fore stry

(includes seed te ch nology articles)

Journal of Tropical Fore st Science

Business Manage r

Forest Research Institute of M alaysia

P.O. Box 201

Kepong, 52109 Kuala Lum pur

Malaysia

Com m onw e alth Fore stry Revie w

Com m onw e alth Forestry Institute

11 Keble Road

Oxford

UK

Pak istan Journal of Fore stry

Editor

P.O. Pak istan Forestry Institute

Pesh aw ar, N.W .F.P.

Pak istan

Canadian Journal of Fore stry Research

Editor

Forestry Canada

P.O. Box 49 0

Sault Ste. Marie, Ontario

P6A 5M7 Canada

Fore st Science

Socie ty of Am e rican Foreste rs

5400 Grose venor Lane

Be th esda, MD 20814-219 8

U.S.A.

R e fe re nce Book s

Th e follow ing re fe rence book s provide inform ation and

te ch nical assistance:

Be w ley and Black 19 82

Ch in and Robe rts 19 80

Murray 19 84a, b

Sch opm eye r 19 74

von Carlow itz 19 86

W illan 19 85

Inte rnational Organizations

Food and Agriculture Organization (FAO) of th e

United Nations

Forest Resources Deve lopm ent Branch

Forest Resources Division

Via de lle Te rm e di Caracalla

I-00100 Rom e

Italy

2
Inte rnational Union of Fore stry Research Organizations (IUFRO)

Se cre tariat

Sch on b runn

A-1131 Vienna

Austria

IUFRO Seed Proble m s Proje ct Group

Current Ch air: Dr. D.G. Edw ards

Forestry Canada

Pacific Forestry Centre

506 W est Burnside Road

Victoria, BC

V8Z lM 5 Canada

In te rnational Seed Te s ti ng As s ociation

(ISTA)

ISTA Se cre tariat

Reck en h olz, P.O. Box 412

CH -8046 Z urich

Sw itz e rland

R e gional Organizations

F/FRED Coordinating Unit

c/o Kase tsart Unive rsity

Faculty of Forestry

P.O. Box 1038, Kase tsart Post Office

Bangk ok 109 03

Th ailand

Organization for Econom ic Coope ration and

Deve lopm e nt (OECD)

Dire ctorate for Agriculture and Food

Paris

France

Research Institute s

ASEAN-Canada Fore st Tree Seed Centre

M auk Le k , Saraburi

Th ailand

Nitrogen Fixing Tree Association

P.O. Box 680

W aim analo, H I 9 6734

U.S.A.

Inte rnational Council for Rese arch in Agrofore stry (ICRAF)

P.O. Box 30677

Nairobi

Keny a

DANIDA Fore st Seed Centre

Kroge rupve j 3A

DK 3050, H um lebae k

Denm ark

Fore st Research Centre

P.O. Box H G 59 5

H igh lands, H arare

Z im babw e

Centre National de Seme nce s Fore stie re s

PB 2682, Ouagadougo

Burk ina Faso

CSIRO Division of Fore st Research

P.O. Box 4008

Queen Victoria Te rrace

ACT 2600, Canbe rra

Australia

Com m onw e alth Fore stry Institute (CFI)

Unive rsity of Oxford

De partm ent of Forestry

Oxford

UK

USDA Fore st Se rvice

Tree Seed Research Unit

Forestry Sciences Laboratory

P.O. Box 9 06

Stark ville, M S 39 759

U.S.A.

USDA Fore st Se rvice

National Tree Seed Laboratory

Rt. 1, Box 182-B

Dry Branch , GA 31020

U.S.A.

USDA Fore st Se rvice

Institute of Tropical Forestry

Unive rsity of Puerto Rico, Agricultural Expe rim ent

Station

P.O. Box 25000

Rio Piedras, PR 009 28-2500

U.S.A.

Centro Agronom ic0 Tropical de Inve stigation y

Ensenanza (CATIE)

Turrialba

Costa Rica

Pe taw aw a National Fore stry Institute

Box 2000

Ch alk Rive r, Ontario

KOJ 1JO Canada

3

Biology
I.

6

Flow e ring, Pollination, and Se e d

M aturation

A. Introduction

Know ledge of th e seed b iology of a tree spe cies

is essential to successful seed production and

h andling. Th e sexual life cycle m ust be k now n

to plan for genetic im provemen t, production,

colle ction, conditioning, storage, and planting of

th e seeds.

B. Obje ctives

1.

2.

3.

4.

5.

Define com m on te rm s used to describe life

cycles of plants.

Describe th e gene ral sexual cycle, flow e r

structure, seed structure, and origin of th e

fruit of gym nospe rm s.

Describe th e gene ral sexual cycle, flow e r

structure, seed structure, and origin of th e

fruit of angiospe rm s.

Iden tify prim ary diffe re n ces be twee n

angiospe rm and gym nospe rm sexual cycles.

Describe th e gene ral deve lopm ent of fruits

and seeds.

C. Key Points

Th e follow ing points are essential for unde rstanding flow e ring, pollination, and seed m aturation :

1. A plant’s life cycle is th e tim e required to

grow from zygote to seed production; th e re

are tw o deve lopm ental cycles -a sexual

cycle and an asexual cycle.

2. Know ledge of th e sexual cycle is required

for:

a. tree-b reeding program s

b . seed orch ard m anagemen t

c. seed colle ction

d. seed conditioning and storage

e. nursery m anagemen t

3. Th e gym nospe rm life cycle follow s th is

ord e r:

it:

Ei.

e.

f.

“h :

i.

j.

k .

1.

m .

nak ed seed

seedling

m ature sporoph yte

strob ili (cones)

m icrospore and m e gaspore m oth e r ce lls

m eiosis

m icrospores and m e gaspores

m ale and fe m ale gam e toph ytes

pollination

single fe rtiliz ation

zygote and gam e toph ytic tissue

emb ryo

nak ed seed on ovulate cone scale

4. Th e angiospe rm life cycle diffe rs from th e

gym nospe rm life cycle in h aving:

a. seeds enclosed in fruit (ripened ovary)

b . true flow e rs rath e r th an strobili

c. double fe rtiliz ation

d. triploid endospe rm tissue rath e r th an

h aploid fe m ale gam e toph ytic tissue in

th e seed

D. De finition of Te rm s

1.

2.

3.

4.

5.

6.

7.

8.

9 .

10‘.

11.

12.

Fruit -a ripened ovary, som e tim es including accessory flow e r parts, th at surrounds th e seed in angiospe rm s.

Seed-a ripened ovule th at consists of an

emb ryo, its stored food supply, and prote ctive cove rings.

M ature seed - a seed th at can be re m oved

from th e tree w ith out im pairing th e seed’s

ge rm ination.

E. Life Cycles

Life cycle -th e tim e required to progress

from zygote to seed production.

Genotype- th e genetic m ak eup of a ce ll

nucleus or an individual.

Ph e notype -th e e xte rnal appe arance of

an organism .

M itosis-nucle ar (and usually ce llular) ce ll

division in w h ich th e ch rom osom es duplicate and divide to produce tw o nuclei th at

are identical to th e original nucleus.

M e iosis - tw o successive nucle ar divisions

in w h ich th e ch rom osom e num be r is h alved

and genetic segre gation occurs.

Pollination-transfe r of pollen grains

from th e anth e r or m icrosporoph yll to th e

stigm a or ovule.

Fe rtiliz ation-fusion of spe rm and egg

(and also spe rm w ith tw o polar nuclei to

form endospe rm in angiospe rm s).

Diploid (2N) - tw o sets of ch rom osom es in

a ce ll nucleus.

H aploid (1N) -one set of ch rom osom es in

a ce ll nucleus.

An understanding of life cycles is needed

b e cause :

1. Se xual and asexual syste m s re produce

genetically diffe rent populations.

2. Know ledge of th e ase xual cycle is needed

be fore ve ge tative propagation can be used.

3. Know ledge of th e sexual cycle is needed for

successful tree b reeding and seed production .

F. Angiospe rm and Gym nospe rm Se xual Cycles

1. AI1 tree spe cies are seed-producing plants

(division, Spe rm atoph yta) and belong to

eith e r th e class Gym nospe rm ae or Angiospe rm ae.

a.

b .

Angiospe rm seeds are enclosed in carpe ls.

Gym nospe rm seeds are borne nak ed on

scales.
c. Seeds of nonconife rous gym nospe rm s

are borne singly.

2. Gym nospe rm life cycle (fig. 1)

a. Sporoph yte

b . Strobilus or cone, including:

(1)

(2)

(3)

(4)

re productive sh ort sh oot

stam inate cone (m ale )

ovulate cone (fe m ale )

gym nospe rm s m ay be eith e r m onoe cious (fe m ale and m ale strobili on

sam e tre e ) or dioe cious (tree h as

only one sex).

c. Meiosis and gam e toph ytes

d. Fe rtiliz ation

e. Seed (fig. 2)

(1) De ve lops from th e fe rtilized ovule.

(2) Contains an emb ryo (cotyledons,

h ypocotyl, radicle ), a seedcoat, storage tissue, and som e tim es a seed

w ing.

f. Fruit

(1) Gym nospe rm s do not h ave true

“fruits.”

(2) Gym nospe rm seeds are enclosed by

th e follow ing structures:

(a) dry ovulate cones (e.g., Abies,

WINTE R

Figure 1. -Life cycle of a gym nosperm (Pinus spp.) (Banner 19 9 1b)

7
SEEDCOAT

STORAGE TISSUE

(FEMALE GAMETOPHYTE)

COTYLEDONS

HYPOCOTYL

RADICLE

Figure Z .-Cross s ection of a typical m ature gym nosperm seed (Pinus ponde rosa) (ad apted from Krugm an and Jenkinson 19 74).

(b)

Cc)

Araucaria, Cupressus, Pinus,

and Tsuga)

flesh y, arillik e structures (e.g.,

Gink go, Tm u s, and lbbrreya)

be rrylik e ovulate cones (e.g.,

Juniperus)

3. An giospe rm life cycle

(1)

(2)

Sporoph yte

Flow e r -a sh ort sh oot w ith ste rile and

re productive le aves.

(1) Ste rile le aves include:

(a) sepals

(b) pe tals

(c) pe rianth

(2) Reproductive le aves include:

(3)

f. Fruits

(1) Deve lop from th e m atured ovary

(2) Enclose th e seed (m atured ovule )

(3) Are difficult to separate from th e

seeds

4. Se xual cycles- Th e gym n ospe rm an d

angiospe rm sexual cycles diffe r in four w ays:

C.

d.

e.

Deve lop from th e double-fe rtilized

ovules

Contain an emb ryo (cotyledons,

h ypocotyl, radicle ), storage tissue,

seed coat, and som e tim es oth e r seed

cove rings

M ay be end ospe rm ic or nonendospe rm ic

(a)_ stam en (m ale )

(b) carpe l (fe m ale )

(c) pistil, a colle ctive te rm th at

describes visible fe m ale structures

(3) Rece ptacle

(4) Th e re are pe rfe ct flow e rs, im pe rfe ct

flow e rs, and polygam ous flow e rs

M eiosis and gam e toph ytes

Fe rtiliz ation

Seeds

a.

b .

c.

d.

-In gym nospe rm s, seeds are not enclosed

in th e ovary, and flow e rs are unise xual;

in angiospe rm s, seeds are borne in a

closed ovary, and flow e rs are pe rfe ct or

im pe rfe ct.

Angiospe rm s h ave true flow e rs, but

gym nospe rm s h ave strob ili (cones).

Double fe rtiliz ation tak es place in

angiospe rm s; single fe rtiliz ation tak es

place in gym nospe rm s.

In gym nospe rm s, th e deve loping emb ryo
1; -.I- * * - ’ : ., s

r f-. :.-

+*i , ,

.

I ;. .

L. .. . .

..

.; -.-. .

. ’

‘?

”. ._..

-_...

.!.g_*?;, _ . . , , *: a.. _

** . _- .

-. .

_ _a.. .-___; , ‘t . ! . -_..-i:---

:‘._ * .-. . -.. .

MICROPYLE

bJICROPYLE

I

RAPHE

IilLUM

Figure 3. -Exte rnal m orph ology of a typical legum e seed of Sch iz olobium parah ybium (ad apted from 7k iuirio and oth ers 1990).

G. Seed

is nourish ed by th e h aploid fe m ale

gam e toph yte ; in angiospe rm s, it is nourish ed from eith e r diploid cotyledons,

h ypocotyl of th e e m b ryo, triploid endospe rm , or diploid nuce llar m ate rial.

and Fruit De ve lopm ent

1. Ph ysical de ve lopm e nt

a. Angiospe rm s

(1) Pollination and fe rtiliz ation trigge r:

(a) form ation of emb ryo and endospe rm

(b) ce ll divisions and enlargemen ts

(2) Le gum es h ave:

(a) a sim ple pistil w ith a supe rior

ovary h aving one cavity (locule )

(fig. 3)

(b) seedcoats com posed of h istologically dense cuticle, radial

colum nar ce lls, scle rench ym atous ce lls, lignin, and oste oscle reid ce lls

b .

(3) Structural te rm s re lating to th e

seedcoat are de fined as follow s

(fig. 4):

(a)

(b)

(4

Cd)

(e>

(0

(iid

cuticle - w axy laye r on oute r

w alls of e pide rm al ce lls

lignin - organic com ponent of

ce lls associated w ith ce llulose

ligh t line -continuous th in

laye r of w ax globules

oste oscle re id - bone-sh aped

scle rench ym a

palisade ce lls - e longated ce lls

pe rpendicular to th e coat surface

pare nch ym a - undiffe rentiated, live ce lls

scle re nch ym a - th ick , lignified ce lls

Gym nospe rm s -Many conife rs flow e r

and ripen seeds in one grow ing season,

som e require tw o seasons, and a fe w

require th ree seasons.

9
PALISADE LAYE R

I

“LIGHT LINE ”

INTERCELLULAR SPAC E

OSTEOSCLEREIDB

CUTICLE LAYE R

-4

EPIDERMIS

I

I

I

SCLERENCHYM A

PARENCHYM A

Figure 4. -Partial s ection th rough th e seedcoat of a h ard seed (legum e).

10
2. Ph ysiological deve lopm ent

a. M oisture content incre ases rapidly afte r

fe rtiliz ation and decre ases at m aturity.

b . H orm one contents are h igh e r w h e re

m e riste m atic activity is gre ate r.

c. Metabolic ch anges are m any; sim ple

sugars, fatty acids, and am ino acids are

conve rted to proteins, oils, and lipids.

3. Classification of m ature fruits (table 1)

H . Sources

For additional inform ation, see Dogra 19 83;

H ardin 19 60; H artm ann and oth e rs 19 83, ch ap.

3, p. 59 -65; Krugm an and oth e rs 19 74; W illan

19 85, p. 7-10,13-15.

II. Se e d Dorm ancy

A. Introduction

Once seeds h ave m atured, survival of th e species requires th at th ey ge rm inate at a tim e and

place favorable for grow th and survival of th e

seedlings. Th e me ch anism th at prevents ge rm ination at undesirable tim es is called dorm ancy. Th e me ch anics of seed dorm ancy m ust

be k now n before nursery practices for ove rcom ing dorm ancy can be deve loped to ensure

tim e ly ge rm ination and uniform grow th of th e

seedlings.

B. Obje ctives

1. Describe th e diffe rent types of seed dorm ancy.

2. Discuss m e th ods for ove rcom ing seed dorm ancy, both for ge rm ination testing and for

nurse ry ope rations.

C. Key Points

Th e follow ing points are essential to understanding seed dorm ancy:

1.

2.

3.

4.

5.

6.

To a large degre e , dorm ancy is under

genetic control.

Environm e n tal conditions during seed

m aturation can influence th e degree of dorm ancy.

Seeds can h ave m ore th an one type of dorm ancy m e ch anism .

Posth arvest environm ent can cre ate secondary dorm ancy.

Th e distinction between “dorm ancy” and

“delayed ge rm ination” is not alw ays cle ar.

Th e le ast seve re tre atm ent to ove rcom e

dorm ancy sh ould be tested first to avoid

dam age to th e seeds.

D. De finition of Te rm s (Banne r 19 84a)

1. Afte rripening- ph ysiological process in

seeds afte r h arvest or abscission th at

occurs before, and is often necessary for,

ge rm ination or resum ption of grow th under

favorable environm ental conditions.

Table 1. -Com m on fruit types for w ood y trees (adapted from H ard in 19 60)

Descrintion Ibe Exam ple

Sim ple Fruit (product of single pistil)

Deh iscent w alls (splitting naturally)

Product of one carpe l

Deh iscing by one suture

Deh iscing by tw o sutures

Product of tw o or m ore carpe ls

W alls indeh iscent (not splitting naturally)

Exocarp flesh y or le ath e ry

Pe ricarp flesh y th rough out

Pe ricarp h e te rogeneous

Exocarp le ath e ry rind

Exocarp flesh y

Endocarp a “stone”

Endocarp cartilaginous

Exocarp dry (pape ry, w oody, or fibrous)

Fruit w inged

Fruit w ith out w ings

One-loculed ovary; th in w all; sm all seed

Se ve ral-loculed ovary; th ick w all; large seed

Com pound Fruit (product of m ultiple pistils)

Pistils of a single flow e r Aggre gate M agn olia

Pistils from diffe rent flow e rs (inflorescence ) Multiple P& anus

Follicle Z anth oxylum

Le gum e Acacia, Prosopis, Robinia

Capsule Eucalyptu s, Populu s

Be rry Vaccinium , Diospyros

H espe ridium Citrus

Drupe Prunus, Vitex, Tectona

Pom e Malu s, Crataegu s

Sam ara Z Yiploch iton, Term inalia, Acer

Achene P& anus, Cord ia

Nut Quercu s

11
2. Dorm ancy- a ph ysiological state in w h ich

a seed disposed to ge rm inate does not, e ven

in th e presence of favorable environm ental

conditions.

3. Ch illing-subje ction of seeds to cold and

m oisture to induce afte rripen ing.

4. Pre ch illi n g- cold , m oist tre atm e n t

applied to seeds to h asten afte rripen ing or

to ove rcom e dorm ancy before sow ing in soil

or ge rm inating in th e laboratory.

5. Pre tre atm e n t-any k ind of tre atm e n t

applied to seeds to ove rcom e dorm ancy and

h asten ge rm ination.

6. Scarification- w e ak e n ing of seed coats,

usually by m e ch anical ab rasion or by brie f

soak s in strong acids, to incre ase th eir pe rm e ability to w ate r and gases or to low e r

th eir m e ch anical resistance to sw e lling

emb ryos.

7. Stratification-placing seeds in a m oist

m edium , often in alte rnate laye rs, to h asten

afte rripen ing or to ove rcom e dorm ancy;

com m only applied to any te ch nique th at

k e e ps seeds in a cold, m oist environm ent.

8. De layed ge rm ination- a gene ral te rm

applied to seeds th at do not ge rm inate

im m ediate ly but are not slow en ough to be

described as dorm ant.

E. Types of Dorm ancy

1.

2.

3.

4.

5.

6.

Se e dcoat (or e xte rnal) dorm ancy

a. Im pe rm e ability to m oisture or gases;

e.g., Acacia, Prosopis, Robinia, an d

oth e r le gum es

b . Me ch anical resistan ce to sw e lling

emb ryo; e.g., Pinus and Quercu s.

Em bryo (or inte rnal) dorm ancy

a. Inh ibiting substances; e.g., Fraxinus,

Rex, and Magnolia

b . Ph ysiological im m aturity; e.g., Juniperus virginiana

M orph ological dorm ancy results from

th e e m b ryo not being com ple te ly deve loped;

e,g., I1c.r opaca, som e Fraxinus spp., and

Pinus spp.

Se condary dorm ancy results from som e

action, tre atm ent, or injury to seeds; e.g.,

Pinus taed a being e xposed to h igh te m pe ratures and m oisture during storage.

Com bined dorm ancy results from tw o or

m ore prim ary factors, such as seedcoat dorm ancy and em b ryo dorm ancy, e.g.; Tilia.

Double dorm ancy results from emb ryo

dorm ancy in both th e radicle and epicotyl;

e.g., Prunus.

F. Ove rcom ing Dorm ancy

1. Seedcoat dorm ancy-Tre atm e nt m u st

incre ase m oisture uptak e and gas exch ange

and ease radicle e m e rgence.

a.

b .

C.

d.

e.

Cold w ate r soak - Soak seeds in w ate r at

room te m pe rature for 24 to 48 h ours.

H ot w ate r soak - Bring w ate r to a boil,

put seeds in, re m ove from h e at, and

allow to stand until w ate r cools.

H ot w ire-Use a h e ated needle or e le ctric w oodburne r to burn a sm all h ole

th rough th e seedcoats.

Acid tre atm ent-Pour a strong m ineral

acid ove r th e seeds and m ix. (Sulfuric

acid is pre fe rred.) Rem ove seeds afte r a

tim e dete rm ined by trials w ith sam ples,

usually 15 to 60 m inutes, and

th orough ly to re m ove acid.

Ph ysical scarification - Crack or

th e h ard seedcoats.

(1) Use h and m e th ods (nick ing).

(2) Use m e ch anical m e th ods for

w ash

b re ak

largescale ope rations.

2. Em b ryo dorm an cy- Tre atm e n t m ust

ove rcom e ph ysiological barrie rs w ith in th e

seeds.

a.

b .

C.

d.

Stratification (ch illing, pre ch illing) -

Refrige rate fully im bibed seeds at 1 to

5 “C for 1 to 6 m onth s (table 2).

(1) Im bibition is com ple ted.

(2) Enzym e syste m s are activated.

(3) Storage foods ch ange to soluble

form s.

(4) Inh ib itor/prom ote r balances ch ange.

In cubation /stratification-For som e

spe cies, provide sh ort, w arm incubation

(15 to 20 “C), follow e d by cold stratification .

Ch e m ical tre atm ent

(1)

(2)

(3)

(4)

H ydrogen pe roxide - Soak for 48

h ours in l-pe rcent solution (e.g.,

Pseudotsuga m enziesii).

Citric acid- Soak for 48 h ours in

l-pe rcent solution, follow e d by 9 0-

day stratification (e.g., Juniperus,

Tarod ium d istich um ).

Gibbe re llins

Eth ylene

Ligh t -Dorm ancy is ove rcom e by th e

red/far-red m e ch anism .

G. Significance

1. Survival strate gy- Dorm ancy allow s ge rm ination during favorable environm ental

conditions.

2. Genetic factor-Dorm ancy in m any seeds

is under genetic control.

3. M u ltiple causes -M any species h ave

probably evolved w ith m ore th an one dorm ancy m e ch anism .

12
Table 2. -Recom m ended prech ill periods for nursery sow ing of som e pines of th e South ern United States (Bonner

19 9 1b)

Pin e

species

Norm al sow ing’

Fresh seed Stored seed

Early

sow ing+

Seed conditions

DWD dorm ancv* Low viaor

Pinus strobus

_.

P taeda

l? palu stris

P rigid a

P serotina

P clausa

var. im m uginata

var. clausa

I? ech inata

P elliottii

var. elliottii

var. densa

l? globra

l? virginiana

.______________________________________________

30-60 60

30-60 30-60

0 0

0 O-30

0 O-30

o-15 o-21

0 0

o-15 O-30

0 O-30

30 O-30

30 30

O-30 30

pp.&ill (Days) ________________________________________________

60-90 60-90 30

60 60-9 0 20-30

. ..* o-15 0

,.. . . . .

. .

. t..

K-30 30-60 0

*Spring sow ing when me an m inim um soil te m pe rature at seed depth is at le ast 10 “C.

+ Early sow ing when s oil te m pe ratures at seed depth m ay be below 10 “C.

*Dorm ancy dem onstrated by paired tests or past pe rform ance of the seedlot.

W onditions not encounte red w ith th is species.

A.

B.

C.

15-30 0

. . . .

... ... ...

30 ... ...

4. Environm e n tal influe n ce -W e ath e r

conditions during m aturation m ay incre ase

th e degree of dorm ancy.

H . Sources

For additional inform ation, see Kh an 19 84;

Krugm an and oth e rs 19 74; Murray 19 8413;

Nik olae va 19 67; W illan 19 85, p. 17-19 , ch ap. 8.

III. Ge rm ination

Introduction

Th e goals of seed te ch nology are successful ge rm ination and seedling establish m ent. Th e tw o

m ajor considerations are th e ph ysiology of th e

seed and th e condition of th e environm ent. In

th e tw o pre ceding sections, seed m aturation and

dorm ancy w e re considered. In th is section,

environm ental factors and h ow th ey control ge rm ination th rough th eir inte ractions w ith seed

b iology w ill be e xam ined.

Obje ctives

1.

2.

3.

4.

Describe th e tw o types of ge rm ination and

th eir im portance in w oody plants.

Review en vironm ental requiremen ts for ge rm ination.

Review ph ysiological ch anges w ith in seeds

th at le ad to ge rm ination.

Discuss h ow seed ph ysiology and environm ental factors inte ract in ge rm ination.

Key Points

Th e follow ing points

standing ge rm ination:

are essential to underD.

E.

1.

2.

3.

4.

5.

Th e tw o types of ge rm ination are e pige ous

and h ypoge ous.

M oisture availability is th e prim ary factor

controlling ge rm ination.

Th e e ffe cts of te m pe rature and ligh t on ge rm ination are strongly re lated.

Constant and alte rnating te m pe rature

re gim es m ay le ad to sim ilar total ge rm ination, but ge rm ination is usually faste r

under alte rnating re gim es.

As ge rm ination begins, th e k ey to inte rnal

processes is th e ch ange from insoluble to

soluble me tabolites. De tails of such m e tabolism are beyond th e scope of th is course.

Types of Ge rm ination

1. Epige ous (e pige al) ge rm ination occurs

w h en cotyledons are forced above th e

ground by elongation of th e h ypocotyl (fig.

5); e.g., Pinus, Acacia, Fraxinus, and Populu s.

2. H ypoge ous (h ypoge al) ge rm ination

occurs w h en th e cotyledons re m ain below

ground w h ile th e e picotyl e longates (fig. 6);

e.g., Juglans, Quercus, and Sh m ea.

3. In Prunus, both types of ge rm ination m ay

be found.

Environm ental Requiremen ts for Ge rm ination

Th e four environm ental requiremen ts for ge rm ination are m oisture, te m pe rature, ligh t, and

gases.

1. M oisture

a. Im bibition is usually considered th e

first ste p in ge rm ination; th us, avail13
HYPOCOTYLS [

2 cm

Figure 5 - Epigeal germ ination sequence of Fraxinus spp. (ad apted from Bonner 19 74).

ability of m oisture is th e first require- 2. Te m pe rature

b .

;.

e.

m ent for ge rm ination.

Uptak e typically occurs in th e follow ing

th ree ph ases:

(1) Rapid initial ph ase, m ainly ph ysical

(2) Extreme ly slow second ph ase

(3) Rapid th ird ph ase th at occurs as

a.

b .

m e tabolism becom es ve ry active

Th e first ph ase is im b ib itional.

A m inim um state of h ydration is

needed .

C.

d.

M inim al requiremen ts for ge rm ination

are frequently studied w ith osm otic

solutions of m annitol or polyeth ylene

gl ycol .

e.

(1)

(2)

(3)

Th e best ge rm ination m ay occur at

sligh t m oisture stress (0.005 to

0.500 bars).

Even sligh tly low e red w ate r potentials w ill slow , but not stop, ge rm ination.

Critical leve ls of w ate r potential

vary by species.

It is difficult to separate th e e ffe cts of

te m pe rature from th ose of ligh t and

m oisture.

For w oody plants, ge rm ination usually

occurs ove r a w ide range of te m pe rature s.

Th e uppe r te m pe rature lim it is around

45 “C.

Th e low e r lim it is around 3 to 5 “C

b e cause ge rm ination processes w ill

occur near freezing.

Optim um te m pe ratures vary little:

(1)

(2)

For Te m pe rate Z one spe cies, alte rnating re gim es of 20 “C (nigh t) and

30 “C (day) h ave proved best for

m any species.

For tropical spe cies, alth ough fe w

critical studies are available, constant te m pe ratures m ay be best for

som e ; e.g., Azadirach ta in d ica, 25

“C;Bom bax ceiba, 25 “C; Eucalyptu s

cam ald ulensis, 30 “C; Le ucaena

14
i

EPICOT YLSI cm

0

/

COTYLEDON

Figure 6. - H ypogeal germ ination sequence of Quercus spp. (ad apted from Olson 19 741.

15
leucoceph ala, 30 “C; Prosopis cineraria, 30 “C!; and Tectona gran d is,

30 “C. Oth e r spe cies do as w e ll or

even bette r unde r alte rnating te m -

pe ratures; e.g., Acacia spp., Ce d rela

spp., and tropical pines.

3. Ligh t

a. Ligh t stim ulates ge rm ination of m any

tree seeds but is necessary for fe w .

b . Ph ytoch rom e is a pigm ent involved in

th e ph otocontrol of ge rm ination.

c. M inim al ligh t le ve ls in ge rm ination testing sh ould be 750 to 1,250 lux.

4. Gas e s

a. Respiration requires a ce rtain supply of

oxygen , and th e carbon dioxide produced

m ust be re m oved.

b . Som e spe cies ge rm inate we ll in anae rob ic conditions.

c. Oxygen uptak e patte rns in seeds are

sim ilar to th ose of m oisture.

d. M any aspe cts of th e influences of gases

on ge rm ination need to be studied.

F. Inte rnal Ph ysiological Ch anges

1.

2.

3.

4.

5.

Structuralch ange s - Im b ib ition is a precursor to necessary m e tabolism .

Enzym e s- Som e syste m s are present in

dry seeds; oth e rs are synth esized as im bibition proceeds.

Rese rve food m obilization - Gene rally,

insoluble form s (carboh ydrates, lipids, and

proteins) are conve rted to soluble form s (in

som e w ays a re ve rse of m aturation trends).

Nucleic acids-Th e se com pounds are

essential for th e form ation of new enzym es.

Translocation - Th e m ovemen t of m aterials w ith in th e e m b ryo is crucial.

G. Sources

For additional inform ation, see Bonne r 19 72,

M aye r and Poljak off-Mayber 19 75, Murray

19 84b , Stanw ood and McDonald 19 89 , W illan

19 85.

16
Collection
I. Genetics and Se e d Source

A. Introduction

Seed quality involves both th e genetic and th e

ph ysiological quality of seeds. In th is section,

th e gene ral principles and m e th ods for sele ction

of seed source and im provemen t of seed quality

th rough genetic sele ction are presented. Genetic

im provemen t of seed quality is based on th e

seeds’ ability to produce trees th at are genetically w e ll suited to th e sites w h e re planted and

for th e products desired. In late r sections,

ph ysiological quality of seeds w ill be considered.

Good seeds are th ose th at h ave both h igh ph ysiological quality and genetic suitab ility.

B. Obje ctives

1. Recognize th e im portance of seed origin

(provenance ) and re com m end gene ral rules

for seed m ovemen t.

2. Review th e advantages and disadvantages

of e xotic tree s pe cies and inte rspe cific

h ybrids for tree im provemen t.

3. De fine factors th at m ust be considered

w h en a tree im provemen t program is

initiate d .

4. Identify th e conditions required for genetic

im provemen t of tree seeds (genetic gain

conce pt).

5. Distinguish between a m inim um initial

strate gy of genetic im provemen t and a m axim um long-te rm strate gy.

6. Identify som e te rm s and conce pts of new

b iote ch nology for genetic im provemen t.

C. Key Points

Th e follow ing points are essential to understanding seed source and genetic im provemen t:

1. A successful tree im provemen t program

sh ould not be tried in anoth e r country or

re gion w ith out considering desired products

and available sites.

2. Know ledge of ph enotype and genotype is

necessary to understand genetic im provem ent of trees.

3. Th e genetic gain equation explains th e

advantages of one im provemen t m e th od

ove r anoth e r.

4. Genetic gains can be obtained from sele ctions am ong spe cies, provenances w ith in

spe cies, and/or trees w ith in provenances.

5. Th e prim ary risk of using e xotics or nonlocal provenances is planting on unsuitable

sites.

6. Test plantings are th e only sure me th od to

dete rm ine genetic quality of seeds.

7. W ith out results of test plantings, th e safest

rule is to use seeds from ph enotypically

sele cted stands or trees in th e local provenance for native spe cies or land race for

e xotic spe cies.

8. Th e seed orch ard conce pt h as tw o partsth e b reeding program and th e production

program .

9 . Seed orch ard b reeding program s involve

progeny tests and sele ction for th e ne xt

advanced gene ration of genetic im provem e n t.

10. Seed orch ard production program s are

m anaged to m axim ize seed production

th rough prote ction and cultural tre atm e n ts.

D. Tree Im provemen t

1. Tree im proveme nt is th e deve lopm e n t

and application of genetically im proved

trees and intensive cultural practices to

en h ance forest productivity th rough artifical re gene ration.

2. Tree im proveme nt program s are plans

of action to b ring about desired obje ctives.

Th e follow ing factors sh ould be considered

w h en a tree im provemen t program is initiated:

a. Products desired

b . Sites to be re gene rated

c. Adaptation to th e planting sites

d. Conservation of forest gene resources

E. Strate gies for Genetic Im provemen t

1. Genetic gain

a. Genetic im provemen t (genetic gain) is

accom plish ed by:

(1) H aving a population of trees w ith

genetic diffe rences

(2) Se le cting th e genetically desirable

tre es

b . Th e am ount of genetic gain (R ) to be

captured from ph enotypic sele ction of

parent trees for a particular trait is:

R =iVph 2

w h e re i = th e intensity of sele ction

h 2 = th e h e ritability of th e trait

VP = th e am ount of ph enotypic

variation.

c. Gain can be captured from sele ction

am ong spe cies CR ,), sele ction am ong

prove n ances w ith in spe cies CR ,), or

sele ction am ong individual trees w ith in

provenances (R I). Th e total gain CR T) is

th e sum :

R , = R ,+ R , + R ,

2. Spe cie s se le ction

a. Spe cies-site studies are needed.

b . Exotic tree s pe cies sh ould be used

sparingly.

18
c. Inte rspe cific h ybridiz ation can be used

to obtain valuable traits.

3. Seed source

a. Provenance re fe rs to w h e re th e m oth e r

trees w e re grow ing and th e seeds w e re

colle cted. Seed source is th e sam e as

provenance. Origin is w h e re th e original

progen itors w e re grow ing in natural forests and w h e re th eir genetic ch aracte ristics w e re deve loped th rough natural

s ele ction .

b . “Local” sources sh ould be used until

provenance test results are available.

c. Purposes of provenance tests include:

(1) M apping patte rns of ge ograph ic

genetic variation

(2) De lineating provenance boundaries

(3) Dete rm ining th e best provenances

d. Th e gene ral results of provenance testing are:

(1) W ide seed transfe r is safe r near th e

cente r of a spe cies’ range th an near

its edge.

(2) W h e re environm ental gradients are

ste e p, m ovemen t of m ate rial m ust

be restricted.

(3) Provenances from h arsh clim ates

(cold or dry) grow m ore slow ly.

4. Im proveme nt strate gie s

a. Th e initial strate gies for a new program

are:

(1) Colle ct available inform ation

(2) Se le ct am ong indigenous tree species

(3) Se le ct seed production are as w ith in

th e “local” seed sources near th e

planting site

(4) R e m ove ph e n otypically infe rior

trees from seed production areas

b . Th e long-te rm strate gies for m axim um

and continued gains are:

(1) Colle ct all e xisting inform ation

(2) Se le ct seve ral spe cies for th e program

(3) Conduct provenance tests

(4) Se le ct th e ph e n otypically “best”

trees

(5) Establish a first-gene ration seed

orch ard

(6) Test progeny

(7) Rem ove genetically poor trees

(8) Se le ct th e best individuals for a

second-gene ration seed orch ard

(9 ) Test th e progeny of th ese secondgene ration sele ctions

c. New strate gies for genetic im provemen t

are:

(1) Gene transfe r

(2) Se le ction of ce lls in a ce ll-suspension

culture

(3) Fusion of protoplasts (w ith out ce ll

w alls)

(4) Som aclonal variation

F. Th e Seed Production Program

Th e production program m ay be com b ined w ith

th e b reed ing program or m ay be k e pt separate.

Th e obje ctive. of a seed production program is to

produce sufficient quantities of genetically

h igh -quality seeds to m e e t seed needs.

1. Seed Production Are as (SPA’s)

a. Existing stands can be m anaged for production of seeds.

b . SPA’s are used on an inte rim basis.

c. SPA’s can utilize supe rior provenances.

d. SPA’s can provide seeds for m inor

spe cies.

e . Th e genetic quality of seeds is im proved

by:

( 1) Rem oving undesirable trees

(2) Establish ing a pollen dilution zone

f. Seed production is incre ased by:

(1) Th inning th e stand

(2) Fe rtilizing

(3) Establish ing access roads

2. Seed orch ards -A seed orch ard is a colle ction of sele cted trees establish ed and

grow n toge th e r unde r intensive m anagem ent for production of genetically im proved

seeds.

a. Th e re are tw o types of orch ards:

( 1) Seedling seed orch ards

(2) Clonal seed orch ards

b . Th e genetic quality of seeds is incre ased

by:

(1) Reducing inbreeding

(2) Establish ing a pollen dilution zone

(3) Se parating provenances into diffe rent orch ards

c. Seed production from orch ards can be

incre ased by:

(1) Ch oosing good soil and clim atic conditions

(2) Spacing w ide en ough for full crow ns

(3) Fe rtilizing

(4) Irrigating

(5) Subsoiling

(6) Prote cting from insects

(7) Prote cting flow e rs from late spring

freezes (cold w ate r irrigation)

(8) Ensuring supplemen tal m ass pollination

G. Sources

For additional inform ation, see Burley and

Styles 19 76, Kh osla 19 82, Nienstadt and

19
Snyder 19 74, Rudolf and oth e rs 19 74, W righ t

19 76, Z obel and Talbe rt 19 84, Z obel and oth e rs

19 87.

II. Production

A. Introduction

M ost tree-planting program s are begun by colle cting seeds from in-country sources, both natural stands and plantations. To plan th ese

colle ctions effe ctive ly, seed m anage rs sh ould

understand th e factors th at affe ct tree seed

crops and gene rally k now wh at seed yields m ay

be e xpe cted. W ith th is basic inform ation, opportunities m ay arise to stim ulate seed production

in k ey areas, such as seed orch ards or m anaged

seed stands.

B. Obje ctives

1. Recognize th e problem of pe riodicity of seed

production in trees.

2. Le arn h ow en vironm ental factors affe ct

seed production.

3. Le arn h ow seed production can be stim ulated in trees.

C. Key Points

Th e follow ing points are essential to understanding seed production:

1. M any tree spe cies bear good crops in cycles.

2. Production is less frequent in h igh latitudes

and h igh altitudes and am ong h e avy predator populations.

3. Environm ental factors influence flow e r production, pollination, and seed m aturation.

4. Seve ral options are available to stim ulate

seed production.

5. Exce pt for seed orch ards of a few spe cies,

production data are e xtreme ly variable.

D. Pe riodicity of Seed Crops

1. Te m pe rate spe cie s

a. M any conife rs bear in cycles.

b . Many angiospe rm s produce good seed

crops eve ry ye ar.

c. As latitude or altitude incre ases, th e

inte rval between good crops and th e frequency of crop failure incre ase.

2. Tbopical spe cie s

a. Pe riodicity m ay de pend on w e t/dry

cycles.

b . Som e spe cies (e.g., Tectona gran d is)

usually flow e r e ach year. Oth e r spe cies

(e.g., Pinus k esiya, Cassia siam ea,

Cupressus lusitanica, and D e lon i x

regia) produce good crops m ost years.

c. Dipte rocarps in Malaysia be ar irre gular

h e avy seed crops at l- to 6-year inte rvals.

d. Som e Eucalyptus spe cies h ave large

crops m ore re gularly w h en grow n in

plantations.

3. Gene tics-Fe cundity is an in h e rite d

trait.

4. Docum e n tation- Th e re are few detaile d

studies and data.

E. Effe cts of Environm ents During Flow e ring

1. Te m pe rature

a. During h ot sum m e rs, trees usually produce h e avy floral bud form ation.

b . Late freezes can destroy flow e rs.

c. Th e com bination of h ot sum m e rs and

late freezes suggests th at orch ards

sh ould be m oved to w arm e r clim ates

(also to escape insects).

2. Ligh t h as not been studied extensive ly. In

th e North e rn Te m pe rate Z one, south e rn

and w e ste rn sides of crow ns h ave th e h e aviest flow e r and fruit crops.

3. Ph otope riod does not appe ar to h ave a

dire ct e ffe ct on trees.

4. M oisture affe cts flow e ring th rough :

a. Drough t

b . Excessive rain during pollination

5. M ine ral nutrie n ts- Th e b alance of nitroge n and ph osph orus can affe ct

flow e ring.

6. Biotic agents - Insects, birds, m am m als,

and m icro-organism s can destroy flow e rs.

Th ese agents are ve ry com m on in th e follow ing tropical tree spe cies:

a. Triploch iton scleroxylon; attack ed by

Apion gh anaense (wee vil)

b . Tectona gran d is; attack ed by Pagyd a

salvaris larvae

c. Pinus m erk usii; attack ed by Dioryctria

spp. (cone w orm s)

F. Pollination Agents

1. W ind pollination occurs am ong all conife rs and m ost Te m pe rate Z one h ardw oods.

a. W ind pollination requires:

(1) Lots of pollen

(2) Pollen sh ed coinciding w ith re ce ptivity

(3) Relative ly close spacing of plants

(4) Good w e ath e r -low rainfall, low

h um idity, and good w inds

b . Supplemen tal m ass pollination (SM P)

h as been used in United States south e rn

pine orch ards.

c. Contam ination in orch ards is a conce rn.

2. Anim al pollination

a. Insects and bats pollinate te m pe rate and

tropical h ardw oods.

b . Anim al pollination is usually com m on in

tropical forests w ith :

20
(1) H igh spe cies dive rsity and w ide

spacing

(2) Abundant foliage to filte r out pollen

(3) H igh h um idity and frequent rainfall

(4) Absence of strong flow e ring stim uli

(5) Abundant anim al ve ctors

G. Stim ulation of Flow e ring

Flow e ring can be stim ulated by seve ral m anagem ent practices.

1. Fe rtiliz ing:

a. Use prim arily nitrogen and ph osph orous, and som e tim es potassium .

b . Irrigation at th e sam e tim e m ay also

h e lp.

c. H ardw oods m ay re act favorably; e.g.,

Acer, Fagu s, and Juglans, but results

h ave been inconsistent.

2. Girdling and oth e r w ounding can produce “stress crops.”

a. Girdling inh ibits dow nw ard translocation of carboh ydrates.

b . Som e h ardw oods also re act favorably.

3. Th inning-Th e bene fits of th inning are

apparent 3 to 4 ye ars afte r tre atm ent.

4. Grow th re gulator tre atm ent - Gibbe re llins (GA) application to conife rs is th e m ost

com m on.

a. A w ate r-based spray is best.

b . A GA 417 m ixture is m ost e ffe ctive.

c. Both pollen and seed cones are induced.

d. Sprays are applied at th e tim e of bud

dete rm ination.

e. Th e m ode of action is still unk now n.

f. Tre atm ents are m ost successful w h en

applied w ith girdling, root pruning, or

m oisture stress.

5. Supplemen tal m ass pollina tion

(SM P) -Th is te ch nique is used in pine

orch ards in th e South e rn United States.

H . Postfe rtiliz ation Proble m s

Postfe rtiliz ation prob le m s include insect

dam age to cones, drough t, cone drop, and h igh

w inds.

I. Sources

For additional inform ation, see Frank lin 19 82;

Owens and Blak e 19 85; Rudolf and oth e rs 19 74;

W h iteh e ad 19 83; W illan 19 85, ch ap. 3; Z obel

and Talbe rt 19 84.

III. Colle ction Ope rations

A. Introduction

Successful colle ction of tree seeds is usually th e

result of quite detailed early planning. Am ple

tim e m ust be allow e d to plan an efficient and

practical colle ction strate gy and to assem ble th e

resources necessary for its im plemen tation. Key

e lemen ts include a good estim ate of crop siz e,

prope r equipm ent, and a w e ll-trained cre w .

Com preh ensive colle ctions for research w ill

alm ost ce rtainly require m ore detailed planning

th an routine bulk colle ctions and m ay require a

le ad tim e of 1 to seve ral years depending on th e

circum stances.

B. Obje ctives

1. Identify sim ple te ch niques for seed crop

estim ation.

2. De te rm ine factors th at sh ould be conside red w h en colle ctions are planned.

3. Understand th e im portance of docum entation.

C. Key Points

Th e follow ing points are essential for planning

colle ction ope rations:

1. Th e best seed sources available m ust be

s ele cte d .

2. Good planning requires advance estim ates

of th e seed crop and, at a late r date, estim ates of seed yield pe r fruit.

3. Planning for large colle ctions m ust include

ch oice of pe rsonnel, training, transportation , colle ction equipm e n t, safe ty of

w ork e rs, labeling of seedlots, description of

sites and stands, e tc.

D. Seed Source

Seed source includes th e follow ing considerations:

1. Origin-th e natural stand location of th e

original m oth e r tre e.

2. Prove n an ce- th e place whe re m oth e r

trees th at produced th e seeds are grow ing.

(Sam e as seed source.)

3. Land race-exotics th at adapt ove r tim e to

provide im proved sources.

4. Seed zone m aps sh ould be deve loped for

all im portant spe cies.

E. Estim ating Seed Crops

Seed crop estim ates are alw ays valuable to th e

colle ctor, espe cially in years w h en seeds are in

sh ort supply. Good crop estim ates h e lp to

stre tch th e available cre w s and equipm ent. Seed

crops can be estim ated by th e follow ing five

m e th ods:

1. Flow e r counts

2. Im m ature fruit and seed counts

3. Fruit counts on standing trees- Th is

m e th od includes total counts and crow n

sam pling.

4. Rating syste m s

5. Cross-section seed counts (table 3).

F. Planning Considerations

Th e ste ps of planning a colle ction are:

1. De fine th e ob je ctive s.

21
Table 3.-Sound seed yield per cone for four Pinus species as estimated from the number of sound

seeds exposed when cones are bisected longitudinally (Derr and Mann 1971)

Sound

seeds

exposed

2

4

6

8

10

12

14

l? elliottii

P. palustris P taeda I? elliottii (Ge orgia- l? echinata

(Louisiana) (Louisiana) (Louisiana) Florida) (Virginia)

______________________________________ Sound see& per cOne _______________________________________

23 31 20 31 12

35 44 35 50 22

47 57 50 69 31

59 70 65 87 41

71 83 80 106 51

83 9 6 9 5 124 60

9 5 109 110 143 70

2. Gath e r back ground data:

a. Se arch th e lite rature.

b . Officially contact th e appropriate forest

services early.

c. Collate and sum m arize all inform ation.

d. Do fie ld re connaissance.

e . De te rm ine th e num b e r of pe rsonne l

needed .

3. Colle ct fie ld data: Inform ation for re location of th e site in future years:

Locality, including latitude and altitude

Aspe ct, slope, clim ate, soils, and associated species

Individual tree descriptions

H e rbarium spe cim ens

Oth e r data and notes

Se curity and labeling

4. Plan th e itine rary:

a. R e ach th e colle ction re gion w e ll in

advance of th e proposed date.

b . Organize th e sequence of ope rations.

c. Mak e th e sch edule fle xible.

5. Organize eq uipm e n t pe rm its and

transportation:

a. Spe cify equipm ent to be used.

b . Identify applicable gove rnm ent re gulations.

c. Use care between th e colle ction of th e

seeds and th eir arrival in th e seed laboratorie s.

G. Colle ction Equipm ent -A Com preh ensive List

Th e follow ing ite m s are ne cessary for m ost colle ction ope rations:

1. Adm inistrative ite m s

::

:.

iF

22

M ovemen t approvals

Colle ction auth orities

Radio transm ission perm its

Drive rs’ licenses

Fire arm pe rm its

Facilities for purch asing stores; e.g.,

gasoline (pe trol) and oil

2. Lite rature

a. Road, topograph ic, and soil m aps to

cove r th e colle ction route itine rary

b . Lite rature on th e gene ra and species to

be colle cted

3. Colle ction equipm e nt

a.

b .

:.

e.

f.

::

i.

j.

k .

1.

m .

n .

0 .

P.

q.

r.

S.

t.

U .

V.

W .

Notebook s, re cording form s, pens, and

pencils

Binoculars

M ark e rs; e.g., colored plastic ribbon

Cam e ra and accessories

Tree-m e asuring instrum ents; e.g., diam e te r tape, h eigh t-m e asuring instrum ent, and length tape

Soil sam ple r, pH testing k it, and soil

ch arts

Com pass

Altim e te r

H and lens

Large colle cting sh e e ts; e.g., 4 by 4 m ,

h e avy plastic or canvas

Sm all colle cting sh e e ts

Seed bags of various sizes

Large grain bags for dispatch ing seeds

Cutting equipm ent

Safe ty ge ar

W e ath e rproof tags

Tags for botanical spe cim ens

Plant presses

Pape rs to dry spe cim ens

Plastic bags

Spe cim en b ottles w ith prese rvative

fluid

Containe rs for soil sam ples

String

H . Sources

For additional inform ation, see Barne r and

Olesen 19 84; Bram le tt and oth e rs 19 77; Doran

and oth e rs 19 83; Ontario M inistry of Natural

Resources 19 83; W illan 19 85, ch aps. 3, 4, 5 +

appendices 1,5,6.
Iv. M aturity

A.

B.

C.

D.

E.

F.

Introduction

Ch oosing good stands and trees for seed colle ction m e ans noth ing if fruit or seed m aturity

cannot be e asily identified on th e trees by

unsk illed w ork e rs. If seeds are dissem inated

im m ediate ly at m aturity, w ork e rs m ust k now

h ow m uch in advance of m aturity seeds can be

colle cted w ith out colle cting seeds th at w ill not

ge rm inate. If predators inflict large losses on

m ature seed crops, a sim ilar problem e xists.

Good m aturity indices are often th e k ey to successful colle ction.

Obje ctives

1. Le arn th e com m on indices of m aturity

e m ployed in tree seed colle ctions.

2. Unde rstand h ow th ese te ch niques can be

adapted for new s pe cies.

Key Points

Th e follow ing points are essential to re cognizing seed m aturity:

1. Seed m oisture content is ve ry im portant,

but dire ct m e asuremen t in th e fie ld is

im practical; indire ct estim ates m ay be substituted.

2. Color ch anges are th e m ost com m on indices.

3. Ch e m ical indices are possible but im practiC d .

4. Artificial m aturation of im m ature seeds is

an option for som e spe cies.

Successful Colle ction

Th e follow ing points are essential to successful

colle ction:

1. Biological ide al-to colle ct at th e pe ak of

ph ysiological m aturity

2. Practical colle ction-In m ost colle ction

ope rations, one m ay:

a. Colle ct seeds from th e ground

b . Colle ct fruits or seeds from logging ope rations

c. Colle ct m ature fruits from standing

trees

d. Colle ct fruits from standing trees w e ll

in advance of m aturity and ripen th e

seeds artificially

Colle ction afte r Disse m ination

Som e seeds can be colle cted afte r disse m ination. Th ese seeds are prim arily large, singleseeded fruits; e.g., spe cies of Q ue rcus an d

Curya. H ow e ve r, th e first seeds to fall are usually bad. W ork e rs m ust quick ly colle ct th e seeds

be fore anim als eat th e m .

Oth e r Colle ction Strate gies

Oth e r colle ction strate gies require dete rm ination of m aturity.

G. M aturity Indices

M aturity indices include ph ysical and ch e m ical

ch aracte ristics.

1. Ph ysical ch aracte ristics include:

a. Color ch ange

b . Moisture content

(1) Th e re are th ree trends during

ripen ing:

(a) In dry, orth odox seeds and fruits,

m oisture decre ases slow ly as

seeds m ature.

(b) In pulpy, orth odox fruits, m oisture de cre ases at first, th en

incre ases.

(c) In re calcitrant seeds, m oisture

incre ases early, th en sligh tly

d ecre ase s.

(2) M oisture content is re lated to protein synth esis.

(3) M oisture content can be m e asured

dire ctly by oven m e th ods; th at is,

cut cones, large fruits, or seeds;

w e igh ; dry for 17 h ours at 103 “C;

and th en we igh again.

(4) Spe cific gravity is usually discussed

separate ly, but it re ally is just an

estim ate of m oisture content (table

4; figs. 7 and 8). Spe cific gravity h as

been m e asured in:

(a) Conife rs (com m only used for

Pinus)

(b) Que rcus (inconsistent results)

(c) Oth e r angiospe rm s (little success )

c. Oth e r ph ysical indices include:

(1) Acorn cup re le ase in Que rcus

(2) Fle x of P strobus cones

(3) W h ite, b rittle e m b ryo in Fruxinus

and oth e r gene ra

(4) Em b ryo size (m inim um pe rcentage

of emb ryo cavity)

Table 4.-Cone specifK gravity values th at indicate seed m aturity in

som e conifers

Spe cies

Abies gran d is

Cunningh nm ia lanceolata

Pinus elliottii

P. m erkusii

I? palu stris

P strobus

P taeda

l? virginiana

Spe cific

gravity R e fe re n ce

0.9 0 Pfiste r 19 67

0.9 5 Jian and Peipei 19 88

0.9 5 Barne tt 19 76

1.00 Daryono and oth e rs 19 79

0.9 0 Barne tt 19 76

0.9 0 Bonner 19 86a

0.9 0 Barne tt 19 76

1.00 Fenton and Sucoff 19 65

23
H . Artificial M aturation

Im m ature seeds can be artificially m atured by

pick ing th e im m ature seeds, th en ripen ing

th em under spe cial storage conditions. H ow -

eve r, seed yields and quality usually suffe r.

Artificial m aturation includes th e follow ing

considerations:

1. Single-seed or m ultiple-seed fruits

24

60

(A) Pinus toeda

5 0 -

40 -

30-

MOISTURE CONTENT q 102.84 - 4 4 79 (

r : 0.92

20 -

(B) Pinus sfrobus

60 -

50 -

40 -

30 _ MOISTUR E CONTENT = 121.37- 53.49 $&$

r = -0.9 8

2 0 -

10- '

I I I I I I 1 I

0.95 0.85 0.75 0 65 0.55

CONE SPECIFIC GRAVITY

Figure 7. -Th e relation sh ip of m oisture conte nt to specific gravity for cones of Pinus

taeda and P. strobus (Banner 19 9 1b).

2. Ch e m ical ch aracte ristics

Ch e m ical indices are b iologically sound but

not practical. Th ey include:

Accum ulation of storage foods (fats and

sugars)

Elemen tal analyses of calcium , m agnesium , and ph osph orus for angiospe rm s

of South e rn United States

Grow th substances

(1) Indole ace tic acid (IAA)

(2) Gibbe re llins

2. Avoiding dorm ancy th rough e arly colle ction

3. Useful for colle cting on re m ote or e xpensive

sites

De layed Colle ctions

For trees w ith serotinous (Pinus and Picea) or

spe cies w ith delayed fruit abscission (Platanus

spp.), th e re is no rush to colle ct th e fruits.

Sources

For additional inform ation, see Bonne r 19 72a,

19 76; Nautiyal and Puroh it 19 85; Redisk e

19 61; W illan 19 85, p. 33-38.

Vi Posth arve st Care

A. Introduction

Th e tim e between colle ction and extraction is

often ove rlook ed as a crucial segm ent of seed

acquisition. Fruits and seeds, often h igh in
I 1

!I 600 ml

775 ml

A B C

CONE WEIGHT (E) 750 - (A)600 = 150 grams

CONE VOLUME: (C) 775 - (Al 600 = 175 grams

SPECIFIC GRAVITY ( WEIGH T ) 150 ‘J i (VOLUM E ) 175~ = 0.66~

Figure 8.-A sim ple tech nique for d eterm ining specific gravity of

pine cones in th e field using a graduated cylinder.

(A) Fill th e cylinder w ith w ater to th e 600 m L m ark .

(B) Float th e cone in th e w ater and record th e w ater level.

(C) Using a pin or needle, subm erge th e cone enough to

com pletely cover th e cone w ith w ater, b ut no m ore. R ecord

th e new w ater level (ad apted from Burnett 19 79 ).

B.

C.

D.

m oisture content, m ust be stored and/or transported for e xtraction and cle aning. Spe cial care

m ust be tak en during th is pe riod to avoid loss of

seed quality, espe cially in tropical and subtropical are as w h e re transportation syste m s do not

allow im m ediate delive ry to e xtraction cente rs.

Obje ctives

1. R e cognize th e crucial tim es w h en seed

quality m ay be lost.

2. Plan storage and transportation syste m s to

m inim ize th e dange r to seed quality.

Key Points

Th e follow ing points are essential to posth arvest care:

1. H igh m oisture contents and h igh te m pe ratures are dange rous for orth odox spe cies.

2. H igh m oisture le ve ls m ust be m aintained in

re calcitrant seeds, but e xcessive h e at is a

problem w ith th ese seeds.

3. Fruit storage can be advantage ous for som e

spe cies because of th e afte rripen ing processes th at occur in th e seeds.

Storage Be fore Extraction

1. Ope ration sch edu le s- Tim e does not

pe rm it seeds from all trees or fam ilies to be

colle cted at pe ak m aturity; th e re fore, som e

m ust be pick ed and stored.

2. Pre d rying- Drying during storage can

re m ove enough m oisture to low e r drying

costs.

3. Com ple tion of m aturation

a. 5 or 6 m onth s in cool, m oist conditions

com ple te m aturation for Abies.

b . Sim ilar bene fits are possible for h igh est

seed quality for som e pines of th e South -

e rn United States.

c. Pre m ature colle ction is suitable for som e

m ultiseed h ardw oods; e.g., Liquid am -

bar, Liriodend ron, and P& anus.

E. South e rn Pines

1.

2.

3.

Storage is usually re lated to ope ration

sch edule s.

Outdoor storage is bette r th an indoor

storage.

Containe rs

a.

b .

:.

All containe rs m ust provide air circulation am ong th e cones.

Burlap bags (about one-th ird h e ctolite r,

loose w e ave ) or w ooden crates are best.

Plastic bags or sack s sh ould not be used.

Pape r sack s are satisfactory for sm all

lots.

4. Tim e

5.

a. Storage can im prove ge rm ination rate.

b . Maxim um length of storage depends on

th e spe cies.

Oth e r factors

a.

b .

Original m aturity of cones is im portant;

m ore m ature cones cannot be stored as

long as less m ature cones.

Local w e ath e r is im portant; w arm ,

rainy conditions incre ase th e risk of

cone m olds.

6.

7.

Im m aturity/Dorm ancy can be ch anged

during cone storage.

H e at and m olds

a. Green cones can gene rate h e at.

b . Exte rnal m olds are com m on in som e

containe rs, but th ese m olds m ay not

cause dam age.

c. Good ae ration is essential; it prevents

m old grow th on cones during drying.

F. Se rotinous Cones

1. Storage is not a m ajor problem for Pinus

glauca, l? contorta, or Ppat& a.

2. Som e pine seeds need to re m ain in th e

cones to re ach m aturity.

G. Oth e r Conife rs

1.

2.

3.

4.

True firs (Ab ies) rn?ust com ple te ripen ing in

th e cones.

Seeds of m ost Picea spe cies sh ould be

e xtracted as e arly afte r colle ction as

possible .

Pseudotsuga cones can be stored for 3 to 4

m onth s in dry, w e ll-ventilated conditions.

Th e re com m endations for tropical pines

are :

25
a.

b .

c.

a.

e .

Cove r w ith good ventilation, w ith te m -

pe ratures between 20 and 35 “C.

Prote ct from rodents and fungi.

In H onduras, Pinus curibaea is pre cured

until all of th e cone ch anges from green

to b row n.

In New Z e aland, im m ature cones of P

rudiatu are stored for 10 week s at 20 to

24 “C.

In Indonesia, green and green /b row n

cones of l? merkusii are stored for 2 to 4

week s.

H . H ardw oods

1. Im m ature fruits of som e s pe cies w ill

respond to artificial ripen ing, but seed

yie lds and quality suffe r.

2. Seeds of som e spe cies sh ould be stored for

as sh ort a pe riod as possible. Orth odox species include:

a. Eucalyptus -Store in tigh tly-w ove n

cloth bags.

b . Le gum es-Storage is e asily m anaged.

c. Drupes - Sh ort storage w ill h e lp to com -

ple te ripen ing.

I. Sum m ary

M ost spe cies fit into one of th ree groups:

1. H arve st dry, k e e p dry-Start drying

im m ediate ly, and k e e p dry afte r e xtraction

(e.g., Pinus, Liquidambar, Liriodendron,

Acacia, and Eucalyptus).

a. Use a slow drying rate.

b . Provide good ae ration.

c. Use suitable containe rs, including:

(1) Burlap bags

(2) Back s

(3) W ooden crates

(4) Canvas or plastic sh e e ts

2. H arve st m oist, th en dry-Ke e p m oist

w h en colle cting and during e xtraction, but

dry seeds for storage (e .g., Nyssa and

Prunus).

a. Spre ad to avoid h e at.

b . Use trays or bags.

c. Avoid oute r coat tough ness.

d. Extract, w ash , and dry for storage.

3. M oist fore ve r-Th is m e th od is used for

re calcitrant seeds because drying decre ases

quality (e.g., Quercus, Aesculus, Shorea,

and Hopea).

a. Neve r dry.

b . Kee p m oisture 2 30 pe rcent.

c. Refrige rate to a safe te m pe rature:

(1) 1 to 3 “C for te m pe rate spe cies

(2) 15 to 20 “C for tropical spe cies

d. Use polyeth ylene-lined containe rs or

bags.

J. Sources

For additional inform ation, see Bonne r 19 87a;

W illan 19 85, p. 78-86.

26
Handling
I. Drying and Extracting

A. Introduction

Lik e agricultural seeds, m any tree fruits dry as

th ey m ature, and seeds are e xtracted best at low

m oisture contents. Oth e r tree seeds are still

ve ry m oist at m aturity, and special considerations are needed for e xtraction. No m atte r w h at

type of fruit is involved, h ow e ve r, th e obje ctive

of e xtraction is to obtain th e m axim um am ount

of seeds in th e best ph ysiological condition in an

e conom ically efficient ope ration. During e xtraction, seed quality can be gre atly reduced by

e xcessive ly h e ating th e fruit to force open ing or

by extracting by h and or m ach ine.

B. Obje ctives

1. R e cognize pote n tial prob le m s of see d

e xtraction re lated to th e type of fruit.

2. Identify th e basic te ch niques of tree seed

drying and extraction.

C. Key Points

Th e follow ing points are essential to seed drying and extracting:

1. For spe cies th at require drying, e xcessive

h e at in th e presence of h igh m oisture content can be deadly.

2. Seed dam age can occur during m e ch anical

separations.

3. Good training of w ork e rs is essential.

4. Extraction strate gy depends on th e type of

fruit involved.

D. Multiseed Fruits

Multiseed fruits include pods; m oist, flesh y

fruits; and cones and capsules. Each type

requires diffe rent ste ps to e xtract th e seeds:

1. Pod s

a. Dry th e fruits.

b . Th resh m anually by:

(1) Flailing w ith poles

(2) Crush ing by tram pling

(3) H itting w ith h e avy m alle ts

c. Th resh m e ch anically by:

(1) Slow , rotating drum s (cemen t m ixe r)

(2) CSIRO flailing th resh e r

(3) Dybvig m ace rator

(4) H am m e r m ills or oth e r flailing

devices

d. Use a series of ste ps for difficult spe cies.

2. M oist, fle sh y fruits

a. Start quick ly to avoid fe rm entation.

b . Soak in w ate r.

c. Extract w ith m ace rators, m ixe rs, coffe e

d e pulpe rs (Gm elina arborea), fe e d

grinders, h am m e r m ills, e tc.

d. Run sm all fruits (e .g., Rubus and

Moru s) in blende rs at slow speeds w ith

lots of w ate r.

3.

e . Use w ate r e xtraction w ith a h igh -

pressure stre am .

Cone s, capsule s, and oth e r m ultiple

fruits

a. Air-dry on flat surfaces.

(1) Canvas is best for large quantities.

(2) Screen trays are good for sm alle r

lots, such as single-tree colle ctions.

(3) Plastic sh e e ts are not strong enough

for Pinus cones (1 h L of con es

equals 35 k g).

(4) Prote ct th e drying fruits from rain

and predators; spre ad th in and stir

frequently.

(5) Dry som e spe cies under sh ade (e.g.,

H opea spp., Triploch iton sch elroxylon, and Pinus oocarpa).

b . Use solar k ilns.

(1) Sim ple type (cle ar polyeth ylene

stre tch ed ove r a fram e )

(2) Solar h e at storage units (m ore

soph isticated)

c. Use h e ated k ilns for large quantities of

cones; som e types are:

(1) Progressive (cone containe rs are

m oved along a gradient of incre asing

te m pe rature )

(2) Large b atch (large, h e ated ch am be rs

in w h ich trays h old cones and rotate

positions)

(3) Sm all batch (w ire-bottom draw e rs

th at h old about one-th ird h L of

cones each )

(4) Stack trays (w ooden trays w ith pe rforated sh e e t-m e tal bottom s, not

w ire screens, in stack s of six w ith

eigh t stack s h e ated w ith one h e ating

syste m )

(5) Tum ble r drie rs (cylindrical batch

k ilns th at rotate w h ile drying)

(6) Oth e r batch k ilns (m any local

designs available )

d. Se t te m pe rature and h um idity param -

e te rs. Th e obje ct is to re m ove m oisture ;

h igh te m pe ratures cre ate a gre ate r

vapor pressure gradient. Som e re com -

m ended param e te rs are 29 to 50 “C for

conife rs, and 8 h ours at 60 “C for

Eucalyptus saligna. Use a 15-second dip

in boiling w ate r for serotinous cones to

m e lt th e resin before placing th em in th e

k iln.

e. Extract th e seeds afte r th e cones are

open w ith :

(1) Tum ble r drie rs

(2) Cemen t m ixe rs

(3) H om e m ade tum ble rs

28
II. Cle aning and Upgrading

E. Single-Seed Fruits

Single-seed fruits include drupes (e.g., Prunus

and V& is) and nuts.

1. For drupes or oth e r flesh y fruits, use m ace rators, m ixe rs, e tc.

2. For nuts w ith h usk s, use m ace rators or

h and rubbing.

F. Sources

For additional inform ation, see W illan 19 85, p.

87-111.

A. Introduction

Cle aning seedlots is a basic ste p in prope r seed

utiliz ation. Cle aning sh ould re m ove w ings or

oth e r seed appendages, e m pty seeds, dam aged

seeds, and nonseed trash . Th is cle aning sh ould

also provide dram atic decre ases in insect and

dise ase proble m s. M any seedlots can be upgraded by re m oving im m ature, dam aged, and

dead seeds afte r th e initial cle aning. M any people view large me ch anical ope rations as th e only

w ay to cle an and upgrade seedlots, but seedlot

quality can be im proved w ith sim ple equipm ent

and te ch niques.

B. Obje ctives

1. Le arn th e advantages of cle aned and

upgraded seedlots.

2. Be com e fam iliar w ith th e principles of seedcle aning equipm ent and te ch niques.

3. Apply th ese principles w h en seed cle aning

and upgrading are planned.

C. Key Points

Th e follow ing points are essential for seed

cle aning and upgrading:

1.

2.

3.

4.

5.

6.

Liquid flotation can be an essential aid for

m any species, espe cially re calcitrant ones.

Screen cle aning is th e basic seed-cle aning

m e th od.

Air se paration, including w innow ing, is a

valuable te ch nique.

Cle aning sm all lots for testing or research

m ay be ve ry diffe rent from cle aning large

lots.

Upgrading seedlots offe rs potential im -

provemen ts in eigh t are as.

Seed sizing can be useful for som e spe cies

or sources but not for oth e rs.

D. Cle aning

1. Flotation- Th e sim plest m e th od of all.

a. Initial m oisture content is crucial.

b . Orth odox seeds are redried afte r flotation, but re calcitrant seeds are not.

c. Flotation

(1) Rem oves ligh t trash .

2.

3.

4.

5.

6.

(2) Rem oves m any em pty, b rok en , dise ased, or insect-dam aged seeds.

(3) Is ve ry good for large seeds w ith

h igh m oisture contents.

Aspirators-Any m ach ine th at uses air to

cle an and separate:

a. Large-scale m ach ines in seed plants

b . Sm all-lot cle ane rs for testing laboratories and research

Som e types are:

(1) Gene ral ER

(2) South Dak ota

(3) stu1ts

(4) Barnes

(5) Oth e r m odels as described by W illan

19 85

(6) H om e m ade fan devices

(7) Carte r Day Duo Aspirator

Scre e ns and sie ve s - Th e re are tw o types

of screen ing devices:

a. H and screens

b . Mech anical screen cle ane rs

Airscre e n cle ane rs - Th ese are th e basic

seed-cle aning m ach ines in m ost seed plants.

Th ey com bine aspiration and screen ing.

Even sm all m odels can cle an 30 to 40 k g of

sm all seeds per h our. Im portant principles

to remembe r are:

a. Th ey pe rform th ree functions:

(1) Scalping

(2) Sizing

(3) Aspiration

b . Th ey are m ore e fficient as cle ane rs, not

as siz e rs.

Ele ctrostatic cle ane rs - Th e H e lm uth

m ach ine is good for ve ry sm all seeds.

De w inging- A spe cial type of cle aning

th at reduces storage volum e, m ak es upgrading possible, m ak es sow ing e asie r, and

re m oves path ogens. Th e re are tw o basic

m e th ods of dew inging - we t and dry.

a. Th e dry m e th od is re com m ended for

tough seeds only because of th e dam age

potential to th in seed coats. Dry dew inge rs include:

(1) Popcorn polish e rs

(2) M issoula Equipm ent Deve lopm ent

Cente r de w inge r

(3) Dybvig

(4) Ele ctric drum

(5) New conife r dry de w inge rs

b . Th e we t m e th od is usually pre fe rred for

conife rs. W e t dew inge rs include:

(1)

(2)

(3)

(4)

Cemen t m ixe rs -

Com m e rcial dew inge rs

Kitch en blende rs (for sm all lots)

Any cylinder w ith gentle agitation

29
E. Upgrading

1. Upgrading is im proving th e potential pe rform ance of a seedlot by re m oving e m pty,

dam aged, w e ak , im m ature, or odd-sized

seeds.

2. Upgrading w ill:

a. Rem ove we ak seeds

b . Rem ove e m pty seeds

c. Reduce ch ances of insect and disease

dam age

d. Im prove control of density in nursery

e. Reduce planting tim e

f Facilitate nursery ope rations

g. Reduce costs and im prove uniform ity

h . Reduce storage space requiremen ts

3. M eth ods and equipm ent

a. Spe cific gravity by flotation uses:

(1) W ate r for som e Pinus, Quercus, and

oth e r large seeds

(2) Organic solvents (usually alcoh ols)

of diffe rent densities for som e sm all

seeds

b . Air-screen cle ane rs:

(1) Se parate by th ree ph ysical prope rties

(2) Upgrade by sizing or by re m oving

e m pties w ith air

(3) Regulate screen patte rn, feed rate,

airflow , screen oscillation (pulleys),

and screen pitch (in som e m odels)

c. Air se parators include:

(1) Large air-colum n separators

(2) Fractionating aspirators

(3) Sm all laboratory blow e rs

d. Gravity separators w e re originally built

to re m ove ore from clay. Th ey:

(1) Can separate seeds of th e sam e size

and diffe rent densities or diffe rent

sizes and th e sam e density.

(2) Are w ide ly used on conife r seeds in

North Am e rica.

(3) R e gulate feed rate , air stre am

th rough deck , deck pitch (side and

end), and eccentric th rust.

e. Ele ctrostatic separators cre ate a ch arge

th at adh e res to th e seed surfaces. Mode ls include:

(1) H e lm uth cle ane r for Eucalyptus and

conife rs of th e We ste rn Unite d

States.

(2) Static e le ctricity for ve ry sm all

seeds. Th e sides of a plastic beak e r

are w iped w ith a nylon cloth .

f. Radiograph y is used for valuable research

lots only.

g. Color separators re m ove ligh t-colored

seeds.

h . Incubation , drying, and separation

(IDS) m e th od-A new m e th od from

Sw e den th at is used on Pinus and Picea.

4. Sizing h e lps w ith som e spe cies or seedlots,

but not w ith oth e rs; e.g., single fam ily seedlots.

G. Sources

For additional inform ation, see Bonne r 19 87b;

Doran and oth e rs 19 83, ch ap. 5; W illan 19 85, p.

87-128.

III. Storage Principle s

A. Introduction

Th e prim ary purpose of storing seeds is to h ave

a viable seed supply w h en it is needed for

re gene ration. Successful storage of w oody plant

seeds m ust be care fully planned, and good planning depends on an understanding of th e purposes of storage, of seed dete rioration, and of

th e e ffe cts of th e storage environm ent on th e

dete rioration processes.

B. Obje ctives

1. Le arn th e obje ctives and rationale of seed

storage .

2. Identify factors th at affe ct seed longe vity in

storage .

3. Review th e gene ral process of seed dete rioration .

C. Key Points

Th e follow ing points are essential to understanding seed storage principles:

1. Longe vity of seeds is a species ch aracte ristic.

2. Prestorage factors affe ct longevity in storage .

3. Th e m ost im portant factors in storage are

seed m oisture content and te m pe rature.

4. Seed dete rioration begins at abscission and

involves com ple x ph ysiological ch anges.

D. Obje ctive of Storage

Th e obje ctive of storage is to delay de te rioration

or decre ase its rate until seeds are used.

E. Rationale for Storage

Storage m ay be sh ort- or long-te rm ; it m ay be

e xtended for long pe riods for ge rm plasm conse rvation .

1. Sh ort-te rm storage :

a. Is used for im m ediate ope rations

b . Typically lasts less th an 5 years

c. Allow s carry-ove r of surplus production

d. Requires m inim um storage space requirem ents

2. Long-te rm storage :

a. Typically lasts from 5 to 10 ye ars

b . Ensures constant seed supply

30
c. Saves special lots th at w ill not be colle cted annually

cl. Requires ve ry good storage environm ents

3. Ge rm plasm conse rvation

a. If storage is planned for 50 ye ars or

m ore

b . Requires th e ve ry best storage environm ent

F. Longevity in Storage

M any factors affe ct seed longe vity in storage:

1. Seed ch aracte ristics

a. Basic ph ysiology

(1) Orth odox seeds are tole rant of desiccation to low m oisture contents.

(2) Recalcitrant seeds are intole rant of

desiccation .

b .

C.

d.

Seed structure-Th ick or h ard seedcoats restrict m oisture uptak e and gas

e xch ange .

Seed ch e m istry - Oily seeds tend to be

h arde r to store th an starch y seeds.

Stage of m aturity- Im m ature seeds

usually w ill not store as w e ll as fully

m ature seeds.

e . Environm e n tal stress-Stress during

m aturation can affe ct longe vity.

2. Seed h andling before storage

a. Ph ysiological m istre atm ent can dam age

storage potential.

b . Processing dam age w ill low e r seed quality.

3. Genetics-Good seed quality is inh e rited

to som e degre e.

4. Storage e nvironm e nt

a. M oisture content

(1)

(2)

(3)

(4)

M oisture content is th e m ost im portant factor.

Potential dam age th resh olds are

outlined in table 5.

Th e best range for orth odox seeds is

5 to 10 pe rcent.

Th e best range for re calcitrant

seeds is full im b ib ition.

Table 5. -Moisture conte nt th resh olds and pote ntial effects on store d

seeds

M oisture

conte n t Effe cts

Perce nt

> 30

18 to 20

10 to 18

> 9

5 to 8

<5

Ge rm ination begins

Ove rh e ating from respiration

Seed fungi b ecome active

Insect activity

Best range for sealed storage

Desiccation dam age possible in some species

Table 6.-Equilibrium m oisture conte nts at 4 to 5 “C and th ree

relative h um id ities (Bonner 19 81b, Ju stice and Bass 19 78)

Relative h um iditv

Spe cies

__ ___________ percent -------------

20 45 :i -

Orth odox trees

Carya ovata

Juglans nigra

Liquidam bar styraciflua

Liriodend ron tulipifera

Picea obies

Pinus sylve stris

I? taeda

Prunus serotina

Orth odox crops

Glycine m ax

Z ea m ays

Recalcitrant trees

Quercus alba

Q. nigra

Sh orea robusta

Moisture content

___________-- perce n t ____ .__~ :. .

I

10 f

11 .,:i

8 20

10 19

6 8

6 8

10 17

9 17

6 8 19

8 12 20

. . . 37 50

13 29

35

*Data not available.

(5) Th e equilib rium m oisture content is

defined as th e seed m oisture content

w h en seed m oisture is in equilibrium w ith th e m oisture in th e

storage atm osph e re (table 6). Equilib rium m oisture content:

(a)

(b)

(cl

is influenced by seed ch e m istry

(fig. 9 )

is rare ly re ach ed w ith re calcitrant seeds

h as sorption and desorption diffe rences

b . Te m pe rature

(1) Gene rally, th e coole r th e seeds, th e

slow e r th e dete rioration rate.

(2) Th e s afe te m pe rature range for

orth odox seeds is re lated to th e

m oisture content of th e seeds:

(a) Orth odox seeds at 5- to lope rcent m oisture can be stored

at m ost te m pe ratures.

(b) Be tween 50 and 0 “C, eve ry 5 “C

low e ring of storage te m pe rature

doubles th e life of th e seeds

(H arrington 19 72).

(3) Th e safe te m pe rature ranges for

re calcitrant seeds are:

(a) Te m pe rate Z one spe cies: - 1 to

3 “C.

(b) Tropical spe cies: usually above

12 to 15 “C.

31
, 4 5 6 0 75 9 0

RELATIVE HUMIDITY (PERCENT)

- -- - Quercus muehlenbergit

- Quercus shumardli

------ Q u e rcus nigr a

,r

I’

,------

/’

Figure 9 .-Equilibrium m oisture conte nt at 25 “C for four

recalcitrant Quercus species (adapted from W illan

19 85).

c. Storage atm osph e re

(1) If oxygen le ve ls are reduced, m e tabolism is slow e d, w h ich can incre ase

longe vity.

(2) Ine rt gases offe r no advantage in

long-te rm storage, but th ey m ay

h e lp in sh ort-te rm storage.

(3) In sealed containe rs, th e COz/O,

ratio ch anges.

G. Ce lls and Tissues During Seed Aging

Th e follow ing ch anges occur in ce lls and tissues

during aging:

1. Loss of food rese rves

2. Accum ulation of m e tabolic byproducts

3. Irreve rsible enzym e deactivation

4. De te rioration of ce ll m emb ranes

5. Lipid pe roxidation

6. Alte rations of DNA

H . Sources

For additional inform ation, see Bonne r and

Voz z o 19 9 0; H arrington 19 72; Justice and Bass

19 78; Tang and Tam ari 19 73; W illan 19 85, p.

129 -160.

IV. Storage Applications

A. Introduction

Th e pre vious section introduced th e principles

and critical factors th at influence seed longevity. Th is section discusses h ow th ese principles

are applied in practice to store tree seeds.

B. Obje ctives

1. Relate seed storage principles to prescriptions for e ach spe cies group.

2. Le arn fe atures of cold storage units.

3. Discuss storage constants and th eir application.

4. Le arn basic principles of seed m anagemen t

in storage.

C. Key Points

Th e follow ing points are essential to storage

applications:

1. Th e re are four classes of seed storage

b eh avior.

2. Cold storage is best but not alw ays necessary for successful seed storage.

3. Each spe cies, and perh aps individual populations w ith in a species, w ill nearly alw ays

respond identically to a given type of storage conditions.

4. Good facilities and good seeds are n ot

enough ; good m anagemen t is essential for

optim um seed storage ope rations.

D. Seed Storage Classes

Th e re are four classes of tree seed storage

beh avior (table 7):

1. ‘lk ue orth odox

a. True orth odox seeds are tole rant of

desiccation (table 8) and:

(1) Can be dried to m oisture leve ls of 5

to 10 pe rcent.

(2) Can be stored at subfreezing te m -

pe ratures.

(3) Are e asily stored for at le ast one

rotation.

(4) H ave gene rally unk now n uppe r

lim its of storage.

b . Exam ples include m ost of th e valuable

te m pe rate gene ra (Pinus, Picea, Betula,

Prunus) and m any tropical gene ra

(Acacia, Eucalyptu s, and Cusuarina).

2. Suborth odox

a. Suborth odox seeds are sim ilar to true

orth odox seeds but are lim ited to sh orte r

pe riods (table 9 ).

(1) Th ey are stored under th e sam e conditions as true orth odox seeds.

(2) Th ey are lim ited in storage potential

because of h igh lipid contents, th in

seedcoats, or genetic m ak eup.

b . Exam ples include th ose w ith h igh lipid

content (Juglans, Carya, som e Abies,

and Pinus), th ose w ith th in seedcoats

(Populu s and Salk ), and som e wh ose

genetic m ak eup requires slow drying

(Fagu s and Citrus).

32
Table I. -Storage conditions for four storage classes of tree seeds

Storage

class

True orth odox

Suborth odox

Storage Se e d

pe riod m oisture Te m pe rature Container type

Years Perce nt “C

15 6-10 o- 5 Airtigh t

=-5 6-10 - 18 Airtigh t

<5 6-10 o- 5 Airtigh t

> 5 6-10 - 1 8 Airtigh t

Te m pe rate

re calcitrant <3 30-45 -1 to- 3 4-m il’ plastic, unsealed

Tropical

re calcitrant <l 30-45 12-20 4-m il plastic, unsealed

‘m il = l/1,000 inch = 0.025 m m .

Table 8. -Storage te st results for true orth od ox species (adapted from Bonner 19 9 0)

Snecies

Storage conditions Storage results

Se e d Tim e Viability

Temne rature m oisture store d loss

Abies procera

Acacia leptopetala

A. m angium

A. pruinocarpa

Acer sacch arum

Albizia falcataria

Araucaria cunningh am ii

A. cunningh am ii

Casuarina equisetifolia

C. torulosa

Liquidam bar styraciflua

Pinus caribaea

var. h ondurensis

l? elliottii

I? m erk usii

I? ponderosa

Tectona gran d is

Ts uga h eteroph ylla

T. h eteroph ylla

“C

0

20-25

4-8

20-25

- 10

4-8

- 15

19

- 3

20-25

3

8

4

4-5

0

O-4

5

- 1 8

Perce n t

9

10

16-23

I

6-16

8-12

5-10

10

<8

8

- 1 2

8

8

Years Perce nt

7.0 11

18.0 1

1.2 6

16.0 20

5.5 5

1.5 10

8.0 few+

0.1 0

2.0 o- 5

18.0 6

9 .0 3

2.7 + 16

50.0 30

4.0 0

7.0 0

7.0 0

2.0 0

2.0 0

*Data not available.

+ Exact value not available from original source.

Table 9 . -Storage te st results for suborth od ox species (adapted from

Bonner 19 9 0)

Spe cies

Storage conditions Storage results

Se e d Tim e Viability

Te m pe rature m oisture stored loss

Citrus lim on

Fagus sylvatica

Gm elina arborea

Populus deltoides

Salix glauca

“C

- 2 0

- 10

- 5

- 2 0

- 10

Perce nt Years Perce nt

o 0.9 *5

10 5.0 34

6-10 2.0 10

6-10 6.0 21

6-10 1.2 0

33
3. Te m pe rate re calcitrant

a. Te m pe rate re calcitrant seeds are intole rant of desiccation (table 10)

(1)

(2)

(3)

Th ey cannot be dried below 20- to

30-pe rcent m oisture ; th e re fore, storage m ust be above freezing.

Th ey h ave me tabolism s so rapid

th at pre ge rm ination com m on ly

occurs in storage.

Th ey cannot be stored in airtigh t

containe rs; th e re m ust be som e gas

e xch ange (table 7).

4.

b . Exam ples include Quercus and Ae sculu s.

Tk opical re calcitrant

a. Tropical re calcitrant seeds are th e sam e

as te m pe rate re calcitrant seeds but are

sensitive to low storage te m pe ratures

(table 11). Th ey e xpe rie n ce ch illing

dam age and death below 12 to 20 “C.

b . Th ey are th e m ost difficult group of all

to store.

c. Exam ples include Sh orea, H opea, and

Dipterocarpus, and even som e le gum es

(F’ith ocellob ium spp. in Costa Rica).

Table 10. -Storage te st results for tem perate recalcitrant species

(ad apted from Bonner 19 9 0)

Soe cies

Storage conditions Storage results

Se e d Tim e Viability

Te m oe rature m oisture store d loss

“C Perce nt M onth s Perce nt

Acer sacch arinurn - 3 50 18 8

Quercus falcata var.

pagod a efolia 3 35 30 6

Q. robur - 1 40-45 29 31-61

Q. rubra -1to - 3 384 5 17 18-46

Q. virginiana 2

I

12 35

*Data not available.

Table ll.-Storage te st results for tropical recalcitrant species

(ad apted from Bonner 19 9 0)

Spe cies

Araucaria

h unsteinii

A. h unsteinii

Azadirach ta

indica

H opea h elferi

Sh orea robusta

S. roxburgh ii

Storage conditions Storage results

Seed

Te m pe rature m oisture

“C Perce nt

19 .0 25-30 54 k 30

2.0 30 365 82

26.0 10-18 56 65

15.0 47 37 2

13.5 40-50 30 60

16.0 40 270 k 30

Tim e Viability

stored loss

Days Perce nt

E.

F.

Cryogen ic Storage

Cryogen ic storage is a m e th od for ve ry longte rm storage for ge rm plasm conse rvation (table

12).

1. Te ch nique s

a.

b .

C.

Pack ages are im m e rsed in liquid nitrogen ( - 19 6 “C) or suspended above it in

th e vapor.

It h as potential for sm all quantities

only.

M axim um tim e lim its are not k now n.

Only a few tests h ave been m ade on tre e

seeds.

2. Costs-Costs are com parable w ith conventional storage in som e cases.

Ph ysical Facilities

1. Cold storage units

Cold storage units require a re liable

pow e r source, sh ould not be built w h e re

floods or e arth quak es are lik e ly, sh ould

be located near oth e r seed activities,

sh ould be rodent proof, and sh ould be on

h igh e levations w h en possible because

am b ient te m pe ratures w ill be coole r.

Units sh ould be built to h old a & year

supply.

a.

b .

C.

d.

e.

f.

g .

h .

i.

For ge rm plasm conservation, about 1

lite r is needed of e ach sam ple ; e.g., 85

m s sh ould h old 22,800 sam ples.

H um idity control is not re com m ended in

th e Tropics.

Dire ct or indire ct vapor-com pression

re frige ration is re com m ended.

Standby gene rators are needed .

Th e rm al tim e constants of 4 to 5 days

sh ould apply in large coole rs.

M odular panel units are e ffe ctive.

Insulation depends on am bient conditions.

Table 12. -Storage te st results for cryogenic trials of forest tree seeds

(ad apted from Bonner 19 9 0)

Spe cies

Abies alba

A. concolor

Fagus sylvatica

Lark decidua

Picea abies

Pinus ech inota

I? ponderosa

P. sylve stris

Populu s trem ula X

trem uloides

Ulm u s pum ila

Seed Tim e Viability

m oisture stored loss

Perce nt Days Perce nt

*

6 5

cl3 180 0

6 100

6 5

6 1

112 0

Cl3 180 0

6 0

. 6 1

. . . 112 0

*Data not available.

34
2. Containe rs

a. Fiber drum s

b . Rigid plastic containe rs, w h ich are bette r th an glass

c. Rectangular containe rs

d. Plastic bags

3. M oisture m anageme nt

a. Seeds w ill re ach an equilibrium m oisture content w h en e xposed to th e storage atm osph e re.

b . W ith h um idity control (50 to 60 pe rcent

re lative h um idity), orth odox seeds need

not be sealed. Re calcitrant seeds cannot

be sealed, so th ey cannot be stored in

such a unit.

c. W ith out h um idity control (> 9 5 pe rcent

re lative h um idity), re calcitrant seeds

store we ll. Orth odox seeds m ust be dried

and stored in sealed containe rs.

d. H um idity controls are not re com m ended

for th e Tropics.

e. Frost-free re frige rators are an inexpensive alte rnative for sm all quantities.

G. Genetic Dam age in Long-te rm Storage

1. W ould be devastating to seeds stored for

ge rm plasm conservation, but th e re is no

strong e vidence to date of lasting dam age.

2. Could cause ch anges in th e population.

H . Retesting in Long-te rm Storage

For re testing in long-te rm storage:

1. Use ISTA rules or com parable procedures.

2. Use th e follow ing test inte rval for orth odox

seeds: initial year, th ird ye ar, and eve ry

fifth year th e re afte r.

3. Ensure nondestructive testing.

4. Regene rate when viability falls to 50 pe rce n t.

I. Viability Constants in Storage

1. Th e ory-Viability re te n tion w ill be th e

sam e for a given spe cies under a given set of

storage conditions.

2. Practice

a. Results are good w ith som e agricultural

seeds.

b . Varie ties of a single spe cies m ay diffe r.

c. One m ust start w ith ve ry good seeds.

d. Th e re are few data for tree seeds.

3. Viability constants-If valid, th ey could

be ve ry use ful in planning long-te rm storage for ge rm plasm conse rvation.

J. Sources

For additional inform ation, see Bonne r and

Voz z o 19 9 0; Ch in and Robe rts 19 80; H arrington 19 72; Inte rnational Board for Plant

Genetic Resources 19 76; Justice and Bass 19 78;

Robe rts 19 73; Tang and Tam ari 19 73; W illan

19 85, p. 129 -160.

35

Evaluating Quality
I. Sam pling

Table 13. -W eigh ts of lots and sam ples for sh rubs and trees (ISTA

19 85)

A. Introduction

B.

C.

D.

Sam pling is th e process of tak ing a sm all part

or quantity of som e th ing for testing or analysis;

it is th e first ste p in seed testing. In sam pling, it

is essential to obtain: (1) a sam ple of prope r siz e

and (2) a sam ple re presentative of th e m ain

seedlot. Th e results of th e laboratory tests can

only sh ow th e quality and ch aracte ristics of th e

sam ple subm itted for th e analysis; th e re fore,

th e validity of test results for a large seedlot is

dete rm ined by th e success of obtaining a re presentative sam ple. Sam pling seedlots for quality

evaluation m ust be done syste m atically, using

appropriate te ch niques, tools, and procedures,

to ensure th at th e seed sam ple re presents th e

entire lot.

Obje ctives

1. Quantify a seedlot according to acce pted

standards,

2. De te rm ine sam pling intensity according to

size and ch aracte ristics of th e seedlot.

3. Le arn appropriate sam pling instrum ents

and te ch niques according to re cognized

st,andards.

Key Points

Th e follow ing points are essential in seed

sam pling:

1.

2.

3.

4.

Laboratories can only m e asure th e prope rties of th e sam ple ; th e sam ple r m ust ensure

th at th e sam ple truly re presents th e seedlot.

Subm itted sam ples sh ould contain at le ast

2,500 seeds (e xce pt for ve ry large seeds of

ce rtain species).

Draw ing th e sam ple m ust be com ple te ly

random .

Prope r pack aging and labeling of th e sam -

ple are essential

De finition of Te rm s

Rele vant te rm s are defined as follow s:

1.

2.

3.

4.

5.

6.

Lot-a spe cified , ph ysically ide n tifiable

quantity of seeds.

Prim ary sam ple -a sm all quantity of

seeds tak en from one point in a seedlot

Com posite sam ple - form ed by com bining and m ixing all th e prim ary sam ples

tak en from a seedlot

Subm itted sam ple-th e sam ple subm itted to th e testing laboratory

W ork ing sam ple -a subsam ple tak e n

from th e subm itted sam ple in th e laboratory

Subsam ple - a portion of a sam ple

obtained by reducing th e sam ple by re cognized m e th ods (table 13).

Soe cies

Maxim um W ork ing sam ple

w e igh t of Subm itte d for purity

seedlot sam nle analysis

Acacia spp.

Ailanth u s altissim a

Alnus rubra

Castanea sativa

Ce d rela spp.

Eucalyptu s

cam ald ulensis

E. glob ulu s

E. tereticornis

M orus spp.

Pinus h alepensis

P. w allich iana

Quercus spp.

Robinia

pseudoacacia

K ilogram s Gram s Gram s

1,000 70 35

1,000 160 80

1,000 15 2

5,000 500 seeds 500 seeds

1,000 80 40

1,000 15 5

1,000 60 20

1,000 15 5

1,000 20 5

1,000 100 50

1,000 250 125

5,000 500 seeds 500 seeds

1,000 100 50

E. Sam pling Intensity

A sam ple is obtained by sele cting sm all portions

at random from various positions in a seedlot

and com b ining th e m .

1. Calculating prim ary sam ple s - Each

com posite sam ple m ust be m ade up of at

le ast five prim ary sam ples.

2. Se e dlot size-For inte rnational trade in

tree seeds, a m axim um size of a seedlot for

m ost spe cies h as been set at 1,000 k g ?5

pe rcent (table 13).

F. Sam pling Procedures

Th e re are th ree com m on sam pling tools or te ch -

niques:

1. ‘IYie rs are used for free-flow ing seeds. Th e

ste ps are:

a. Close th e gates before inse rting th e trie r

into th e drum .

b . Insert th e trie r into th e drum .

c. Open th e gates.

d. Close th e gates.

e. Rem ove th e trie r.

f. Dum p th e seeds.

2. Soil divide rs are used prim arily for sm all

lots. Th e ste ps are:

a. Pour th e seeds th rough th e divide r seve ral tim es for m ixing.

b . Divide th e sam ple into h alves, quarte rs,

e tc.

3. Extended h and m e th od is used for

ch affy, w inged, or oth e r nonflow ing seeds.

Th e ste ps are:

a. Extend th e finge rs, and insert th e h and

straigh t into th e seeds.

b . Close th e h and, and w ith draw a prim ary

sam ple.

38
G. Pre paration of th e Sam ple

1. Com posite sam ple -All prim ary sam ples

are com b ined and m ixed (table 13).

2. W ork ing sam ple-Th e subm itted sam ple

is reduced to a w ork ing sam ple by:

a. Me ch anical divide r m e th od

b . Random cups m e th od

c. Modified h alving m e th od

d. Spoon m e th od

e. Manual h alving m e th od

3. Extra seeds-Th e re m ainde r of th e subm itted sam ple sh ould be stored to pe rm it

re testing if necessary. Inte rnational Seed

Testing Association (19 85) re com m ends

storing for 1 ye ar.

H . Sources

For additional inform ation, see Association of

Official Seed Analysts 19 88, Edw ards 19 87,

Inte rnational Seed Testing Association 19 85.

II. Moisture Content

A.

B.

C.

D.

Introduction

Th e first m e asuremen ts tak en in seed testing

are m oisture, purity, and w e igh t. All of th ese

m e asuremen ts are im portant, but m oisture is

th e m ost critical one. Seed m oisture leve ls can

influence or indicate seed m aturity, longe vity in

storage , and th e am ount of pre tre atm e n t

needed for rapid ge rm ination.

Obje ctives

1. Le arn th e principles of official seed testing

for m oisture.

2. Apply th ese principles in practical e xe rcises.

Key Points

Th e follow ing points are essential to testing for

m oisture content:

1.

2.

3.

Official testing procedures are prescribed in

detail.

M any tests m ay be unofficial, and diffe rent

m e th ods m ay be used, but accuracy and

pre cision are still essential.

Large re calcitrant seeds present spe cial

proble m s th at official testing rules h ave not

yet adequate ly addressed.

De finition of Te rm s

Rele vant te rm s are defined as follow s:

1.

2.

3.

Sam ple, subm itted-th e sam ple of seeds

subm itted to a seed-testing station; it

sh ould be tw ice th e size of th e w ork ing

sam ple.

Sam ple, w ork ing- a reduced seed sam ple

tak en from th e subm itted sam ple in th e

laboratory

Se e dIot -a spe cified quantity of seeds of

re asonably uniform quality

E. M oisture Me asuremen ts

1. Im portan ce

a. Is th e m ost im portant factor in viab ility

re tention.

b . Controls insect and disease activity

(table 5).

c. Affe cts th e re lationsh ip of w e igh t to

num be r of seeds.

2. Frequen cy-Moisture is m e asured :

a. Afte r e xtracting and cle aning.

b . W h en seeds are placed in storage.

c. Pe riodically during storage.

d. W h en seedlots are sh ipped.

3. Procedures -Accurate results are ensured

by:

a. Using th e subm itted sam ple.

b . Measuring im m ediate ly on re ceipt.

c. Expressing results as a pe rcentage of

fresh we igh t (w e t w e igh t), not dry w e igh t.

4. M e th ods-Moisture conte n t can be m e asured by four m e th ods:

a. Ovendrying m e th od- Critical points are:

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9 )

H e at sam ples for 17 + 1 h ours at

103 t2 “C.

Use forced-draft ovens.

Place sam ples in glass or m e tal containe rs.

Le ave space between cans in th e

ove n .

Cool th e sam ples in desiccators.

Kee p am b ient h um idity less th an 70

pe rcent in th e laboratory if possible.

W eigh to th e ne arest m illigram .

Grind or cut large seeds or seeds of

h igh m oisture content.

Predry if seed m oisture e xceeds 17

pe rce n t in seeds th at m ust b e

ground, or 30 pe rcent in oth e r species.

(10) Ch e ck tole rance on results. M oisture tole rances for tree seeds are

m ore liberal th an th ose for agricultural seeds (table 14).

Table 14. -Tole rance levels for d ifferences betw een tw o d eterm ination s

of m oisture conte nt of tree and sh rub seeds (ISTA 19 85)

Seed size Seeds per

class k iloaam Initial m oisture Tole rance

Sm all seeds

Sm all seeds

Large seeds

Large seeds

Large seeds

N um b e r

> 5,000

> 5,000

<5,000

<5,000

<5,000

______________ perce nt ---------------

Cl2 0.3

> 12 0.5

Cl2 0.4

12-25 0.8

> 25 2.5

39
b . Ele ctric m e te rs:

(1)

(2)

(3)

(4)

Are not allow e d for official ISTA

tests but are ve ry use ful.

Are b ased on ele ctrical resistance or

capacitance and are accurate to

w ith in I+ _ 1 pe rcent on free-flow ing

seeds.

Require construction of calibration

ch arts.

Are available in various m odels:

(a)

(b)

Cc)

Cd)

M otom co - based on capacitance

and ve ry accurate

Radson (Dole or Seedburo) -a

re liable m odel in th e United

States

Dick ey-Joh n or Insto- based on

capacitance

Supe r-Beh a - w ide ly used in

Europe

c. Infrared instrum ents are sm all, infrared ovens w ith built-in balances, w h ich

use a gravim e tric m e th od based on drying tim e.

F.

G.

d. Laboratory m e th ods for research :

(1) Karl Fisch e r m e th od

(2) Toluene distillation

(3) Nucle ar m agnetic resonance (nondestructive )

(4) Infrared spectroscopy

Sum m ary-See table 15.

Sources

For additional inform ation, see Bonne r 19 81b;

Inte rnational Seed Testing Association 19 85,

sections 9 , 9 A, W illan 19 85, p. 227-230.

III. Purity and W eigh t

A. Introduction

Afte r m oisture content h as been dete rm ined,

th e subm itted sam ple is re ady for purity and

w e igh t dete rm inations. Th ese dete rm inations

are a vital part of official seed testing and practical seed use, w ith le gal ram ifications in both

dom estic and inte rnational seed trade.

Table 15.-Suggested test procedures for tree seed m oisture (Banner 19 81b)

Seed size class

Accurate me asuremen t or

ISTA official test Rapid estim ate

Sm all seeds, low oil content

(e.g., P& anus, R obinia)

Sm all seeds, h igh oil

content (e.g., Abies,

Pinus, Tsuga,

Z anth oxylum )

Large seeds, low oil content,

m oisture <20% (e.g.,

Nyssa)

Large seeds, low oil content,

m oisture > 20%, (e.g.,

Aesculus, Quercus)

Large seeds, h igh oil

content (e.g., Carya,

Fagus, Juglans)

Oven : 103 k 2 “C for 17 k 1

h ours.

Sam ple: 4 to 5 g

Oven : 103 rt 2 “C for 17 2 1

h ours.

Sam ple: 4 to 5 g

or

Toluene distillation

(1)

(2)

(1)

(2)

(3)

(1)

(2)

Grind or equivalent

Oven : 103 k 2 “C for 17

+ 1 h ours.

Sam ple: 4 to 5 g or

enough to equal w e igh t

of five seeds

Predry to <20% at 130

“C for 5 to 10 m inutes

Grind or equivalent

Oven : 103 22 “C for 17

k 1 h ours.

Sam ple: enough to

equal w e igh t of five

seeds

Grind or equivalent

Oven : 103 k 2 “C for 17

? 1 h ours.

Sam ple: enough to

equal w e igh t of five

seeds

or

Toluene distillation

Ele ctric m e te r

Sam ple: 80 to 200 g,

depending on type

Ele ctric m e te r

Sam ple: 80 to 200 g,

depending on type

Microw ave drying

Sam ple: 4 to 5 g or enough

to equal w e igh t of five

seeds

Microw ave drying

Sam ple: enough to equal

w e igh t of iive seeds

Microw ave drying

Sam ple: enough to equal

w e igh t of five seeds

40
B. Obje ctives

1. Le arn th e principles of official seed testing

for purity and w e igh t.

2. Apply th ese principles in practical e xe rcises.

C. Key Points

Th e follow ing points are essential to dete rm ine

seed purity and w e igh t:

1. Th e line between true seeds and trash can

be am biguous for som e tree seeds, especially th ose th at are dew inged.

2. Patience and good eyesigh t are needed .

3. Th e sm alle r th e seeds, th e m ore difficult th e

purity test w ill be.

D. De finition of Te rm s

Rele vant te rm s are defined as follow s:

1. Purity-proportion of cle an , intact seeds

of th e designated species in a seedlot, usually expressed as a percentage by w e igh t

2. Sam ple, subm itted-th e sam ple of seeds

subm itted to a seed-testing station; it

sh ould be tw ice th e size of th e w ork ing

sam ple

3. Sam ple, w ork ing- a reduced seed sam ple

tak en from th e subm itted sam ple in th e

laboratory on w h ich som e test of seed quality is m ade

4. Se e dlot -a spe cified quantity of seeds of

re asonably uniform quality; m axim um lot

size is 1,000 k g (5,000 k g for Fugus and

large r seeds)

E. Purity

1. Procedu re - Th e ISTA (19 85) rules are

follow e d for purity testing. Th e ste ps are:

a. Reduce th e subm itted sam ple (afte r

m ixing) to th e w ork ing sam ple by:

(1) M e ch anical divide rs

(2) Random cups

(3) M odified h alving

(4) Spoon m e th od

(5) M anual h alving (ch affy, w inged ,

and large seeds)

b . Divide th e w ork ing sam ple into fractions

of

(1) Pure seeds

(2) Oth e r seeds

(3) Ine rt m atte r

c. W eigh and express each as a pe rcentage

of th e total sam ple we igh t

2. Pure seed com ponen t-Th is com ponen t

contains:

a. Intact seed units of th e desired species

b . Pie ces of seed units large r th an one-h alf

th e original siz e, e ven if th ey are brok en

3. Tree seed s pe cifics

a. Seeds of Le gum inosae, Cupressace ae,

Pinace ae, and Taxodiace ae w ith seedcoats entire ly re m oved are inert m atte r.

b .

C.

d.

e .

f.

g .

In Abies, Lark, Libocedrus, Pinus

elliottii, P. echinata, P. rigida, P. taeda,

and Pseudotsuga, w ings or w ing fragm ents are detach ed and re m oved and

placed in th e inert m atte r fraction.

Oth e r Pinus spp. re tain w ing fragm ents

(see “a” above ).

For sam aras, w ings are not re m oved

(e .g., Acer, Fraxinus, Cedrela, a n d

Swietenia).

For drupes, th e flesh y cove rings are not

re m ove d .

In Eucalyptus, for spe cies w ith sm all

seeds, a sim plified procedure is used;

only oth e r seeds and inert m atte r th at is

obviously of nonseed origin are rem oved.

For Le gum inosae, if any portion of th e

testa is present, it m ust be classified as

pure seed.

If spe cies distinctions are im possible,

only th e genus nam e is given on th e

ce rtificate.

F. Seed W eigh t

1. De te rm ination- Th e ISTA (19 85) rule s

are used to prope rly dete rm ine seed w e igh t.

Eith e r th e wh ole w ork ing sam ple or re plicates from it are used.

a. W ork ing sam ple-W eigh th e entire pure

seed fraction.

b . Replicates- Count and w e igh 8 re plicates of 100.

2. Reporting results - Results are re ported

in one of tw o w ays:

a. l,OOO-seed we igh t

b . Seeds pe r gram (or pe r k ilogram , ounce,

or pound)

G. Sources

For additional inform ation, see Inte rnational

Seed Testing Association 19 85, se ct. 3, 3A, 10;

W illan 19 85, p. 19 8-202, 221.

IV. Ge rm ination Te sts

A. Introduction

Good seed testing is th e corne rstone of any seed

program , no m atte r w h at k ind of seeds: agricultural, forestry, agroforestry, or ornam ental.

Th e quality of th e seeds used m ust be m e asured

and described. Seed testing m ay h ave le gal ram -

ifications because of its connection to seed sales.

For th is re ason, th e Inte rnational Seed Testing

Association (ISTA) coordinates inte rnational

e fforts to standardize seed testing. Th e quality

of seeds m ust be k now n to m ak e e fficient and

e ffe ctive use of th em in re forestation or

afforestation program s.

41
B. Obje ctives

1. Identify th e inte rnational organizations

th at deal in tree seed testing and h ow th ey

derive th eir prescriptions.

2. Le arn th e principles of ge rm ination testing

and h ow th ey are applied in th e laboratory

for standard conditions.

3. Practice actual ge rm ination testing in th e

laboratory.

4. Le arn proven te ch niques to analyze ge rm ination data and h ow th ese data can be

e xpressed .

5. Le arn th e application of ge rm ination test

results to practical nurse ry and fie ld conditions.

6. Le arn te ch niques for rapid estim ates of

seed quality w h en tim e and/or prope r facilities are absent or lim ited.

C. Key Points

Th e follow ing points are essential for conducting ge rm ination tests:

1. Laboratory ge rm ination tests are designed

to provide th e optim um conditions for ge rm ination and to dete rm ine th e full ge rm ination potential of th e seeds under th ese

conditions.

2. Th e prim ary conditions to be considered are

te m pe rature, ligh t, ae ration, and m oisture.

3. Rapid estim ates of ge rm ination are just

th at -estim ates; th ey are not as accurate as

ge rm ination tests.

4. If m ore th an 60 days are required for a

ge rm ination test, analysts sh ould use a

rapid estim ate for official testing.

5. Ge rm ination te s ting in th e course of

research m ay require diffe rent m e th ods and

equipm ent from official testing.

6. No m atte r h ow standardized th e test prescriptions are, th e judgm ent of th e analyst

m ust pre vail in th e laboratory.

D. De finition of Te rm s

Relevant te rm s in ge rm ination testing w ill be

defined according to th e glossary de ve loped by

th e Seed Proble m s Proje ct Group of th e Inte rnational Union of Forestry Research Organiz ations (IUFRO) (Bonne r 19 84a). Th ese te rm s

are defined as follow s:

1. Abnorm al seedlings - in seed testing,

seedlings th at do not possess all norm al

structures required for grow th , nor sh ow

th e capacity for continued deve lopm ent

2. Filled seed-a seed w ith all tissues essential for ge rm ination

3. Ge rm ination-re sum ption of active grow th

in an em b ryo, w h ich results in its em e rgence

from th e seed and deve lopm ent of th ose

structures essential to plant deve lopm ent

42

4.

5.

6.

7.

8.

9 .

Ge rm ination capacity-proportion of a

seed sam ple th at h as ge rm inated norm ally

in a specified test pe riod, usually expressed

as a pe rcentage (synonym : ge rm ination percentage )

Ge rm ination ene rgy-proportion of ge rm ination th at h as occurred up to th e tim e

of pe ak ge rm ination, th e tim e of m axim um

ge rm ination rate, or som e presele cted point,

usually 7 test days. (Th e critical tim e of m e asuremen t can be ch osen by seve ral m e ans.)

Ge rm ination pe rce ntage - (see ge rm ination capacity)

H ard se e ds - seeds th at re m ain h ard and

unge rm inated at th e end of a prescribed

test pe riod because th eir im pe rm e able seedcoats h ave pre vented absorption of w ate r

Pe ak ge rm ination-th e spe cific tim e

w h en rate of ge rm ination is h igh est. It m ay

be de rived in m any w ays (see ge rm ination

ene rgy).

Pre tre atm e n t-any k ind of tre atm e n t

applied to seeds to ove rcom e dorm ancy and

h asten ge rm ination

10. Purity-proportion of cle an, intact seeds

of th e designated species in a seedlot, usually expressed as a percentage by w e igh t

11. Sam ple, subm itted- th e sam ple of seeds

subm itted to a seed-testing station

12. Sam ple, w ork ing-a reduced seed sam ple

tak en from th e subm itted sam ple in th e

laboratory, on w h ich som e test of seed quality is m ade

13. Se e dlot -a spe cified quantity of seeds of

re asonably uniform quality

14. Seed quality-a gene ral te rm th at m ay

re fe r to th e purity, ge rm ination capacity, or

vigor of a seedlot

15. Sound seed- a seed th at contains in viable

condition all tissues necessary for ge rm ination

16. Tole rance-a pe rm itted deviation (plus or

m inus) from a standard. In seed testing,

th e pe rm itted diffe rence between or am ong

re plicated m e asuremen ts beyond w h ich th e

m e asuremen ts m ust be re pe ated

17. Vigor-seed prope rties th at dete rm ine th e

potential for rapid, uniform eme rgence and

deve lopm ent of norm al seedlings under a

w ide range of fie ld conditions.

E, Quality Evaluation

For satisfactory e valuation of ge rm ination, th e

follow ing principles are fundam ental:

1. Sam pling m ust be good; tests describe th e

sam ple only.

2. Testing at standard, optim um conditions

ensures th at:
a. Absolute m axim um potential of th e lot

is de te rm ined.

b . Standard conditions can be duplicated

by all laboratories for test com parison.

F. Meth odology

Satisfactory ge rm ination testing depends on

prope r m e th ods:

1. Pure seed com pone nt -Only th e pure

seed com ponent is used in th e test (4 re plications of 100 seeds each ).

2. Environm e ntal conditions-Te m pe rature, ligh t, m oisture, and m edium m ust be

care fully controlled.

a. Te m pe ratu re requi remen ts diffe r

according to spe cies. Inte rnational Seed

Testing Association prescriptions sh ould

be follow e d.

b . Ligh t requiremen ts are also spe lled out

in th e ISTA rules.

c. Th e ge rm ination m edium m ust be nontoxic; it can be eith e r natural or synth e tic m ate rial.

(1) Natural m ate rials include soil, sand,

pe at, and oth e r organic m ate rials.

(2) Synth e tic m ate rials include blotte rs,

pape r tow e ls, ce llulose w adding

(Kim pak s), filte r pape r, agar, and

cloth .

3. M oisture - Excessive m oisture is a com -

m on problem in m any tests.

4. Equipm e n t -Ge rm ination equipm e n t

m ust be dependable.

a. Cabinet ge rm inators

b . Jacobsen tables

c. W alk -in room s

d. Sm all containe rs (pe tri dish es and

plastic boxes)

5. Te st Procedures

a. Pre tre atm ent

(1) M icro-organism /path ogen tre atm ent.

(2) Ove rcom ing dorm ancy (de layed ge rm ination)

(a) stratification (pre ch ill)

(b) ch e m ical tre atm ent (nitrates,

h ydrogen pe roxide, and grow th

re gulators)

(c) scarification for h ard seeds

b . Placemen t of sam ples

c. Counting

(1) Define “ge rm inated seed”

(2) Count frequency; week ly is th e m inim um

(3) Recognize abnorm al seedlings; com -

m on abnorm alities are albino seedlings, stunted roots, n e gative

ge otropism , “endospe rm ” collars,

and necrotic are as

d. Length of test

e. De te rm ining condition of unge rm inated

seeds

6. Tole rance and re te sting

a. Review th e conce pts for official testing.

b . Analysts sh ould be aw are of oth e r re asons for a re test:

(1) Too m uch dorm ancy; additional prech ill needed.

(2) Too m uch fungal infe ction; incre ase

distance between seeds on blotte r or

test in sand or soil.

(3) Norm al/abnorm al distinction uncle ar.

(4) Evidence of h um an e rror.

G. Additional Testing Considerations

1. Th e rm ogradient plates

2. Green h ouse or nurse ry bed tests

3. Testing by w e igh t (e.g., Eucalyptus an d

Betula)

4. Quercus and oth e r large seeds can be cut in

h alf

H . Re porting Results

1. Ge rm ination capacity

2. Rate of ge rm ination

a. Ge rm ination ene rgy

b . Mean ge rm ination tim e (MGT)

c. Tim e for a ce rtain proportion of ge rm ination to occur (e.g., num be r of days

for 50 pe rcent of th e seeds to ge rm inate )

d. Ge rm ination Value (GV)

e. Pe ak Value (PV)

I. Practical revie w

J. Sources

For additional inform ation, see Bonne r 19 84a,

19 8413; Cz abator 19 62; Edw ards 19 87; Inte rnational Seed Testing Association 19 85, se ct. 5,

5A, 11; W illan 19 85, p. 202-227.

Rapid Te sts: Cutting, Vital Stains,

Excised Em byro, and H ydroge n

Pe roxide

A. Introduction

Th e standard for judging seed quality is alw ays

a ge rm ination test under optim um conditions.

Under ce rtain circum stances, h ow e ve r, ge rm ination tests are not possible, and so-called

“rapid tests” m ust be used to estim ate seed

quality. W h en pe rform ed prope rly, rapid tests

can furnish valuable inform ation to seed users,

analysts, and m anage rs.

B. Obje ctives

1. Le arn th e diffe rent types of rapid tests and

h ow to pe rform th e m .

2. Recognize th e lim itations of e ach test and

w h en it sh ould be used.

43
3. Exam ine th e inte rpre tation of test results.

C. Key Points

Th e follow ing points are essential to pe rform

rapid tests:

1. Th e cutting test is th e quick est and sim -

plest and can be e xtreme ly useful w ith

fresh seeds.

2. Tests w ith vital stains re ve al m ore th an just

potential ge rm ination, but inte rpre tation is

subje ctive .

3. X-ray radiograph y is th e m ost e xpensive,

but not necessarily th e best, of th e rapid

tests. It is ve ry e ffe ctive for som e situations.

4. Le ach ate conductivity is a new and prom ising m e th od.

D. Use of Rapid Tests

Rapid tests are used w h en one of th e follow ing

conditions occurs:

1. 60-day rule of ISTA- If a ge rm ination

test requires m ore th an 60 days to com -

ple te, th en a rapid test sh ould be used.

2. Requested by user

3. Seed supply is lim ited

4. A quality ch e ck is needed during colle ction

5. Th e re are oth e r te st obje ctive s

E. Sam pling

Th e sam e sam pling principles and pre cautions

apply for rapid tests as for standard ge rm ination tests.

F. Test M eth ods

Th e re are six rapid tests th at h ave applications

w ith tree seeds.

1. Cutting

a. Te ch nique: Seeds are cut in h alf length -

w ise and all tissues are exam ined.

b . Evaluation: Good seeds are firm , w ith

good color.

c. Advantages

(1) Quick est and ch e apest

(2) Can be pe rform ed in th e fie ld

(3) Is accurate on fresh seeds

d. Disadvantages

(1) H as size lim itations

(2) Produces poor results w ith stored

seeds

(3) Is a destructive test

2. Vital stains

a. Te ch nique: Em b ryo and storage tissues

are e xposed by cutting and th en stained.

Location and intensity of staining indicate viable or dead tissue.

b . Stain options:

(1) Te traz olium ch loride (TZ ) (m ost

w ide ly used) stains live tissues red.

(2) Indigo carm ine stains de ad tissues

blue.

(3) Se len ium or te llurium salts.

c. Evaluation (TZ only):

(1) Sound tissue sh ould stain carm ine.

(2) “Topograph ic stain” analysis is th e

m ost accurate, but it is th e m ost

difficult to standardiz e.

(3) Th e ISTA (19 85) prescribes TZ for

ce rtain dorm ant spe cies.

d. Advantages

(1) Fast, stains can be re ad w ith in 24

h ours

(2) Inexpensive

(3) Equipm ent needs are sim ple

e. Disadvantages

(1) Labor-intensive

(2) Difficult to obtain uniform penetration of th e stain

(3) Difficult to inte rpre t th e stain

(4) Requires practice and expe rience

(5) Destructive test

3. Excised e m bryo

a. Te ch nique: Seeds are cut open , and th e

emb ryos are incubated in dish es.

b . Evaluation

(1) Viable seeds are green and w h ite,

w ith som e grow th .

(2) Nonviable seeds are dark or m oldy,

w ith no grow th .

c. Advantages

(1) Sim ple equipm ent needs

(2) Actual seed perform ance is tested

(3) Easy to evaluate

d. Disadvantages

(1) Labor-intensive

(2) Requires practice for prope r e xcision

(3) Slow (10 to 14 days)

(4) Destructive test

4. H ydrogen peroxid e

a. Te ch nique: Seedcoats are cut to e xpose

th e radicle, and th e seeds are incubated

in l-pe rcent h ydrogen pe roxide. Radicle

grow th is m e asured.

b . Evaluation: Based on radicle grow th .

c. Advantages

(1) Inexpensive test

(2) Partially obje ctive

(3) Sim ple pre paration

d. Disadvantages

(1) Not practical for ve ry sm all seeds

(2) Tested only on conife rs

(3) Destructive test

(4) Slow (7 to 8 days)

5. X-ray radiograph y

a. Te ch nique: Intact seeds are e xposed to

soft x rays, and th e im ages th at are captured on film are e xam ined.

b . Evaluation: Evaluation is ve ry subje ctive.

44
c. Advantages

(1) Fast

(2) Provides a pe rm anent im age

(3) Nondestructive

d. Disadvantages

(1) Equipm ent is e xpensive

(2) Extensive training is required

(3) Inte rpre tation is subje ctive

6. Le ach ate conductivity

a. Te ch nique: Seeds are le ach ed in deionized w ate r for 24 to 48 h ours; e le ctrical

conductivity of th e le ach ate is th en m e asure d .

b . Evaluation: Re lationsh ip of conductivity

to ge rm ination m ust be establish ed for

e ach spe cies.

c. Advantages

(1) Requires no e xpensive equipm ent

(2) Fast and sim ple

(3) Obje ctive me asuremen t

(4) Nondestructive

d. Disadvantages

(1) Indire ct m e asuremen t of seed quality

(2) Unk now n factors still cause trouble

G. Sources

For additional inform ation, see Inte rnational

Seed Testing Association 19 85, annex to ch ap.

6, app. B; Le ade m 19 84; W illan 19 85, p. 221-

226.

VI. Rapid Te sts: X Rays and Le ach ate

Conductivity

A. Introduction

B.

Lik e oth e r rapid tests, radiograph y offe rs a

quick estim ate of seed quality w h en th e re is no

tim e for a com ple te ge rm ination test. Th e

application of x-ray radiograph y in seed science

is one of th e few te ch nologies th at originated

w ith tree seeds inste ad of agricultural seeds. It

h as not yet fulfilled its early prom ise, but th e re

are m any applications w ith seeds. Many rapid

estim ates of seed quality h ave m ajor draw -

back s: h igh cost, subje ctive inte rpre tations,

e xcessive tim e, e tc. Th e le ach ate conductivity

m e th od offe rs a test th at m e e ts all requirem ents: low cost; fast, obje ctive me asuremen ts;

e asy procedures; and nondestructive. Alth ough

re lative ly new , it sh ow s gre at prom ise.

Obje ctives

1. Review x-ray th e ory, and see h ow x rays can

be used in seed radiograph y.

2. Le arn th e principles of seed radiograph

inte rpre tation.

C.

D.

3.

4.

5.

Exam ine th e ph ysiological basis for le ach -

ate testing.

Le arn th e le ach ate m e th odology.

Recognize th e advantages and th e disadvantages of both te ch niques.

Key Points

Th e follow ing points are essential to an understanding of th ese tw o m e th ods:

1.

2.

3.

4.

5.

6.

7.

8.

M any types of seed dam age can be dete cted

by x-ray testing.

Em b ryo de ve lopm ent can be m e asured precise ly, but e xact corre lations w ith ge rm ination are not possible.

Th e use of contrast agents can incre ase th e

am ount of inform ation obtained from radiograph s; h ow e ve r, m any of th ese agents k ill

th e seeds.

M any special radiograph ic te ch niques are

available, but m ost require equipm ent associated w ith m edical x-ray te ch nology.

As seeds dete riorate, ce llular m emb ranes

are dam aged, allow ing th e le ach ing of m any

substances from th e seeds.

M any ch e m ical groups can be dete cted, but

e le ctrolytic activity is th e e asiest to m e asure .

Good estim ates of quality are possible w ith

m any species, but ge rm ination tests are

still pre fe rred as th e standard m e asurem ent of seed quality.

Th e conductivity m e th od is prom ising, but

m ore research is needed.

X rays

1. Th e orie s

a.

b .

X rays are e le ctrom agnetic ene rgy of

ve ry sh ort w ave length s. X rays penetrate

m ate rials th at absorb or re fle ct ligh t,

and are th e m selves absorbed by th e targe t obje ct.

Radiograph s are pictures of th e obje ct

form ed by th e x rays th at pass th rough

th e obje ct and strik e a ph otograph ic

m ate rial.

(1) Radiograph quality is de fined by

contrast, density, and definition of

th e im age.

(2) Quality is controlled by k ilovoltage

(k V), m illiam pe rage (m A), e xposure

tim e , focus-film -distance (FFD),

and obje ct-film -distance (OFD).

_ _

2. M e th od s

a.

b .

Equipm e n t: Se ve ral types of x-ray

equipm ent are available com m e rcially.

Film : Se ve ral film ch oices are available,

including

( 1) Conventional film

45
(2) Polaroid film

(3) Radiograph ic pape r

c. Contrast agents: Contrast agents are

used to incre ase density of ce rtain seed

tissue im ages on th e radiograph .

(1) Aqueous agents are prim arily solutions of h e avy cation salts (e.g.,

barium ch loride and silve r nitrate ).

(2) Vaporous age n ts: ch loroform or

oth e r h alogen de rivatives of alk anes.

d. Safe ty is an im portant aspe ct of seed

radiograph y.

3. Spe cial Te ch niques-Mainly for research

application, th ey include:

a. Ste re oradiograph y

b . Tom ograph y

c. Xe roradiograph y

4. Applications in seed te s ting-X rays

w e re first used on seeds in Sw e den in 19 03.

a. Th e m ost e ffe ctive uses are:

(1) Dete rm ining seed anatom y

(2) Dete rm ining insect dam age

(3) Dete rm ining m e ch anical dam age

b . X rays h ave lim ited usefulness in dete rm ining viab ility.

E. Le ach ate Conductivity

1. M ajor points-As seeds dete riorate, substances can be le ach ed in proportion to th e

degree of dete rioration . Sugars, am ino

acids, and ele ctrolytes are just som e of th e

m ate rials th at can be m e asured.

2. Te ch niques- Le ach ate conductivity can

be m e asured in tw o w ays:

a. Multiple-seed analyze rs

(1) Advantages

(a) Fast

(b) Receives input from individual

seeds

(c) Data are printed on pape r tape

(d) Som e m odels can calculate statistics

(2) Disadvantages

(a) H igh cost (US $6,500)

(b) Som e equipm ent not re liable

(c) Influence on th e conductivity/

ge rm ination re lationsh ip unk n ow n

b . Single probe te ch niques

(1) Th e ISTA h andbook on vigor testing

(Pe rry 19 81) includes th is m e th od

for pe as.

(2) Advantages

(a) Fast

(b) Ine xpensive equipm ent

(c) Com ple te ly obje ctive

(d) Accuracy for som e spe cies w ith -

in 10 pe rcent of ge rm ination

(3) Disadvantage: Som e factors h ave an

unk now n e ffe ct.

F. Sources

For additional inform ation on x rays, see Voz z o

19 78, 19 88; W illan 19 85, p. 224-226. For additional inform ation on le ach ate conductivity, see

Bonne r 19 9 1a, Pe rry 19 81.

VII. Vigor Te sts

A. Introduction

Standard ge rm ination tests do not adequate ly

m e asure th e ability of seeds to ge rm inate and

produce norm al seedlings under fie ld conditions

because ge rm ination tests are conducted in th e

laboratory under optim um conditions. Such

conditions are seldom encounte red in th e fie ld,

so ge rm ination and em e rgence m ay be m uch

low e r th an in th e laboratory. Th e re fore, a m ore

sensitive me asuremen t of seed quality h as been

sough t by th ose conce rned w ith th e planting

quality of a seedlot. Th is m e asuremen t of seed

quality h as been re fe rred to as seed vigor. Seed

vigor tests add supplemen tal inform ation about

th e quality of seeds to inform ation obtained

th rough oth e r tests.

B. Obje ctives

1. Le arn th e conce pt of seed vigor and re aliz e

h ow it can h e lp th e seed users.

2. Be com e fam iliar w ith th e types of seed

vigor tests and k now wh ich ones are m ost

suitable for tree seeds.

C. Key Points

Th e follow ing points are essential to an understanding of vigor tests:

1. Vigor is a seed quality th at m ay or m ay not

be indicated by a standard ge rm ination test.

2. Vigor is m ost im portant under adve rse fie ld

conditions, and it can also indicate th e storage potential of a seedlot.

3. Vigor tests usually involve eith e r dire ct or

indire ct m e asuremen ts.

4. For m any tree seeds, rate of ge rm ination is

th e best e xpression of vigor.

D. De finition of Te rm s

1. Vigor

a. Association of Official Seed Analysts:

“Th ose seed prope rties w h ich dete rm ine

th e potential for rapid, uniform eme rgence, and deve lopm ent of norm al seedlings unde r a w ide ran ge of fie ld

conditions” (AOSA 19 83).

b . Inte rnational Seed Testing Association:

“Th e sum of th e prope rties w h ich dete rm ine th e potential leve l of activity and

pe rform ance of th e seed or seedlot dur46
ing ge rm ination and seedling eme rgence ” (Pe rry 19 81).

c. Inte rnational Union of Forestry Research

Organizations: “Th ose seed prope rties

w h ich dete rm ine th e potential for rapid,

uniform eme rgence, and deve lopm ent of

norm al seedlings under a w ide range of

fie ld conditions” (Bonne r 19 84a).

2. Seed quality-“A gene ral te rm th at m ay

re fe r to th e purity, ge rm ination capacity, or

vigor of a seedlot” (Banne r 19 84a).

E. Seed Vigor Conce pts

1. Ph ysiological quality- Seedlots vary

tremend ously in ph ysiological quality. Th is

is e xe m plified by th e diffe rent rates of ge rm ination w ith in a seedlot, th e variation in

th e grow th rates and sizes of seedlings produced, and th e ab ility of som e seeds to produce seedlings under adve rse conditions

w h ile oth e rs do not. Th e ph ysiological quality of seeds is com m only called seed vigor.

2. Ph ysiological m aturity- Seeds re ach

th eir m axim um ge rm ination capacity and

vigor during th e m aturation process at

th eir m axim um dry w e igh t, or th e “ph ysiological m aturity” stage. Once ph ysiological

m aturity h as been re ach ed, dete rioration

begins and continues until th e death of th e

seed. Th e process cannot be stopped, but

th e rate of dete rioration can be controlled to

som e e xtent. Diffe rent seeds decline in vigor

at diffe rent rates.

3. Dete rioration - Seed vigor declines m ore

rapidly th an does th e ab ility to ge rm inate.

Th e first sign of dete rioration is a loss of

vigor. Th us, a seed m ay ge rm inate even

th ough som e of its ph ysiological functions

m ay h ave been im paired. Th e ab ility to produce seedlings under stress conditions and

th e grow th and yield of plants m ay be

affe cted as vigor declines. Vigor is th us a

m ore encom passing m e asuremen t of seed

quality th an th e standard ge rm ination test.

4. Strate gy-Th e gene ral strate gy in dete rm ining seed vigor is to m e asure som e

aspe ct of th e seed pe rform ance or condition

th at re fle cts th e stage of dete rioration or

genetic deficiency. Deve loping a good test

for th is strate gy is not e asy. A practical seed

vigor test sh ould:

a. Be re producible

b . Be e asily inte rpre ted

c. Indicate fie ld pe rform ance potential

d. Tak e a re asonable length of tim e

e. Not require e xpensive equipm ent

f. Not require e xtensive training

F. Com m on Seed Vigor Tests

Vigor tests can be grouped into four cate gories:

1. Seed ling grow th and evaluation

a. Seedling vigor classification

b . Seedling grow th rate

2. Stress te s ts

a. Acce le rated aging

b . Cold test

c. Cool ge rm ination test

d. Osm otic stresses

e. Meth anol tre atm ent

3. Bioch e m ical te s ts

a. Te traz olium ch loride (TZ ) staining

b . Adenosine triph osph ate (ATP) activity

c. Glutam ic acid de carboxylase activity

(GAIN

d. Oxygen uptak e (respiration)

e. Le ach ate tests

(1) Sugars

(2) Am ino acids

(3) Ele ctrolytes

4. Ge rm ination data

a. M ath e m atical m odeling of ge rm ination

response

(1) Norm al distribution

(2) Polynom ial re gressions for curve

fitting

(3) Logistic function

(4) Prob it transform ation

(5) W eibull function

b . Ge rm ination rate

(1) Early counts

(2) Pe rcentiles

(3) Me an ge rm ination tim e (MGT)

(4) Ge rm ination value (GV) and Pe ak

value (PV)

G. Recom m endations For Tree Seeds-Th e follow -

ing tests h ave th e m ost potential for tree seeds:

1. Ge rm ination rate param e te rs

2. Seed ling grow th te s ts

3. Te traz olium staining for large seeds

4. Acce le rated agin g

5. Le ach ate condu ctivity

H . Sources

For additional inform ation on vigor tests, see

Association of Official Seed Analysts 19 83;

Blanch e and oth e rs 19 88; Bonne r 19 86b; Pe rry

19 81; W illan 19 85, ch ap. 9 .

47

Protection
I. Inse cts

A. Introduction

Insects are one of th e gre atest destroyers of tre e

fruits and seeds. Th ey reduce both quality and

quantity of seeds and affe ct angiospe rm s and

gym nospe rm s equally. Dam age is done th rough

all re productive stages, from deve loping buds to

cle aned seeds in storage. Losses to seed insects

are h uge, and m uch is ye t to be le arned about

th eir com ple te role in th e re productive cycle of

w oody plants.

B. Obje ctives

1. Le arn th e orders of insects th at cause th e

m ost dam age to tree seeds and th e spe cies

th ey attack .

2. Recognize th e types of injury th at insects

cause .

3. Le arn som e me th ods of insect control and

m anagemen t.

C. Key Points

Th e follow ing points are essential in prote cting

seeds from insects:

1. Insects of th e orders H ym enopte ra, Dipte ra, Le pidopte ra, H e m ipte ra, Cole opte ra,

H om opte ra, and Th ysanopte ra do th e m ost

dam age to flow e rs, fruits, and seeds of

w oody plants.

2. Dam age ranges from causing re productive

structures to abort to causing loss of seeds

in storage.

3. Gene ral types of dam age include:

a. Destroying th e seeds only, H ym enopte ra

(w asps).

b . Form ing galls and m ine scales, Dipte ra

(flies).

c. Free feeding, Le pidopte ra (m oth s).

d. Consum ing end ospe rm , H e m ipte ra

(true bugs).

e. Mining cone axes, Cole opte ra (beetles).

f. Causing cone ab ortion , H om opte ra

(aph ids, e tc.) and Th ysanopte ra (th rips,

e tc.).

4. Control m e th ods depend on identifying and

k now ing th e insect’s life cycle and th e h ostplant re lationsh ip.

5. Som e me th ods for reducing dam age are

preventive me asures, insecticides, natural

biological control agents, and prope r m anagemen t te ch niques.

D. Dam age

1. Gene ral conce pts

a. Insects reduce seed production by infesting buds, flow e rs, cones, and seeds.

b . Th e m ost dam aging insects are large ly

restricted to six orders: Le pidopte ra

(m oth s and butte rflies), Dipte ra (flies),

Cole opte ra (bee tle s ), H ym e n opte ra

(w asps), H e m ipte ra (true bugs), and

Th ysanopte ra (th rips).

2. Spe cific conce pts

a. Cole opte ra (bee tle s ) are th e m ost

dam aging group in arid and se m iarid

z ones.

(1) Bruch idae (b ruch id beetles) are th e

m ost im portant by far for Le gum inosae ; e.g., beetles in th e gene ra:

Am blycorus, Bruch id ius, and Caryedon.

(2) Curculionidae (wee vils) lay th eir

e ggs on deve loping fruits:

(a) Conotrach elu s

(b) Curculio and Conotrach elus

(c) Th ysanocn em is

(d) Nanoph yes

(e) Apion gh anaense

b . Le pidopte ra (m oth s and butte rflies )

dam age stored seeds:

(1) Pyralidae

(2) M elissopus and Vale ntinia

(3) Agath iph aga

(4) Ge le ch iidae

c. H e m ipte ra (true bugs) feed on seeds

w ith spe cialized suck ing m outh parts:

(1) Coreidae attack Eryth rina seeds in

India and som e Acacia spe cies in

Africa

(2) Pentatom idae

d. H ym enopte ra (w asps) feed on seeds:

(1) Torym idae (Megastigm us spp.) larvae

feed on Pinus, Abies, and Pseudotsuga

(2) Eurytom idae (Bruch oph agu s)

e. H om opte ra (aph ids, cicadas, and scales)

are not a m ajor th re at to seeds.

f. Th ysanopte ra (th rips) cause som e dam -

age to tree seeds.

E. Controlling insects

Control m e asures m ust be guided by th e spe cies

and ecology of th e insect.

1. Pre vention-Th e insect m ay be prevented

from re ach ing th e seeds.

2. Ch e m ical control-Include s foliar sprays,

syste m ic poisons, ligh t traps, ch e m ical

traps, and carbon dioxide.

3. Natural enem ie s - Th e targe t insect’s life

cycle and h istory sh ould reve al its natural

ene m ie s.

4. Colle ction practice s - Colle cting good

seeds is th e first ste p in k e e ping dow n

losses incurred in storage.

F. Sources

For additional inform ation, see Cib rian-Tovar

and oth e rs 19 86, Joh nson 19 83, Sch opm eye r

19 74, South gate 19 83.

50
II. Path ogens

A. Introduction

Path ogen ic organism s (fungi, bacte ria, and

viruses) cause gre at e conom ic losses. Not only

are seeds th e victim of path ogens, but th ey also

are passive carrie rs (ve ctors) of path ogens th at

m ay not dire ctly affe ct th e seeds but m ay

endange r oth e r organism s. Th is fact is th e b asis

of plant quarantine re gulations th at include

seeds in th e im port and export restrictions on

plant m ate rial.

B. Obje ctives

1. Le arn th e m ajor types of seed path ogens

and th e typical dam age th at th ey cause.

2. Identify ste ps to decre ase losses to seed

path ogens.

3. Review docum ented occurrence of m icroorganism s associated w ith tree seeds.

C. Key Points

Th e follow ing points are essential to pre venting

seed path ogens:

1. Th e m ajor dise ase-causing organism s are

fungi, bacte ria, and viruses.

2. All tree seeds carry m icro-organism s, prim arily on th e surface of th eir seedcoats.

3. All seed m icro-organism s are not path ogen ic; som e m ay e ven be bene ficial.

4. Path ology of tree seeds h as not been studied extensive ly; m uch w ork re m ains to be

don e.

D. Types of Path ogens

1. Virus e s

a. Viruses account for seven k inds of seed

dam age :

(1) Abortion of seeds

(2) Flow e r ste rility

(3) Seedcoat w rink ling

(4) Sh rive ling

(5) Ch alk y endospe rm

(6) Staining

(7) Ne crosis

b . In le gum es, emb ryo-borne viruses reduce

viability.

c. A h igh incidence of triploidy can result

from viral infe ction.

d. M ark e t value of seeds can be reduced.

e. A virus can outlive th e seed.

2. Bacte ria- Bacte rial infe ctions account for

four k inds of seed dam age:

a. Abortion

b . Rot

c. Discoloration

d. Slim e disease

3. Fungi are a se rious th re at to seed h e alth

sim ply because of th e gre at num be rs of re presentative spe cies k now n as seed path ogens. Fungi account for eigh t k inds of seed

dam age:

a. Abortion

b . Sh runk en seeds and reduced seed size

c. Rot

d. Scle rotiz ation and strom atiz ation

e. Ne crosis

f. Discoloration

g. Low e red ge rm ination capacity

h . Ph ysiological alte rations

E. Control M e ch anism s

Seed path ogens can be controlled by reducing

infe ction and by tre ating seeds in laboratories,

storage facilities, and nurse ries.

1. Infe ction redu ction-Infe ctions i n

orch ards can be reduced by:

a. Locating seed orch ards in are as of low

infe ction risk

b . Rem oving alte rnate h ost plants

c. Sanitizing orch ards

d. Applying fungicides

e. Using good cone- and fruit-h andling

m e th ods

2. Seed tre atm ent in laboratorie s

a. Surface ste riliz ation

b . Fungicides

c. H ot w ate r soak s

3. Se e d tre atm e nt in storage

4. Seed tre atm ent in nurse rie s

a. Dam ping-off

b . Seedling dise ases

F. Micro-organism s Found on Tree Seeds

See th e ch e ck list of Anderson (19 86a).

G. Sources

For additional inform ation , see Anderson

19 86a, Inte rnational Seed Testing Association

19 66, Ne e rgard 19 77, Suth e rland and oth e rs

19 87.

51

Basics for Nurseries
I. Production Syste m s

A. Introduction

Th is course is not intended to cove r all aspe cts

of nursery establish m ent and m anagemen t.

H ow e ve r, a few nu rse ry proble m s involve seeds

and seed m anagemen t practices. Th e type of

nursery syste m , size of th e nursery, and location are im portant for seeds. It w as once

belie ved th at all seedling production and planting in th e Tropics h ad to be done in containe rs.

Th is is not true; in gene ral, h ow e ve r, bare-root

production syste m s predom inate in th e Te m perate Z ones and containe r production syste m s

predom inate in th e Tropics.

B. Obje ctives

1. Recognize diffe rent nursery syste m s and

th e conditions m ost favorable for e ach .

2. Le arn th e re lationsh ip of nursery syste m s

to national seed program m anagemen t.

3. Review b asic seed te ch nology for sow ing in

e ach syste m .

C. Key Points

Th e follow ing points are essential in understanding seed basics for nurse ries:

1.

2.

3.

4.

5.

Bare-root syste m s are m ore com m on in

Te m pe rate Z ones; containe r syste m s are

m ore com m on in th e Tropics.

Bare-root production is possible in th e

Tropics w ith som e pines and for stum p production of sele cted species.

In containe r syste m s, large seeds are usually sow n dire ctly in containe rs, w h ile sm all

seeds are sow n in ge rm ination beds or trays

and transplanted (prick ed out).

Tray m ob ility is an advantage in caring for

and prote cting young seedlings.

In sm all nurse ries, seed tre atm ents for ge rm ination are usually done by h and.

D. Core M ate rial

1. Type of Nurse ry

a. Bare-root syste m s

(1) Are suitable w ith large-scale planting program s.

(2) Can produce seedlings or stum ps.

(3) Require sam e-day planting in tropical environm ents.

(4) Used w ith Pinus cczribueu in Venezue la and stum p plantings of Gm elina,

Dalb ergia sissoo, and Cassia siam ea.

b . Containe r production syste m s

(1) Are pre fe rred in m ost tropical locations because:

(a) Th ey can be sm all, labor-intensive ope rations.

(b) Containe rized seedlings can

stand th e transport stress.

(2) System options include:

(a) A large centralized nurse ry w ith

production of 0.5 to 1.0 m illion

seedlings

(b) Num e rous sm all nurseries w ith

production of 10,000 to 100,000

seedlings

(3) Th is system can be used for “w ildings.”

c. Seed program conside rations

(1) In a large, centrally located nurse ry,

seed cle aning and storage sh ould be

located nearby.

(2) In sm all, dispe rsed nurse ries, cle aning and sh ort-te rm storage sh ould

be in a regional cente r.

(3) In sm all nurseries, m uch seed colle ction, e xtraction, and cle aning are

pe rform ed locally.

(4) Localized colle ction forces th e use of

local seed sources.

(5) For tropical re calcitrants, sm all

local nurse ries m ust be used to

avoid viab ility loss in seeds.

(6) A com bination of approach es w ill

probably evolve.

2. Bare-Root Production

a. Sm all seeds- For sm all seeds, use

m e ch anized sow ing and culture.

b . Large seeds - For large seeds, sow by

h and.

c. Cove ring

(1) Sm all seeds-Press into th e soil surface and cove r w ith a ligh t m ulch (2

to 3 m m ).

(2) Large seeds-Place on th eir sides,

press into th e soil, and cove r w ith 5

mm of soil.

3. Containe r Production

In containe r production, eith e r sow dire ctly

into containe rs, or sow in seedbeds or seed

trays and transplant late r (prick ing out).

a. Sow ing into containe rs

(1) Is good for th e root syste m s.

(2) Is used for

(a) Large seeds th at can be h andled

individually

(b) Seedlots w ith e xpe cted h igh ge rm ination

(3) Sustained sow ing rates are sh ow n

in table 16.

(4) Allow s prick ing out of “doubles.”

(5) Aim s for one seedling pe r containe r.

(6) Calculation of seed needs.

54
b . Sow ing into seedbeds or seed trays

(1) Concentrates ge rm ination in sm all

are as.

(2) Is used for

(a)

(b)

Cc)

(d)

(e )

Seedlots w ith e xpe cted ge rm ination of less th an 40 pe rcent

Seedlots w ith slow ge rm ination

Spe cies th at h ave seve ral seedlings pe r seed unit

Ve ry sm all seeds

Scarce or e xpensive lots

Table 16.-Suggested sow ing rates for seedling prod uction in containers (Napier and Robbins 19 89 )

Expe cted ge rm ination Seeds per container

Perce nt

80

60-79

40-59

<40

N um b e r

1 or 2’

2

3

use seedbeds

D.

‘Sow h alf th e containers w ith one seed and h alf th e containers

w ith tw o seeds.

(3)

(4)

(5)

Sources!‘)

Provides th e advantages of seed tray

m ob ility.

Follow th ese ste ps:

(a) Sand:topsoil m ix of 1:l.

(b) Pure sand for Pinus, Eucalyptus, and oth e rs.

(c) Press seeds into m edium , barely

cove r w ith w ash ed sand, and

m ulch ligh tly.

(d) M onitor closely to m aintain

prope r m oisture le ve l.

Sow ing into seedbeds is m ost com -

m on.

(a) Provide we ll-drained seed beds.

(b) Broadcast sm all seeds, pressing

th em into soil, and cove ring

ligh tly.

(c) Prote ct from rodents.

(d) Sow ve ry sm all seeds by m ixing

seeds and fine sand.

Calculate sow ing rates.

For additional inform ation, see Lantz 19 85,

Lie ge 1 and Venator 19 87, Napie r and Robb ins

19 89 , W illan 19 85.

55

Seed Programs
I. National Program s

A. Introduction

B.

C.

D.

E.

National seed program s are necessary to support national re forestation and afforestation

e fforts by ensuring an adequate supply of h igh -

quality seeds of suitable spe cies and sources.

Countries of th e Association of South e ast Asian

Nations are losing ove r 1.2 m illion h e ctares of

forest lands annually to oth e r uses. De forestation of “officially” designated forest lands in

India h as not been e xcessive since th e 19 50’s

(about 3 pe rcent of th e lands under th e Forest

De partm ent), but m ore th an 10 tim es th is are a

of w aste lands, sm all groves, e tc., h as been

denuded. W ith in th e fram e w ork of national program s, State or Provincial seed program s m ay

also be needed.

Obje ctives

1. Le arn th e gene ral functions of a national

forest-seed program .

2. Exam ine possible adm inistrative structures of a national program .

3. Exam ine an e xisting national program as a

case study.

Key Points

Th e follow ing points are im portant in national

seed program s:

1. Th e prim ary function of a national forestseed program is to ensure an adequate supply of suitable tree seeds.

2. National program s can serve m any oth e r

im portant functions.

3. National program s sh ould serve th e needs

of all tree planting: industrial w ood plantations, w ate rsh ed prote ction, social forestry

plantings, agroforestry, e tc.

Tree-planting activities sh ould be served by a

national forest-seed program .

1. Industrial w ood products

2. Fue lw ood and ch arcoal

a. Village forests

b . Individual landow ne rs

c. Com m e rcial production

3. W ate rsh ed prote ction

a. Gene ral prote ction

b . Prote ction in specific are as (i.e., rese rvoirs, m ine spoils, dune stabiliation)

4. W indbre ak s or sh e lte rbe lts

5. Urban planting

6. W ildlife h abitat and food plantings

7. Agrofore stry planting

8. Social fore stry

9 . Conse rvation of genetic re source s

Scope of th e Program

1. Population distribution

2. Ph vsioPranh ic ch aracte ristics

3. Available land are a and ow ne rsh ip

4. R e alistic annual goals

5. Seed storage needs

6. Use of indigenous spe cie s

F. Spe cies Ch oices

1. Indigenous spe cie s and land race sTh ese m ay be best.

2. Exotics- Caution sh ould be used w ith

e xotics.

3. Seed source-Provenance tests are needed.

4. Natural plant succe ssion sh ould be

follow e d.

G. Adm inistrative Structure-Many gove rnm ent

agencies or m inistries m ay be involved, such

as:

1. Fore stry m inistry le ve ls

a. National

b . Provincial or State

c. Village or oth e r local structures

2. Com pre h ensi ve n atural re s ource

age ncie s

3. Agricultural agencie s

4. M ilitary de partm ents

5. Division of re sponsibilitie s

a. Ove rall planning

b . Seed acquisition and distributionTh e re m ay be a central location or

re gional cente rs for th e follow ing:

(1)

(2)

(3)

(4)

(5)

(6)

(7)

Colle cting and cle aning

Testing

Storage

Ce rtification

Record k e e ping

Sales to oth e r countries

Sales w ith in country

H .

c. Seedling production can be based in:

(1) National or State nurseries

(2) Village nurseries

(3) Private nurseries (farm e rs)

(4) Com m e rcial nurseries

d. Plantation care- Tw o factors m ust be

considere d :

(1) Prote ction, prim arily from anim als,

fire, and people

(2) Me asuremen t of survival and early

grow th

e. Research -Many proble m s m ay require

research , such as:

(1) Seed proble m s

(2) Spe cies, site, and seed source evaluations

Critical Ste ps

Seve ral critical ste ps in th e planning process

call for good decisions:

1. Planting goals- w h at, w h e re, and h ow

m uch ?

2. Availability of seed supply

58
a. Indigenous species

b . Com m e rcial sources

3. Colle ction cre w s

a. Equipm ent and transport

b . Training

c. Le gal obstacles

4. Nurse ry adm inistration

a. Site

b . Pe rsonnel

c. Equipm ent

5. Colle ction goals

6. Seed ce n te rs-National, State , or

re gional?

I. Oth e r Considerations

1. Continuity of ope rations

2. Tk aining

3. Multiple functions

a. Some fore ste rs also grow and distribute

fruit tre e s.

b . In som e countries, only one seed laboratory is available to test both agricultural

and tree seeds.

4. Inte rnational organiz ations th at can

h e lp in planning:

a. ISTA - ISTA Se cre tariat

Reck en h olz, P. 0. Box 412

CH -8046 Z urich

Sw itz e rland

b . IUFRO - IUFRO Se cre tariat

Sch on b runn

A-1131 Vienna

Austria

c. FAO - Forest Resources Deve lopm ent

Branch

Forest Resources Division

Forestry De pt., FAO

Via de lle Te rm e di Caracalla

I-00100 Rom e

Italy

d. ICRAF - Inte rnational Council for

Research in Agroforestry

P.O. Box 30677

Nairob i

Kenya

J. Case Study

K. Sum m ary

Th e functions of a national seed cente r are:

1. Furth e r de ve lop taxonom y and aids to species identification.

2. Colle ct and dissem inate data on th e e cology

of individual spe cies, th us enh ancing understanding of th e pe rform ance of spe cies.

3. Prom ote me asures, as necessary, to conserve th e genetic resources of im portant

spe cies.

4. Deve lop optim um seed colle ction strate gies

based on k now ledge of b reed ing syste m s.

5. M aintain e xisting seed colle ctions and

ensure th eir future deve lopm ent as program s evolve to utilize prom ising spe cies

and provenances.

6. Assist colle ctors from oth e r countrie s

w ith in th e fram e w ork of national policy;

som e countries restrict colle ctions by foreign nationals.

7. Provide inform ation on th e ph ysical and

ph ysiological ch aracte ristics of seeds, and

any diseases th at m igh t be borne by seeds.

8. Encourage quarantine practices th at m inim ize th e ch ances of dom estic insects

becom ing establish ed in oth e r countries.

9 . Disse m inate inform ation by providing

appropriate training, sym posia, and publications.

10. Disse m inate seed sam ples for research or

spe cies trials to oth e r institutions or countries on a cost or e xch ange b asis.

L. Sources

For additional inform ation see Gre gg 19 83,

H e llum (in press), Robb ins and Sh resth a (in

press), Rudolf 19 74.

II. Se e d Cente rs

A. Introduction

National forest-seed program s require som e

sort of national tree-seed cente r, institute, or

laboratory. Dedicated facilities and som e centralized auth ority are suggested for tree-seed

cente rs. Th eir le ve l of te ch nology m ay vary w ith

th e country’s needs, but th ese cente rs sh ould

serve as th e focal point of seed activities.

B. Obje ctives

1. Le arn th e gene ral functions of national

tree-seed cente rs and h ow th ey support

national seed program s.

2. Exam ine seve ral options for cente r design.

C. Key Points

Th e follow ing points are essential to seed cente r

deve lopm ent :

1. Th e prim ary function of a seed cente r is to

support th e national forest-seed program .

2. Seed cente rs provide seed se rvices, research

on seed proble m s, training of seed w ork e rs,

and extension activities for seed users.

3. M any countries w ill require re gional or subcente rs for e fficient ope ration.

D. Functions

1. Se rvice s

a. Coordinate s seed colle ction

b. Conditions seed colle ctions

(1) All ope rations at a m ain cente r

59
(2) Drying and extracting at re gional

ce nte rs

c. Storage of se e ds

(11 Operational storage

(2) Long-te rm storage of surplus stock s

(3) Ve ry long-te rm storage

d. Testing

(1) National seed program colle ctions

(2) Oth e r in-country use rs

(3) Th ird-party testing

e. Ce rtification

2. Se e d rese arch

a. Applied research

b . Basic research

3. ‘bak ing and e xtension

a. Train seed colle ctors, analysts, and

oth e rs

b. Extension program s for nurse ry

w ork e rs and farm e rs

E. National or Regional Cente rs

1. National cente rs can be m ore responsive

to political re alities.

2. R egional cente rs can e xpand th e scope of

ope rations.

3. Com prom ise -National cente rs are best

for storage, testing, and research ; re gional

ce nte rs are good for colle cting and cle aning.

F. Location Conce rns

1. Proxim ity to se e ds

2. nansportation

3. Isolation

4. Te ch nical h e lp

5. Disaste r potential

G. Cente r Design

1. Activity zone s include th e follow ing are as:

a. Loading dock

b . Drying are a

c. Extraction equipm ent

d. Cle aning equipm ent

e. Conditioning equipm ent

f. Se e d storage

g. Testing laboratory

h . Offices for re cords and supervision

i. Supply store room .

2. Building de sign- Suggested floor plans

and designs are available from ISTA.

3. Equipm ent

a. Com m e rcial sources are best, but m uch

can be m ade locally.

b . Spare part sources are crucial.

c. Maintenance m ust be available.

d. Ele ctrical supply m ust be dependable.

4. Staffing- Supe rvisors sh ould h ave

defined are as of w ork :

a. Dire ctor of th e cente r

b . Colle ction supervisor

c. Extraction and cle aning supe rvisor

d. Testing supe rvisor

e. Inventory and sh ipping supe rvisor

5. Training-All staff m e m be rs sh ould be

trained in th e ir spe cialties by unive rsity

staff, spe cial sh ort courses, or on-th e -job

training at an establish ed ce nte r. If pe rsonnel ch ange jobs, th e new pe ople m ust b e

trained im m ediate ly. Th e sk ills of long-tim e

staff sh ould be updated as new me th ods are

deve loped.

III. Labe ling and Ce rtification

A. Introduction

W h en forest re productive m ate rials (se e ds,

seedlings, and ve ge tative propagules) are not

colle cted or grow n by th e use r, th at user sh ould

h ave re asonable assurance of th e identity and

quality of th e m ate rial h e is buying. M any seedlab eling law s require detailed lab eling to assure

th e buyer of th e seeds’ ide ntity, purity, viab ility,

and freedom from pests; i.e., th e ph ysiological

quality of th e seedlot. Ce rtification is m ore th an

lab eling required by seed law s; it is a statemen t

about th e genetic quality and identity of th e

seedlot.

B. Obje ctives

1. Understand th e purpose of ce rtification.

2. Identify th e gene ral e lemen ts of a ce rtification program .

3. Describe th e four ce rtification cate gories

used in th e Organiz ation for Econom ic

Coope ration and De ve lopm ent (OECD)

standards for inte rnational trade.

C. Key Points

Th e follow ing points are essential to understanding lab eling and ce rtification of forest

re productive m ate rials:

1. Ce rtification is th e guarantee b y an officially re cognized organization th at forest

re productive m ate rials of identified varie ties h ave been grow n, colle cted, processed,

and distributed in a m anne r to m aintain

h igh quality and genetic ide ntity.

2. A ce rtification program requires a ce rtification agency, a produce r w h o w ish es to sell

ce rtified m ate rial, re cords of th e b reeding

program , ce rtification standards, independent inspections, and ce rtification lab els.

3. Th e four ce rtification cate gories used by

OECD are:

a. source-identified (ye llow tag)

b . se le cted (green tag)

c. untested seed orch ards (pink tag)

d. tested re production m ate rial (blue tag)

60
4. Ce rtification usually requires inspections of

th e production unit prior to pollination, a

crop inspection before h arvest, inspections

during th e colle ction-to-storage ph ases, and

inspections at th e tim e of pack aging m aterials for sale.

D. Ce rtification

1. De finition- Ce rtification is th e guarante e

of ch aracte r and quality of re productive

m ate rials by an officially re cognized organiz ation.

2. Purpose- Ce rtification is m ore th an just

labeling. Its purpose is to m aintain and

m ak e available to th e public h igh -quality

seeds and propagating m ate rials of superior

crop plant varie ties.

3. Inte rnational aspe ct -An inte rnational

sch eme for ce rtifying forest re productive

m ate rial h as been deve loped by OECD.

E. De finition of Te rm s

Th e follow ing definitions are for te rm s used in

th e OECD Sch eme (Organiz ation for Econom ic

Coope ration and Deve lopm ent 19 74):

1. Fore st re productive m ate rial

a. Seeds: cones, fruits, and seeds intended

for th e production of plants

b . Parts of plants: ste m , le af, and root

cuttings, scions and laye rs intended for

th e re production of plants

c. Plants: plants raised by m e ans of seeds

or parts of plants; also includes natural

re gene ration

2. Clone-a genetically uniform assemb lage

of individuals derived originally from a single individual by ve ge tative propagation,

such as by cuttings, divisions, grafts, laye rs,

or apom ixis

3. Cultivar - an assem blage of cultivated

individuals, w h ich is distinguish ed by any

ch aracte rs (m orph ological, ph ysiological,

cytological, ch e m ical, or oth e rs) significant

for th e purposes of agriculture, forestry, or

h orticulture and w h ich , w h en re produced

(se xually or asexually), re tains its distinguish ing fe atures

4. Prove n ance - th e place in w h ich any

stand of trees is grow ing; th e stand m ay be

indigenous or nonindigenous. (Th is is th e

location of th e seed source.)

5. Origin - for indigenous stands of trees, th e

origin is th e place in w h ich trees are grow -

ing; for nonindigenous stands, th e origin is

th e place from wh ich th e seeds or plants

w e re originally introduced

6. De signated auth ority- an organization

or institution designated by and responsible

to th e gove rnm e n t of a country participating in th e OECD sch eme for th e purpose of im plemen ting th e rules of th e

sch eme on its beh alf

F. Gene ral Elemen ts of a Ce rtification Program

1. De signated auth ority- Th e designated

auth ority m ust h ave le gal standing.

2. Produce r-Th e re m ust be qu alified produce rs.

3. H istory of th e m ate rial- Th ese d ata

cove r provenance, seed source, and breed ing

h istory.

4. Supe rvised production-Th e designate d

auth ority does th is.

5. Standards-Th e m ate rial m ust m e e t m inim um standards.

6. Ce rtification labels - Labels are attach ed

to all products.

G. Standards for Ce rtification

1. Ce rtification classes- Fore s try program s typically use th e follow ing OECD

standards:

a. Source-identified re productive m ate rial

(ye llow tag). Conditions are:

(1) Seed source and/or provenance m ust

be defined.

(2) Seeds m ust be colle cted, processed,

and stored under inspection.

b . Se le cted re productive m ate rial (green

tag). Conditions are:

(1) Isolated

(2) Norm al variation

(3) Sufficient siz e

(4) Sufficient age and stage of deve lopm e n t

(5) Ph enotypic supe riority

c. Mate rial from untested seed orch ards

(pink tag).

d. Tested re productive m ate rial (blue

tag).

2. Seed colle ction z one s h ave s pe cial fe atures:

a. Th ey are delim ited by adm inistrative

and ge ograph ic boundaries,

b . Boundaries and re fe rence num be rs of

seed colle ction zones sh ould be establish ed and publish ed.

c. Seed colle ction zones are necessary for

“source-identified re productive m aterial.”

3. Oth e r requi remen ts of ce rtification

a. Th e originator, deve lope r, ow ne r, agent,

or produce r m ust request ce rtification

and furnish :

(1) Nam e of th e varie ty

(2) Statemen t of th e varie ty’s origin

(3) Detailed description of ch aracte ristics th at distinguish th e varie ty

61
(4) Evidence of pe rform ance

(5) Statemen t on th e suggested are a of

adaptation

b . Inspections m ay include:

(1)

(2)

(3)

(4)

Initial fie ld inspections

M ature crop inspections

Inspections during colle ction, conditioning, and storage

Inspection at th e tim e of pack aging

for sale

c. Fees are paid by produce r to support th e

syste m .

H . Oth e r Docum entation

1. Labels- Som e countries or oth e r political

entities require labels for com m e rcial sales

w ith identity (spe cies), purity, ge rm ination,

e tc., on th e labels. No ce rtification is im plied.

2. Ph ytosanitary ce rtificate - Ph ytosanitary ce rtification is required by m ost

countries to stop th e spre ad of insects and

path ogens. It ce rtifies th at th e seeds h ave

been inspected and/or tre ated.

I. Sources

For additional inform ation, see Bonne r 19 81a,

Organization for Econom ic Coope ration and

De ve lopm ent 19 74, Rudolf 19 74.

IV. Ge rm plasm Conse rvation

A. Introduction

B.

C.

Loss of forests around th e w orld is w ide ly

deplored for m any re asons. One consequence of

deforestation is th e loss of valuable ge rm plasm

th at could be used in artificial re gene ration and

future b reed ing program s. Th e Food and Agriculture Organiz ation (FAO) lists m ore th an 300

tree spe cies or provenances as endange red. Fortunate ly, th e re are ste ps th at can be tak en to

conse rve th is ge rm plasm .

Obje ctives

1. Recognize th e consequences of e xcessive

loss of ge rm plasm of forest trees.

2. Le arn th e strate gies available to conserve

ge rm plasm .

Key Points

Th e follow ing points are essential to understanding ge rm plasm conse rvation:

1.

2.

3.

4.

Th eiie al practice w ould be extensive in situ

prese rvation.

Ex situ conservation is w ide ly practiced

alre ady, but “passport data” on planted

m ate rial need to be m aintained.

Seed storage can play a critical role in

ge rm plasm conse rvation.

National program s of conservation sh ould

be care fully planned and establish ed.

62

D. Im portance of th e Proble m

1. De fore station

2. Inse ct and dise ase losse s

3. Global clim ate ch ange s

4. Endange red specie s

E. Available Te ch nologies for Conse rvation

Th e follow ing strate gies are options for

ge rm plasm conse rvation:

1. In situ conse rvation

2. Ex situ conse rvation

3. Conventional seed storage

4. Cryogenic storage

5. Storage of pollen

6. M icropropagation tissue s

F. Current Efforts

Th e follow ing organizations engage in som e

ge rm plasm conse rvation (table 17):

1. Food and Agriculture Organization (FAO)

2. Inte rnational Board for Plant Gene tic

Resources (IBPGR)

3. Central Am e rica and Mexico Conife rous

Resource Coope rative (CAM CORE)

4. Oxford Forestry Institute (OFI)

5. Danish Inte rnational Deve lopm ent Agency

(DANIDA)

6. Centre Te ch nique Forestie r Tropical (CTFT)

7. Com m onw e alth Scientific and Industrial

Research Organiz ation (CSIRO)

8. M any oth e r countries h ave national seed

storage facilities

G. Recom m endations for Action

1. Incre ased e fforts in in situ conse rvation

2. Inte rnational e fforts for m ore e x situ

conse rvation plantings

3. M ore re se arch on conventional seed

storage

4. Incre ased re se arch w ith re calcitrant

se e ds

5. Establish m ore seed bank s

K Applied Research

A. Introduction

M any seed proble m s can be solved locally w ith out

soph isticated research equipm ent th at is costly to

acquire and ope rate. Som e investigations furnish

answ e rs w ith out statistical tre atm ent; oth e rs

need statistical w ork to dem onstrate th eir re liab ility. Sim ple designs are usually satisfactory in

seed w ork , including com ple te ly random ized

tre atm ents and factorials. Th e m ain requirem ents are curiosity and dedication.

B. Obje ctives

1. Le arn a few principles of sim ple research

studies.
Table 17. -Som e m ajor inte rnational seed storage cente rs’

Cente r Country

Approxim ate

size of colle ction

Spe cies Sources R e fe re n ce

United States Forest

Tree Seed Cente r

National Seed

Storage Laboratory

Pe taw aw a National

Forestry Institute

DANIDA Forest

Seed Centre

CSIRO Tree Seed

Centre

O F1

Oxford, UK

Banco Latinoam e ricano

de Se m illas Forestales

Banco de Se m illas

COH DEFOR

_____ Number _____

United States 67 19 7

United States 18 41 Bass (19 85)+

Canada 118 2.130 Janas ( 19 84)

Denm ark 46 187 Anonym ous (19 85)

Australia

Unite d

Kingdom

9 00 4,000

i *

Costa Rica 153 308 Anon. (19 83)

H onduras 4 46 Gustav0 (19 85)+

Karrfalt (19 85)+

Turnbull and Doran (in press)

*

‘Bonne r, F.T. 19 86. Unpublish ed re port. On file w ith : USAID Science and Te ch nology Office,

W ash ington, DC. [Num b e r of pages unk now n ].

+ Pe rsonal com m unication from cente r dire ctors.

*Data not available.

2. Review case study exam ples of applied seed

research .

C. Key Points

Th e follow ing points are essential to applied

seed research :

1.

2.

3.

4.

Proble m s can often , but not alw ays, be

solved w ith sim ple tests and experim ents.

Standard procedures are alw ays used w h en

th ey are available ; e.g., ISTA (19 85) rules

for ge rm ination testing.

Tre atm ents are alw ays re plicated w ith

seve ral seed sources or in diffe rent seed

y e ars.

Lim itations of th e procedures in use m ust

be re cognized; e.g., e le ctric seed m oisture

m e te rs cannot be accurate to 0.1 pe rcent.

D. Gene ral Considerations

1.

2.

3.

R e plication - Th e “standard” is 4 re plicates of 100 seeds each (typically designated 4 x 100).

Docum entation - Com ple te re cords are

essential.

Statistics

a. Studies m ust be designed to allow statistical analysis.

b . Sim ple designs are used w h ene ve r

possible .

c. Com m on sense cannot be re placed w ith

statistics.

4. Publication - Good results sh ould be publish e d .

E. Case Studies

1. M aturity indice s of fruits or se e dsdete rm ine by:

f.

g .

Using a m inim um of five trees.

Sam pling for a re asonable tim e pe riod.

Colle cting 10 to 15 fruits pe r tre e.

Tak ing color ph otograph s if possible.

Testing for th e best param e te rs:

(1) Size (length and diam e te r)

(2) W eigh t (w e t and dry; dried at 103 “C

for 15 to 24 h ours)

(3) M oisture content

(4) Ge rm ination

(5) Ch e m ical analyses

Colle cting data and plotting m e ans on a

tim e scale.

Repe ating at le ast tw ice to cove r th re e

seed crops.

2. Extracting and cle aning m e th ods

a. Possible tests include:

(1)

(2)

Sun drying vs. sh ade drying

H and extraction vs. m ach ine e xtraction

63
(3) Any m e ch anical action vs. h and

cle aning

(4) De te rm ining seed size effe cts by sizing into th ree groups and testing

ge rm ination

(5) Dew inging vs. sow ing w inged seeds

b . Each tre atm ent sh ould be re plicated 5

tim es; e ach re plicate is tested w ith 4

sam ples of 50 seeds each .

c. Unusual results are alw ays re tested.

d. Suggested statistical designs are “t”

tests for tw o tre atm ents and com ple te

random iz ation for m ore th an tw o tre atm e n ts.

3. Pre tre atm ent for ge rm ination

a. Possible me th ods to test include:

(1) H and scarification vs. m e ch anical

scarification

(2) H ot vs. cold w ate r soak

(3) Stratification tim e and te m pe rature

(4) Ch e m ical stim ulation

b . Th e s am e gene ral dire ctions as

described in “2.b” th rough “d” above

apply in th ese tests also.

4. Storage conditions

a. Possible tests include:

(1) Room te m pe rature vs. re frige rated

conditions

(2) Diffe re n t re frige ration te m pe ratures

(3) Seed m oisture leve ls

(4) Type of storage containe rs

b . Replicates sh ould be large enough to

allow sam pling ove r tim e.

c. Frequency of testing for orth odox seeds

is 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 ye ars,

and for re calcitrant seeds, frequency is

1, 2, 4, 8, 12, 18, and 24 m onth s, th en

e ve ry 6 m onth s th e re afte r.

d. At le ast four re plicates sh ould be used.

5. Te sting for re calcitrance -A good test

for re calcitrance is:

C.

d.

e.

f.

g.

h .

Bring th e seedlot to full im bibition.

Start drying w ith at le ast tw o rates

(slow and fast).

Tak e pe riodic sam ples for m oisture content and ge rm ination.

M aintain th e drying range from full

im bibition to lo-pe rcent m oisture or

until death of th e seeds.

Designate seeds th at cannot be dried

below 20 pe rcent 3s re calcitrant.

Repe at th is test to confirm re calcitrance ; neve r trust just one m e asurem ent cycle. Tests of additional seedlots

are desirable.

Ch e ck ch illing injury at 0 to 5 “C by

e xposing fully im bibed seeds to th is

te m pe rature for 24 h ours.

Kee p statistics in pe rspe ctive. Realiz e

th at th ey are not as im portant as com -

m on sense in inte rpre tation of results.

64
Laboratory Exercises

1.

2.

3.

4.

5.

6.

7.

8.

9 .

10.

11.

12.

Title Page

Seed Structure ................................................. . 66

Seed Crop Estim ation ............................................66

Cone Drying and Seed Extraction

a. Central Am e rica .............................................67

b . India/Pak istan ............................................... . 67

Storage Space Requiremen ts ..................................... . 68

Sam pling....................................................... . 68

M oisture, Purity, and W eigh t..................................... . 69

Calib ration of Ele ctric M oisture Mete rs ............................ 70

Ge rm inationTests.. ............................................ . 71

Scarification .................................................... . 71

Rapid Test: Te traz olium Staining. .................................72

Rapid Tests: Cutting and Excised Em b ryo. ......................... 72

SeedH e alth Testing ............................................. . 73

65
Exe rcise 1- Se e d Structure

Obje ctive :

To le arn basic seed structures and th eir function in im portant seed types.

M e th ods:

1. Presoak seed sam ples in tapw ate r at room te m pe rature (or 27 “C) for 15 to 24 h ours. Th e im bibition w ill

soften tissues and facilitate dissection.

2. Using a k nife, clippe rs, or a single-edged raz or blade and depending on type of seeds, care fully cut th e seeds

in one of tw o diffe rent w ays:

cross section (transve rse )

L: length w ise (longitudinal)

Se ve ral cuts m ay be necessary to e xpose th e e m b ryo and oth e r inte rnal tissues.

3. Exam ine th e tissues exposed by th e cuts and label th em on fre eh and sk e tch es of th e cut m ate rial. De te rm ine

w h ich tissues are for emb ryo prote ction, storage of food rese rves, e tc. Look for abnorm al structures, insect

dam age, e tc.

4. On at le ast one seed of e ach spe cies, try to re m ove th e e m b ryo w ith out dam age, sk e tch it, and label th e parts.

Supplie s:

Clippe rs (or k nives), single-edged raz or blades, dissecting needles, a sm all m agnifying glass (or h and lens), pencil

and pape r, and seed sam ples of five tree spe cies.

Exe rcise 2 - Seed Crop Estim ation

Obje ctive :

To predict seed crops in advance of colle ction by estim ating th e num be r of:

1. Good seeds per fruit

2. Fruits pe r tre e

M e th ods:

1. Good seeds per fruit

a. Ch oose a m ultiple-seed fruit and colle ct 15 fruits prior to m aturity.

b . Cut fruits in h alf length w ise and count good seeds visible on each h alf.

c. Dry th e fruit h alves in an oven (40 to 50 “C) to e xtract seeds and to obtain actual counts of good seeds.

d. Calculate re gression equations to predict total seeds from fruit cross-section counts.

2. Fruits pe r tree-Visit nearby trees and estim ate fruit crops by:

a. Total count

b . One-fourth crow n count

c. Sam ple b ranch count

d. Any oth e r k now n w ays

3. Com bine results of both m e th ods to estim ate size of th e seed crop.

Supplie s:

Cone cutte rs or sh arp blades, an oven , drying containe rs, and binoculars.

66
Exe rcise 3a-Cone Drying and Se e d Extraction (Central Am e rica)

Obje ctive :

To le arn h ow to calculate seed and fruit needs for a planting program .

Assum ptions:

Are a to plant-2,000 h e ctares (h a) at 1,700 trees pe r h e ctare

Spe cies - Pinus caribaea

1. All m oisture contents are pe rcentage of w e t w e igh t.

2. Th e re are 800 closed cones pe r h e ctolite r (h L) (40 k g).

3. M oisture content of closed cones is 40 percent.

4. Cones double in size w h en open .

5. Yield ave rages 400 gram s (g) of pure seeds pe r h e ctolite r of closed cones.

6. Th e re are 68,200 seeds pe r k ilogram (k g).

7. Laboratory ge rm ination is 80 pe rcent; 50 pe rcent of th e ge rm inated seeds produce plantable seedlings.

8. Cones are put into th e k iln w h en th ey re ach 25pe rcent m oisture content.

9 . Drying trays h old 0.5 h L of closed cones; e ach stack of eigh t drying trays h olds 4.0 h L; eigh t stack s can fit in

th e k iln at once.

10. It tak es 12 h ours to dry a full ch arge to th e lo-pe rcent cone m oisture needed for th e cones to open fully.

11. It tak es 700 k ilocalories (Kcal) to h e at 1 h L of cones for 1 h our.

12. Fuel value for w ood of Casuarina equisetifolia is 4,9 50 Kcal pe r k ilogram ; for P caribaea cones, 4,500.

13. Open P caribaea cones w e igh 104 g pe r lite r (L).

Que stions:

1. H ow m any cones m ust be colle cted to m e e t th e planting goal?

2. H ow m uch total m oisture m ust be lost in predrying (prior to ente ring k iln)?

3. H ow m any drying stack s w ill be needed to predry e ve ryth ing at once ?

4. H ow m any k iln ch arges w ill be needed?

5. H ow long w ill it tak e to open all cones?

6. H ow m uch fuel w ill be needed w ith C. equisetifolia w ood? w ith P caribaea cones?

7. H ave en ough cones been colle cted to h e at th e k iln?

Exe rcise 3b - Cone Drying and Se e d Extraction (India/Pak istan)

Assum ptions:

Are a to plant-2,000 h a at 1,700 trees pe r h e ctare

Spe cies - Pinus roxb urgh ii

1. All m oisture contents are e xpressed as a percentage of w e t w e igh t.

2. Th e re are 400 closed cones pe r h e ctolite r (h L) (40 k g).

3. M oisture content of closed cones is 40 percent.

4. Cones double in size w h en open .

5. Yield ave rages 1.2 k ilogram s (k g) pure seeds pe r h e ctolite r of closed cones.

6. Th e re are 12,000 seeds pe r k ilogram .

7. Laboratory ge rm ination is 80 pe rcent; 50 pe rcent of th e ge rm inated seeds produce plantable seedlings.

8. Cones are put into th e k iln w h en th ey re ach 25-pe rcent m oisture content.

9 . Drying trays h old 0.5 h L of closed cones; e ach stack of eigh t drying trays h olds 4.0 h L; eigh t stack s can fit in

th e k iln at once.

10. It tak es 12 h ours to dry a full ch arge to th e lo-pe rcent cone m oisture needed for cones to open fully.

67
11. It tak es 700 Kcal to h e at 1 h L of cones for 1 h our.

12. Fuel value of w ood of Casuarina equisetifolia is 4,9 50 Kcal pe r k ilogram ; for P roxb urgh ii cones, 4,500. Open

P. roxburgh ii cones w e igh 110 g pe r lite r (LX

Que stions:

1. H ow m any cones m ust be colle cted to m e e t th e planting goal?

2. H ow m uch total m oisture m ust be lost in predrying (prior to ente ring k iln)?

3. H ow m any drying stack s w ill be needed to predry e ve ryth ing at once ?

4. H ow m any k iln ch arges w ill be needed?

5. H ow long w ill it tak e to open all cones?

6. H ow m uch fuel w ill be needed w ith C. equisetifolia w ood? W ith l? roxb urgh ii cones?

7. H ave en ough cones been colle cted to h e at th e k iln?

Exe rcise 4 - Storage Space Requireme nts

Obje ctive s:

Once annual seed requiremen ts are k now n, space requiremen ts for cold storage m ust be calculated. A de cision

m ust also be m ade as to h ow m any years’ supply of seeds w ill be m aintained as a safe ty m argin: 1 ye ar’s?

3 ye ars’?

Assum ptions:

1. You m ust grow 2 m illion Acacia nilotica and 3 m illion Pinus w allich iana seedlings each year.

2. A 3-ye ar supply of seeds w ill be stored.

3. Num be r of seeds per k ilogram (k g) is 7,000 for A. nilotica and 26,000 for l? w allich iana.

4. For e ve ry th ree seeds planted, only tw o w ill produce a plantable seedling.

5. Th e seeds w ill be stored in large plastic bottles th at h old 10 k g e ach . Th e bottles are 80 centim e te rs (cm ) tall

and 40 cm in diam e te r.

6. Ten pe rcent of th e cold storage space is in aisles, e tc.

Calculate :

1. H ow m any k ilogram s of e ach spe cies are to be stored?

2. H ow m any bottles and cubic m e te rs of storage space w ill be required?

3. Repe at calculation 2 if storage is in boxes 40 by 40 by 40 cm . Each box w ill h old 6.5 k g of seeds.

4. W h at are th e m inim um cold storage dim ensions needed to store th e seeds in calculations 2 and 3 above ?

Exe rcise 5 - Sam pling

Obje ctive :

To le arn th e b asic m e th ods of sam pling bulk lots and som e spe cial applications for tree seeds.

M e th ods:

1. M ix e ach lot th orough ly eith e r w ith a m e ch anical m ixe r or by h and. To do th e latte r, spre ad th e seeds out on a

sm ooth surface and m ix by scooping from side to side. Th en pour back and forth between tw o containe rs.

2. De te rm ine th e prope r size of th e subm itted sam ple (tw ice th e w ork ing sam ple ).

68
3. Draw sam ples using th e follow ing equipm ent/m e th ods:

a. Seed trie r

b . Mech anical divide r

c. Division

d. Extended h and

4. W eigh e ach sam ple to th e ne arest gram , place in a plastic bag, and label.

5. Save th ese bagged sam ples for late r m e asuremen ts of purity, w e igh t, and m oisture.

Supplie s:

A seed trie r, a m e ch anical divide r, a spatula, a spoon, plastic bags (15 by 15 cm ), m ark ing pens, and laboratory

balance s.

Exe rcise 6 -M oisture, Purity, and W eigh t

Obje ctive :

To carry out th e b asic ste ps in m e asuring m oisture, purity, and w e igh t of a subm itted sam ple.

M e th ods:

1. M oisture

a. Use subm itted sam ples draw n in th e sam pling e xe rcise.

b . Use a spoon or spatula to draw tw o subsam ples of 4 to 5 g e ach . Place sam ples in drying cans.

Follow guidelines in Bonne r (19 81b).

c. W eigh to th e ne arest 0.01 g and dry in ovens for 17 h ours at 103 “C.

d. Cool in desiccators and rewe igh . If desiccators are not available, use rapid-w e igh te ch niques to

obtain dry w e igh t.

e. Calculate m oisture as a pe rcentage of w e t w e igh t:

pe rcent m oisture = w e t w t. - dry w t. (100)

w e t w t.

2. Pu rity

a. Reduce th e re m ainder of th e subm itted sam ple to th e prope r w ork ing sam ple size. To dete rm ine th e

prope r siz e, tak e at le ast 2,500 seeds up to a m axim um of 1,000 g.

b . W eigh th e w ork ing sam ple (see 3.5.1.A in ISTA 19 85).

c. Divide th e sam ple into th e follow ing com ponents:

(1) Pure seeds

(2) Oth e r seeds (oth e r spe cies)

(3) Ine rt m atte r (includes seed parts)

d. W eigh e ach com ponent and express as a percentage of th e w ork ing sam ple we igh t:

pe rcent pure seed =

:$;;;IIZ ple (loo)

3. W e igh t

a. Use th e pure seed com ponent from th e purity test.

b . Eith e r w e igh and count th e entire pure seed com ponent or use sm alle r re plicates (th e usual

m e th od).

c. Replicate me th od:

(1) Random ly count out 8 re plicates of 100 seeds each .

(2) W eigh e ach re plicate to th e sam e num be r of decim al places used in th e purity dete rm ination.

(3) Obtain th e me an w e igh t of 100 seeds and m ultiply by 10 for l,OOO-seed we igh t.

69
(4) Conve rt to pure seeds per k ilogram as follow s:

1,000,000 = seeds pe r k g

wt. of 1,000 seeds

d. In official testing, variation w ould be estim ated as follow s:

(1)

(2)

(3)

(4)

Supplie s:

Variance = i(C3c2) -(Xx)2

n(n - 1)

Standard deviation (a) = & ri& e

Coe fficient of variation (CV) = 4 (100) ( 1

-

(X = m e an w t. of 100 seeds, see 3.c above )

If CV is 4.0 or less, th e answ e r in “3.~ ” above is acce ptable. If CV is m ore th an 4.0, tak e 8 m ore

re plicates and re pe at th e process, using all 16 re plicates in th e calculations.

A spatula, a spoon, laboratory balances, an oven , drying cans, desiccators, force ps, and pencil and pape r.

Exe rcise 7 - Calibration of Ele ctric M oisture Mete rs

Obje ctive :

To dem onstrate a sim ple me th od for deve loping calib ration ch arts for e le ctric m oisture me te rs. Th ese m e th ods

w ill w ork w ith any type of m e te r.

M eth ods:

1. Draw 4 to 5 k g of seeds from a bulk lot of th e desired species; m ix w e ll.

2. Se parate into 10 random sam ples of about 400 g e ach .

3. Adjust m oisture in th ese sam ples to span th e range of m oisture content th at w ill be encounte red

(approxim ate ly 5 to 20 pe rcent). Do th is by drying seve ral sam ples (vary drying conditions) and by

adding w ate r to oth e rs (vary th e am ount of w ate r).

4. Place e ach sam ple in a plastic bag and place th e bag in a coole r for 1 week to allow com ple te m oisture

equilib ration.

5. Afte r 1 week , re m ove th e sam ples and le t th em com e to room te m pe rature (2 to 3 h ours).

6. Tak e a m e te r re ading on th e driest lot according to th e m anufacture r’s instructions. Record th e value

and im m ediate ly draw tw o 5-g subsam ples for oven dete rm inations of m oisture content. Follow

pre vious instructions.

7. Repe at ste p 6 w ith th e oth e r sam ples in th e order of ascending m oisture content.

8. Plot data on a graph : oven m oisture pe rcentage vs. m e te r re ading. Use th is curve to re late future

m e te r re adings to actual m oisture content for th is particular spe cies only.

9 . For m ore accurate calibration, fit a re gression curve (oven m oisture pe rcentage on m e te r re adings) and

calculate values for a calib ration table. More th an 10 obse rvations sh ould be available for a re gression,

so anoth e r 10 sam ples sh ould be draw n for a re pe at of th e entire process.

10. Th is procedure m ust be done separate ly for e ach spe cies to be tested.

Supplie s:

An ele ctric m e te r, a spoon, laboratory balances, an oven , w e igh ing dish es, desiccators, plastic bags, and graph

pape r.

70
Exe rcise 8 - Ge rm ination Te sts

Obje ctive :

To le arn th e b asic ste ps of a ge rm ination test and to carry out sim ple tests on som e im portant spe cies. Be cause of

th e length of th is course, a full test m ay not be possible. By starting a few sam ples before talk ing about testing at

length , som e ge rm ination sh ould occur and be available for e valuation.

M e th ods:

1.

2.

3.

4.

5.

6.

7.

Presoak seed sam ples in tapw ate r at room te m pe rature (27 “0 for 15 to 24 h ours.

Divide sam ples into 2 re plicates of 20 to 50 seeds each , depending on th e spe cies. If Eucalyptus spp.

seeds are used, w e igh out tw o re plicates according to ISTA (19 85) rules.

H alf th e class w ill surface-ste rilize th eir sam ples w ith a lo-pe rcent ch lorine ble ach solution, and th e

oth e r h alf w ill not tre at th eirs. Tre atm ent w ill consist of a 5- to lo-m inute soak , follow e d by rinsing in

running tapw ate r.

H ard-seeded species (e.g., Acacia) w ill be scarified w ith a k nife, file, or sandpape r on th e radicle ’end as

dete rm ined in Exercise 1.

Place re plicates in glass or plastic dish es on m oist filte r pape r or oth e r suitable me dia. Pape r sh ould be

m oist, but not m oist enough to le ave “free w ate r” in a de pression m ade by m ash ing dow n on th e pape r

w ith a finge r. Put dish cove rs on; if th e re are no cove rs, use plastic w rap.

Label all dish es and place th em in a ge rm inator or constant te m pe rature room if available. If th ese

facilities are not available, place th e dish es on a table under ligh ts in th e cente r of th e room . If good

ligh ts are not available, place th e dish es near w indow s th at allow good natural ligh t.

Ch e ck dish es eve ry day for m oisture ; add w ate r if th ey dry out. Ge rm ination m ay becom e e vident in

about 7 days. Re cord norm al ge rm ination, abnorm al ge rm ination, and evidence of insect or dise ase

proble m s.

Supplie s:

A k nife, sm all file, or sandpape r for scarification, ch lorine ble ach , ge rm ination blotte rs, dish es (10 pe r student), a

glass-m ark ing pen , and laboratory balances. A ge rm inator or constant te m pe rature room is desirable but not

n e cessary.

Sugge sted spe cie s:

Pinus, Acacia or anoth e r le gum e, Eucalyptu s, and tw o indigenous species,

Exe rcise 9 - Scarification

Obje ctive :

To dem onstrate th e re lative e ffe ctiveness of sim ple scarification te ch niques th at can be used in seed testing.

M eth ods:

1. Count out 120 seeds of a h ard-seeded species and divide th em into 8 sam ples of 15 seeds each .

2. Scarify 2 re plicates of 15 by each of th e follow ing procedures:

a. Rub h and files or sim ilar ab rasive devices across th e seedcoat enough to cut a notch in th e seed.

b . Use h and clippe rs, sh e ars, or a k nife to cut th rough th e seedcoat along one side.

c. Sandpape r th e seed enough to cut th rough th e seedcoat on th e radicle end.

d. Th e oth e r tw o sam ples w ill be th e untre ated controls.

71
3. Place th e scarified sam ples on m oist blotte rs in dish es and cove r as in th e ge rm ination test. Place all

dish es in th e ge rm inator if th e re is space. If not, place th em on a table w ith good ligh ting and le ave

th em for obse rvation th rough th e rest of th e course.

4. Pe riodically count th e num be r of ge rm inating seeds and th e num be r of sw ollen seeds. Th is latte r

condition confirm s th at w ate r uptak e h as occurred, but som e th ing e lse m ay be block ing ge rm ination.

Report both conditions as a pe rcentage of th e total num be r of seeds in th e test.

Supplie s:

Four glass or plastic dish es, ge rm ination blotte rs, a h and file, clippe rs or sh e ars, rough sandpape r, and m ark ing

pens.

Exe rcise 10 -Rapid Te st: Te trazolium Staining

Obje ctive :

To le arn basic te ch niques of th e te traz olium (TZ ) stain test for viab ility.

M e th ods:

1.

2.

Draw tw o 50-seed sam ples from seeds th at h ave been soak ing in tapw ate r for 24 h ours.

Pre pare a l-pe rcent solution of TZ by dissolving 10 g of a TZ salt (ch loride or b rom ide ) in 1,000 m L of

distilled w ate r (pH 6.5 to 7.0). If th e pH of th e w ate r is outside th is range, a buffe red solution m ust be

pre pared as follow s:

a. Pre pare tw o solutions:

(1) Solution 1 -Dissolve 9 .078 g KH ,PO, in 1,000 m L of w ate r.

(2) Solution 2-Dissolve 11.876 g Na,H PO, - 2H ,O in 1,000 m L of w ate r.

3.

4.

5.

b . Mix tw o parts of solution 1 w ith th ree parts of solution 2.

c. Dissolve 10 g of TZ salt in 1,000 m L of th e buffe r solution to m ak e a l-pe rcent solution.

Care fully cut open th e im bibed seeds to fully expose th e e m b ryo. Th e e m b ryo m ay be com ple te ly

re m oved, as in th e e xcised em b ryo test.

Com ple te ly im m e rse th e e m b ryos in TZ solution in dish es and incubate th em in th e dark at 30 “C for

15 to 24 h ours (de pending on th e spe cies and seed condition).

For e valuation, decant th e TZ solution, rinse seeds in w ate r, and exam ine th e e m b ryos on a w e t

surface. Moderate red staining gene rally indicates viable tissues, h e avy red staining indicates dam aged

tissues, and th e absence of any staining indicates nonviable tissues. Stain inte rpre tations m ay vary by

spe cies. See ISTA (19 85) for guidelines.

6. Com pare results w ith oth e r rapid test results or ge rm ination test results.

Supplie s:

Dissecting equipm ent, dish es, te traz olium salt, buffe rs (if necessary), and a constant-te m pe rature dark incubator.

Exe rcise ll-Rapid Te sts: Cutting and Excised Em bryo

Obje ctive :

To le arn te ch niques of th e cutting test for viability estim ation and of emb ryo re m oval for th e e m b ryo e xcision

te st.

72
M e th ods:

1. Cutting Test

a. Draw sam ples of 50 seeds from e ach of seve ral seedlots and divide th em into sublots of 25 seeds each .

b . Cut th e seeds in h alf, using a transve rse cut th rough th e cente r of th e seeds. Cate gorize seeds as eith e r

viable, dam aged by insects or dise ase, or e m pty. Ave rage th e results from th e sublots.

2. Excised Em b ryo Test

a. Draw sam ples of 50 seeds from e ach of seve ral seedlots th at h ave been soak ing in tapw ate r for 24 to 48

h ours at room te m pe rature and divide into sublots of 25 as be fore.

b . Using raz or blades or scalpe ls, care fully cut th rough e ach seedcoat and endospe rm (if present), and

e xpose th e e m b ryo.

c. Care fully “te ase ” th e e m b ryo out of th e surrounding tissues w ith dissecting needles or oth e r sh arppointed instrum ents. Avoid dam aging th e e m b ryo.

d. Care fully place th e e xcised em b ryo on m oist filte r pape r in a cove red dish , such as a pe tri dish .

M aintain at 20 “C in ligh t until an evaluation can be m ade (usually w ith in 14 days).

e. Dise ased or dam aged em b ryos sh ould not be placed in th e dish es. Em pty seeds sh ould be cate gorized as

such and not re placed in th e test.

f. Th e w ork ing surface and all instrum ents sh ould be disinfe cted to reduce m old infe ctions w ith a 50-

pe rcent e th anol solution. Instrum ents sh ould be “dipped” between e ach dissection.

g. Em b ryos sh ould be cate gorized w ith in 14 days as follow s:

(1) Viable

(a) ge rm inating emb ryos

(b) emb ryos w ith one or m ore cotyledons exh ib iting grow th or green ing

(c) emb ryos re m aining firm , sligh tly enlarged, and eith e r w h ite or yellow according to spe cies

(2) Nonviable

(a) emb ryos th at rapidly deve lop seve re m old, dete riorate, and decay

(b) degene rated em b ryos

(c) emb ryos e xh ib iting e xtreme b row n or black discoloration, an off-gray color, or w h ite w ate ry

appe arance

(d) seeds in w h ich th e e m b ryo is de ad, m issing, or deform ed

h . Com pare your results w ith th e cutting test results.

Supplie s:

Single-edged raz or blades, scalpe ls, dissecting needles, dish es, filte r pape r, and eth anol.

Exe rcise 12 - Seed H e alth Te sting

Obje ctive :

To le arn basic te ch niques of seed h e alth testing.

Back ground:

H e alth testing of seeds is im portant for th ree re asons:

1. Seed-borne inoculum m ay cause diseases in th e fie ld.

2. Im ported seedlots m ay introduce new d ise ases, so tests to m e e t quarantine re gulations m ay be required.

3. Seed h e alth testing m ay aid in seedling e valuation and h e lp de te rm ine causes for poor ge rm ination or fie ld

establish m ent. It supplemen ts th e ge rm ination test.

Se e d H e alth re fe rs prim arily to th e presence or absence of dise ase-causing organism s (e.g., fungi, bacte ria,

and viruses) and anim al pests (e.g., e e lw orm s and insects). H ow e ve r, ph ysiological conditions such as trace

e lemen t deficiency m ay be involved.

Incubation m aintains seeds in an environm ent favorable to th e deve lopm ent of path ogens or sym ptom s.

73
Pre tre atm ent is any ph ysical or ch e m ical laboratory tre atm ent of th e w ork ing sam ple pre ceding incubation th at is

done sole ly to facilitate testing.

Tre atm e nt is any process, ph ysical or ch e m ical, to w h ich a seedlot is subm itted.

Sam ple :

1. Entire subm itted sam ple m ay be th e w ork ing sam ple, depending on th e test.

2. Th e w ork ing sam ple is norm ally 400 pure seeds or an equivalent w e igh t.

3. Sam pling rules are follow e d.

4. Replicates containing a spe cified num be r of seeds, if required, are tak en at random for a subsam ple afte r

th orough m ixing.

Gene ral dire ctions:

1. Use diffe rent m e th ods of testing depending on factors such as path ogen or condition being investigated,

spe cies of seeds, and purpose of test. See ISTA (19 66, 19 85).

2. Exam ine th e w ork ing sam ple w ith or w ith out incubation.

a. Exam ine w ith out incubation. (Th is m e th od provides no indication of th e viab ility of th e path ogen .)

(1) Exam ine th e sam ple w ith a ste re om icroscope for gene ral e vidence of dise ases or pests.

(2) Exam ine im b ibed seeds. Im m e rse th e w ork ing sam ple to m ak e fruiting bodies, sym ptom s, or pests

m ore e asily visible and to encourage th e re le ase of spores. Exam ine w ith ste re om icroscope afte r

im b ib ition.

(3) Exam ine organism s re m oved by w ash ing. Im m e rse th e w ork ing sam ple in w ate r w ith a w e tting

agent, or in alcoh ol, and sh ak e to re m ove spores, h yph ae, nem atodes, e tc. Exam ine th e e xcess

liquid w ith a com pound m icroscope.

b . Exam ine afte r incubation.

(1) Afte r a spe cific pe riod of incubation, e xam ine th e w ork ing sam ple. Note th e presence of dise ase

organism s or pests on or in seeds or seedlings. Use blotte rs, sand, or agar for incubation m edia.

(2) Use blotte rs w h en required to grow th e path ogens from th e seeds or to e xam ine th e seedlings.

Seeds m ay or m ay not be pre tre ated. Space w ide ly to avoid se condary spre ad of organism s. Use

ligh t as necessary to stim ulate sporulation. Exam ine w ith a m icroscope.

(3) Sand or artificial com posts can be used for ce rtain path ogens. Seeds are not usually pre tre ated,

but th ey are w ide ly spaced on th e me dium . Incubation is favorable for sym ptom e xpression.

(4) Use agar plates to obtain identifiable grow th of organism s from seeds.

(a) Ste rility is required; seeds are norm ally pre tre ated and spaced.

(b) Identify ch aracte ristic colonies and spores by m icroscopy.

(c) Use ligh ting and ge rm ination inh ib itors.

3. Exam ine grow ing plants. Grow plants from seeds and exam ine th em for dise ase sym ptom s to dete rm ine

th e presence of bacte ria, fungi, or viruses. Use inoculum from th e test seedlot to test for infe ction of

h e alth y seedlings.

Calculations and Expre ssion of Results:

1. Express results as a pe rcentage of seeds affe cted or as num be r of organism s in th e we igh t of sam ple

e xam ined.

2. Report results on th e ISTA ce rtificate.

a. Report test m e th od.

b . Report pre tre atm ents.

c. Absence of h e alth test does not im ply satisfactory h e alth condition.

Spe cific Te st Exam ple-Pitch Cank e r Fungus:

1. Adapted from Anderson (19 86b).

2. Blotte r M eth od, 400-seed sam ple.

74
a. Pentach loronitrobenzene (PCNB) Broth

Com bine pe ptone, 15 g; M gSO, 7H z 0, 5 g; RI& PO,, 1 g; te rraclor, 1 g w ith 1 L of distilled H zO. Stir

w e ll using m agnetic stirre r. Autoclave for 15 m inutes. Afte r autoclaving, place flask on m agnetic stirre r

and stir slow ly until solution cools to room te m pe rature or sligh tly w arm e r. Add 1 g of stre ptom ycin

sulfate and 1 to 2 g of neom ycin sulfate under ste rile conditions, and stir.

b . Place 25 seeds on blue blotte r pape r in plastic containe rs. Crush th e seeds w ith a ste rilized pie ce of

plastic cut to fit th e plastic box open ing. Spray seeds and blotte r pape r w ith PCNB b roth .

c. Incubate 14 days at 20 “C or until colonies are 2 cm in diam e te r.

d. Inspect all seeds for slow -grow ing, granular w h ite colonies. Ch e ck e ach suspected colony using a ligh t

m icroscope at 100 to 400 m agnification for m icroconidia and polyph ialids. Se le ct fungus from seed

surface, not th e blotte r surface. Split th e seeds into 4 groups of 100 for re porting purposes.

3. Agar Meth od

a. Pre pare fresh potato dextrose agar (PDA) (m ak es 1 L)

(1) Cle an and dice one m edium -sized potato.

(2) Put diced potato in beak e r w ith 500 m L of distilled H zO. Run th rough autoclave.

(3) In flask , add 20 g of dextrose and 17 g of agar to 500 m L of distilled H ,O.

(4) Put dextrose/agar solution on m agnetic stirre r and low h e at.

(5) Strain cook ed potatoes th rough tw o laye rs of ch eesecloth to obtain at le ast 200 m L of slurry.

(6) Note am ount of slurry, and pour slurry in flask w ith dextrose/agar solution.

(7) Add enough distilled H a0 to m ak e total slurry solution am ount to 500 m L (i.e., if th e re are 200

m L of slurry, add 300 m L of distilled H ,O).

(8) Put solution in autoclave and run for 15 m inutes. To acidify m edia, add 20 drops of 50 pe rcent

lactic acid to obtain a pH of 4.7.

b . Isolating e xte rnal seed fungi, 25-seed sam ple

(1) Place th e wh ole seeds on acidified PDA (pH 4.7).

(2) Incubate 14 days at 20 “C.

(3) If possible, obse rve fungal grow th daily and identify th e fungi.

c. Isolating inte rnal seed fungi, 25-seed sam ple

(1) Surface ste rilize th e wh ole seeds in 70 percent e th anol for 10 m inutes. Stir th e seeds eve ry 2

m inutes.

(2) Under ste rile conditions, cut e ach seed open and re m ove h alf th e cente r m ate rial.

(3) Place th e seed h alf (cente r m ate rial) on acidified PDA (pH 4.7) using ste rile te ch nique.

(4) Incubate 14 days at 20 “C.

(5) If possible, obse rve fungal grow th daily and identify th e fungi.

75

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