Mini-cuttings: an effective technique for the propagationof Pinus pinaster Ait.

Juan Majada • Celia Martınez-Alonso • Isabel Feito • Angelo Kidelman •

Ismael Aranda • Ricardo Alıa

Received: 30 March 2010 / Accepted: 8 November 2010 / Published online: 21 November 2010� Springer Science+Business Media B.V. 2010

Abstract Pinus pinaster Ait. is one of the main forest tree species planted in Spain,

Portugal and France. Due to its high economic relevance, there is considerable interest in

developing techniques for vegetative breeding aimed at mass propagation. In this study we

present a mini-propagation protocol in order to define an efficient method to propagate

families or clones of P. pinaster. We carried out three experiments using mini-cuttings of

3–5 cm in length with the aim of evaluating the effects of temperature (4�C vs. 25�C),

plant growth regulator (IBA) and shoot age on rooting ability. Percentage of rooted cut-

tings and morphological root variables were recorded. The percentage of rooted cuttings

per treatment ranged from 68 to 97%. Treatment with IBA significantly influenced the

rooting process at 25�C but not at 4�C. The number of apexes, length, area and volume of

roots were all positively affected by temperature treatment. Shoot age also had a positive

effect on rooting capacity of cuttings, with the cuttings from the youngest shoots (70 days

after pruning) having higher rooting percentages, ranging from 84.7 to 98.3%. The use of

juvenile material, good environmental conditions and IBA all benefited the rooting of

clonal material, resulting in high rooting capacity. This study presents an innovative

propagation protocol for P. pinaster that can be used as a tool in breeding programs.

Keywords Maritime pine � Vegetative propagation � Rooting � Shoot age �Indolebutyric acid � Temperature

J. Majada � I. Feito � A. KidelmanSeccion Forestal, SERIDA, Finca Experimental ‘‘La Mata’’,33820 Grado Principado de Asturias, Spain

J. Majada � C. Martınez-Alonso (&)Seccion Forestal, CETEMAS, Finca Experimental ‘‘La Mata’’,33820 Grado Principado de Asturias, Spaine-mail: cmartinez@cetemas.es

I. Aranda � R. AlıaDepartamento de Sistemas y Recursos Forestales, CIFOR, Instituto Nacional de Investigacion yTecnologıa Agraria (INIA), Carretera de La Coruna, Km. 7.5, 28040 Madrid, Spain

123

New Forests (2011) 41:399–412DOI 10.1007/s11056-010-9232-x

Introduction

Maritime pine (Pinus pinaster Ait.) is one of the most important forest species in France,

Portugal and Spain, and it is widely distributed in the Western Mediterranean region

(Southern Europe, Northern Africa and the Atlantic coasts of Portugal, Spain and France).

The main uses of this species are related to wood production (construction, chipboard,

floorboards and palettes), pulp and paper production, resin production and soil protection,

all of which explain its use in afforestation and genetic improvement programs (Alıa and

Martın 2003). Furthermore, maritime pine populations from the Iberian Peninsula show

high levels of genetic diversity in growth and survival traits as the result of adaptations to

local ecological conditions and fragmented distribution (Alıa et al. 1997). In order to

conserve this genetic resource and put it to good use in breeding programs focusing on

different traits, it is necessary to establish detailed information about the genetic variability

of specific maritime pine populations.

Genetic improvement programs play an important role in the commercial breeding of

maritime pine, although there is still little information about vegetative propagation of this

species. However, clonal forestry for some other conifers is now a commercial reality, for

example in Australia, New Zealand, Canada or Northern Europe (Foster et al. 2000;

Burdon et al. 2004). Propagation by cuttings is a well-known method for the vegetative

propagation of species such as the conifers Pinus radiata, Pinus taeda, Pinus sylvestris,Picea abies or Pseudotsuga menziessi (Clair et al. 1985; Ritchie 1991; Hogberg 2005;

Aparicio-Renterıa et al. 2008). As such, considerable information is available regarding the

relationship between the morphological features of traditional cuttings and their rooting

although conflicting results exist regarding the effects of cutting length on rooting (e.g.

Foster et al. 2000 for P. taeda).

One limitation of traditional techniques is that only young juvenile plants can be

propagated at high rates. When plants get older, their rooting ability decreases and rooted

plant cuttings taken from them display plagiotropic growth and asymmetric branching

(Dietrichson and Kierulf 1982). The stage of maturation of the donor plant is one of the

most important factors that influence rooting capacity in conifers. To maintain the donor

plant in a juvenile condition or rejuvenate the donor plant is therefore of paramount

importance in this type of propagation program, and the principal method of doing so is

hedging (Anderson et al. 1999). The use of juvenile material increases the capacity of

rooting and decreases the time required to produce viable plants.

However, somatic embryogenic methods could provide a complete and cost-effective

solution to this ‘physiological ageing’ problem, a necessary first step before clonal forestry

becomes a workable reality for P. pinaster. Nowadays it is possible to improve rooting

capacity and solve some ageing problems using micro-cuttings (cuttings from plants

rejuvenated in vitro) (Miguel et al. 2004; Harvengt 2005; Lelu-Walter et al. 2006) and/or

mini-cuttings (cuttings from mini-hedges not previously rejuvenated by in vitro tech-

niques) (Wendling et al. 2000): both techniques are currently being used with very positive

results in the commercial cloning of Eucalyptus for example. The advantages of these

methods over conventional cutting techniques are the small size of clonal mini-hedges,

high productivity, better rooting indices, lower cost, reduced need for plant growth reg-

ulators and low variation in rooting (Assis and Mafia 2007; Wendling et al. 2010). The lack

of knowledge of the effects of hedge maturation in P. pinaster, as well as few higher breed

genotypes has resulted in reluctance among Southern European foresters to plant cuttings

of this species.

400 New Forests (2011) 41:399–412

123

In spite of maritime pine being a well studied species in the Mediterranean region

hormone, etc., on rooting for several other conifers (Alıa et al. 1997; Correia et al. 2008;

Eveno et al. 2008; Aranda et al. 2010; Gaspar et al. 2009), there are no published studies

relating to the techniques for and capacities of vegetative propagation by mini-cuttings.

Therefore, the aims of the present study are to determine (1) the effects of temperature and

indolebutyric acid (IBA) on mini-cutting root initiation and development in P. pinaster, (2)

the effect of shoot age and IBA on rooting capacity and (3) the effect time of cutting

collection has on the production of cuttings and their rooting capacity, thereby defining an

efficient protocol for the vegetative propagation of maritime pine clones.

Materials and methods

Plant material and culture conditions

Seeds from P. pinaster Ait. were collected from selected stands (Galicia Coast Provenance,

NW of Spain (E 01a)). The experiments were carried out at SERIDA in the experimental

station ‘‘La Mata’’ in Grado, Asturias, Spain, located at 43� 230N 6� 40W at 60 m a.s.l. In

April 2005, half-sib family seeds were sown in 250 ml containers (Cetap 54-Universal), in

a 4:1 (v:v) mixture of peat (PINSTRUD) and vermiculite (VERLITE), and following

germination, seedlings were grown in a greenhouse for 9 months. At the end of January

2006, the leader shoot of 750 seedlings was pruned at least 10 cm from the tap root. After

pruning, seedlings were fertilized weekly for 6 weeks with N2 (9.27 mg L-1), P2O5

(4.25 mg L-1), K2O (7.46 mg L-1), MgO (0.038 mg L-1), Mo (0.006 mg L-1), Fe

(0.064 mg L-1), Mn (0.042 mg L-1), Zn (0.006 mg L-1), B (0.006 mg L-1) and Cu

(0.032 mg L-1) solution, and for a further 2 weeks were fertilized twice a week. Apical

cuttings of 3–5 cm in length were taken 60 days after pruning of the leader shoot as using

only apical cuttings avoids development of quiescent meristems in needle fascicles. Cut-

tings were soaked in a solution of 3 ml l-1 of fungicide (Iprodiona 25%) for 10 min,

before setting into trays (77 pots of 60 cm3 per tray, 1,023 pots m-2) filled with a 1:1 (v:v)

mixture of perlite and peat. The trays were placed in an air-conditioned glasshouse on an

ultra-fog bench and maintained at 25 ± 2�C and 90% relative humidity (RH). Plants were

allocated to treatment conditions using a completely randomized nested design.

In addition, in April 2005, nine half-sib families of donor plant seeds from six plus-trees

from Asturias and Galicia (Alto la Llama 4, Alto la Llama 8, Armayan 5, Cadavedo 4,

Castropol 9, Lamuno 2, Sergude 11, Sergude 13 and Sergude 19) (Table 1) were shown in

the same conditions as cited above (same containers and mixture), and following germi-

nation, seedlings were grown in a greenhouse for 9 months. At the end of January 2006, the

leader shoot of 2,000 seedlings was pruned at least 10 cm from the tap root. After pruning,

seedlings were fertilized weekly with N2 (76.87 mg L-1/day), P2O5 (35.22 mg L-1/day),

K2O (61.79 mg L-1/day), MgO (0.82 mg L-1/day), Mo (0.14 mg L-1/day), Fe

(1.37 mg L-1/day), Mn (0.91 mg L-1/day), Zn (0.14 mg L-1/day), B (0.14 g L-1/day)

and Cu (0.69 mg L-1/day) solution for 1 year. All ministumps able to provide one or more

cuttings of 3–5 cm in length were taken every month during 1 year (April 2006–April

2007), although only randomly selected apical cuttings were selected for use in the trial.

Selected mini-cuttings were treated as described above (same fungicide, trays and glass-

house conditions).

New Forests (2011) 41:399–412 401

123

Experimental design

Experiment 1

To evaluate the effects of temperature and growth regulator on rooting ability, we selected

240 cuttings (10 cuttings per treatment 9 4 replications per treatment 9 6 treatments). In

the temperature treatment, all plants were kept at environmental temperature

(25�C ± 2�C) but half the plants were subjected to preconditioning in the first week at 4�C

without light. The bases (bottom 1 cm) of all prepared mini-cuttings were dipped (for 10 s)

into different solutions of IBA concentrations (0, 10 and 40 g L-1) dissolved in 50%

ethanol and water. Cuttings were assessed for adventitious rooting induction at approxi-

mately 2 week intervals over 8 weeks, following treatment with IBA. After this 8 weeks in

the glasshouse, most rooting was completed and cuttings were transferred to an open mist

bench and kept at 25 ± 2�C and 75% RH. After 4 months in the rooting environment, the

rooting of the cuttings was assessed. A cutting with one or more roots in excess of 1 mm in

length (i.e. where a root primordium was evident) was considered as rooted in accordance

with Goldfard et al. (1998).

Rooting capacity, expressed as a percentage of rooted cuttings per experimental unit, in

addition to number of roots and shoots that elongated during the rooting period and root

morphological variables (length, surface area, diameter, volume and number of apexes)

were recorded for each successfully rooted cutting. Upon completion of data collection, the

rooted cuttings were transplanted into 200 mL pots.

Experiment 2

To evaluate the effects of shoot age and growth regulator on rooting capacity, 3–5 cm long

apical cuttings were taken at 70, 90 and 120 days after leader shoot pruning. Thus, shoot

age refers to the time elapsed between leader shoot pruning and the time cuttings were

harvested from the subsequent regrowth. The trial consisted of 15 cuttings per treatment

and each treatment was represented by 4 replications per shoot age, a total of 720 cuttings

(12 treatments 9 15 cuttings 9 4 replications). The bases (1 cm) of all prepared mini-

cuttings were dipped (for 10 s) into different solutions of IBA concentrations (0, 250, 500

and 1,000 mg L-1) dissolved in 50% ethanol and water. After 4 months in the rooting

Table 1 Geographic and climatic information of different families employed in experiment 3

Family code Province Latitude andlongitude

Mean altituderange (m)

Mean annualrainfall (mm)

Mean annualtemperature (�C)

Alto la Llama 4 Asturias 43� 280 N 6� 490 W 503 1155 11.5

Alto la Llama 8

Armayan 5 Asturias 43� 180 N 6� 290 W 532 1160 11.4

Cadavedo 4 Asturias 43� 320 N 6� 250 W 180 1316 13.2

Castropol 9 Asturias 43� 500 N 6� 980 W 391 1179 12.6

Lamuno 2 Asturias 438 330 N 6� 130 W 85 1282 13.4

Sergude 11 Galicia 275 1741 13.1

Sergude 13

Sergude 19

402 New Forests (2011) 41:399–412

123

environment, rooting success and morphological evaluation was conducted as in experi-

ment 1.

Experiment 3

To evaluate the effect of collection time on potential production of mini-cuttings (pro-

duction of 3–5 cm cuttings per square meter), cuttings were taken every month during

1 year (April 2006–April 2007). In total, twelve cutting times were evaluated at 30, 60, 90,

120, 150, 180, 210, 240, 270, 300, 330 and 360 days after leader shoot pruning. During the

winter season (cycles 240 and 270) we were not able to take any mini-cuttings due to the

vegetative growth stop (December 2006 and January 2007). We selected 5 plants per half-

sib family and each cycle was represented by 3 replications, a total of 1350 cuttings (5

plants 9 9 families 9 3 repetitions 9 10 cutting times). All cuttings were dipped in a

40 g L-1 IBA solution for 10 s before planted 90 days. We evaluated the percentage of

rooted cuttings, number of roots, root dry biomass (RDB), length of mini-cuttings and

aerial dry biomass (ADB).

Data collection and analysis

Morphological evaluation of roots was made with the interactive scanner-based image

analysis programme WinRhizo 2002 software (�Regent Instruments, Canada) version

WinPro. Prior to conducting the analysis of variance, data were checked for normality. All

further analyses were conducted using the untransformed data. Analyses of variance

(ANOVA) were performed on the number of shoots produced per plant, number of roots,

number of apexes, root diameter, root length, root surface area, root volume, RDB and

ADB. As a post hoc test, we used Turkey’s HSD. On percentages of rooted cuttings, we

performed a pair-wise chi-squared analysis. All the statistical analyses were performed

using SPSS Inc.�, Win TM, version 12.

Results

Effects of temperature and IBA

The IBA treatment significantly improved cutting rooting ability at 25�C but this effect was

not observed when the cuttings were preconditioned for 1 week at 4�C without light where,

in fact, the highest percentages of rooting were observed without IBA. A significant

interaction between IBA and temperature was found on the percentage of rooted cuttings,

ranging from 68 to 97%, but the highest percentage was observed with IBA levels of

40 g L-1 at 25�C. The morphological root variables analyzed (number of apexes, length,

area and volume) were significantly influenced by the increase in temperature (P \ 0.05),

although this effect was not found for the root diameter. The growth regulator effect was

significant only for surface area and volume of roots (Table 2).

Effects of shoot age, collection time and mini-cuttings production

A significant interaction was detected between IBA and the shoot age in relation to the

rooted cuttings. Earlier time of collection significantly influenced rooting capacity and

New Forests (2011) 41:399–412 403

123

Ta

ble

2E

ffec

tso

fte

mp

erat

ure

and

ind

ole

bu

tyri

cac

id(I

BA

)o

nth

ep

erce

nta

ge

of

roo

ted

cutt

ing

s,n

um

ber

of

roo

tsan

dap

exes

,d

iam

eter

,le

ng

th,

surf

ace

area

and

vo

lum

eo

fro

ot

per

roo

ted

min

i-cu

ttin

g(m

ean

±S

E)

Tre

atm

ents

Roo

ted

cutt

ing

(%)

Nu

mb

ero

fro

ots

Nu

mb

ero

fap

exes

Roo

td

iam

eter

(mm

)R

oo

tle

ngth

(mm

)R

oo

tsu

rfac

ear

ea(c

m2)

Ro

ot

vo

lum

e(c

m3)

Tem

per

atu

re(�

C)

IBA

(gL

-1)

40

92

.53

.1±

0.2

ab3

7.0

±3

.7c

1.3

6±

0.0

4a

28

.5±

2.7

b1

1.2

9±

0.9

c0

.36

±0

.03

c

10

86

.53

.7±

0.2

ab4

4.5

±4

.2b

c1

.33

±0

.05

a3

6.2

±3

.4ab

13

.77

±0

.9b

c0

.43

±0

.03

bc

40

67

.63

.7±

0.3

ab4

3.7

±4

.1b

c1

.35

±0

.04

a3

2.9

±3

.9b

13

.46

±1

.3b

c0

.45

±0

.04

bc

25

±2

09

2.1

2.9

±0

.2b

50

.2±

5.0

ab1

.43

±0

.04

a3

3.4

±3

.1b

14

.21

±1

.1b

0.4

6±

0.0

4b

10

94

.73

.5±

0.2

ab5

2.1

±4

.6ab

1.3

9±

0.0

5a

34

.6±

2.9

b1

4.1

5±

1.0

b0

.45

±0

.03

bc

40

97

.43

.8±

0.3

a5

9.7

±4

.4a

1.4

0±

0.0

4a

43

.8±

2.6

a1

8.5

7±

0.9

a0

.59

±0

.04

a

Tem

p.

0.0

14

0.0

73

[0

.000

10

.08

60

.017

[0

.000

1[

0.0

00

1

IBA

0.5

75

0.5

58

0.2

76

0.7

81

0.0

60

0.0

13

0.0

15

Tem

p.

9IB

A0

.00

80

.67

10

.89

80

.156

0.1

14

0.2

51

Mea

nv

alues

foll

ow

edb

yth

esa

me

lett

erar

en

ot

sig

nifi

can

tly

dif

fere

nt

at5

%le

vel

404 New Forests (2011) 41:399–412

123

survival of cuttings in the experiments. The youngest cuttings (70 days after pruning) had

the highest rooting percentages (nearly 90%). On the contrary, the percentage dropped to

32.8 after 120 days of pruning in experiment 2 (Table 3).

Later shoot age at time of collection significantly and negatively influenced morpho-

logical root variables (number of apexes, diameter, length, area and volume). The youngest

mini-cuttings had roots with high surface area, volume, diameter and length (Table 3).

With regards to mini-cutting production, the collection time significantly influenced

(F = 85.203, P \ 0.001) the production of mini-cutting per square meter, and the highest

value (1,363 in trays) was obtained at 330 days after planting. The spring was the most

favourable season for the collection of mini-cuttings. On the contrary, the lowest pro-

duction values were observed in winter time (cuttings planted in autumn) (Fig. 1). Vari-

ation in rooting between families increased with more favourable seasonal conditions,

though family rankings changed during the study period. There was a highly significant

difference in rooting ability between the 9 half-sib families (P \ 0.001), but the effect of

repetitions was not significant (P \ 0.2). The family 9 repetition interaction was used as

the error term for both effects.

The annual productivity per square meter was around 6,435 mini-cuttings. The average

rooted cutting percentage during the year was 75%, with minimum values (54%) in winter

time and a maximum of 97% at the end of summer. The months of least mini-cutting

growth were December and January, when mean values were: 1.13 ± 0.08 for number of

roots, 0.02 ± 0.001 g for RDB, 29.25 ± 0.82 mm for mini-cutting length and

0.18 ± 0.02 g for ADB. On the other hand, the mini-cuttings pruned before spring time

(cycle 300) had the highest values for length and ADB. There were statistically significant

differences between cycles for all variables evaluated (rooted cuttings, number of roots,

RDB, length and ADB) (Table 4).

Discussion

The rooting capacity found in the present study indicates that the mini-cutting management

and induction treatments imposed significantly influenced the rooting ability of P. pinaster.

The high rate of rooting percentage ([97%) with IBA application at environmental tem-

perature (25�C) shows that maritime pine can be successfully cultivated under these

conditions, demonstrating the efficacy of our protocol. It is worth noting that the appli-

cation of growth regulator affected root development, with the highest morphological

development being associated with the highest IBA levels (40 g L-1 or 1,000 mg L-1) at

environmental temperature. This behaviour is similar to that found by Wendling et al.

(2000) for Eucalyptus, and Titon et al. (2006) for Ilex paraguariensis. However, in others

species such as Sweetgum (Liquidambar styraciflua), IBA application has no effect on

rooting response (Wendling et al. 2010), which is an advantage in that it reduces overall

production cost (Rocha and Niella 2002). However, it is important to consider that growth

regulator not only influences percentage of rooting, it may also accelerate the onset of the

rooting process and increase the number and quality of roots (Hartmann et al. 2002), an

important factor since a good radical system favours increased survival (Duryea 1984;

Rose et al. 1998). Also, the use of IBA has a strong effect on root initiation: not only does it

affect morphogenetic factors, but it also increases the transport of carbohydrates formed

during photosynthesis from needles to the site of root formation at the base of a cutting

(Cameron and Thomson 1969).

New Forests (2011) 41:399–412 405

123

Ta

ble

3E

ffec

tso

fsh

oot

age

(SA

)an

din

do

leb

uty

ric

acid

(IB

A)

on

the

per

cen

tag

eo

fro

ote

dcu

ttin

gs,

nu

mb

ero

fro

ots

and

apex

es,

dia

met

er,

len

gth

,su

rfac

ear

eaan

dv

olu

me

of

roo

tp

erro

ote

dm

ini-

cutt

ing

(mea

n±

SE

)

Tre

atm

ents

Roote

dcu

ttin

g(%

)N

um

ber

of

roots

Num

ber

of

apex

esR

oot

dia

met

er(m

m)

Root

length

(mm

)R

oot

surf

ace

area

(cm

2)

Root

volu

me

cm3

S(d

ays)

IBA

(mg

L-

1)

70

084.7

2.0

7±

0.1

9b

48

±4

ab1.3

5±

0.0

4a

32.1

±2.5

0b

12.9

±0.8

6b

0.4

3±

0.0

3b

250

88.1

2.1

9±

0.2

1b

46

±4

ab1.3

7±

0.0

3a

32.5

±2.1

7b

13.3

±0.7

5b

0.4

4±

0.0

2b

500

96.6

2.2

0±

0.1

6b

45

±±

5b

1.3

3±

0.0

3a

31.7

±2.6

0b

12.4

±0.8

3b

0.3

8±

0.0

2b

1,0

00

98.3

3.1

2±

0.2

1a

58

±4

a1.2

8±

0.0

3a

44.6

±2.4

7a

17.5

±0.9

1a

0.5

2±

0.0

3a

0.0

17

[0.0

001

0.1

22

0.2

16

[0.0

001

[0.0

001

0.0

01

90

058.8

1.8

2±

0.3

0a

42

±4

a1.4

5±

0.0

4a

23.2

±2.7

3a

10.4

5±

1.2

3a

0.3

8±

0.0

5a

250

56.0

1.2

4±

0.2

7a

30

±3

b1.4

8±

0.0

4a

18.5

±2.6

7a

8.1

5±

1.0

4a

0.2

9±

0.0

3ab

500

71.7

1.6

5±

0.2

0a

34

±3

ab1.3

9±

0.0

3a

19.9

±2.2

9a

8.2

4±

0.8

6a

0.2

8±

0.0

3b

1,0

00

65.5

1.4

5±

0.2

0a

32

±3

b1.4

4±

0.0

3a

19.1

±2.4

5a

8.4

2±

1.0

4a

0.3

0±

0.0

4ab

0.3

77

0.3

69

0.0

98

0.3

41

0.5

90

0.3

81

0.2

05

120

021.7

0.3

7±

0.1

0c

23

±5

a1.4

0±

0.0

9a

7.2

±1.2

9a

2.9

2±

0.5

1a

0.1

0±

0.0

2ab

250

33.9

0.5

3±

0.1

1bc

19

±3

a1.3

6±

0.0

5a

6.9

±1.1

7a

2.8

3±

0.4

5a

0.0

9±

0.0

1b

500

37.0

0.5

2±

0.1

0bc

21

±3

a1.5

4±

0.0

7a

8.5

±1.1

3a

3.9

9±

0.5

5a

0.1

5±

0.0

2a

1,0

00

39.0

0.7

8±

0.1

5ab

24

±3

a1.5

2±

0.0

9a

9.1

±1.4

4a

3.9

1±

0.5

8a

0.1

4±

0.0

2ab

0.1

81

0.0

95

0.3

75

0.5

73

0.2

71

0.2

82

0.0

99

IBA

054.7

1.3

9±

0.1

3b

42

±3

a1.3

9±

0.0

3a

25.5

5±

1.8

3ab

10.6

4±

0.7

0ab

0.3

6±

0.0

2a

250

59.5

1.3

2±

0.1

3b

37

±2

a1.4

0±

0.0

2a

23.6

±1.7

1b

9.8

4±

0.6

4b

0.3

3±

0.0

2ab

500

69.2

1.4

7±

0.1

1b

37

±3

a1.3

9±

0.0

2a

23.9

±1.7

4b

9.6

5±

0.5

9b

0.3

1±

0.0

2b

1,0

00

67.6

1.7

8±

0.1

3a

43

±3

a1.3

8±

0.0

2a

29.6

±2.0

1a

11.9

7±

0.7

6a

0.3

8±

0.0

2a

SA

70

91.9

2.3

9±

0.1

0a

49

±2

a1.3

3±

0.0

2b

35.4

±1.2

8a

14.1

2±

0.4

4a

0.4

5±

0.0

1a

90

62.9

1.5

4±

0.1

2b

34

±2

b1.4

4±

0.0

2a

20.2

±1.2

6b

8.7

9±

0.5

2b

0.3

1±

0.0

2b

120

32.8

0.5

5±

0.0

6c

22

±2

c1.4

6±

0.0

4a

8.0

6±

0.6

4c

3.4

8±

0.2

7c

0.1

2±

0.0

1c

IBA

0.0

18

0.0

15

0.2

87

0.9

32

0.1

22

0.0

92

0.2

03

SA

[0.0

001

[0.0

01

[0.0

001

[0.0

001

[0.0

001

[0.0

001

[0.0

001

SA

xIB

A0.0

07

0.3

24

0.0

39

0.0

13

0.0

05

0.0

20

Mea

nval

ues

foll

ow

edby

the

sam

ele

tter

sar

enot

signifi

cantl

ydif

fere

nt

at5%

level

406 New Forests (2011) 41:399–412

123

Another important variable is the combined influence of temperature and light on the

rooting process. There is evidence in literature about the effect of keeping shoots in the

dark as a pre-severance treatment that could benefit root initiation by mobilizing nitrogen

within the shoot. However, our experiments show high rates of rooting even when the

cuttings were not preconditioned for 1 week at 4�C without light, and were kept at

environment temperature (25�C) from the start.

Our data suggest that the use of juvenile cuttings considerably increased rates of rooting

capacity and positively influences morphological root variables (e.g. length, surface area

and volume), which concurs with results of other studies using species like Eucalyptus, Ilexor Pinus virginia P. Mill (Foster et al. 2000). Our experiments also show that the use of

juvenile cuttings with growth regulator favoured the increase of these morphological

variables as IBA concentration increased. Other studies, in contrast, have found that use of

juvenile material reduced hormone requirements for the rooting process, thereby

decreasing propagation cost (Higashi and Goncalves 2000; Wendling and Souza Junior

2003).

Hamann (1998), working with Pinus taeda, found that in cuttings aged 90 days, from

seedlings and hedge donor plants, 80% showed development of roots. However, the

incidence of root development in cuttings from 3 years-old trees ranged from 0 to 20%.

Moreover, it was further observed that callus development in cuttings from 3 year-old trees

was less vigorous and also that emerging roots were thicker and more brittle compared to

those of cuttings from hedges or seedlings.

Nº

min

i-cut

tings

3-5

cm

/ m2

0

500

1000

1500

2000

Mea

n m

onth

ly h

umid

ity (

%)

0

20

40

60

80

100

Mea

n m

onth

ly te

mpe

ratu

re (

ºC)

5

10

15

20

25

Alto 4Alto 8Arma 5Cada 4Cast 9Lamu 9

Segu 11Segu 13Segu 19HumidityTemperature

Time

Jun 06Jul 0

6

Aug 06

Sept 06

Oct 06

Nov 06

Dec 06Jan 07

Feb 07

Mar 07

Apr 07

Fig. 1 Production of mini-cuttings of 3–5 cm in length per square meter during 1 year (from June 2006 toApril 2007), for nine half-sib families of Pinus pinaster Ait. and mean monthly temperature and humidityunder glasshouse conditions (SigmaPlot 10.0)

New Forests (2011) 41:399–412 407

123

Ta

ble

4E

ffec

tso

fco

llec

tio

nti

me

(CT

)o

nro

ote

dcu

ttin

gs

(%),

nu

mb

ero

fro

ots

,ro

ot

dry

bio

mas

s(R

DB

),m

ini-

cutt

ing

len

gth

and

aeri

ald

ryb

iom

ass

(AD

B)

of

nin

efa

mil

ies

of

Pin

us

pin

ast

erA

it.,

ov

er1

yea

r(6

0,

90

,1

20

,1

50

,1

80

,2

10

,3

00

,3

30

and

36

0d

ays)

Coll

ecti

on

tim

es(C

T)

Dat

eof

mea

sure

men

tR

oote

dcu

ttin

gs

(%)

Num

ber

of

roots

RD

B(g

)L

ength

(mm

)A

DB

(g)

60

(Ju

n0

6)

Sep

t0

65

7.7

7±

6.4

8cd

2.5

6±

0.0

7a

0.0

7±

0.0

0a

61

.85

±0

.92

b0

.27

±0

.01

cd

90

(Ju

l0

6)

Oct

06

75

.56

±9

.10

abc

2.4

7±

0.0

6a

0.0

6±

0.0

0a

54

.91

±1

.06

c0

.26

±0

.01

cd

12

0(A

ug

06

)N

ov

06

88

.15

±5

.53

abc

1.7

3±

0.0

6b

0.0

4±

0.0

0b

44

.60

±0

.83

de

0.2

4±

0.0

1d

15

0(S

ept

06

)D

ec0

69

7.0

4±

1.6

1ab

c1

.69

±0

.06

bc

0.0

4±

0.0

1cd

f4

1.9

5±

0.8

3e

0.1

9±

0.0

1e

18

0(O

ct0

6)

Jan

07

65

.93

±5

.49

bcd

1.1

3±

0.0

8d

0.0

2±

0.0

0e

29

.25

±0

.82

f0

.18

±0

.02

e

21

0(N

ov

06

)A

pr

07

54

.81

±4

.27

d0

.95

±0

.07

cd0

.03

±0

.00

fe3

2.0

2±

0.8

2f

0.2

2±

0.0

1d

30

0(F

eb0

7)

May

07

82

.96

±3

.16

abc

1.6

8±

0.0

7b

0.0

4±

0.0

0b

cd6

8.4

2±

0.8

5a

0.3

6±

0.0

1a

33

0(M

ar(0

7)

Jun

07

74

.07

±4

.90

abcd

2.0

7±

0.0

7b

0.0

3±

0.0

0d

f4

3.9

9±

0.8

7d

e0

.29

±0

.01

bc

36

0(A

pr

07

)Ju

n0

78

0.7

4±

3.5

9ab

c1

.87

±0

.06

bc

0.0

4±

0.0

0b

c4

6.3

2±

0.8

3d

0.3

3±

0.0

1ab

Mea

n±

SE

75

.23

±2

.22

1.7

9±

0.0

70

.04

±0

.00

46

.42

±0

.84

0.2

6±

0.0

1

CT

\0

.000

1\

0.0

00

1\

0.0

00

1\

0.0

00

1\

0.0

00

1

Fam

.0

.474

6\

0.0

00

1\

0.0

00

1\

0.0

00

10

.06

CT

xF

am.

0.1

32

7\

0.0

00

10

.087

\0

.000

1\

0.0

00

1

Mea

n(±

SE

)v

alu

esfo

llo

wed

by

the

sam

ele

tter

sar

en

ot

sig

nifi

can

tly

dif

fere

nt

at5

%le

vel

408 New Forests (2011) 41:399–412

123

Importantly, the mini-propagation techniques used herein provided a regular production

of cuttings over the time evaluated (1 year): an aspect that has been widely studied by

many authors in many species (Greenwood and Hutchison 1993; Rosse et al. 1997;

Hogberg 2005; Alcantara et al. 2007; Munoz-Gutierrez et al. 2009).

However, there is currently still little information about the effect of time of collection

on vegetative propagation of conifers by mini-cuttings (Greenwood and Hutchison 1993)

and there is no information at all for maritime pine (Majada and Alıa 2004). Our results

show that the time of collection was a significant factor affecting rooting capacity. The data

obtained in this work suggest that the number of times cuttings are collected can be

increased without any loss in total productivity of each donor plant (Wendling et al. 2010).

Originally the mini-cutting system was based on mini-hedges established out-doors

through rooted mini-cuttings grown in small containers (dibble tubes). This system pro-

vided a series of technical and economic benefits as well as good root quality (Assis and

Mafia 2007). Despite representing a great advance over clonal hedges in the field, mini-

hedges had some limitations. The outdoor mini-hedges were still hostage to climate, and

problems related to adequate maintenance of nutritional status and leaf diseases continued,

especially during winter. These limitations led to the development of an indoor mini-hedge

system. In the present study, the use of indoor mini-hedges enabled the doubling of the

number of cutting collections per year (data not shown). The main reasons for such

increases are related to, firstly, higher levels of juvenility and, secondly, optimal nutritional

content of the tissues, both of which improve rooting predisposition and speed of root

initiation. The rooting speed of mini-cuttings has two other important consequences in a

commercial cloning program: the time that the plants are kept indoors is usually reduced

by half compared to rooting of stem-cuttings, Moreover, the mini-cuttings produce better

quality root systems with a tendency for development of a taproot-like system where the

connection between roots and stem tissues in the mini-cuttings is apparently more suitable

due to reduced lignification of the tissues involved. However, further long-term studies

need to be conducted to optimize the number of collections that can be made from the

mini-clonal indoor hedge without significant loss in productivity.

Comparing the average mini-cutting production rate of maritime pine (2.02 per 90 days)

with other species like Ilex paraguariensis (3.4 per 30 days), Liquidambar styraciflua (2.5

per 30 days) or Eucalyptus (9.7 per 30 days), it can be seen to be lower. In addition, the

annual productivity of P. pinaster mini-cuttings per square meter can be estimated at

around 6,435 less than the average of 9,762 for Eucalyptus grandis x E. europhylla (Titon

et al. 2006) hybrid but higher than the 2,953 for L. styraciflua. The applicability of the

mini-cutting technique has also been tested on other broadleaf and coniferous woody

species. In general, the methods used in this work are similar to those used with Euca-lyptus, though with minor adaptations. A review of available literature showed this tech-

nique to be fully applicable to species like Pinus taeda, P. elliiottii, Acacia mearnsii and

Ilex paraguariensis as well as other woody and non-woody species.

Managing pine mini-hedges is an efficient method of providing mini-cuttings with a

good nutritional status. The production per square meter of the pine mini-propagules,

ranged from 4,447 to 8,581 per year depending on the rooting capacity of each family.

Similar results of 9,600/m2/year for Pinus (P. taeda and P. elliiottii), have been cited in

Assis et al. (2004).

One question that arises is the influence of plant spacing in mini-hedges on the rooting

of mini-cuttings and mini-stump productivity. In our case, trials showed that 5 9 10 cm

spacing between plants does not interfere significantly in the rooting capacity of the mini-

cuttings. Moreover, the development of special nutritive solutions for P. pinaster (work in

New Forests (2011) 41:399–412 409

123

progress) tends to improve the results and it is clearly possible to establish efficient clonal

programs for this species.

Compared to the traditional rooting procedure by stem-cuttings, mini-cuttings have

many advantages, leading to operational, technical, economic, environmental and quality

benefits. Operationally, the labour and cost demands are markedly reduced, due to the

elimination of labour intensive treatment with growth substances, and other operations

required. Many field operations such as soil preparation, fertilization, cultivation, pest and

disease control, etc. are replaced by intensive activities in smaller indoor areas at much

lower costs.

The creation of the technologies used in this investigation is a land-mark in the evo-

lution of the cloning systems used for certain species (e.g. Eucalyptus, Assis et al. 2004),and has resulted in profound changes in operational procedures, marking the beginning of a

new cycle in the propagation of woody species.

Conclusions

Vegetative propagation by mini-cuttings provided a high rooting percentage in P. pinasterAit. The use of juvenile material, good environmental conditions and the application of

IBA benefited the rooting of clonal material and resulted in high rooting capacity. The

propagation protocol developed in this study proved to be very effective and is easy to

implement on a large scale. The authors consider this protocol to be a valuable innovative

propagation technique for P. pinaster that has been used to produce a reference collection

of the different genotypes for the Mediterranean distribution area of this commercially and

environmentally important species.

Acknowledgments This study was supported by funding from the European Project TREESNIP-QLK3-CT2002-01973 and the project ‘‘Heterogeneidad ambiental y adaptabilidad en respuesta a la sequıa encolecciones clonales de Pinus pinaster’’, ref RTA-2007-00084-00-00. Enrique Fernandez, Dolores Busto,Inmaculada Concepcion and Manuel Zapico provided technical support during the experiments.

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