Scientia Horticulturae, 51 ( 1992 ) 335-342 335

Elsevier Science Publishers B.V., Amsterdam

Short Communication

Root development of in vitro hybrid walnut

microcuttings in a vermiculite-containing gelrite
medium

C. Jay-Allemand, P. Capelli and D. Cornu

Station d'A m~lioration des A rbres Forestiers, INRA, 45160 A rdon, Frarce
(Accepted 29 January 1992 )

ABSTRACT

Jay-Allemand, C., Capelli, P. and Cornu, D., 1992. Root development of in vitro hybrid walnut mi-
crocuttings in a vermiculite-containing gelrite medium. Scientia Hortic., 51" 3"~5-342.

Quantitative and qualitative improvements in root development have been obtained in six clones
of hybrid walnut trees propagated by tissue culture and selected for multiplication and rooting abili-
ties. A mixture of diluted gelified medium (DKW, macroelements 1/4) and vermiculite, used in the
proportions 250/200 (v/v), strongly promoted root elongation (two to seven-fold) and the devel-
opment of secondary roots of induced shoots (IBA 24.6 #M ) after 2 weeks of culture. Furthermore,
rooting rates were enhanced (from 15 to 50%) and the number of primary roots per rooted explant
was from two to six-fold higher. Distilled water added to vermiculite always gave the poorest rooting.
Vermiculite promoted penetration and aeration of the roots more than gelrite alone, and its effect was
better than that of perlite. This procedure resulted in 80-100% rooting for five hybrid clones.

Keywords: Juglans; micropropagation; root development; rooting, substrate; vermiculite; walnut.

Abbreviations: BA = Benzyl-adenine; GM = DKW gelified medium with macroelements diluted to I /

4; IBA = indole-butyric acid.

INTRODUCTION

The vegetative propagation of walnut trees has not yet been totally per-
fected for efficient commercial applications in spite of important technical
improvements (Driver and Kuniyuki, 1984; Cornu and Jay-Allemand, 1989 ),
in vitro clonal selection (Jay-Allemand et al., 1989) and the j uvenility of plant
material (Cossio and Minotta, 1983; Jay-Allemand and Comu, 1986). We
Correspondence to: C. Jay-Allemand, Station d'Am61ioration des Arbres Forestiers, INRA, 45160
Ardon, France.

© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4238/92/$05.00

336 C. JAY-ALLEMAND ET AL.

have shown the possibility of producing 60 shoots per month suitable for
rooting from 100 bud clusters (Cornu and Jay-Allemand, 1989 ) of interspe-
cific hybrids (Juglans nigraXJuglans regia), and maintaining them in vitro
for several years. Some promising results have also been obtained with J. re-
gia (McGranahan et al., 1988) and Juglans hindsii×J, regia (Driver and
Kuniyuki, 1984), all of which suggests a significant potential for organoge-
nesis in Juglans. However, Rodriguez et al. (1989) recently concluded that
walnut micropropagation is still an unsolved problem. The main reasons are
irregular and often low rooting rates (Liu and Hun, 1986; Jay-Allemand et
al., 1989; Rodriguez et al., 1989 ), and high mortalities of rooted plants during
acclimatization (Schwarz, 1988). The work reported here was undertaken to
develop new tissue culture techniques for promoting root development and
successful propagation in vitro of selected clones of interspecific hybrid wal-
nut trees.

MATERIALS AND METHODS

Plant material. - Six clones of interspecific hybrid walnut were established

from different embryonic axes isolated axenicaUy (Jay-Allemand and Cornu,
1986) from mature nuts collected on J. nigra or hybrid walnuts. The embryos
from black walnuts were identified as hybrids 3 weeks after their in vitro in-
troduction by means of typical morphological characteristics previously de-
scribed in young seedlings (Jay-Allemand et al., 1990). These hybrids were
obtained by open pollination of female flowers of black walnuts or interspe-
cific hybrids by pollen of J. regia. The origins of the clones are detailed in
Table I.
Tissue culture (Fig. l ). - Shoots for rooting were obtained during the multi-
TABLE I

Origin and identification of hybrid walnut clones. All mother trees were pollinated by J. regia

Mother trees

J. t~igra J. major J. major No. 209 X J. seboldiana ×

J. regia J. regia

Tree (No.) 23 38 209 513 522

Locality INRA Nursery Nurser3 ONF ONF

Bordeaux AILeac Albenc Gap Gap
(33) (38) (38) (05) (05)

Clone (No.) D152 G! MR8 HA3-1 HA2-13

MR9
IN VITRO ROOT DEVELOPML "OF HYBRID WALNUT CLONES 337

MULTIPLICATION ROOTING PHASE

PHASE INDUCTION DEVELOPMENT
IN VITRO IN VIVO
il t !
2 week,,, 2 weeks ]i

N t
i
Fig, 1. The different steps of the in vitro culture of hybrid walnut clones in order to produce
rooted plants after several subcultures (multiplication phase) and three rooting phases, includ-
ing induction (5 days), in vitro root development (2 weeks) followed by in vivo root develop-
ment (2 weeks) for acclimatization. Root formation and elongation were mainly obtained 2
weeks after root induction.

plication phase from elongated shoots on bud clusters in jars (750 ml) con-
taining DKW gelified medium (Driver znd Kuniyuki, 1984) with 4.4 uM
BA, 0.005/zM IBA and 0.25% w/v gelrite after 3 weeks. The numbers of sub-
cultures of Clones D152, G1, MR8, MR9, HA3-1 and HA2-13 were 37, 19,
2 l, 19, 32 and 3 l, respectively, before the experiments. The rooting phase was
divided into three steps: (a) root induction of shoots in DKW gelified me-
dium with macroelements diluted to 1/4 (GM) and 24.6/zM IBA for 5 days;
(b) in vitro root development normally occurred in 2 weeks in GM without
growth regulator, from auxin-treated shoots; (c) rooted and unrooted shoots
were then systematically transferred to vermiculite: sand: water (2: l : l, v / v /
v ) under mist produced by an ultrasonic system (Biotop 161, SOFRAXAIR,
France). A temperature of 28°C with a light intensity of 70 # E m -2 s-~ and
a photoperiod of 16 h day: 8 h night were used, except for the root-induction
phase which was conducted in darkness.

Experiments. These were concentrated on the in vitro root development

-
phase (Fig. 1 ) by modifications of the substrate quality in order to stimulate
root expression. The different support media used in these experiments were
GM without growth regulator, vermiculite (Fertil-Vermagri, Size M, France)
or perlite (Fertile-Perlagri, France), mi~,ed or unmixed with the same me-
dium solidified by 0.2% w/v gelrite. Gelified medium (200 ml) was added
to 250 ml of vermiculite or perlite. Two other series of shoots from Clones
MR9 (60 shoots) and HA3-1 (66 shoots) were used to check the effects on
root development of both the different amounts of GM ( 100, 200 and 300
ml) added to 250 ml of vermiculite, and the medium composed mainly of
sucrose~ mineral salts and gelrite, in comparison with the control vermicu-
338 C. JAY-ALLEMAND ET AL.

lite-distilled water (250-200 ml). The substrate was prepared by gradually

pouring hot medium (liquid medium containing gelrite) into ajar containing
250 ml of vermiculite or pedite. Measurements were taken mainly at the end
of the in vitro root development phase, e.g. 15 days after the induction phase,
using shoots carefully removed from the substrate and then soaked in distilled
water. Numbers of primary and secondary roots and the length of primary
roots were determined just before the last transfer phase. Complementary ob-
servations were made on roots of Clone HA3-1 cultivated for 4 weeks in GM
in order to show its effect on root development. Standard errors were deter-
mined on the average length of primary roots and the number of primary
roots per rooted explant.

RESULTS

Vermiculite The addition of vermiculite to GM during the in vitro

effects. -
rooting phase improved the rooting rates of the six clones. Five clones reached
80-100% rooting and the number of primary roots per shoot increased two to
six-fold in four clones (Tables 2 and 3). Most clones formed numerous sec-
ondary roots after 2 weeks of culture (Table 2; Fig. 2). In addition, the strol g-
est effect concerned the elongation of roots, which increased two to three-fold
in Clones MR9, HA3- I, D 152 and MR8, and four to seven-fold in Clones G 1
and HA2-13, compared with the controls (Tables 2 and 3 ). The stimulation
of root development is shown in Figs. 2(A), 2(C) and 2(E), 2(G) for two
clones after 2 weeks ofculture, including uniformity in the number and length
TABLE 2

Efl}ct of vermiculite or perlite (250 ml) mixed with gelified medium (GM; 200 ml) on in vitro
rooting rates and root development of two hybrid walnut clones after 2 weeks. Standard errors were
determined for the length and number of primary roots

Clone No. of No. ofrooted Total no. Total no. Averagelength No. of primary
explants explants ofprimary of secondary of primary roots per
(% rooting) roots roots roots (ram) rooted explant

MR9 GM 16 14(88) 63 0 11.7±! 4.5+__0.6

GM with 16 11 (69) 82 6 21.9±1.2 7.5±0.9
perlite
GM with 16 16 (100) 89 72 34.3+_ 1.8 5.6____.0.7
vermiculite

HA3-1 GM 16 7 (44) II 0 8.1 ± 1.2 1.6+0.2

GM with 32 26 (81) 120 0 18.1 ± 1.3 4.6+0.5
perlite
GM with 32 29 (91) 139 14 26.6+__ 1.3 4.8±0.5
vermiculite
IN VITROROOT DEVELOPMENTOF HYBRIDWALNUTCLONES 339

TABLE 3

Effect of vermiculite (250 ml ) mixed with gelified medium (GM; 200 ml ) on in vitro rooting rates and root
development of four hybrid walnut clones after 2 weeks. Standard errors were determined for the length and
number of primary roots

Clone No of No. of rooted Total no. Total no. Average length No. of primary
explants explants of primary of secondary of primary rootsper
(% rooting) roots roots roots (mm) rooted explant

D152 GM 24 13 (54) 69 0 9.5_+0.8 5.3+_ 1

GM with 24 19 (79) 97 13 20.6-+ I.I 5.1 ___0.7
vermiculite

GI GM 16 1 (6) I 0 5 I
GM with 24 11 (46) 20 9 35.2_+5.1 1.8_+.0.6
vermiculite

MR8 GM 12 9 (75) 22 0 8.4+0.8 2.4+0.7

GM with 17 17 (100) 92 18 21.8+ 1.3 5.4_+0.7
vermiculite

HA2-13 GM 21 10 (48) 14 0 6.5+3.5 1.4+0.2

GM with 28 28 (100) 231 0 23.5+ I.I 8.6+0.7
vermiculite

of roots (Figs. 2 (D) and 2 (H) ). The root quality was also strong!y affected
by the substrate: in GM roots remained short and thick, as shown in Fig. 2 (F)
after 4 weeks of culture, whereas roots which developed in the vermiculite-
GM mixture were thinner (Figs. 2(C) and 2(G) ).

P e r l i t e effects. - Perlite (250 ml) mixed with GM 1200 ml) was tested and
compared with GM alone or associated with vermiculite in two clones (Table
2 ). Perlite was less effective than vermiculite in increasing the length of pri-
mary roots as well as the number of secondary roots. However, we observed
an increase in root number per rooted explant with perlite for Clone MR9.

Other medium effects. - In the additional experiments, vermiculite mixed with

200 ml of GM gave the best results for root elongation: 21-22 +_ 1.3-1.4 mm
for Clones HA3-1 and MR9. The average primary root length in GM at 100
ml for Clone HA3-1 was 10.3 +_7.2 mm and, in this mixture, no root was de-
veloped for Clone MR9. Root elongation with GM at 300 ml was 15 + 2.7 mm
for Clone HA3-1 and 8.9_+0.8 m m for Clone MR9. Furthermore, a small
amount of GM ( 100 ml) drastically reduced the rooting rates: from 60 to
30% for Clone HA3-1 and from 90 to 0% for Clone MR9. The control with
water was always the worst treatment with only 8% and 25% of rooting and
short roots, respectively, for Clones HA3-1 and MR9.
340 C. JAY-ALLEMAND ET AL.

Fig. 2. Roots of two hybrid walnut clones, HA3-1 (A-D) and MR9 (E-F), developed in vitro
in different substrates 2 or 4 weeks after root induction (A and E) in gelified medium after 2
weeks; (F) in gelified medium after 4 weeks; (B) in perlite with gelified medium after 2 weeks;
(C, D, (3 and H) in vermiculite with gelified medium after 2 weeks. On (C, L" G and H ), note
t.t ~ relative uniformity of primary roots and the presence of secondary roots (arrows).
IN VITRO ROOT DEVELOPMENT OF HYBRID WALNUT CLONES 341

DISCUSSION AND CONCLUSIONS

The proposed method favouring the in vitro root development of walnut

increased both the rooting rates and the number of roots per rooted shoot tip.
The semi-solid medium of gelrite with vermiculite is a reliable substrate for
root expression, giving more regular and homogeneous results than those ob-
tained in GM. For example, from three series of shoot tips of HA3-1 corre-
sponding to three different subcultures, rooting rates were 60-90%, whereas
with the conventional procedure they were 10-80% (unpublished results)
often with low rooting rates. Moreover, these results suggest that the induc-
tion phase by IBA in darkness is effective (five roots and more per rooted
shoot in most of the clones).
Possible explanations for the improved results include aeration, which is
strongly enhanced by this procedure. Aeration plays an important role in
raspberry root development with foam substrates (Gebhart, 1985 ). In addi-
tion, the emergence and development of adventitious roots of artichoke were
directly associated with 02 uptake and activation of the alternative pathway
in respiratory metabolism, showing the importance of available 02 in these
physiological processes (Hase, 1987 ). Secondly, vermiculite mixed with GM
in the right proportions seems to give a good balance between aeration and
the availability of water (humidity) at the bottom of the shoot tip, which
promotes root initiation and development. A small amount of medium ( 100
ml) added to vermiculite (250 ml) showed low rates of rooting and short
roots. Similar effects on root length were found with a large amount of me-
dium (300 ml). Changes of medium pH cannot be excluded, in view of pos-
sible interactions with rooting in woody species in vitro (Williams et al.,
1985 ). Moreover, perlite associated to be a gelified medium, which was suc-
cessfully used to convert globular somatic embryos into plantlets (Nadel e~
al., 1990), was less effective than vermiculite in our experiments. This result
could be connected with the factors mentioned above, including also the
hardness of the substrate (penetration) and the availability of components
from the culture medium, such as sucrose which promotes root development
(Pal and Nanda, 1981; Haissig, 1982; Jay-Allemand and Cornu, 1986).
This new substrate, which can easily be used during the first phase of root
development, will aid basic research on walnut rhizogenesis based on reliable
criteria, including rapid root expression. In addition, this method should pro-
mote commercial application, still depending on acclimatization studies,
which could be realized with rooted explants of high quality. This support
system could be also extended to other woody species.

ACKNOWLEDGEMENTS

We would like to thank William J. Mattson (USDA Forest Service, East

Lansing) for the English correction of this article.
342 C. JAY-ALLEMAND ET AL.

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