Root development of in vitro hybrid walnut microcuttings in a vermiculite-containing gelrite medium

Scientia Horticulturae, 51 ( 1992 ) 335-342 Elsevier Science Publishers B.V., Amsterdam

335

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 microcuttings 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 abilities. 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 development 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 perfected 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

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have shown the possibility of producing 60 shoots per month suitable for rooting from 100 bud clusters (Cornu and Jay-Allemand, 1989 ) of interspecific hybrids (Juglans nigraXJuglans regia), and maintaining them in vitro for several years. Some promising results have also been obtained with J. regia (McGranahan et al., 1988) and Juglans hindsii×J, regia (Driver and Kuniyuki, 1984), all of which suggests a significant potential for organogenesis 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 walnut 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 introduction by means of typical morphological characteristics previously described in young seedlings (Jay-Allemand et al., 1990). These hybrids were obtained by open pollination of female flowers of black walnuts or interspecific 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 multiTABLE 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. regia

J. seboldiana × J. regia

Tree (No.)

23

38

209

513

522

Locality

INRA Bordeaux (33)

Nursery AILeac (38)

Nurser3 Albenc (38)

ONF Gap (05)

ONF Gap (05)

Clone (No.)

D152

G!

MR8 MR9

HA3-1

HA2-13

IN VITRO ROOT DEVELOPML

337

"OF HYBRID WALNUT CLONES

ROOTING PHASE

MULTIPLICATION PHASE

INDUCTION

il

t

DEVELOPMENT IN VITRO

IN VIVO

!

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, including induction (5 days), in vitro root development (2 weeks) followed by in vivo root development (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) containing 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 subcultures 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 medium 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. 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 medium 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 vermicuExperiments.

-

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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 observations 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 determined on the average length of primary roots and the number of primary roots per rooted explant. RESULTS

The addition of vermiculite to GM during the in vitro 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 secondary roots after 2 weeks of culture (Table 2; Fig. 2). In addition, the strol gest 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 Vermiculite

effects. -

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

MR9

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 GM 16 GM with 16 perlite GM with 16 vermiculite

HA3-1 GM 16 GM with 32 perlite GM with 32 vermiculite

14(88) 11 (69)

63 82

0 6

11.7±! 21.9±1.2

4.5+__0.6 7.5±0.9

16 (100)

89

72

34.3+_ 1.8

5.6____.0.7

7 (44) 26 (81)

II 120

0 0

8.1 ± 1.2 18.1 ± 1.3

1.6+0.2 4.6+0.5

29 (91)

139

14

26.6+__ 1.3

4.8±0.5

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 GM with 24 vermiculite

13 (54) 19 (79)

69 97

0 13

9.5_+0.8 20.6-+ I.I

5.3+_ 1 5.1 ___0.7

GI

GM 16 GM with 24 vermiculite

1 (6) 11 (46)

I 20

0 9

5 35.2_+5.1

I 1.8_+.0.6

MR8

GM 12 GM with 17 vermiculite

9 (75) 17 (100)

22 92

0 18

8.4+0.8 21.8+ 1.3

2.4+0.7 5.4_+0.7

HA2-13 GM 21 GM with 28 vermiculite

10 (48) 28 (100)

14 231

0 0

6.5+3.5 23.5+ I.I

1.4+0.2 8.6+0.7

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 vermiculiteGM 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 primary 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 developed 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.

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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 obtained in GM. For example, from three series of shoot tips of HA3-1 corresponding 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 induction 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 addition, 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 medium (300 ml). Changes of medium pH cannot be excluded, in view of possible interactions with rooting in woody species in vitro (Williams et al., 1985 ). Moreover, perlite associated to be a gelified medium, which was successfully 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 promote 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.

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