Micropropagation of <i>Lupinus Mutabilis</i>

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Reference

Micropropagation of Lupinus Mutabilis

HARDY, I., et al.

Abstract

Desinfected seeds of Lupinus mutabilis were allowed to germinate in vitro. The seedlings were early transferred on a Murashige and Skoog medium in the presence of BAP or TDZ, which allowed axillary proliferation from the cotyledonary buds. The shoots raised were further used for continued multiplication in the presence of 10 μM BAP. Higher concentrations of BAP and TDZ induced vitrification. Before rooting, the shoots were elongated in the absence growth of regulators. Rooting was achieved through the use of two successive rooting media:

an "induction" one with IBA or NAA in dark for 7 days, followed by an "expressive" medium without auxin, in light. There was a relationship between the rooting rate and the degree of shoot elongation.

HARDY, I., et al . Micropropagation of Lupinus Mutabilis . Mededelingen van de Faculteit Landbouwwetenschappen , 1995, vol. 60, no. 3b, p. 1107-1111

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1107 Med. Fae. Landbouww. Univ. Gent. 60/3b. 1995

MICROPROPAGA TION OF LUP!NUS MUTABILIS

I. Hardy, I. Grange, L. Jouve* & Th. Gaspar*

Laboratoire de Biotechnologies Vegetales, Faculte des Sciences Universite Catholique de Lyon, 25 rue du plat, F - 69288 Lyon cedex 02, France,

*

SUMMARY

Desinfected seeds of Lupinus mutabi!is were allowed to germinate in vitro. The seedlings were early transferred on a Murashigc and Skoog medium in the presence of BAP or TDZ, which allowed axillary proliferation from the cotyle- donary buds. The shoots raised were further used for continued multiplication in the presence of 10 µM BAP. Higher concentrations of BAP and TDZ induced vitrification. Before rooting, the shoots were elongated in the absence growth of regulators. Rooting was achieved through the use of two successive rooting media: an "induction" one with IBA or NAA in dark for 7 days, followed by an "exlJressive" medium without auxin, in light. There was a relationship be- tween the rooting rate and the degree of shoot elongation.

Key words: axillary shooting, Leguminosae, in vUro, rooting Abbreviations:

BAP = N6-benzy!aminopurine,

TDZ = N-phenyl-N'-1,2,3,3-thiadiazol-5-ylurea (Thidiazuron), IBA = indolebutyric acid,

NAA = naphtaleneacetic acid,

MS= Murashige and Skoog (1962) medium.

INTRODUCTION

Several lupin species (Lupinus a/bus, L.mutabilis, L. angustifolius) have been cultivated for cen- turies but assays of breeding or varietal amelioration for intensive cultures are relatively recent. The control of in vitro cultures will be helpful for such purposes. Lupinus is a legume which has not been extensively investigated in in vitro cultures. Pioneering work by Ball (1946) and Lee (1955) on L. a/bus and L. hartwegii showed the possibility to grow stem tips and excised portions of embryos in vitro. Sroga (1983, 1987) reported some regeneration of L. angustifolius and L. polyphyllus through callus cultures. Satar (1985) described limited organogenesis in four Lupinus species. More recently Nadolska-Orczyk (1992) demonstrated the possibility to induce somatic embryos in some Lupinus species and Upadhyaya et al. (1992) developed a micropropagation system for L. texensis starting from cotyledonary node explants. The present work reports a quite similar technique of mi- cropropagation of L. mutabilis through axillary proliferation, and also describes the conditions found for rooting the shoots raised.

MATERIAL AND METHODS Seeds desinfection and germination

Seeds of Lupim1s mutabilis were obtained from A Bellio (LUPSEM, Lusignan, France). They were surface sterilized by soaking in 70 % ethanol for 3 min followed by a treatment with sodium dichloroisocyanurate solution (Bayrochlor from BA YER) plus 0.1 % Tween 80 for 20 min with gentle agitation. The sterilant was removed by washing the seeds three times with sterile distilled water. Seeds were placed on filter paper for germination in a petri dish in darkness at 25°C. After 3

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days, seedlings were treated with I % sodium hypochlorite for I 0 min and rinsed with sterile distilled water.

Seedling culture

Seedlings were cultured in Magenta culture vessels (Magenta Corp., Chicago, USA). Three seed- lings were placed in a 300 ml box containing 45 ml of basal MS medium (Murashige and Skoog,

1962) with 3% sucrose, 0.75 % Roland agar,and 0 - 50 µM ofBAP or TDZ; pH was adjusted to 5.8 prior to autoclaving at I20°C for 20 min. All cultures were incubated at 25°C in darkness for the first 2 weeks and later transferred to light for 4 additional weeks (16 h photoperiod, photosynthetic pho- ton flux of25 W.m·² provided by Sylvania Gro-Lux fluorescent lamps). The number of cotyledonary shoots per culture were determined after a 6-week culture period.

Shoot proliferation

Shoots from one of the previous conditions (BAP 30 µM) were excised from the mother seed- lings and subcultured each 4 weeks on MS basal medium supplemented with various concentrations of BAP. The quality and the number of shoots per culture were then determined after 4 weeks on these various media.

Rooting

Four weeks old shoots produced on the above multiplication media and transferred directly to an auxin-based rooting medium or to the two successive rooting media indicated below never rooted.

Preliminary investigations indicated the necessity of a prior elongation of the shoots through a 3- week passage on an elongation medium (MS without growth regulators but with 2.5 g.r¹ activated charcoal, in rectangular (10.8 x 8.2 x 5.5 cm) plastic containers, with plastic adjustable lids (from Reynolds Film Inc.). Then, the shoots were transferred for a week in darkness on a MS medium with different concentrations of IBA or NAA, and afterwards for 4 weeks under light on an "expressive"

rooting medium (without auxins, plus 2.5 g.r¹ active charcoal). Thirty shoots were used per assay.

RESULTS

Seed germination and seedling growth

Seed germination was about 60 %. BAP and TDZ did not affect seedling growth drastically, ex- cept that they severely reduced growth of the root system. BAP and TDZ on the contrary favoured shoot development from cotyledonary axillary buds. 30 µM BAP and 0.4 µM TDZ allowed the high- est percentages of axillary shoot-producing seedlings (Table I). The highest number of shoots per seedling however were obtained using BAP and TDZ at the respective concentrations of 50 µM and 0.4 µM. Under the latter conditions, 11 and 9.5 shoots respectively were collected per seedlings. On TDZ-containing media, the shoots were somehow abnormally developed because highly vitrified.

Apparently normal shoots from the 6-weeks old 30 µM BAP seedlings cultures were taken as ex- plants for further shoot proliferating cultures.

Shoot proliferation

Shoots developed axillary from seedlings were subcultured on MS medium with BAP only. The results after three subcultures of 4 weeks, as reported in Table 2, indicate an increase of the multipli- cation rate together with the increase of BAP concentration. However, higher was BAP concentra- tion, higher were the vitrification symptoms and callus formation at the stem bases. Further routine multiplication was performed on BAP 10 µM (because vitrification was restricted to 20 % of the shoots only) and limited to three subcultures. Apparently normal shoots (without basal callus) were

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separated and then transferred to the elongation medium for three weeks, to be used afterwards ei- ther in another multiplication series or for rooting assays.

Table I: Effect of different concentrations of BAP and TDZ on axillary proliferation from cotyle- donary buds of Lupinus mutabilis.

Cytokinin Concentation Seedlings with axillary shoots Number of axillary

(µM) (%) shoot/seedling

None 0 0 0

BAP 10 20 3

20 14 2

30 50 3

40 22 5

50 20 l l

TDZ 0.2 3 5

0.3 47.5 6.3

0.4 71 9.5

55 7

3 52.5 4.2

10 55 5

50 57 8

Table 2: Effect of BAP concentration on the rate of shoot proliferation, presence of callus at the shoot bases and occurrence of vitrification.

Concentration Mean number of Callus Vitrification

(µM) shoots I cluster

0 1 0 0

1.2 0 0

3 2.3 + ++

10 2.6 + ++

30 2.9 +++ +++

Rooting

Adventitious root formation was observed only on shoots which had been cultured for 3 weeks on the elongation medium. Such shoots further Table 3: Effect of IBA and NAA concentration on rooted for 5% in the absence of auxin (Table the percentage of rooting of lupin shoots (after 35 3). The passage of the shoots on an auxin days on the rooting expressive medium) (IBA or NAA) based medium for 1 week under darkness induced the shoots to further Auxin ( µM ) Rooting ( % ) root, up to 85% on the expressive rooting

None 0 5 medium, in the absence of auxin. Observa-

IBA 14. 7 85 tions from other experimental series indicated

4. 9 61 that a maximum rooting rate was associated _ _ _ _ _ _ _ _ 0_._5 _ _ _ _ _ 5_2 _ _ _ _ with the highest elongation rate of the shoots

NAA 16 63 ( 45 % and 90 % rooting rates for 53 and 97

mm long shoots, respectively).

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DISCUSSION

For so-called recalcitrant plants, for instance leguminous trees (Hossain et al., 1993 ), seedlings provide explants which readily respond to the classical growth regulators used in micropropagation protocols. Upadhyaya et al. (1992) similarly used cotyledonary nodes explants to initiate a propaga- tion system for Lupinus texensis. The system used here for Lupim1s mutabilis was somehow differ- ent in that the cotyledonary nodes reacted to BAP or TDZ without being separated from the seed- lings. Axillary shoots were developed but not somatic embryos as obtained by Malik and Saxena (1992) in bean and Saxena et al. (1992) in peanut. Lupin shoots raised in the presence ofBAP and TDZ were very susceptible to vitrification as are many species and that may be a hindrance for fur- ther rooting (Gaspar, 1991).

A satisfactory rooting rate of L. mutabilis shoots could be obtained provided that (apparently) non-vitrifying BAP doses were used at the proliferating stage and that, prior to the rooting media, an elongation stage was allowed. Such an elongation stage before rooting is necessitated for many spe- cies and might correspond to a "detoxication" of the cytokinins or cytokinin-like-compounds used at the multiplication step (George, 1993). This relationship between the rooting ability and former and/or continued shoot elongation will be further investigated. The requirement of lupin shoots of two successive rooting media as for woody species (Ripetti et al., 1994) also might be interpreted as a need of such shoots to be detoxified from the exogenously supplied auxin before expressive root formation induced priorily in the presence of auxin. Such an external auxin also might simply be in- hibitory for the growth of root primordia.

ACKNOWLEDGEMENTS

I.H. and L.J. are greatly indebted to the E. C for the award of the contract ERBA IR 1 CT 925 105 which allowed them to perform the present work at the University of Liege.

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