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Castanea sativa plantlets proliferated from axillary buds cultivated in vitro
Scientia Horticulturae, 18 (1982/83) 343--351
343
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
C A S T A N E A S A T I V A PLANTLETS PROLIFERATED FROM AXILLARY BUDS CULTIVATED IN VITRO
A.M. VIEITEZ' and M.L. VIEITEZ 2
'Plant Physiology, C.S.I.C., Apartado 122, Santiago de Compostela (Spain) 2Department of Plant Physiology, Faculty of Biology, University of Santiago de Compostela (Spain) (Accepted for publication 10 March 1982)
ABSTRACT Vieitez, A.M. and Vieitez, M.L., 1983. Castanea sativa plantlets proliferated from axillary buds cultivated in vitro. Scientia Hortic., 1 8 : 3 4 3 - - 3 5 1 , Chestnut plants were proliferated in vitro from axillary buds of juvenile shoots. N6Benzyl-aminopurine (BAP) at 0.1--0.5 mg l - ' was optimal for shoot multiplication. The important role played by the macronutrient formula on shoot multiplication, and especially on the rooting-stage, is emphasized. The MS (1/2 NO3) macronutrients gave the best rooting percentage as well as the highest number of roots per rooted shoot. In these experiments, shoots remained in the 3 mg l" indole-3-butyric acid (IBA) medium for 12 days, after which they were transferred to an auxin-free medium where roots developed fully. Optimum rooting was achieved by immersing the 1 cm basal end of shoots in concentrated IBA solutions (0.5--1 mg m l - ' ) f o r periods ranging from 2 to 15 rain.
INTRODUCTION
The induced proliferation of shoot apices or axiUary buds to produce multiple shoots that can eventually be rooted is now recognised as a highly useful technique for the propagation of forest species (De Fossard et al., 1977; Boulay, 1979; Minocha, 1980; Grewal et al., 1980). In previous papers, we have reported our initial results on in vitro multiplication of axillary buds from juvenile chestnut and subsequent clonal population, but with only a very limited rooting-response (Vieitez and Vieitez, 1980a, b). The purpose of the present paper is to describe the tissue-culture conditions developed to improve both the shoot-multiplication stage and the rooting in vitro. Various mineral media and auxin treatments were tested with regard to their effect upon the quantity and quality of the shoot-multiplication cultures and upon in vitro rooting.
0304-4238/83/0000--0000/$03.00
© 1983 Elsevier Scientific Publishing Company
344 M A T E R I A L AND METHODS
The initial explants were 1 cm-long nodal sections from 3-month-old seedlings and embryonic axes dissected from mature embryos of C a s t a n e a s a t i v a Mil., which were cultured in a BAP (1 mg 1-1) medium as described previously (Vieitez and Vieitez, 1980a, b). The proliferated shoots were used as the source of explants for subsequent shoot multiplication and rootingexperiments. The experimental explants for shoot-multiplication experiments were shoot tips 0.5--1 cm long cut from the multiple shoots arising from axillary buds. Three basal media were tested, those of Heller (1953), Murashige and Skoog (1962) and Lepoivre (Quoirin and Lepoivre, 1977), to be designated as H, MS and L, respectively. MS was used with nitrates at half strength and will be referred to below as the MS-1/2 NO3. All media contained Fe EDTA (Fe salt of ethylenediaminetetracetic acid) and the micronutrients described by Murashige and Skoog (1962) as well as the following additives: 100 mg 1-1 m-inositol; 1 mg 1-1 thiamine; 0.1 mg 1-1 nicotinic acid; 0.1 mg 1"1 pyridoxineHC1; 2 mg 1-1 ascorbic acid; 30 g 1-1 sucrose; 6 g 1"1 agar. Concentrations of BAP ranging from 0.05 to 2.0 mg 1"1 were added to induce shoot multiplication. The pH was adjusted to 5.5--5.6 with 0.1 N NaOH before autoclaving. The explants were cultured in 25 ml of medium in 30 X 150-mm test tubes. To induce rooting, single shoots of 2 cm or longer, isolated from the multiplication cultures, were placed in 20 X 150-mm culture tubes containing 15 ml of medium to which varying quantities of indole-3-butyric acid (IBA) or naphthalene acetic acid (NAA) had been added. After 28 days, the percentage of rooted shoots and the number of roots per shoot were recorded. The effect of immersing the base of shoots in concentrated IBA solution for short periods of time was also studied. All cultures were incubated in a growth chamber set for a 14-h photoperiod with temperatures of 25°C (day) and 18°C (night). Cool white fluorescent tubes gave an irradiance of 353 ~W cm -2. Experiments on multiplication rate consisted of 12 replicates, those for rooting had 18 or 24 replicates. All experiments were repeated at least twice. RESULTS
p r o l i f e r a t i o n . - - After 6 weeks, the initial cultures were well established and sub-cultures were repeated at monthly intervals to obtain clonal populations. The influence of 5 levels of BAP and 3 macronutrient formulae upon the mean shoot multiplication rate was determined. Explants from all the clonal populations responded similarly to the microculture conditions. In medium without BAP, shoot multiplication was practically zero, only slight shoot and leaf growth having been observed in a few cases. In all 3 basal media tested, the number of proliferated shoots per shoot tip tended to increase and the length of the main shoot to decrease with increasing concen-
Shoot
345
trations of BAP {Table I). The maximum number of shoots attained was with 1 or 2 mg 1-1. These shoots failed to elongate, however, and often assumed the form of tiny rosettes or closed buds. They generally proved unsatisfactory in subsequent attempts at in vitro rooting. The leaves developed at these concentrations were thickened and chlorotic compared with those produced at lower levels of BAP, and their edges were somewhat curled. Concentrations of BAP at 0.1--0.5 mg 1-1 were found most suitable for optimum proliferation together with the healthy growth necessary for subsequent rooting (Fig. 1).
Fig. 1. Effect of BAP (rag 1-1 ) on shoot proliferation in medium containing Lepoivre macronutrients.
Of the 3 macronutrient formulae tested, Lepoivre's gave a slightly higher mean multiplication rate than the other 2 {Table I). When successive subcultures were carried o u t in MS-l/2 NO3 media, there was a tendency towards the formation of succulent shoots with flaccid tips and elongated dark green leaves, the capacity for multiplication rapidly being lost. The basal zone of the multiplication cultures generally formed a callus composed of very firm and compact tissue with external nodular outcrops. This callus was lacking in the succulent form of cultures. Heller's macronutrients gave rise to a minim u m of succulent shoots, but these were t o o short for rooting. The degeneration of sub-cultures into the succulent form was also found to depend upon the genotype of the seedling line. In a supplementary experiment, proliferated shoots were found to develop from most axillary buds on long shoots {4--6 cm) placed upside d o w n in the multiplication medium with the tips removed. Every 4 weeks, the new shoots were removed to rooting-media and the proliferating shoots were subcultured. This cycle could be successfully repeated 3 or 4 times. Lane (1979) found a similar response to orientation in pear plants.
346 TABLE I Effect of BAP and 3 basal media on numbers of shoots formed per culture (N) and height (cm) of tallest shoot (H). Mean separation within columns by Duncan's multiple range test, 5% level BAP (mg 1-1 )
Heller
0.05 0.1 0.5 1 2
MS-1/2 NO 3
Lepoivre
N
H
N
H
N
H
4.3a 4.2a 4.8ac 5.3bc 5.0bc
2.3a 1.5b 1.2cd 1.3bd 1.0cd
3.5a 3.6a 4.3b 5.1c 5.0c
3.5a 3.9b 2.5c 2.5c 2.2c
3.2a 4.8b 4.9b 5.9b 5.4b
3.2a 2.4ac 2.2bc 1.5b 1.4b
induction. - - For the rooting-experiments, shoots of 2 cm or longer were separated from the shoot-multiplication cultures and transferred to rootingmedia, the remaining shoots being used for further multiplication in media with 0.1--0.5 ml 1-1 of BAP. After numerous sub-cultures, some seedling
Root
70
~50
o
o $BA
•
.NAA
/
/o
o
3O
I0 -- "~///" I
I
i
I
i
a)
I
2
3
5
my~ l
'- 3 ck
~7 0.!
0.5
Fig. 2. Influence of IBA and NAA on adventitious root formation. Rooting in Lepoivre macronutrients 1 month after transfer. (a) Percentage rooting. (h) Roots per rooted culture. Bars denote SE of the mean.
347
lines would sporadically root on their multiplication media, b u t in general the application of auxin was necessary to obtain routine r o o t initiation. Previously, only very limited rooting was achieved using Heller's macronutrient formula with 3 mg 1-1 of IBA (Vieitez and Vieitez, 1980a). The results described here are obtained using Lepoivre's formula and the MS-1/2NO3 (both at 1/2 concentration). In these experiments, shoots remained in the auxin medium for 12 days, after which they were transferred to an auxin-free medium for root development. Figure 2 shows the effect of different levels of IBA and NAA used in conjunction with 1/2 conc. Lepoivre's macronutrients. IBA was more effective than NAA at the levels tested, both for percentage rooting and number of roots per r o o t e d shoot. R o o t morphogenesis varied according to the auxin. IBA-induced roots were longer and more fibrous than the short thick NAA-induced roots. NAA also gave rise to the formation of more callus tissue at the base of the shoot than IBA (Fig. 3). The o p t i m u m auxin for the induction of root initials was 3 mg 1-1 of IBA (58% and 4.8 roots per shoot); 5 mg 1-1 produced a similar percentage of rooted shoots, but these also had considerable callus at the base.
Fig. 3. Morphogenetic d i f f e r e n c e s b e t w e e n IBA-induced roots (left) and NAA-induced roots (right).
348 When the optimal concentration of IBA (3 mg 1-1) was used with 1/2 conc. MS-1/2NO3, the rooting-rate rose to 74% with an average of 5.6 roots per rooted shoot. Root primordia generally emerged between the second and third week, although a few shoots had 1--3-mm roots on being transferred to the auxinfree medium after 12 days. This was especially so when the half-strength MS-l/2 NO3 was used. With this medium, there even occurred some root emergence after 9 or 10 days. Varying the period spent in the auxin medium (7, 12 or 28 days) yielded no great difference in the rooting-rate, but it did affect the number and length of the roots developed, the highest values occurring with 7 days in the IBA medium (Table II). Keeping the shoot-multiplication cultures in darkness for 2 weeks prior to rooting had a negative effect upon the rooting-rate, with values of 25% for the half-strength Lepoivre medium and 29% for the half-strength MS-l/2 NO3. TABLE II Effect on the number of roots per rooted shoot (N) and the average length (ram) of the longest root (L) of different periods spent in media containing 3 mg l-' of IBA. Results at the end of 4 weeks culture. Mean separation within columns by Duncan's multiple range test, 5% level Days in IBA medium 7 12 28
Lepoivre
MS-l/2 NO3
N
L
N
L
5.0 a 3.9 bc 4.5 ac
43.6 a 21.5 bc 18.4 bc
7.2 a 6.2 a 4.6 b
17.3 a 14.4 a 5.8 b
In a further series of experiments, the rooting-procedure was modified. The shoots were separated from the multiplication cultures and treated as cuttings by dipping the basal ends of 1 cm in concentrated (0.1, 0.5 or 1.0 mg m1-1) IBA solution for periods ranging from 2 to 15 min, after which t h e y were transferred to auxin-free agar media, MS-1/2NO3 and Lepoivre. The response to the 0.1 mg m1-1 was unsatisfactory. The most effective treatment was 2 min in 1.0 mg m1-1 IBA, which gave 90% rooting (Fig. 4) and an average of 12.3 roots per shoot. Fifteen minutes in 0.5 mg m1-1 IBA gave 81% rooting (Fig. 4) and 11.0 roots per rooted shoot. Both these treatments were followed by transfer to the 1/2 conc. MS-l/2 NO3. Fifteen minutes in 1.0 mg m1-1 IBA was effective for rooting, but the swellings which appeared t h r o u g h o u t the length of the shoots seemed to indicate that toxic levels were being reached. There were differences between the clones in their tolerance to strong IBA-levels. A large number of shoots developed roots half way up the stem. These aerial roots grew rapidly and developed normally upon reaching the surface of the agar.
349
[]
M S- I / 2 NO 3 Lepoivre macronutrients
90
7O w
50
minutes
5
tO
IBA rng/ml
0.5
15
2
1
5
10
15
t.O
Fig. 4. Effect of basal treatment with IBA for periods ranging from 2 to 15 min. i n s o i l . - - After 4 weeks, the rooted plantlets were strong enough to be transferred to soil. A mixture of peat and perlite was found to cause necrosis of the roots and base of the plantlets. Better results were obtained when the plantlets were transferred to pots containing equal quanti-
Establishment
Fig. 5. Potted plant of in vitro propagated chestnut.
350 ties of soil and perlite. For the first 15 days in the glasshouse, plastic covers were used to prevent dehydration and a month later the plantlets were transplanted to larger pots (Fig. 5). DISCUSSION The experiments described above demonstrate the potential of micropropagation techniques for chestnut. We found that the shoot multiplication rate induced by BAP was inversely related to the growth of the shoots. This was also observed in apple shoots (Lundergan and Janick, 1980). The advisability of reducing nitrates to half strength when Murashige and Skoog's macronutrient medium is used in shoot multiplication cultures (determined by ourselves in preliminary experiments) was also followed for Eucalyptus by Grewal et al. (1980). The tendency of the MS macronutrients to cause degenerate growth after repeated use throughout several generations of subculture was noted for the genus Prunus by Quoirin and Lepoivre (1977) and Tabachnik and Kester (1977). In our work, the best results from this last point of view were obtained using the low-mineral-content formula of HeUer, although in this medium the development of shoots and leaves was inferior to that achieved with the other 2 media. These results, together with the clear superiority to the MS-1/2 NO3 formula in the rooting stage, suggest that when numerous successive sub-cultures are desired, Heller's formula should be used during the multiplication stage except in the sub-culture immediately preceding the rooting-stage. MS-l/2 NO3 formula should be employed prior to rooting. The nutritional balance of shoots thus prepared by a single cycle in the MS-1/2 NO3 would appear to ensure a physiological state optimal for root differentiation. Lane's observation (I978) that high auxin levels sometimes interfere with rooting and inhibit full root development is borne out in our present work by the finding that longer than 7--12 days in the relatively concentrated auxin medium inhibits root growth. On the other hand, immersion of the base of the shoots in concentrated IBA solution for a short period has proved to be a very effective method of root induction. Transport of IBA up the shoot leads to root differentiation above the level of the medium, perhaps due to suitable dilution of the auxin. There is also no lack of aeration in these upper tissues, which can be a limiting factor in rooting. Abbott and Whiteley (1976) brought about an increased rooting-rate in apple shoots by placing the IBAtreated shoots upside down in the medium, so that the base remained in the air. The immediate advantage offered by repeated sub-culturing and shoot multiplication is the rapid production of hundreds of plants of a single genotype, which can be used for studies on pathogenicity and nutrition, and for direct planting in the field. However, work still has to be done in applying this technique to mature trees selected for their economically important characteristics from populations growing in the wild. The adaptation of the
351 t e c h n i q u e here described t o m a t u r e trees is s o m e t h i n g we are n o w w o r k i n g on, e n c o u r a g e d b y t h e successful r e g e n e r a t i o n r e c e n t l y r e p o r t e d for m a t u r e trees o f forest species such as Sequoia sempervirens, Sequoiadendron giganteum, Pseudotsuga menziesii, Eucalyptus, a n d the h y b r i d s Populus tremula X P. alba a n d Juglans regia X J. nigra (Boulay, 1 9 8 0 ) . ACKNOWLEDGEMENTS We t h a n k Mr. E. F e r r o for technical help. T h e s t u d y was s u p p o r t e d in part b y a grant f r o m t h e U . S . A . - - S p a i n C o o p e r a t i v e Project I I I - 0 3 6 0 .
REFERENCES Abbott, A.J. and Whiteley, G., 1976. Culture of Malus tissues in vitro. I. Multiplication of apple plants from isolated shoot apices. Scientia Hortic., 4: 183--189. Boulay, M., 1979. Propagation in vitro du Douglas par micropropagation de germination aseptique et culture de bourgeons dormants. AFOCEL, Etud. Rec., 12:67--75. Boulay, M., 1980. La micropropagation des arbres forestiers a l'Association Forest Cellulose. In: L'arbre in vitro: Connaissance de la biologie des arbres par les cultures in vitro. Abstr. 2e R~union de la Section Francaise de L'I.A.P.T.C., Paris, 14--15 November. De Fossard, R.A., Barker, P.K. and Bourne, R.A., 1977. The organ culture of nodes of four species of Eucalyptus. Acta Hortic., 78: 157--165. Grewal, S., Ahuja, A. and Atal, C.K., 1980. In vitro proliferation of shoot apices of Eucalyptus citriodora Hook. Ind. J. Exp. Biol., 18: 775--777. Heller, R., 1953. Recherches sur la nutrition min~rale des tissus v~g~taux cultiv~s in vitro. Ann. Sci. Nat. Bot. Biol. V~g., 14: 1--223. Lane, W.D., 1978. Regeneration of apple plants from shoot meristem-tips. Plant Sci. Lett., 13: 281--285. Lane, W.D., 1979. Regeneration of pear plants from shoot meristem-tips. Plant Sci. Lett., 16: 337--342. Lundergan, C.A. and Janick, J., 1980. Regulation of apple shoot proliferation and growth in vitro. Hortic. Res., 20: 19--24. Minocha, S.C., 1980. Cell and tissue culture in the propagation of forest trees. In: F. Sala, B. Parisi, R. Cella and O. Ciferri (Editors), Plant Cell Cultures: Results and Perspectives. Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 295--300. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 437--497. Quoirin, M. and Lepoivre, Ph., 1977. Etude de milieux adapt~s aux cultures in vitro de Prunus. Acta Hortic., 78: 437--442. Tabachnik, L. and Kester, D.E., 1977. Shoot cultures for almond and almond--peach hybrid clones in vitro. HortScience, 12 : 545--547. Vieitez, A.M. and Vieitez, E., 1980a. Plantlet formation from embryonic tissue of chestnut grown in vitro. Physiol. Plant., 50: 127--130. Vieitez, A.M. and Vieitez, M.L., 1980b. Culture of chestnut shoots from buds in vitro. J. Hortic. Sci., 55: 83--84.
343
Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
C A S T A N E A S A T I V A PLANTLETS PROLIFERATED FROM AXILLARY BUDS CULTIVATED IN VITRO
A.M. VIEITEZ' and M.L. VIEITEZ 2
'Plant Physiology, C.S.I.C., Apartado 122, Santiago de Compostela (Spain) 2Department of Plant Physiology, Faculty of Biology, University of Santiago de Compostela (Spain) (Accepted for publication 10 March 1982)
ABSTRACT Vieitez, A.M. and Vieitez, M.L., 1983. Castanea sativa plantlets proliferated from axillary buds cultivated in vitro. Scientia Hortic., 1 8 : 3 4 3 - - 3 5 1 , Chestnut plants were proliferated in vitro from axillary buds of juvenile shoots. N6Benzyl-aminopurine (BAP) at 0.1--0.5 mg l - ' was optimal for shoot multiplication. The important role played by the macronutrient formula on shoot multiplication, and especially on the rooting-stage, is emphasized. The MS (1/2 NO3) macronutrients gave the best rooting percentage as well as the highest number of roots per rooted shoot. In these experiments, shoots remained in the 3 mg l" indole-3-butyric acid (IBA) medium for 12 days, after which they were transferred to an auxin-free medium where roots developed fully. Optimum rooting was achieved by immersing the 1 cm basal end of shoots in concentrated IBA solutions (0.5--1 mg m l - ' ) f o r periods ranging from 2 to 15 rain.
INTRODUCTION
The induced proliferation of shoot apices or axiUary buds to produce multiple shoots that can eventually be rooted is now recognised as a highly useful technique for the propagation of forest species (De Fossard et al., 1977; Boulay, 1979; Minocha, 1980; Grewal et al., 1980). In previous papers, we have reported our initial results on in vitro multiplication of axillary buds from juvenile chestnut and subsequent clonal population, but with only a very limited rooting-response (Vieitez and Vieitez, 1980a, b). The purpose of the present paper is to describe the tissue-culture conditions developed to improve both the shoot-multiplication stage and the rooting in vitro. Various mineral media and auxin treatments were tested with regard to their effect upon the quantity and quality of the shoot-multiplication cultures and upon in vitro rooting.
0304-4238/83/0000--0000/$03.00
© 1983 Elsevier Scientific Publishing Company
344 M A T E R I A L AND METHODS
The initial explants were 1 cm-long nodal sections from 3-month-old seedlings and embryonic axes dissected from mature embryos of C a s t a n e a s a t i v a Mil., which were cultured in a BAP (1 mg 1-1) medium as described previously (Vieitez and Vieitez, 1980a, b). The proliferated shoots were used as the source of explants for subsequent shoot multiplication and rootingexperiments. The experimental explants for shoot-multiplication experiments were shoot tips 0.5--1 cm long cut from the multiple shoots arising from axillary buds. Three basal media were tested, those of Heller (1953), Murashige and Skoog (1962) and Lepoivre (Quoirin and Lepoivre, 1977), to be designated as H, MS and L, respectively. MS was used with nitrates at half strength and will be referred to below as the MS-1/2 NO3. All media contained Fe EDTA (Fe salt of ethylenediaminetetracetic acid) and the micronutrients described by Murashige and Skoog (1962) as well as the following additives: 100 mg 1-1 m-inositol; 1 mg 1-1 thiamine; 0.1 mg 1-1 nicotinic acid; 0.1 mg 1"1 pyridoxineHC1; 2 mg 1-1 ascorbic acid; 30 g 1-1 sucrose; 6 g 1"1 agar. Concentrations of BAP ranging from 0.05 to 2.0 mg 1"1 were added to induce shoot multiplication. The pH was adjusted to 5.5--5.6 with 0.1 N NaOH before autoclaving. The explants were cultured in 25 ml of medium in 30 X 150-mm test tubes. To induce rooting, single shoots of 2 cm or longer, isolated from the multiplication cultures, were placed in 20 X 150-mm culture tubes containing 15 ml of medium to which varying quantities of indole-3-butyric acid (IBA) or naphthalene acetic acid (NAA) had been added. After 28 days, the percentage of rooted shoots and the number of roots per shoot were recorded. The effect of immersing the base of shoots in concentrated IBA solution for short periods of time was also studied. All cultures were incubated in a growth chamber set for a 14-h photoperiod with temperatures of 25°C (day) and 18°C (night). Cool white fluorescent tubes gave an irradiance of 353 ~W cm -2. Experiments on multiplication rate consisted of 12 replicates, those for rooting had 18 or 24 replicates. All experiments were repeated at least twice. RESULTS
p r o l i f e r a t i o n . - - After 6 weeks, the initial cultures were well established and sub-cultures were repeated at monthly intervals to obtain clonal populations. The influence of 5 levels of BAP and 3 macronutrient formulae upon the mean shoot multiplication rate was determined. Explants from all the clonal populations responded similarly to the microculture conditions. In medium without BAP, shoot multiplication was practically zero, only slight shoot and leaf growth having been observed in a few cases. In all 3 basal media tested, the number of proliferated shoots per shoot tip tended to increase and the length of the main shoot to decrease with increasing concen-
Shoot
345
trations of BAP {Table I). The maximum number of shoots attained was with 1 or 2 mg 1-1. These shoots failed to elongate, however, and often assumed the form of tiny rosettes or closed buds. They generally proved unsatisfactory in subsequent attempts at in vitro rooting. The leaves developed at these concentrations were thickened and chlorotic compared with those produced at lower levels of BAP, and their edges were somewhat curled. Concentrations of BAP at 0.1--0.5 mg 1-1 were found most suitable for optimum proliferation together with the healthy growth necessary for subsequent rooting (Fig. 1).
Fig. 1. Effect of BAP (rag 1-1 ) on shoot proliferation in medium containing Lepoivre macronutrients.
Of the 3 macronutrient formulae tested, Lepoivre's gave a slightly higher mean multiplication rate than the other 2 {Table I). When successive subcultures were carried o u t in MS-l/2 NO3 media, there was a tendency towards the formation of succulent shoots with flaccid tips and elongated dark green leaves, the capacity for multiplication rapidly being lost. The basal zone of the multiplication cultures generally formed a callus composed of very firm and compact tissue with external nodular outcrops. This callus was lacking in the succulent form of cultures. Heller's macronutrients gave rise to a minim u m of succulent shoots, but these were t o o short for rooting. The degeneration of sub-cultures into the succulent form was also found to depend upon the genotype of the seedling line. In a supplementary experiment, proliferated shoots were found to develop from most axillary buds on long shoots {4--6 cm) placed upside d o w n in the multiplication medium with the tips removed. Every 4 weeks, the new shoots were removed to rooting-media and the proliferating shoots were subcultured. This cycle could be successfully repeated 3 or 4 times. Lane (1979) found a similar response to orientation in pear plants.
346 TABLE I Effect of BAP and 3 basal media on numbers of shoots formed per culture (N) and height (cm) of tallest shoot (H). Mean separation within columns by Duncan's multiple range test, 5% level BAP (mg 1-1 )
Heller
0.05 0.1 0.5 1 2
MS-1/2 NO 3
Lepoivre
N
H
N
H
N
H
4.3a 4.2a 4.8ac 5.3bc 5.0bc
2.3a 1.5b 1.2cd 1.3bd 1.0cd
3.5a 3.6a 4.3b 5.1c 5.0c
3.5a 3.9b 2.5c 2.5c 2.2c
3.2a 4.8b 4.9b 5.9b 5.4b
3.2a 2.4ac 2.2bc 1.5b 1.4b
induction. - - For the rooting-experiments, shoots of 2 cm or longer were separated from the shoot-multiplication cultures and transferred to rootingmedia, the remaining shoots being used for further multiplication in media with 0.1--0.5 ml 1-1 of BAP. After numerous sub-cultures, some seedling
Root
70
~50
o
o $BA
•
.NAA
/
/o
o
3O
I0 -- "~///" I
I
i
I
i
a)
I
2
3
5
my~ l
'- 3 ck
~7 0.!
0.5
Fig. 2. Influence of IBA and NAA on adventitious root formation. Rooting in Lepoivre macronutrients 1 month after transfer. (a) Percentage rooting. (h) Roots per rooted culture. Bars denote SE of the mean.
347
lines would sporadically root on their multiplication media, b u t in general the application of auxin was necessary to obtain routine r o o t initiation. Previously, only very limited rooting was achieved using Heller's macronutrient formula with 3 mg 1-1 of IBA (Vieitez and Vieitez, 1980a). The results described here are obtained using Lepoivre's formula and the MS-1/2NO3 (both at 1/2 concentration). In these experiments, shoots remained in the auxin medium for 12 days, after which they were transferred to an auxin-free medium for root development. Figure 2 shows the effect of different levels of IBA and NAA used in conjunction with 1/2 conc. Lepoivre's macronutrients. IBA was more effective than NAA at the levels tested, both for percentage rooting and number of roots per r o o t e d shoot. R o o t morphogenesis varied according to the auxin. IBA-induced roots were longer and more fibrous than the short thick NAA-induced roots. NAA also gave rise to the formation of more callus tissue at the base of the shoot than IBA (Fig. 3). The o p t i m u m auxin for the induction of root initials was 3 mg 1-1 of IBA (58% and 4.8 roots per shoot); 5 mg 1-1 produced a similar percentage of rooted shoots, but these also had considerable callus at the base.
Fig. 3. Morphogenetic d i f f e r e n c e s b e t w e e n IBA-induced roots (left) and NAA-induced roots (right).
348 When the optimal concentration of IBA (3 mg 1-1) was used with 1/2 conc. MS-1/2NO3, the rooting-rate rose to 74% with an average of 5.6 roots per rooted shoot. Root primordia generally emerged between the second and third week, although a few shoots had 1--3-mm roots on being transferred to the auxinfree medium after 12 days. This was especially so when the half-strength MS-l/2 NO3 was used. With this medium, there even occurred some root emergence after 9 or 10 days. Varying the period spent in the auxin medium (7, 12 or 28 days) yielded no great difference in the rooting-rate, but it did affect the number and length of the roots developed, the highest values occurring with 7 days in the IBA medium (Table II). Keeping the shoot-multiplication cultures in darkness for 2 weeks prior to rooting had a negative effect upon the rooting-rate, with values of 25% for the half-strength Lepoivre medium and 29% for the half-strength MS-l/2 NO3. TABLE II Effect on the number of roots per rooted shoot (N) and the average length (ram) of the longest root (L) of different periods spent in media containing 3 mg l-' of IBA. Results at the end of 4 weeks culture. Mean separation within columns by Duncan's multiple range test, 5% level Days in IBA medium 7 12 28
Lepoivre
MS-l/2 NO3
N
L
N
L
5.0 a 3.9 bc 4.5 ac
43.6 a 21.5 bc 18.4 bc
7.2 a 6.2 a 4.6 b
17.3 a 14.4 a 5.8 b
In a further series of experiments, the rooting-procedure was modified. The shoots were separated from the multiplication cultures and treated as cuttings by dipping the basal ends of 1 cm in concentrated (0.1, 0.5 or 1.0 mg m1-1) IBA solution for periods ranging from 2 to 15 min, after which t h e y were transferred to auxin-free agar media, MS-1/2NO3 and Lepoivre. The response to the 0.1 mg m1-1 was unsatisfactory. The most effective treatment was 2 min in 1.0 mg m1-1 IBA, which gave 90% rooting (Fig. 4) and an average of 12.3 roots per shoot. Fifteen minutes in 0.5 mg m1-1 IBA gave 81% rooting (Fig. 4) and 11.0 roots per rooted shoot. Both these treatments were followed by transfer to the 1/2 conc. MS-l/2 NO3. Fifteen minutes in 1.0 mg m1-1 IBA was effective for rooting, but the swellings which appeared t h r o u g h o u t the length of the shoots seemed to indicate that toxic levels were being reached. There were differences between the clones in their tolerance to strong IBA-levels. A large number of shoots developed roots half way up the stem. These aerial roots grew rapidly and developed normally upon reaching the surface of the agar.
349
[]
M S- I / 2 NO 3 Lepoivre macronutrients
90
7O w
50
minutes
5
tO
IBA rng/ml
0.5
15
2
1
5
10
15
t.O
Fig. 4. Effect of basal treatment with IBA for periods ranging from 2 to 15 min. i n s o i l . - - After 4 weeks, the rooted plantlets were strong enough to be transferred to soil. A mixture of peat and perlite was found to cause necrosis of the roots and base of the plantlets. Better results were obtained when the plantlets were transferred to pots containing equal quanti-
Establishment
Fig. 5. Potted plant of in vitro propagated chestnut.
350 ties of soil and perlite. For the first 15 days in the glasshouse, plastic covers were used to prevent dehydration and a month later the plantlets were transplanted to larger pots (Fig. 5). DISCUSSION The experiments described above demonstrate the potential of micropropagation techniques for chestnut. We found that the shoot multiplication rate induced by BAP was inversely related to the growth of the shoots. This was also observed in apple shoots (Lundergan and Janick, 1980). The advisability of reducing nitrates to half strength when Murashige and Skoog's macronutrient medium is used in shoot multiplication cultures (determined by ourselves in preliminary experiments) was also followed for Eucalyptus by Grewal et al. (1980). The tendency of the MS macronutrients to cause degenerate growth after repeated use throughout several generations of subculture was noted for the genus Prunus by Quoirin and Lepoivre (1977) and Tabachnik and Kester (1977). In our work, the best results from this last point of view were obtained using the low-mineral-content formula of HeUer, although in this medium the development of shoots and leaves was inferior to that achieved with the other 2 media. These results, together with the clear superiority to the MS-1/2 NO3 formula in the rooting stage, suggest that when numerous successive sub-cultures are desired, Heller's formula should be used during the multiplication stage except in the sub-culture immediately preceding the rooting-stage. MS-l/2 NO3 formula should be employed prior to rooting. The nutritional balance of shoots thus prepared by a single cycle in the MS-1/2 NO3 would appear to ensure a physiological state optimal for root differentiation. Lane's observation (I978) that high auxin levels sometimes interfere with rooting and inhibit full root development is borne out in our present work by the finding that longer than 7--12 days in the relatively concentrated auxin medium inhibits root growth. On the other hand, immersion of the base of the shoots in concentrated IBA solution for a short period has proved to be a very effective method of root induction. Transport of IBA up the shoot leads to root differentiation above the level of the medium, perhaps due to suitable dilution of the auxin. There is also no lack of aeration in these upper tissues, which can be a limiting factor in rooting. Abbott and Whiteley (1976) brought about an increased rooting-rate in apple shoots by placing the IBAtreated shoots upside down in the medium, so that the base remained in the air. The immediate advantage offered by repeated sub-culturing and shoot multiplication is the rapid production of hundreds of plants of a single genotype, which can be used for studies on pathogenicity and nutrition, and for direct planting in the field. However, work still has to be done in applying this technique to mature trees selected for their economically important characteristics from populations growing in the wild. The adaptation of the
351 t e c h n i q u e here described t o m a t u r e trees is s o m e t h i n g we are n o w w o r k i n g on, e n c o u r a g e d b y t h e successful r e g e n e r a t i o n r e c e n t l y r e p o r t e d for m a t u r e trees o f forest species such as Sequoia sempervirens, Sequoiadendron giganteum, Pseudotsuga menziesii, Eucalyptus, a n d the h y b r i d s Populus tremula X P. alba a n d Juglans regia X J. nigra (Boulay, 1 9 8 0 ) . ACKNOWLEDGEMENTS We t h a n k Mr. E. F e r r o for technical help. T h e s t u d y was s u p p o r t e d in part b y a grant f r o m t h e U . S . A . - - S p a i n C o o p e r a t i v e Project I I I - 0 3 6 0 .
REFERENCES Abbott, A.J. and Whiteley, G., 1976. Culture of Malus tissues in vitro. I. Multiplication of apple plants from isolated shoot apices. Scientia Hortic., 4: 183--189. Boulay, M., 1979. Propagation in vitro du Douglas par micropropagation de germination aseptique et culture de bourgeons dormants. AFOCEL, Etud. Rec., 12:67--75. Boulay, M., 1980. La micropropagation des arbres forestiers a l'Association Forest Cellulose. In: L'arbre in vitro: Connaissance de la biologie des arbres par les cultures in vitro. Abstr. 2e R~union de la Section Francaise de L'I.A.P.T.C., Paris, 14--15 November. De Fossard, R.A., Barker, P.K. and Bourne, R.A., 1977. The organ culture of nodes of four species of Eucalyptus. Acta Hortic., 78: 157--165. Grewal, S., Ahuja, A. and Atal, C.K., 1980. In vitro proliferation of shoot apices of Eucalyptus citriodora Hook. Ind. J. Exp. Biol., 18: 775--777. Heller, R., 1953. Recherches sur la nutrition min~rale des tissus v~g~taux cultiv~s in vitro. Ann. Sci. Nat. Bot. Biol. V~g., 14: 1--223. Lane, W.D., 1978. Regeneration of apple plants from shoot meristem-tips. Plant Sci. Lett., 13: 281--285. Lane, W.D., 1979. Regeneration of pear plants from shoot meristem-tips. Plant Sci. Lett., 16: 337--342. Lundergan, C.A. and Janick, J., 1980. Regulation of apple shoot proliferation and growth in vitro. Hortic. Res., 20: 19--24. Minocha, S.C., 1980. Cell and tissue culture in the propagation of forest trees. In: F. Sala, B. Parisi, R. Cella and O. Ciferri (Editors), Plant Cell Cultures: Results and Perspectives. Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 295--300. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15: 437--497. Quoirin, M. and Lepoivre, Ph., 1977. Etude de milieux adapt~s aux cultures in vitro de Prunus. Acta Hortic., 78: 437--442. Tabachnik, L. and Kester, D.E., 1977. Shoot cultures for almond and almond--peach hybrid clones in vitro. HortScience, 12 : 545--547. Vieitez, A.M. and Vieitez, E., 1980a. Plantlet formation from embryonic tissue of chestnut grown in vitro. Physiol. Plant., 50: 127--130. Vieitez, A.M. and Vieitez, M.L., 1980b. Culture of chestnut shoots from buds in vitro. J. Hortic. Sci., 55: 83--84.