Seasonality in rooting of Prosopis chilensis cuttings and in-vitro micropropagation

Forest Ecology and Management, 40 ( 1991 ) 163-173

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Elsevier Science Publishers B.V., Amsterdam

Seasonality in rooting of Prosopis chilensis cuttings and in-vitro micropropagation Patricio Arcea and Orlando Balboa b* aprograma de Micropropagacibn Vegetal, Pontificia Universidad CatOlica de Chile, Casilla 6177Santiago, Chile bFacultad de Agronomia UNLPam 6300, Santa Rosa, La Pampa, Argentina (Accepted 25 September 1989)

ABSTRACT Arce, P. and Balboa, O., 1991. Seasonality in rooting ofProsopis chilensis cuttings and in-vitro micropropagation. For. Ecol. Manage., 40: 163-173. Seasonality in rooting of cuttings and its relationship with some environmental conditions and growth parameters of Prosopis chilensis (Mol. Stunz ) were studied. It was observed that rooting could be induced in cuttings taken from field collections only during the dry season, the period corresponding to the highest vegetative growth and reproductive activity (September to March); however, percentages did not exceed 15%. The rooting response does not occur during the dormant period (May to September). When using cuttings from clonal material growing in the glasshouse, rootings exceeding 80% were obtained in liquid aerated media. In-vitro micropropagation was assessed in P. chilensis using nodal and apical segments collected both from the field and from clonal material and plantlets grown from seed in the glasshouse. Juvenile material gave an 80% regeneration rate of complete plants in Murashige-Skoog medium fortified with 5 mg naphtaleneacetic acid 1-t and 10 mg cysteine 1-~. In this same medium, the regenerative response of segments obtained from rooted cuttings was 60%. The material collected from the field showed no rooting responses.

INTRODUCTION

Prosopis chilensis (Mol. Stunz) is a very important tree species of the Mimosoidae (Leguminosae) family in Chile; it ranges from latitude 22 ° 54'S to 33 ° 00' S and longitude 69 ° 04' W to 70 ° 39'W, corresponding to hyper-arid, arid and semi-arid zones. Foliage is used as fodder for livestock, mainly sheep and goats, while its wood is used for fuel and for making charcoal (Anonymous, 1980). Due to symbiotic associations with Rhizobium bacteria, Prosopis trees fix nitrogen and can improve soils with N deficiences (Torres, 1984; Arce and CAuthor to w h o m correspondence should be addressed.

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© 1991 - - Elsevier Science Publishers B.V.

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Balboa, 1986). As Prosopis species are self-incompatible (Simpson, 1977; Balboa et al., 1986 ) they show large inter- and intra-specific variability regarding biomass productivity, vigour, growth habits, size and number of spines, N-fixing capacity, and protein and sugar content of the fruit (Felker, 1984; Hunziker et al., 1986; Oduol et al., 1986; Saunders et al., 1986). As plants obtained from seeds show extreme variability, future reforestation programmes involving large-scale plantings must use vegetatively propagated elite material. Consequently, the development of quick and easy propagation methods is necessary for large-scale clonal multiplication (Felker and Clark, 1981; Goyal and Arya, 1984; Jordfin et al., 1985a; Klass et al., 1985; Arya and Shekhawat, 1986; Burley et al., 1986; Tabone et al., 1986; Balboa et al., 1987 ). The purpose of this work is to relate the seasonal growth pattern of P. chilensis to its rooting ability, and to evaluate the regenerative potential of this species by in-vitro micropropagation. MATERIAL AND METHODS

Vegetative propagation by cuttings i) Propagationfrom field-grown trees. Cuttings collected monthly during 1985 and 1986 were used; these cuttings were obtained from adult individuals (over 40 years old) of the Chacabuco-Peldehue populations, located 70 km northwest of Santiago. The branches selected were from the previous year's growth, and were treated with fungicide (0.15% Captan) for 24 h, prior to treatment with phytohormones. The propagation technique used was a modification of that described by Arce and Balboa (1986) and Balboa et al. (1987). Cuttings were placed in an aqueous solutions of 100 mg 3-indole-3-butyric acid (IBA) 1-i before being placed in bubbled tap water (composition detailed later) containing 10 mg H3BO31-1. This treatment was repeated after 96 and 192 h. Throughout this period the cuttings were kept in a growth chamber (Lab-Line Biotronette, Mark IV) in which the temperature was 29.5 ° C + 1.5 ° C and the light regime was 100 ~mol m - 2 s- ~for 12 or 16 h. Ten replicates of five cuttings per treatment were made monthly between May 1985 and October 1986.

ii) Serial propagation of rooted field cuttings. Prosopis chilensis plantlets obtained from cuttings rooted and grown in pots were used for re-propagation tests of adult plants. Cuttings 20 cm in length were taken from the plants and then recut under water to a length of 15 cm, with an average of ten nodes per cutting. The leaves of the three first nodes from the bottom were removed, and the cuttings treated with 0.15% Captan solution for 10 min. The cuttings were then treated with IBA and H 3 B O 3 and maintained in growth cabinets as previously described.

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iii) Composition of the rooting medium (tap water). The Water Quality Control of Metropolitan Water Works (EMOS) provided the analyses of the characteristics and ionic composition of the rooting medium. pH, 7.6; filterable residue, 755 mg 1-1; hardness, 376 mg CaCO3, 1-1; alkalinity, 102 mg CO3; 1-l; SO4-, 275 mg 1-i; C12, 141 mg 1-l; Mg, 13 mg 1-1; plus trace elements. Phenology and growth habit in the field i) Phenology. The phenological stages of 20 P. chilensis trees were recorded when the material was collected and while the rooting tests were in progress (May 1985 to October 1986). Eight branches were chosen per tree and labelled. These were oriented to the north, south, east and west and intermediate points. The phenophases, vegetative growth, flower bud formation, flowering, fruiting and leaf-fall were recorded every 15 days. The percentage of bud development in laboratory conditions was also recorded. ii) Xylem water-potential. A Scholander bomb was used to measure xylem water-potential following the method described by Tyree and Hammel ( 1972 ). Six twigs of the previous year's growth were collected from five P. chilensis trees on the 14-15th day of each month, between 13 : 00 and 14: 00 h. iii) Dormancy. To determine whether dormancy was endogenous or imposed by environmental conditions, ten cuttings each were collected from 20 trees between the 14th and 16th of each month. These cuttings were placed with their bases in tap water (four cuttings per pot) at 30-32°C, (50-60% RH) under a 12-h photoperiod of 200/tmol m - 2 s- 1. Bud growth measurements were made weekly. iv) Temperature and rainfall. Precipitation was registered by means of a rain gauge from May 1985 to October 1986; minimum and maximum daily temperatures were also recorded during this period. In-vitro micropropagation of juvenile and adult material i) Micropropagation of juvenile material. The material employed in in-vitro culture was obtained from juvenile P. chilensis seedlings 1-4-months old growing in pots in the glasshouse at 30°C, 60% RH and maximum radiation 400/~mol m-2 s-~ for 16-h photoperiod. Two leaf nodal segments, 20-25 mm in length and 2-3 m m diameter, were used. The explants were surface-sterilized in commercial sodium hypochlorite solution (10% v/v) for 5 min, and then were well rinsed in sterile distilled water. The explants were placed on Whatman filter paper bridges in culture tubes 14 cm long and 22 m m in di-

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ameter, containing 10 ml of Murashige and Skoog (MS) ( 1962 ) nutrient solution. The culture tubes were previously sterilized for 20 min at 121 °C and 15 psi. The effects of naphthalenacetic acid (NAA), (5 and 10 mg 1-l ) and cysteine (5 and 10 mg 1- ~) concentrations were evaluated. Finally, pH was adjusted at 5.5. Once sealed, the culture tubes were kept in a growth chamber (25°C), illuminated by fluorescent tubes, 18-h photoperiod and radiation 100/~mol m - 2 s- 1. Four replicates of 12 h per treatment were used. When explants developed new leaves and roots they were transferred to plastic pots filled with sterile vermiculite. They were watered daily with a Hoagland's solution (Hoagland and Arnon, 1950) and covered by polyethylene bags, which were removed after one month.

ii) Micropropagation of adult material. Nodal and apical segments were removed from adult P. chilensis trees growing in the Chacabuco-Peldehue population and also from glasshouse-grown potted plants obtained from rooted cuttings. The dimensions of explants and culture procedure were the same as for micropropagation of juvenile material. Murashige-Skoog medium, with and without 5 mg NAA 1- ~and 10 mg cysteine 1-1, was used. Eight replicates of 12 tubes per treatment were made and kept in a growth chamber, as previously described; rooted explants were also handled as previously described. RESULTS In 1985-1986, the vegetative growth and reproductive development took place from September to March (Fig. 1 ). The phenophases overlapped such that trees began one phase before finishing the previous one. The period of leaf-fall was the longest, lasting from February to November, and growth came to a total stop between May and September, when average temperatures fell below 15 ° C. Xylem water-potential in the plants changed dramatically from -1.2 MPa in July 1986 (corresponding to the period of rain fall and low average temperatures). The lowest period of bud activity (March to June) concurred with the period of greatest water stress at the end of the dry season, while the period of highest activity (over 90%) occurred at the start of the dry season (October to December; Fig. 1 ). Rooting in adult cuttings of P. chilensis, varied between 6 and 9%, and only occurred at the end of the wet season (Fig. 1). The evaluation of the effects of photoperiod and bubbling air through the rooting solution on field cuttings (Table 1 ) showed that, under long days ( 16 h ), improved aeration did slightly increase rooting percentage, although there was no effect under short days. Photoperiod alone had no effect on rooting. Similarly, daylength had no effect on the rooting of serially propagated glasshouse-grown plants, although aeration did enhance rooting. This material rooted very much better than that from the field (Table l, Fig. 2d, e).

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Fig. 1. P h e n o l o g y a n d physiological b e h a v i o r o f Prosopis chilensis d u r i n g t h e year, c o m p a r e d with e n v i r o n m e n t a l p a r a m e t e r s . U p p e r graph; t e m p e r a t u r e ( . . . . ), rainfall ( b a r s ) ; M i d d l e graph: w a t e r status ( ), b u d b r e a k ( - - - ) ; L o w e r graph: p h e n o l o g y . TABLE1 Effect o f aeration and p h o t o p e r i o d on rooting o f Prosopis chilensis adult, material collected during december 1985

Rooting(%) @ p h o t o p e f i o d ( h ) : AERATED 12

NON-AERATED 16

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Different letters indicate significant differences according to Tukeys's test ( P < 0.01 ).

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TABLE2 Effect of naphthaleneacetic acid (NAA) and cysteine on the regeneration of complete plants (%) by in-vitro micropropagation of Prosopis chilensis juvenile material (nodal segments ) Cysteine (mg 1-* )

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Different letters indicate significant differences according to Tukey's test (P < 0.01 ). TABLE 3 Regeneration of complete plants by in-vitro micropropagation in Murashige-Skoog medium (MS) of

Prosopis chilensis adult material (nodal and apical segments ) Control Modified* Field explants 0.0 a 0.0 a Explants from rooted material kept in the greenhouse 50.6 _+6.2 b 60.0 + 5.6 b *MS solution plus 5 mg NAA 1- ~ and 10 mg cysteine 1-~. Different letters indicate significant differences according to Tukey's test ( P < 0.01 ).

was found that 5 mg NAA 1- t and 10 mg cysteine 1- 1 gave the best rooting (78%; Table 2 ). However, under similar conditions, no regeneration was obtained from adult nodal explants collected directly from the field (Table 3 ). By contrast, glasshouse material rooted satisfactorily (50-60%). When the cysteine concentration was below 10 mg 1-1, browning occurred within the first 7 days of culture. DISCUSSION

During the past years, interest has grown in forestation of arid and semiarid zones with highly productive species, such as Prosopis, resistant to drought and tolerant to salinity (Felker and Bandurski, 1979; Habit, 1981, 1985; Kohl et al., 1981; Felker et al., 1983; Arce and Balboa, 1986). To increase productivity further by clonal selection, several vegetative propagation techniques have been tested. Most of the work on vegetative propagation has been done in soil, sand, vermiculite, or various mixtures of these. We reported previously that, by using tap water as a rooting medium, IBA-treated juvenile cuttings of P. chilensis rooted well (80-90% success) with many long roots (Arce and Balboa, 1987; Balboa et al., 1987 ). Although previously we reported the impossibility of inducing rooting in adult field-grown material using concentrated hormone

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solutions (Arce and Balboa, 1986; Arce and Balboa, in press), there was limited success (14%) in the present study (Fig. 1, Table 1 ) this being greatest with aeration. This success seems to be attributable to the use of lower auxin concentrations with longer periods of treatment. Concentrated hormone solutions have been beneficial in other Prosopis species (Felker and Clark, 198 l; Souza and Nascimento, 1984; Klass et al., 1985; Souza and Felker, 1986) including other provenances ofP. chilensis growing in other environments. It appears, however, that provenances of this species from the Metropolitan area of Chile are particularly difficult to propagate. The low rooting percentages found in adult cuttings of P. chilensis agree with Felker and Clark ( 1981 ) but not with results for other species of this genus, such as P. juliflora (Souza and Nascimento, 1984 ) and P. alba (Klass et al., 1985 ), who reported that when the cuttings were exposed to high hormonal concentration, it was possible to induce high rooting percentages. Nevertheless, although the rooting of field-grown cuttings was low, it was not disappointing, since once this material has rooted it can be used as source of new cuttings, which then root well (over 80%) with aeration. In the present study, rooting of I'. chilensis was seasonal, occurring in the dry season, from September to February, when vegetative growth, flowering, and fruiting were in progress (Fig. 1 ). The absence of rooting during the dormant period, when trees were leafless, is not surprising, considering the known requirement of leaves in many species for good rooting (Hartmann and Kester, 1981 ). The simultaneous occurrence of low vegetative activity in the laboratory, even under ideal conditions, indicates that other endogenous factors may also be limiting. Interestingly, good rooting was strongly related to periods of high bud activity. Water status does not seem to play an important role in the rooting of P. chilensis cuttings, as water-potential was both highest and lowest during the period when cuttings did not root (Fig. 1 ). However, the cuttings were able to root, albeit in low percentages, when xylem water-potential values were between - 2 . 7 and - 3 . 7 MPa, which shows the remarkable capacity of this species to tolerate water deficits. Not surprisingly, xylem water-potential was positively correlated with temperature ( r = 0 . 9 9 ) and negatively correlated with rainfall ( r - -0.91 ). Micropropagation of juvenile material reached an 80% regeneration rate using the MS medium modified with 5 mg NAA l- 1 and 10 mg cysteine 1-1. Similar success, using plantlets from seeds and juvenile trees from experimentals plantations, has been described before (Goyal, 1982; Goyal and Arya, 1984; Jordan and Balboa, 1985; Jordan et al., 1985a,b; Tabone et al., 1986; Balbao et al., 1987 ). However, the micropropagation of field material has not been previously reported. Tests to regenerate adult P. alba in vitro only resulted in the induction of multiple buds, but not roots (Tabone et al., 1986).

SEASONAL1TY lN ROOTING OF PROSOPIS AND 1N-VITRO M1CROPROPAGA'I ION

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In the present study, adult material was regenerated ( 60% ) in vitro from previously rooted mature cuttings, giving plantlets with strong roots. This technique was not successful with material obtained directly from the field. However, when juvenile segments of other Prosopis species were cultured in vitro, complete plant regeneration was also achieved, although the percentages did not exceed 14% and 40% for P. tamarugo and P. alba respectively (Jord~in et al., 1985a,b). Complete plants have also been regenerated from explants of P. cineraria (Goyal, 1982; Goyal and Arya, 1984; Arya and Shekhawat, 1986), P. chilensis (Jord~in and Balboa, 1985; Jord~in et al., 1985a: Balboa et al., 1987 ) and P. alpataco (Arce, unpublished data, 198?). As all the above reports have used the Murashige-Skoog medium, the different morphogenetic responses cannot be attributed to differential nutrient availability. Similarly, use of the same hormonal concentrations indicates that the different levels of regeneration are attributable to species differences, not to hormone treatments. As no micropropagation from adult field-grown material has been obtained, it may be that the physiological condition of these tissues is similar to that of more-difficult-to-propagate species. Juvenile tissues generally show better rizogenic responses (Pierik, 1969; Pierik and Steegmans, 1975 ), and it is assumed that these related to their physiologically active growth perhaps being controlled by endogenous regulators. Ageing in plants is thought to be accompanied by morphologic and metabolic changes, and by the accumulation of secondary metabolites, which inhibit regenerative potentialities of the tissues, causing oxidation and inactivating enzymes and phytohormones. Factors such as these may be one of the causes of the low propagation percentages when using cuttings from adult material, and the impossibility of obtaining direct regeneration by in-vitro culture. ACKNOWLEDGEMENTS Financial support for this research was provided by the U.S. National Academy of Sciences/National Research Council by means of a grant from the U.S. Agency for International Development.

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Pierik, R.L.M. and Steegmans, 1975. Analysis of adventitious root tbrmation in isolated stem explants ofRhodendron. Sci. Hort., 3: 1-20. Saunders, R.M., Becker, R., Meyer, D., Marzo, E. and Torres, M.E., 1986. Identification of commercial milling techniques to produce high sugar high fiber protein and high galactomannan gum fraction from Prosopis pods. For. Ecol. Manage., 16: 169-180. Simpson, B.B., 1977. Breeding system of dominant perennial plants of two disjunct warm desert ecosystems. Oecologia (Berlin), 27: 203-226. Souza, S.M. and Felker, P., 1986. The influence of stock plant fertilization on tissue concentration of NPK and carbohydrates and the rooting ofProsopis alba cuttings. For. Ecol. Manage., 16: 181-190. Souza, S.M. and Nascimiento, C.E., 1984. Propagacaeo vegetativa de algarroba a trav6s de estaquia. Petronila EMBRAPA-CPATSA, Circ. T6c. No. 27. Tabone, T.J., Felker, P., Bingham, R.L., Ryes, I. and Lughrey, S., 1986. Techniques in the shoot multiplication of the leguminous tree Prosopis alba clone B2Vs,,. For. Ecol. Manage., 16: 191-200. Torres, M.E., 1984. Fijaci6n biol6gica de nitr6geno en Prosopis tamarugo y Prosopis alba. Estudios correspondientes al Proyecto "Zonas Aridas" de la Gerencia de Desarrollo de la Corporaci6n de Fomento de la Producci6n Chile. Tyree, M.T. and Hammel, M.T., 1972. The measurement of the turgot pressure and the water relations of the plants by the pressure-bomb technique. J. Exp. Bot., 23: 267-282.