African Crop Science Journal African Crop Science Society ISSN: 1021-9730 EISSN: 2072-6589 Vol. 7, Num. 4, 1999, pp. 303-311

African Crop Science Journal, Vol. 7. No. 4, pp. 303-311, 1999

M. B. Kwapata, F. Kalengamaliro, J. Bakuwa and S. Manyela
University of Malawi, Bunda College of Agriculture, P.O. Box 219, Lilongwe, Malawi

Code Number: CS99019

ABSTRACT

The formation and growth of roots and axillary shoots from Faidherbia albida (Del.) A. Chev. terminal shoots cultured in vitro were studied. Shoot sections of 5-10 mm were cultured in root induction media of Murashige and Skoog (MS) basal salts supplemented with 0, 5.0 and 25.0 µM of 1H-Indole-3-butyric acid (IBA), and incubated in the dark or light conditions for seven days before being transferred into free IBA root development media. The other experiments involved varying levels of IBA (0, 2.5, 5.0, 10.0 and 20.0 µM ), using three different basal salts media formulations (MS, Gamborg and McCown) and supplementation with N6-Benzyladenine (BA) at concentrations of 0, 2.5, 5.0, 10.0 and 20.0 µM. The final experiments evaluated rooting of axillary shoots under 16-hours of incubation light intensities of 0, 45, 90, 135 and 180 µmoles m -2 s -1 . Additionally, axillary shoots proliferation from seedling (juvenile) stem segments of F. albida. was evaluated using cytokinin species : N6-Benzyladenine (BA), N6-Furfuryladenine (Kinetin) and N6-Isopentenyladenine (2iP) at 0, 2.5, 3.75, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 25.0, 37.5, and 50.0 µM and Coconut water (at 10% of full strength) in combination with different concentration of BA. The formation of roots did not require induction treatment. Root numbers and growth rate were highest in the explants exposed to the basal salt medium containing 10.0 µM IBA. There were no significant differences in rooting responses among the three basal salts media formulations. The number of roots per shoot increased significantly with increasing incubation light intensity, and an optimum of 10 roots per shoot was obtained at 135 µmoles m -2 s -1 light conditions. Multiple axillary shoots regenerated from a single stem segment (explant) with different cytokinin species. The best cytokinin species was BA at concentrations of 5.0 to 25.0 µM, with 25.0 µM being optimum level that gave 3.8 shoots per explant after 8 weeks of culturing. Addition of coconut water to the media stimulated more multiple axillary shoots growth, with new shoots per culture (explant) ranging from 9.0 to 13.0 and the highest number of new shoots were obtained from the 12.5 µM BA plus 10% Coconut water treatment. The axillary shoots proliferation level obtained in the study was acceptable for rapid multiplication of F. albida propagules.

Key Words: BA-benzyladenine, Faidherbida, 2iP-N6-isopentenyladenine, IBA- 1H-indole-3-butyric acid, Kinetin-N6furfuryladenine, light intensity, media formulations, regeneration

RÉSUMÉ

La formation et la croissance des racines et de pousses axillaires des feuilles terminales de Faidherbia albida (Del.) A. Chev. cultivées in vitro ont été étudiées. Des sections de pousses de 5-10 mm étaient cultivées dans le milieu d’induction de racines de Murashige et Skoog (MS) à base de sels complémenté avec 0, 5.0 et 25.0 µM d’acide 1H-Indole-3-butyrique, et incumbé dans des conditions noires ou de lumière pendant sept jours avant d’être tranferré dans le milieu de développement de racines IBA libre. Les autres essais impliquaient la variation des niveaux d’IBA (0, 2.5, 5.0, 10.0 et 20.0 µM), utilisant trois differentes formulations de milieux à base de sel (MS, Gamborg et McCown) et la complémentation avec N6-Benzyladenine (BA) à des concentrations de 0, 2.5, 5.0, 10.0 et 20.0 µM. Les derniers essais évaluaient l’enracinement des pousses axillaires pendant 16 heures d’incubation à des intensités de lumière de 0, 45, 90, 135 et 180 µmoles m -2 s -1 . Additionellement, la prolifération de pousses axillaires venant des segments de tiges de plantules de F. albida, a été evaluée utilisant des espèces de cytokinine: N6-Benzyladenine (BA), N6-Furfuryladenine (Kinetin) et N6-Isopentenyladenine (2iP) à 0, 2.5, 3.75, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5, 25.0, 37.5, et 50.0 µM et l’eau de noix de Coco (à10% de puissance totale) en combinaison avec différentes concentrations de BA. La formation de racines n’a pas exigé l’induction du traitement. Le nombre de racines et le taux de croissance étaient les plus élevés dans les explants exposés au milieu à base de sel contenant 10.0 µM IBA. Il n’yavaient pas de differences significatives de réponses à l’en racinement parmi le differentes formulations de milieux à base de sel. Le nombre de racines par pousse a augmenté significativement avec l’augmentation de l’intensité de lumière d’incubation, et l’optimum de racines par pousse a été obtainu à une condition de lumière de 135 µmolesm -2 s -1 . Multiple pousses axillaires ont regénéré à partir d’un seul segment de tige (explant) avec differentes espèces de cytokinine. La meilleure espèce de cytokinine était BA à des concentration de 5.0 à 25.0µM, avec 25.0 µM étant le niveau optimal qui a donné 3.8 pousses par explant après 8 semaines de culture. L’addition d’eau de noix de coco au milieu a plus stimulé la croissance de multiple pousses axillaires, avec des nouvelles pousses par culture (explant) variant de 9.0 à13.0 et le plus haut nombre de nouvelles pousses a été obtainu de 12.5 µM de BA plus un traitement de 10% de noix de coco. Le niveau de prolifération des pousses axillaires obtenu dans cette étude était un niveau acceptable pour la multiplication rapide des propagules de F. albida.

Mots Clés: BA-benzyladenine, Faidherbida, 2iP-N6-isopentenyladenine, IBA- 1H-indole-3-butyrique, Kinetine-N6furfuryladenine, intensité de lumière, formulation de milieu, regénération

INTRODUCTION

Viable agroforestry practices for adoption by smallholder farmers hinge upon the availability of large quantities of good planting materials (propagules). However, large quantities of good quality propagules and field survival of transplanted propagules is depended to a large extent on the rapid multiplication of axillary shoots and formation of normal and functional roots before transplanting. Attempts to use conventional vegetative propagation methods and micropropagation techniques have been unsatisfactory for large scale production of good quality Faidherbia albida propagules (David, 1985; Danthu, 1992 ). One of the main reasons for producing low quantities of poor quality F. albida propagules is the low rate of axillary shoots and root formation and subsequent poor root growth and development from the explants used in the various methods of vegetative propagation.

The problems of poor axillary shoots regeneration and rooting of propagated plant tissues are common for most tropical woody perennial tree species. In several in-vitro propagation experiments, Gassama (1989), Detrez et al. (1992) and Ahee and Duhoux (1994) found only 1 to 3 roots regenerating per explants and less than 30% of explants developed normal roots. In contrast, seedlings arising from seed develop many normal roots ranging from 5 to 20 in the first three to six weeks of germination. In other studies with several woody perennial tree species, it was also found that inadequate light quality exposure of in-vitro propagated plant tissues resulted in delayed root formation and fewer and weak roots formed which contributed to high mortality rate of transplants in greenhouse (Torrey, 1952; Capite, 1955; Scott et al., 1961; Maliro, 1997). It is well established that the survival of young seedlings or propagules depends on having normal and functional roots for water and nutrients uptake (Hartman and Kester, 1983; Brown and Sommer, 1985; Diaz-Perez et al., 1995). Therefore, it is necessary to find an asexual propagation technique which promotes rapid axillary shoots regeneration and increases formation of the normal and functional roots of propagated F. albida explants to enhance production and field survival of propagules.

The common propagation method of F. albida is by seed. Sexual propagation, however, does not retain the desirable and superior parental traits in progeny. Attempts to use conventional vegetative propagation methods and micro-propagation have been unsatisfactory for large scale production of F. albida propagules (David, 1985; Gassama, 1989; Danthu, 1992; Detrez et al., 1992). One of the main problems is the low number of multiple shoots regeneration from explants used, as reported by Muhamed and Muhamed (1988), Gassama (1989), Detrez et al. (1992), Ruredzo and Hanson (1993) and Ahee and Duhoux (1994).

Our objectives for carrying out the study were: 1) to determine whether rooting of F. albida shoots can be enhanced by in vitro root induction treatment; 2) to determine IBA level that promote root formation and growth rate; 3) to determine the most suitable basal salts culture media for rooting F. albida explants; 4) to ascertian whether supplementation with low levels of BA is necessary to promote root formation and development in F. albida species; 5) to determine optimum incubation light intensity that induce formation of normal and functional roots on axillary shoots of F. albida; and 6) to determine the best culture media formulation that induces high axillary shoots proliferation in F. albida shoots explants for use in rapid propagation of propagules.

MATERIALS AND METHODS

Rooting studies. Several experiments were carried out at Bunda College Tissue Culture laboratory to assess rooting responses of F. albida shoots, from adult and juvenile plants, subjected to various treatments. Each treatment had 20 culture tubes filled with 20 ml of media, which were sterilised by autoclaving at about 100 0 C under 121 psi pressure for 10 minutes. All experiments were replicated three times and repeated two times.

The first experiment studied root induction and used a randomised block experimental design with the following treatments: Control, Dark + 5.0 µM IBA, Dark + 25.0 µM IBA, Light + 5.0 µM IBA, and Light + 25.0 µM IBA. Terminal shoots (20-30 cm) from field-grown five-year old F. albida trees were used as source of explants. They were collected, washed in running tap water, put in small beakers and vacuum-sterilised in 5% sodium hypochloride solution for 20 minutes. The sodium hypochloride solution was decanted and the explants rinsed with distilled water under sterile laminar air-flow hood. The surface sterilised explants (shoots) were cut into smaller 5-10 mm long sections and transferred into sterile culture tubes containing 20 ml of Murashige and Skoog (MS) media (Huang and Murashige, 1976). The induction period was 7 days and thereafter the cultures were transferred into IBA free MS root development media. All cultures in root development media were incubated under 16-hour daily illumination with 45 µmoles m -2 sec -1 light from cool-white long fluorescent lamps and at 23-28 0 C incubation room temperature. Data on number of shoots forming roots (%), number of roots per shoot and root length (mm) were collected over a period of 12 weeks and analysed

The second experiment evaluated IBA influence on rooting. A completely randomised block experimental design was used. Plain MS basal salts media was supplememted with IBA at 0, 2.5, 5.0, 10.0 and 20.0 µM as treatments. Stem sections (explants), of 5-10 mm long were cut from terminal shoots collected from 5-year old field-grown F. albida trees. The explants were prepared as described in the root induction study, but received no root induction treatment. The sterile explants were transferred into culture tubes filled with media, under sterile laminar air-flow hood and incubated in a culture room under 16-hours daily illumination with 45 µmoles m -2 sec -1 light from cool-white fluorescent lamps and at 23-28 0 C. Data on number of roots per explant and root length were collected over a period of 12 weeks and analysed.

The third experiment compared the influence of media formulations at different BA levels on rooting. A factorial experimental design was used and the following three basal salts media formulations evaluated: MS (Huang and Murashige, 1976); Gamborg (Gamborg et al., 1968) and McCown (McCown and Lloyd, 1981) in combinations with 0, 2.5, 5.0, 10.0 and 20.0 µM cytokinin (BA). A 2.5 µM IBA (auxin) was added to each treatment.

Two sets of trials were set up with one using nodal stem section as explants and the other using internodal stem sections as explants. In both experiments 5-10 mm long stem sections from aseptically raised juvenile (4 weeks old seedlings) plants of F. albida were used as explants. The explants were prepared, sterilised, inoculated into culture tubes filled with 20 ml of media and incubated as described in the IBA influence experiment. Data on number of explants forming roots, number of roots per explant and root length were collected over a period of 12 weeks and analysised.

The final rooting study was on light influence. A completely randomised block experimental design was used. The treatments were five incubation light intensities of 0 (dark), 45, 90, 135, and 180 µmoles m -2 s -1 of 16-hours of light exposure supplied by cool-white long fluorescent lamps. The light intensities were achieved by using 0, 1, 2, 3, 4, and 5 lamps per shelf compartment in the incubation room.The incubation room temperature was controlled by air-conditioner set at 23-28 0 C range. Each culture tube contained MS basal salt media supplemented with 10 µM IBA. Eight-week old F. albida shoots obtained from axillary shoots proliferation study were cultured and incubated under the different light intensities for observation. Data on number of roots per culture were collected after 4-weeks of incubation period and analysed.

Axillary shoots proliferation studies. Several experiments on in vitro axillary shoots proliferation of F. albida were done following randomised block experimental design. For these studies, each treatment had 20 test tube cultures and were replicated 2 times. The test tubes contained 20 ml of growth media and were sterilised as in rooting study. The cultures were incubated under 16 hours light exposure (45 µmoles m -2 s -1 ) from cool-white long fluorescent lamps and 8 hours dark conditions with incubation (growth) room temperature maintained at the range of 23-28 0 C.

Explants were obtained from aseptically raised seedlings of F. albida. Seeds (purchased from Forestry Research Institute of Malawi) were washed and put into boiling water for 20 minutes. After cooling the seeds were surface sterilised by placing them in 2% sodium hypochlorite solution for 10 minutes. The sterilant was decanted and the seeds rinsed in cool sterile water before transfering them to inoculation room. Under sterile laminar air-flow hood, the seed coat of each individual seed was removed using sterilised surgical tools. The naked seeds were inoculated into sterile test tubes containing Murashige and Skoog (MS) basal salts culture media (Huang and Murashige, 1976). The cultures were transfered into the incubation room for growth. Seedlings were ready for use in several experiments after two months of growing. Each experiment was ran for a period of 8 weeks.

The first experiment studied BA (N6-benzyladenine) influence on axillary shoot proliferation. Stem nodal sections, each 1 cm long, were inoculated in MS basal salts media with BA concentrations of 0, 2.5, 3.75 and 5.0 µM as treatments. Cultures were incubated in the incubation room for 8 weeks and evaluated for regeneration of shoots from explants.

Another trial studied Kinetin (N6-furfury-ladenine) influence on axillary shoot profiration using stem nodal sections (1 cm long) inoculated in MS basal salt media containing Kinetin concentrations of 0, 7.5, 10.0, 12.5, 15.0 and 17.5 µM as treatments.

The third experiment compared effect of Cytokinin species (BA, Kinetin, 2iP) influence on axillary shoot proliferation. Stem nodal sections of 1 cm long were inoculated in MS basal salts media containing BA (N6-benzyladenine), Kinetin (N6-furfuryladenine) and 2iP (N6-isopenteny-ladenine) at concentration levels of 0, 2.5, 5.0, 12.5, 25.0, 37.5, 50.0 µM.

Finally, the influence of BA plus coconut water on axillary shoot proliferation of two month-old seedlings was also studied. The stem nodal sections were first inoculated in MS basal salts media containing 15.0 µM BA, and incubated in an incubation room for 3 weeks. The callus formed were subcultured into another MS media containing BA at concentrations of 0, 2.5, 5.0, 12.5 and 25.0 µM, plus 10% (100 ml l -1 ) coconut water per BA concentration level as treatments. The subcultures were incubated for another 5 weeks in an incubation room for observation and evaluated for axillary shoots regeneration from callus.

The data from the various experiments were subjected to analysis of variance using the Msta-c computer software, and mean separation was by LSD.

RESULTS AND DISCUSSION

Rooting of F. albida . The rooting responses of induced and non-induced shoot sections of F. albida are shown in Table 1. There were no significant differences in root formation between induction and non-induction treatments. The number of roots per shoot (explant) was higher in IBA induced shoots. But dark or light induction conditions had little effect on the number of roots formed per shoot.

The presence of auxin (IBA) depressed root growth (increase in length) and high IBA concentration (25 µM ) reduced the number and growth of roots. These results indicate that for F. albida, the root induction step is not necessary for root formation, and higher levels of auxin (IBA) do not suppress root initiation, but do inhibit subsequent root growth and development.

Root induction studies with other woody perennials have shown that higher concentrations of the orders of 50.0 µM IBA or above were necessary to induce more root formation in species such as Gerbera, Eucalyptus and Citrus, but induction treatment may not be necessary in some tree species (Murashige et al., 1974; Le Roux and van Staden, 1991), as is the case with F. albida.

Table 1. In-vitro rooting of F. albida shoot explants as influenced by root induction treatment

ns = not statistically different at P = 0.05

The effects of varying concentration of IBA on rooting of F. albida shoots are presented in Table 2. The percentage of explants (shoot sections) forming roots and number of roots per shoot section increased with increasing concentration of IBA in the media up to 10.0 µM, and beyond this level the rooting intensity began to decline. The decline may be due to inhibitory effects of high levels of IBA concentrations (Lennox, 1995; Maliro, 1997). The growth of roots as measured by root length corresponded closely with rooting intensity (number of roots) response pattern. The IBA levels of 5.0 to 10.0 µM promoted more root growth than the other levels, and 10.0 µM was optimum for promotion of in-vitro rooting of shoots of five year old F. albida trees. In comparison with findings from studies of other woody perennials such as Eucalyptus species and Uapaka species, the optimum range of IBA concentration promoting root formation and growth was 5.0-10.0 µM (LeRoux and van Staden, 1991; Maliro, 1997).

Table 2. In-vitro rooting of F. albida shoot explants as influenced by IBA

The rooting of F. albida shoots was not influenced by use of different rooting media formulations and increasing cytokinin (BA) levels (Table 3). There was no rooting difference between nodal and internodal shoot sections at all concentration levels of BA. This suggests that for rooting of F. albida shoots, any of the three basal salt formulations and nodal or internodal stem sections may be used without BA supplementation. Although appropriate balance of exogenous auxin (NAA, IBA, etc.) and cytokinin ( BA, Kinetin, etc.) concentrations has been reported to be necessary for root growth and development for some woody perennial species, in this study there was no indication that cytokinin is required for rooting F. albida shoots.

Table 3. In-vitro rooting of F. albida shoot explants in different media formulations and varying concentration of BA

The results of light experiments showed that more roots per culture were formed with increasing light intensity upto 135 µmoles m -2 s -1 and beyond this the root numbers per shoot declined (Table 4). Although at high light intensities the plantlet shoots appeared yellow and several leaflets defoliated, the roots appeared normal and healthy. The condition of the plantlets was typical of well hardened in-vitro propagated propagules reported by others (Capite, 1955; Muhamed and Muhamed, 1988; Diaz-Perez et al., 1995). Based on the results of the several rooting experiments discussed, in-vitro formation of normal and healthy roots on young F. albida shoots can be enhanced using MS basal salts media with supplimentation with no or low IBA and incubation under 135 µmoles m -2 s -1 of light and 23-28 0 C room temperature.

Table 4. Number of F. albida roots formed per culture under different light intensities

Axillary shoots proliferation of F. albida. The results of BA influence on axillary shoots regeneration are presented in Table 5. More cultures regenerated at higher BA concentration. Out of the regenerating cultures more explants formed callus as opposed to shoots at lower levels of BA. The number of new shoots per explant increased with high BA levels. A similar pattern of results was obtained with Kinetin study (Table 6 ). In Table 7, BA gave the highest number of shoots per culture than Kinetin and 2iP with 25.0 µM BA being optimum level for axillary shoots proliferation.

Table 5. Effect of BA concentration on F. albida shoot proliferation after 8 weeks of incubation

TABLE 6. Effect of Kinetin concentration on F. albida shoot proliferation after 8 weeks of incubation

The results reported in Tables 5-7 demonstrated that axillary shoots proliferation responses of juvenile F. albida stem section explants is influenced by both the species and concentration levels of cytokinin. Among the cytokinin species compared BA was superior to Kinetin and 2iP; and the concentration levels ranging from 5.0 to 25.0 µM BA induced axillary shoots proliferation, with 25.0 µM BA being optimum. Although Table 7 indicated the potential of using BA for axillary shoots proliferation work, the number of new shoots per explant (culture) was low (ranging from 1-4), to meeting the high demand for F. albida propagules.

Table 7. Effect of cytokinin species and concentration on shoot proliferation (shoots/explant) of F.albida after 8 weeks of incubation

ns= not statistically different at P=0.05

Table 8 shows the combined effect of BA and coconut water on inducing axillary shoots proliferation from juvenile (seedling) stem segments of F. albida. The addition of coconut water ( 10% of full strength) enhanced axillary shoots proliferation at every concentration level of BA. The optimum combination was 12.5 µM BA plus 10% Coconut water which gave 13 shoots per explant (stem segment or culture). The quality of developing shoots was much better with the combined BA and Coconut water than with BA alone (see Tables 5-7). The effect of adding coconut water clearly demonstrated an improvement in axillary shoots proliferation over that of cytokinin species alone. The addition of coconut water enhanced induction of multiple shoots per stem segment. The number of new shoots per subculture of 9 to 13 is fairly reasonable to significantly contribute to the rapid multi-plication of F. albida propagules to meet the demand by farmers.

Table 8. Effect of BA and Coconut water on shoot proliferation of F. albida after 8 weeks of incubation

ACKNOWLEDGEMENT

The authors acknowledge the financial assistance provided by the Rockefeller Foundation under the Forum programme, technical back-stopping by Prof. T. Murashige and the logistical support given by Drs. M. Blackie and B. Patel of Rockefeller Foundation Office in Lilongwe, Malawi.

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