On-line version ISSN 1678-4685
Genet. Mol. Biol. vol. 21 n. 3 São Paulo Sept. 1998
Micropropagation of rubber trees (Hevea brasiliensis Muell. Arg.)*
Asseara Batista Leitão Mendanha1, Roberto Augusto de Almeida Torres1 and Adelson de Barros Freire2
1Laboratório de Cultura de TecidoVegetal, Departamento de Biologia Geral, Universidade Federal de Goiás, Caixa Postal 131, 74001-970 Goiânia, GO, Brasil. Send correspondence to R.A.A.T. and A.B.L.M.
2Centro Nacional de Arroz e Feijão (CNPAF - Embrapa). Fazenda Palmital, Santo Antônio de Goiás, Caixa Postal 179, 74001-970 Goiás, GO, Brasil.
Tissue cultures were established from newly expanded leaves and axillary buds of rubber trees (Hevea brasiliensis Muell. Arg.). Calli formed from these explants, but no regeneration occurred. Shoots were obtained from axillary buds cultured on Murashige and Skoog's (MS) medium (Physiol. Plant. 15: 473-497, 1962) supplemented with 1.0 mg/l kinetin, 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 20 g/l sucrose and 4 g/l Difco agar. Formation of a root similar to a tap root was induced on MS medium supplemented with 5.0 mg/l naphthaleneacetic acid (NAA), 3.0 mg/l indolylbutyric acid (IBA), 50 g/l sucrose and 4 g/l Difco agar. Several types of explants were used in attempts to recover complete rubber tree plants with well-developed tap roots. Leaf explants and axillary buds formed calli on MS basic medium with different combinations of kinetin, benzylaminopurine (BAP), 2,4-D, IBA, NAA and indolylacetic acid (IAA). The antibiotic tetracycline was also used to control possible bacterial infections. However, no antibiotic effect was noted. Calli formation was abundant, but no regeneration was observed when the calli from different media was transferred to MS medium without growth hormones. On this basic medium, callus cultures became necrotic and died. Shoots developed from axillary buds, rooted vigorously when cultured on MS medium with NAA, IAA, and IBA. Based on these results, further studies with commercially important clones should lead to a feasible micropropagation technique.
Rubber trees are traditionally propagated by grafting buds from selected clones on seedlings or plants from seed orchards. This process is lengthy, since one to two years are required before the plants can be transplanted to the field. Furthermore, no evaluation of the interaction between rootstock and scion is involved. For this reason the grafted plants sometimes do not produce at the expected levels.
New methods of propagation would be recommended if one could eliminate possible incompatibility between rootstock and scion, as well as reduce the cost and time required for plant production. Micropropagation, or propagation in vitro of complete plants, is a promising technique for large scale multiplication of selected clones. It has the advantages of small space requirements and reduced costs when compared to conventional vegetative propagation techniques.
One of the major problems with rubber tree micropropagation is the recovery of complete plants with well-developed tap roots, which is fundamental for successful transplantation to the field. Paranjothy and Ghandimathi (1975) induced rooting in plantlets derived from tissue cultures but were unable to root clonal material, although they regenerated shoots from axillary buds of some clones.
MATERIAL AND METHODS
Experiments were designed to test regeneration via organogenesis from petioles, stems and leaves as well as micropropagation from axillary buds. Explants were derived from Hevea brasiliensis seedlings initially germinated in sand and then transferred to plastic sacks with 10 kg soil + organic fertilizer (1:1 v/v). Seeds were donated by the Estação Experimental de Pindorama of the Secretaria de Agricultura de São Paulo, and were harvested in February; the plants were maintained in a greenhouse, where they were watered once a day.
The explants were harvested between nine and 10 in the morning. The material was washed with Tween 80 (two to three drops per liter) followed by immersion in 70% (v/v) ethanol for one minute. Disinfestation was made with commercial hypochlorite solution (2% active chlorine) followed by four to five rinses in sterile distilled water. All disinfestation procedures were carried out in a laminar flow hood.
Explants measured approximately 5 mm in length. After inoculation in the different nutrient media, the cultures were incubated at 27 + 1oC with a photoperiod of 16 h.
The nutrient medium of Murashige and Skoog (1962; MS) was used as the basic medium for all experiments. Different components were tested for callus development (Table I): kinetin, benzylaminopurine (BAP), indolylbutyric acid (IBA), indolylacetic acid (IAA), naphthaleneacetic acid (NAA), mio-inositol and sucrose. The cultures were incubated in the dark at 27oC. After 30 days, the proliferating cells were transferred to fresh medium. After an additional 30 days, the cells were transferred to MS medium without hormones in order to induce formation of somatic embryos. The cultures were maintained in the light with a photoperiod of 16 h (2000 lux). Four repetitions of the experiment were carried out.
The media in Table II were tested for the induction of callus in axillary buds. Seven-hundred explants were distributed on these five media. The cultures were incubated at 27 + 1oC in the dark. After 35 days the cell masses were transferred to MS medium, without hormones, in order to induce embryos. This stage was conducted with a photoperiod of 16 h (2000 lux) at 27 + 1oC.
When axillary buds were induced to develop shoots directly, the media in Table III were used. A total of 1108 explants were inoculated in these media, with 10 ml per test tube. The cultures were maintained in the light with a photoperiod of 16 h (2000 lux). The resulting 244 shoots were transferred to four different treatments for rooting (Table IV). Rooting was conducted in the light with a photoperiod of 16 h (2000 lux) at 27 + 1oC.
The pH of all nutrient media was adjusted to 5.8 + 0.1 with NaOH or HCl, before adding agar. The media were autoclaved at 121oC for 20 min.
The c2 test was used for analysis of the results (Steel and Torrie, 1960).
RESULTS AND DISCUSSION
Various concentrations of sodium hypochlorite were tested for disinfestation of the explants. The best results were seen with 20% active chlorine.
Rubber tree leaf explants were wrinkled after 15 days. Between 25 and 30 days after inoculation, abundant cell growth was observed on the edges of the leaf explants. No regeneration of shoots or embryos was observed when these cells were transferred to MS medium without growth hormones. Similar results were reported by Chua (1966), Wilson and Street (1974) and Paranjothy and Ghandimathi (1975).
Nutrient medium "C" produced the highest percentage of leaf explants with calli (Table V). The presence of the antibiotic tetracycline did not produce any significant modifications in the aspect of the callus.
The c2 test showed a highly significant difference between the nutrient medium "C" and the other media. Thus, medium "C" is the medium of choice for callus production from leaf explants.
Callus formation was initiated from axillary buds between 15 and 20 days after inoculation. After 35 to 40 days, abundant calli had formed on the surface of the explants. When transferred to MS medium without growth hormones, all the callus cultures died. Similar results were reported with axillary buds by Enjarlic and Carron (1982), Wilson and Street (1974), Carron and Enjarlic (1982) and Enjarlic (1983).
The highest percentage of axillary buds formed calli on medium "A" (Table VI). The difference between the nutrient media was highly significant, based on the c2 test (P < 0.01). Addition of tetracycline to medium "D" apparently had no effect on explant development.
Axillary buds were also used to develop shoots on the four media described in Table III. On the 15th day, shoots began to emerge from the buds. After 50 days, the shoots had reached approximately 4 cm in length, and had three leaves. The c2 test showed a significant difference in shoot development between the nutrient media (P < 0.01). The best nutrient medium was "A" (Table VII). According to Skoog and Miller (1957), the development of plant organs is regulated by the interaction of growth hormones in nutrient medium, which was demonstrated once again in the present experiment.
Shoots from this experiment were submitted to four different treatments for rooting (Table IV). After 30 days, roots began to appear. The root system was fully developed after 50 days, with a well-developed tap root in all plants. Nutrient medium "D" presented the highest percentage of rooted shoots (Table VIII).
Previous studies reported root formation on hypocotyl explants from rubber tree seedlings in vitro (Thideman and Hawker, 1982; Chua, 1966; Wilson and Street, 1974). In the present study, a vigorous tap root with numerous secondary roots developed. There was no significant difference in percentage of shoots which rooted on the different nutrient media tested in the present study (Table VIII), although the highest percentage of shoots rooted on medium "D".
The authors thank Dr. Paulo de Souza Gonçalves of the Instituto Agronômico de Campinas for suggestions presented during the current study and Dr. Antônio Lúcio Martins, head of the Estação Experimental de Pindorama (São Paulo) for the seeds, without which this study could not have been conducted. They are also grateful for the financial support of CNPq.
Culturas de tecidos in vitro foram estabelecidas de folhas recém-expandidas e de gemas axilares de seringueira (Hevea brasiliensis Muell. Arg.). Houve formação de calos nestes explantes mas a regeneração destes calos em embrióides não ocorreu. Brotos foram obtidos de gemas axilares cultivadas no meio de cultura básico de MS (Murashige and Skoog (Physiol. Plant. 15: 473-497, 1962)), suplementado com 1,0 mg/l de cinetina, 1,0 mg/l de ácido 2,4-diclorofenoxiacético (2,4-D), 20 g/l de sacarose e 4 g/l de ágar Difco. Para o desenvolvimento do sistema radicular com raiz pivotante o meio de cultura usado foi o MS, suplementado com 5,0 mg/l de ácido naftalenoacético (NAA); 3,0 mg/l de ácido indolilbutírico (IBA); 50 g/l sacarose e 4,0 g/l ágar Difco.
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(Received September 8, 1997)
*Part of a thesis presented by A.B.L.M. to the Universidade Federal de Goiás (UFG) in partial fulfillment of the requirements for the Master's degree.