Plant Cell, Tissue and Organ Culture 55: 79–83, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.
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In vitro regeneration from leaf explants of Neoregelia cruenta (R. Graham) L.B. Smith, an endemic bromeliad from Eastern Brazil L.A. Carneiro1,2 , R.F.G. Ara´ujo1 , G.J.M. Brito1 , M.H.P.B. Fonseca1 , A. Costa3 , O.J. Crocomo2 & E. Mansur1,∗ 1 Laborat´ orio
de Micropropagação e Transformação de Plantas, Instituto de Biologia – DBCG, Universidade do Estado do Rio de Janeiro. Rua São Francisco Xavier 524 - PHLC sala 505, Rio de Janeiro, Brasil; 2 Centro de ´ Biotecnologia Agricola /ESALQ, USP; 3 Depto. de Botânica, Museu Nacional, UFRJ (∗ requests for offprints; Fax: 21 587 7377; e-mail:
[email protected]) Received 29 October 1997; accepted in revised form 28 October 1998
Key words: bromeliad, endangered species, micropropagation, shoot formation
Abstract An efficient plant regeneration system was developed for the induction of direct shoot formation from leaves derived from seedlings of Neoregelia cruenta, an endemic Bromeliaceae of Eastern Brazil. Shoot differentiation occurred directly from the leaf bases. In vitro responses were influenced by seedling age and growth regulator combinations. Highest regeneration rates were obtained from explants excised from 7-week-old seedlings cultured in the presence of 22 µM BA and 2.5 µM NAA. Shoot conversion to whole plants was most effective in shoots formed in response to 4.4 or 8.8 µM BA combined with 2.5 µM NAA.
Abbreviations: BA – 6-benzylaminopurine; NAA – naphthaleneacetic acid; MS – Murashige and Skoog Introduction Eastern Brazil and especially the state of Rio de Janeiro are very rich in bromeliad species. As a consequence of the increase of human populations, the coastal ecosystems of this region are being degraded at an accelerating rate. Since several bromeliads are highly endemic, some species may be irreversibly lost following the destruction of their natural habitats (Leme and Marigo, 1993). Neoregelia cruenta is an endemic bromeliad found exclusively in sandy coastal plains of Rio de Janeiro (Leme, 1993). As in other species of the family, its tank-shaped rosette forms a micro-habitat for several groups of organisms, conferring an important ecological role on this species in the maintainance of ecosystem complexity and diversity (Oliveira et al., 1994). Moreover, it is considered a pioneer species during ecological succession because it colonises totally bare
sand, providing organic material and alterations of soil pH which allow the development of other plant species (Leme, 1983). N. cruenta is widely used in landscape design because of its beautiful red-tipped leaves which are up to 70 cm long (Figure 1A). In spite of being one of the most common bromeliad species on the coastal plains, it may be viewed as endangered since its natural habitat is under intense pressure from man’s activities. In addition to this, it is extensively and illegally collected in order to supply the landscape design market. In view of these problems, the aim of this work was to develop a tissue culture methodology that can provide an alternative means of production to supply the market and be used for reintroduction programs of impacted areas. Taking into account that the conservation of a wide range of endangered species has been achieved through the use of micropropagation techniques (Fay and May, 1990; Sharma and Chan-
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Figure 1. In vitro regeneration of N. cruenta. (A) Adult plant kept under greenhouse conditions. (B) Shoot-bud cluster forming on the protuberance at the cut end of the leaf blade after 5 weeks of culture on MS medium supplemented with 22 µM BA and 2.5 µM NAA. (C) Multiple shoot formation after 4 months of culture. (D) Rooted plantlet three months after shoot excision and transfer to MS0.
Material and methods
(NAA) were performed prior to autoclaving (120 ◦ C for l5 min) in 250 ml flasks. Cultures were maintained in a growth chamber at 28± 2 ◦ C with a total irradiance of 46 µmol m−2 s−1 provided by cool-white and Growlux fluorescent lamps (3:1), in a 16-h day light regime.
Culture media and conditions
Explant culture
The basal culture medium consisted of MS mineral salts and vitamins (Murashige and Skoog, 1962) plus 3% sucrose, solidified with 0.7% agar. The adjustment of pH to 5.8 and the addition of the growth regulators 6-benzyladenine (BA) and α-naphthaleneacetic acid
Seeds of N. cruenta were collected in Massambaba, an Environmental Protection Area in the Saquarema municipality (Rio de Janeiro). The seeds were surface sterilised by dipping in 70% ethanol followed by immersion in 5% sodium hypochlorite plus 0.01%
del, 1992; Martín and Pérez, 1993), this methodology also provides a basis for the establishment of in vitro conservation systems for N. cruenta.
81 Tween-80, for 30 min with agitation, at room temperature. After three rinses with sterile distilled water, seeds were inoculated on solidified MS salts and vitamins plus 3% sucrose (MS0) for germination, which occurred at a rate of 74.3% and resulted in the production of one plant per seed. Seedlings were subcultured to the same medium after 14 weeks. Whole leaves (2– 3 cm long) and the remaining stems containing the exposed axillary buds were excised from seedlings and used as explants at 7, 14 and 24-weeks of age in order to examine the optimum age of tissues. Four concentrations of BA (2.2, 4.4, 8.8 and 22 µM) and four concentrations of NAA (0, 0.5, 1 and 2.5 µM) were used in all combinations to determine the optimum concentration of growth regulators. Subcultures to medium with the same composition were performed at bimonthly intervals, over eight months. Five explants were cultured per flask. In vitro regeneration efficiency was calculated as the percentage of explants that regenerated shoots as well as the mean number of shoots and whole plantlets produced per explant. The number of shoots formed per explant was recorded for eight months and those with a minimum length of 1 cm were transferred to MS0 for rooting. Whole plantlets were transplanted to pots containing a mixture of the agricultural substrate Plantamaxr and sand (1:1) and maintained in an area shaded by Sombriter screens with 50% sunlight reduction. Each experiment was performed at least twice, using fifteen explants per treatment. The data were analysed using Student t test at a 0.05 level of significance among the means.
Results and discussion Shoot regeneration from axillary buds The development of axillary buds present in the stem explants was rather poor for all growth regulator combinations tested both in terms of percent regeneration and number of shoots per explant (data not shown). The mortality of explants was high, due to high oxidation rates. Preliminary results showed that liquid culture prevents explant oxidation and may therefore be a good alternative to induce shoot regeneration from stem explants of N. cruenta. Shoot regeneration from leaf explants Adventitious bud proliferation was evident at the base of leaf explants 3–4 weeks after culture initiation.
Shoots originated directly from protuberances formed at the cut end of the leaf blade (Figure 1B) and no intermediate callus phase was observed. Bud formation at the base of leaf explants has been described in other species of Bromeliaceae (Hosoki and Asahira, 1980; Vinterhalter and Vinterhalter, 1994) and the formation of protuberances on leaf bases prior to bud development was also found in V. fosteriana (Mercier and Kerbauy, 1992) and Dyckia macedoi (Mercier and Kerbauy, 1993). Shoot regeneration occurred in response to all growth regulator combinations tested regardless of tissue age. However, percent regeneration and mean number of shoots produced per explant were inversely correlated with the age of the donor plant. Maximum percent regeneration in leaves excised from 7-weekold seedlings was 64.28% in response to 22 µM BA plus 2.5 µM NAA, whereas 22-week-old tissues had rates in the range of 22 to 33%. The first shoots were observed after five weeks (Figure 1B) and intense multiplication was observed after four months of culture (Figure 1C). Maximum production of shoots per explant was also achieved from 7-week-old seedlings in response to 22 µM BA combined with 2.5 µM NAA (Table 1). Higher regeneration rates of young tissues has been reported to occur in other species (Baker and Bhatia, 1993). It is possible that new shoots originate from axillary meristems with high potential for bud formation, which are left at the base of the leaves upon excision from the donor plant (Hosoki and Asahira, 1980; Kukulczanka and Czastka, 1989). The number of shoots ≥ 1 cm long was significantly higher in the presence of 4.4 µM BA and 8.8 combined with 2.5 µM NAA, resulting in percent conversions of 67.18 and 79.03%, respectively (Table 1). By contrast, in the growth regulator combination which induced maximum shoot production (22 µM BA and 2.5 µM NAA), only 25.84% of the shoots grew long enough to be transferred to root induction medium during the observation period. All shoots rooted after transfer to MS0, resulting in the production of whole plants (Figure 1D). In vitro regeneration was also obtained from leaves excised from in vitro-grown plantlets upon inoculation on MS supplemented with 22 µM BA and 2.5 µM NAA. Plantlets did not require any treatment to avoid desiccation before transfer to the greenhouse. Plants showed normal phenotypes during an observation period of one year in greenhouse conditions. During this period plants did not reach maturity and hence the normality of flowers and fruits was not observed.
82 Table 1. Effect of explant age and BA/NAA combinations on regeneration frequency, number of shoots and whole plants per regenerating leaf from N. cruenta, after eight months of culture. Growth regulator concentration (µM) BA NAA
Responsive explants (%)
Mean number of shoots/explant
Mean number of plantlets/explant
Shoot-to-plant conversion (%)
4.4 4.4 4.4
0 1 2.5
15/30 (50.00) 16/29 (55.17) 17/30 (56.66)
Explant age 7 weeks 17.5 ± 4.0d 6.2 ± 2.4a e 20.1 ± 4.2 10.2 ± 3.3c g 32.3 ± 4.0 21.7 ± 3.8d
35.42 50.74 67.18
8.8 8.8 8.8
0 1 2.5
13/26 (50.00) 17/29 (58.62) 13/27 (48.14)
15.0 ± 4.1d 25.8 ± 4.5f 24.8 ± 3.0f
4.9 ± 2.1a 9.7 ± 1.8c 19.6 ± 2.9d
32.66 37.59 79.03
0 1 2.5
13/26 (50.00) 15/27 (55.55) 18/28 (64.28)
18.7 ± 2.4e 18.5 ± 1.9e 50.3 ± 6.2h
10.3 ± 2.0c 11.1 ± 1.4 c 13.0 ± 2.4d
55.08 60.00 25.84
4.4 4.4 4.4
0 1 2.5
10/29 (34.48) 12/30 (40.00) 11/28 (39.28)
Explant age 14 weeks 9.2 ± 1.2b 5.5 ± 1.2a d 15.0 ± 1.4 9.0 ± 1.3c d 11.0 ± 1.6c 16.2 ± 1.5
59.78 60.00 67.90
8.8 8.8 8.8
0 1 2.5
11/30 (36.66) 13/29 (44.82) 12/30 (40.00)
9.3 ± 1.7b 14.0 ± 1.6d 16.0 ± 1.8d
5.0 ± 1.4a 12.0 ± 2.0 c 11.0 ± 1.8c
53.76 85.71 68.75
0 1 2.5
8/28 (28.57) 14/30 (46.66) 11/28 (39.28)
12.5 ± 1.6c 11.8 ± 3.2c 15.6 ± 1.5d
7.0 ± 1.3a 5.9 ± 1.7a 8.0 ± 1.6a
56.00 50.00 51.28
4.4 4.4 4.4
0 1 2.5
7/28 (25.00) 8/28 (28.57) 9/28 (32.14)
Explant age 22 weeks 4.2 ± 1,1a 3.0 ± 1.2b b 10.5 ± 1.9 6.0 ± 1.3a b 11.4 ± 1.1 6.0 ± 1.2a
71.42 57.14 52.63
8.8 8.8 8.8
0 1 2.5
6/27 (22.22) 10/30 (33.33) 8/29 (27.5)
7.0 ± 1.4a 10.6 ± 1.7b 12.2 ± 1.4c
2.0 ± 1.4b 7.0 ± 1.4a 7.0 ± 1.8a
28.57 66.03 57.37
0 1 2.5
6/27 (22.22) 9/28 (32.14) 8/28 (28.57)
8.0 ± 1.4b 9.6 ± 1.8b 11.0 ± 1.5b
4.0 ± 1.4b 3.0 ± 1.4b 4.0 ± 1.6b
50.00 31.25 36.36
22 22 22
22 22 22
22 22 22
a-g Means within each column followed by the same superscript are not significantly different by Student t test at 0.05% probability level.
The figures in Table 1 refer to an 8-month observation period. However, intense shoot multiplication continued to occur for at least five months following the transfer of the tissue mass which remained after the removal of shoots for rooting (data not shown).
Outgrowth of lateral shoots did not occur when rooted shoots were left on MS devoid of growth regulators. Outgrowth of lateral shoots has been correlated to ethylene production in culture medium supplemented with IAA and BA in Aechmea. This effect
83 was attributed to the synergistic effect of cytokinins and auxins on the synthesis of ACC-synthase (Dijck et al., 1988). This is in contrast to other bromeliad species, such as Cryptanthus sinuosus, which do not require the presence of exogenous growth regulators for lateral bud emission (Carneiro et al., 1998). As far as in vitro conservation of endangered species is concerned, there are several advantages of regeneration from leaves derived from young seedlings when compared to methods involving the culture of apical meristem or lateral buds. First, since seedlings are the tissue source, this method allows for the representation of genetic variability without sacrificing entire plants. Second, surface sterilisation of explants is not required since they are obtained from seedlings grown under aseptic conditions. Third, the use of young tissues can reduce the frequency of somaclonal variation (Mercier and Kerbauy, 1993). The experiments carried out here resulted in highly efficient plant production, providing a means of propagation of more than 3000 plants a year from a single seed. Hence, this method is satisfactory both for large scale propagation and in vitro conservation programs. Although not recommended for the propagation of selected clones, a methodology for propagation of N. cruenta based on seedlings as starting material may be equally suitable to supply the market, thus reducing the need for extractivism.
Acknowledgements We thank Mrs Telma Telles for the technical assistance, Dr. Carlos Tadeu S. Dias for statistical analysis and Renata Garcia for help with manuscript preparation. Fellowships were received from CAPES (L.A.
Carneiro), CNPq (E. Mansur and M.H.P.B. Fonseca) and FAPERJ (G.J.M. Brito).
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