In vitro regeneration of Haplophyllum patavinum (L.) G. Don fil., a rare and endangered plant

R. Filippini1, R. Caniato1, E.M. Cappelletti1, A. Piovan2, G. Innocenti2 and G. Cassina3

1. Dipartimento di Biologia, Via Trieste, 75, 35121 Padova

2. Dipartimento di Scienze Farmaceutiche, Via Marzolo, 5, 35131 Padova

3. Orto Botanico, Via Orto Botanico, 15, 35123 Padova Italy


Haplophyllum patavinum, an endangered plant which faces extinction in its Italian range, was regenerated from nodal stem segments. Best shoot induction occurred on MS + 0.2mg/l K and 1mg/l 2,4-D within 5-6 weeks. Rooting occurred on MS + 0.5mg/l BAP and 1mg/l IAA. Plantlets were hardened and subsequently weaned to the glasshouse.

Abbrevations: 2,4-D, 2,4-dichloro-phenoxyacetic acid; K, kinetin; IAA, indol-3-acetic acid; BAP, 6-benzylamino-purine; MS, Murashige and Skoog medium (1962); B5, Gamborg et al. B5 medium (1968).


Recent data (Davis et al. 1986; Anon, 1991) show that the percentage of endangered species is increasing and that conservation measures are a matter of urgency (Fay, 1992).

Haplophyllum patavinum (L) G. Don fil. (Rutaceae) has a discontinuous distribution with a wide illyric range extending from Albania to Slovenia and a punctiform relict disjointed range on the Euganean Hills near Padua (Dolcher, 1957; Pignatti, 1982), where it forms endangered and highly unstable populations confined to open habitats on well-drained calcareous soils. These populations are facing slow extinction as the result of habitat modification by man's activities and the peculiar propagation features of the species. Natural propagation is mainly vegetative by shoots arising from the rhizome, leading to disappearance after deep ploughing (Cappelletti, 1957). The germination percentage of seeds is extremely low owing to embryo abortion at different developmental stages (Cappelletti, 1929; Guzzo et al., 1991). Slightly greater amounts of viable seeds are produced by specimens transplanted to different soil, which supports the hypothesis of a sterility of mycotic origin put forward by Cappelletti (1929). However, transplanted specimens do not survive for a long time.

H. patavinum could have pharmaceutical interest as a natural source of coumarin compounds, the wide occurrence of which in Rutaceae, including several Haplophyllum spp., is well documented (Murray, 1982).

In vitro techniques have been successfully used in recent years as a non-conventional method of plant propagation and conservation (Kumary and Sadadhi, 1991; Rathore, 1991; Lin and Griffin, 1992). This paper deals with the factors affecting in vitro regeneration of H. patavinum from stem segments of in vitro raised seedlings.

Material and Methods

Seeds were collected from plants grown in the Botanical Garden of the University of Padova and scarified by immersion in concentrated H2SO4 for 30 min. Several rinses removed the acid. After a surface sterilization in a 7% (w/v) Ca(OCl)2 solution for 20 min, seeds were rinsed three times with sterile distilled water and sown on B5 + 3% sucrose, pH 5.7, and solidified with 0.3% (w/v) Gelrite. They were kept at 25° C in a 12h photoperiod for germination. After two weeks, every seed was wetted with 20 µl K (1mg/ml). In vitro raised seedlings, 10-12 weeks old, were used as a source of explants (leaf, nodal shoot and root segment).

Explants of 0.5-1.5 cm were cultured on MS and B5 + 3% sucrose and 0.8% agar. pH of the medium was adjusted to 5.7 before autoclaving. Media were supplemented with 0.2mg/l K and 0.1 or 1mg/l 2,4-D (Tisserat, 1985). Cultures were cultured in a 12h photoperiod at 25°C. Differentiated shoot segments were sub-cultured to fresh medium after 6 weeks for further multiplication and for rooting of individual shoots. MS + 0.5mg/l BAP and 1mg/l IAA was evaluated for root induction. Cultures for root induction were kept in the dark for 12h and then under a 12h photoperiod. After two weeks, they were transferred to ½MS. Hardening was achieved by transferring intact plantlets to distilled water for 10 min and spraying with 0.5% (w/v) Benlate fungicide followed by transfer to pots containing sterile sandy soil. They were enclosed with two interlocking clear plastic polystyrene tumblers and kept in a culture room at 25°C, 12h photoperiod. They were periodically irrigated with liquid B5 without sucrose and sprayed with Benlate. Hardened plants were finally transferred to glasshouses at the Botanical Garden, Padova.

Results and Discussion

In spite of very low seed germination percentages, seeds are preferred to vegetative material as the source of propagation material because a wider genetic base can thus be maintained. In addition, germination of seeds in some species can be greatly increased by the use of in vitro methods (Fay, 1992).

Our results show that in vitro conditions and the addition of K to the seeds greatly increased percentage germination. These results prove the presence of normally developed embryos in at least some seeds from plants cultivated in edaphic conditions different from those occurring in the Euganean habitat (Cappelletti, 1957).

Nodal shoot segments were found to be the best explants for multiple shoot induction. Leaf and root segments produced only callus but no shoots. Abundant shoots were induced from the nodal segments on MS + 0.2mg/l K and 1mg/l 2,4-D. On B5 and/or the other growth regulator concentrations, shoot formation was observed but the incidence was much lower (Table 1). About 70% of isolated shoots rooted on MS + 0.5mg/l BAP and 1mg/l IAA. The percentage root induction was low on other media. Hardening of plants before transplantation was found to be essential.

These results prove that H. patavinum can be multiplied in culture using this protocol which involves regeneration from existing meristems. This pathway of regeneration is recognized as the most genetically conservative propagation method, and it has been used extensively with rare and endangered plants (Krogstrup et al., 1990; Clemente et al., 1991; Rathore et al., 1991). Research on H. patavinum multiplication through plant regeneration from callus is also in progress, the possibility of widening the genetic base of a species by inducing somaclonal variation having been suggested as a method of generating new vigor into populations (Bramwell, 1990; Jacobsen and Dohmen, 1990).


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