Note IN VITRO CULTURE FROM MATURE SEEDS OF PASSIFLORA SPECIES

The genus Passiflora comprises hundred species, mainly native of the South American tropics and rainforests, which are grouped into 21 subgenera. Some species are widely studied for their economic importance and are chiefly cultivated for production of fruit juice. To obtain a continuous source of material for a screening of secondary metabolites, zygotic embryo culture was attempted for 62 Passiflora species, starting from seeds mainly collected in the wild. Twenty nine of these species produced calli, which had very different growth rates. Plants were successfully regenerated from calli of 13 different species. For 25 of the responsive species this is the first report of in vitro culture.


INTRODUCTION
The genus Passiflora comprises several hundred species, mainly native of the South American tropics and rainforests, which are grouped into 21 subgenera.Some species (P.edulis, P. quadrangularis, P. ligularis) are widely studied for their economic importance and are chiefly cultivated for production of fruit juice.P. incarnata is reputed for its sedative properties and several other species are known for their ethnobotanical uses (see The Phytochemical and Ethnobotanical Databases, http://www.ars-grin.gov/duke/).However, the molecules responsible for these various activities are known only for a few species.Also, some Passiflora species have been described only recently (e.g.P. trialata, Feuillet & MacDougal, 1996).
For systematic study of the activities of secondary metabolites, a continuous source of material is necessary.For some Passiflora species, dehydrated seeds can be purchased, but for most, seeds have to be collected in the wild.Nonetheless, dehydrated seeds of many Passiflora species may require from many months up to two years to germinate (Vanderplank, Passiflora Society International Meeting, Rome, 15-16 September 2001).To alleviate these difficulties, an in vitro collection of Passiflora species, together with a greenhouse collection of regenerated plants was established.

MATERIAL AND METHODS
Passiflora seeds, collected in the wild or produced in greenhouse, were kindly furnished by Dr. Maurizio Vecchia (Ripalta Cremasca, Cremona, Italy).For each species, four to 20 seeds were available.Mature seeds were surface sterilized with 70% ethanol for 10 min, followed by immersion in a sodium hypochlorite solution, containing 5% active chlorine for 70 min.During the first 10 min, samples were kept under vacuum.Seeds were extensively washed with sterile water, soaked overnight at 35°C, and then for 24 h at room temperature.They were then treated with sodium hypochlorite for 10 min and extensively washed prior to dissection.Embryos were extracted from seeds under a dissection microscope in a flow cabinet, and placed on media A and B; for some species, C or BG medium was also used (for media composition see Table 1).When solid parts of endosperm were present, they were explanted as well.
The following treatments depended on the response of the embryo: a) when embryo produced calli, these were maintained on B5+ or B5-2 medium (Table 1) depending on the species, and subcultured every 30 days; part of these calli were transferred to regenerating medium (REM) to regenerate shoots; rooting medium (RM) was used to obtain roots from shoots; b) when germination from explanted embryos occurred, part of the plantlets where cut into pieces and transferred to B5-2 medium to obtain undifferentiated calli, which were treated as above.
Part of the regenerated or germinated plantlets were transferred into Magenta vessels and grown in vitro in hormone-free MS medium (Murashige & Skoog, 1962) until both shoots and roots were well developed.The small plants were acclimatized in autoclave-sterilized soil for about three weeks at 100% humidity, and then transplanted into pots in a greenhouse.

Embryo and endosperm culture: early events
Seeds of Passiflora genus vary greatly in size and shape.However, several common features are apparent, including hard seed coats surrounding a white, well-developed, straight embryo, with large flat cotyledons.A thin layer of endosperm, which can be ruminated, surrounds the embryo.
Endosperm and embryos extracted from seeds (Table 2) were grown in two different media, A and B (Table 1).The plant growth regulator and sucrose concentration of these media have been reported to induce undifferentiated callus formation (medium A) and to stimulate in vitro germination of zygotic embryos (medium B) in rice (Ko et al., 1983).Twenty six species responded to either A or B medium with embryo germination or callus formation (Table 2).
When a sufficient number of seeds were available, embryos of species that did not respond to A or B media were treated with medium containing gibberellic acid (BG medium, Table 2).Embryos of P. mayarum, P. morifolia and P. subpeltata germinated on this medium.P. foetida and P. palmeri were also tested in medium C, which induced embryo germination (P.foetida) and callus production from embryos (P. palmeri).
In most of the responsive species, the earlier modification observed in responding embryos was cotyledon greening, which occurred within one-three weeks of culture (data not shown), followed by cotyledon enlargement and opening (Figure 1a).This effect was independent of the composition of the growth medium.
Embryo germination occurred at a higher frequency on medium B (17 of 20 responsive species), while medium A ordinarily induced embryos to produce undifferentiated calli (17 of 18 responsive species), as expected (Ko et al., 1983).However, 7 of the 27 responsive species presented embryo germination in medium A, while 8 of the 27 responsive species presented calli on medium B. In particular, P. kawensis responded only to medium A with embryo germination.
giving rise to shoots without roots and vice-versa (data not shown).Shoots grew independently from the root even in the case of incomplete excision of the embryo from seed coats, when residual seed coat pieces embedded the root (Figure 1c).It is reasonable to surmise that the failure of root germination depended on some dormancy factors within the seed coats that were still in tight contact with the root.The shoots to root on medium containing IBA (indole-3-butyric acid) (RM, see Table 1) (Figure 1d); the independent roots were cut and used to obtain calli in high 2.4-D (2.4-dichlorophenoxyacetic acid) medium (B5-2, Table 1).
Fragments of endosperm from mature seeds were also explanted in A, B, and BG media, resulting in undifferentiated white or yellow callus production for 7 of the 27 species in A, 2 of the 27 species in B, and 1 of the 4 species in BG (Table 2).Several species did not respond to any of the tested media (Table 2).However, for some unresponsive species only a very few seeds were available.The responsiveness of species to our culture conditions appeared to be independent on the taxonomic position, at least for the Decaloba and Passiflora subgenera, in which 8 of 19 and 12 of 27 species, respectively, showed some kind of response.

Callus culture and plant regeneration
Undifferentiated calli were spontaneously produced especially from embryos grown on medium A. Alternatively, pieces of embryo-derived hypocotyl or root were cut and transferred to high 2.4-D (B5-2 medium, Table 1) to obtain calli.Different embryos from a single species and even individual embryos produced calli with different characteristics (Table 3).
These calli were separated and grown on B5 medium containing 2.25 µmol L -1 2.4-D and several homogeneous cell lines with different features were obtained (Table 3).Media with high 2.4-D (9 µmol L -1 ) were chosen for species with very high morphogenetic capabilities, such as P. foetida, in order to maintain the undifferentiated callus.
Preliminary characterization has shown that calli have different morphogenetic potential, even within the same species.The growth rate of calli varied greatly among different species.The increase in weight of very low and fast growing species are detailed in Table 4. Green and compact calli were chosen as the best candidates to induce shoot regeneration (Figure 1e).For this purpose, calli were treated with medium containing 6-BAP (6-benzylaminopurine) (REM, Table 1).Regeneration was obtained after a transition period varying from a few weeks to several months.During this period, green round masses that gradually developed an epidermis-like tissue appeared at the callus surface (Figures 1f, g).Successively, shoots emerged from these masses (Figure 1 h, i).Shoot regeneration was successful in 13 callus-forming species.Calli of P. foetida and P. tenuifila developed shoots spontaneously even without 6-BAP treatment.Shoot regeneration was accompanied or followed by root regeneration in either MS hormone-free or IBA containing medium (RM, Table 1).In only one case (P.palmeri) the shoots did barely differentiate into roots, even in rooting medium and over a long period of time (several months).
In callus-maintaining medium, P. mayarum had spontaneous formation of long and branched roots from white friable calli (Figure . 1j).These roots were subsequently able to develop shoots in hormone-free medium.for their regeneration capabilities.However, regeneration from endosperm-derived callus has been described in P. foetida (Mohamed et al., 1996).

Plantlet acclimatization
Plantlets obtained from embryo germination or by regeneration were acclimatized and transplanted into pots and grown in the greenhouse, with a near 100% success rate, with the exception of P. vespertilio.In the greenhouse, mature plants of P. foetida, P. tenuifila and P. coriacea appeared to be fertile, spontaneously producing seed-containing fruits.P. apetala and P. palmeri produced normal flowers, although no fruits have yet been observed.Since plants were not hand-pollinated, this could simply be attributed to the absence of the proper pollinator in self-incompatible species.The other species have not reached sexual maturity yet.
In conclusion, embryo culture allowed the production of calli from 29 Passiflora species, and plant regeneration from 13 of these.For 25 of the responsive species, tissue culture had not been previously reported.Embryo culture represents a possible strategy to obtain Passiflora plants not only to provide a continuous source of material, but also for the conservation of endangered species.

Figure 1 -
Figure 1 -In vitro culture of Passiflora embryos.Embryos in vitro germinated from a) P. trialata and b) P. incarnata, c) P. foetida: shoot germination in an embryo with partially coated root, d) P. cincinnata: isolated embryo shoot, with root induced on IBA containing medium, e) P. garkei: green, compact callus, f) P. trialata: green compact callus transferred to REM showing early regeneration response, g) P. garkei: green compact callus transferred to REM, showing the development of organized masses with epidermislike purple layers.Well-developed shoots from regenerating calli of h) P. palmeri and i) P. foetida, j) P. mayarum branched roots emerging from white friable callus in callus maintaining B5+ medium.Bar a-e, h-j = 500 µm; f, g = 100 µm.

Table 2 -
Type of response of Passiflora species to different inducing media EG: embryo germination; CFE: callus formation from endosperm; CFEM callus formation from embryo; -: no response; nt: not tested; * tissue culture not previously reported.a +: plant regeneration was obtained under the conditions described in the results from material induced in the medium indicated in parentheses.
Calli obtained from endosperm have not yet been tested

Table 3 -
Characteristics of calli obtained from Passiflora responsive species

Table 4 -
Weight (g) increase of selected Passiflora calli after 20 days of culture a ratio between final and initial weight.P. rufa and P. apetala are representative of low-growing species, while P. nitida, P. garkei and P. palmeri are representative of fast growing species.