The seeds were rinsed four times in sterile distilled water, and 5 seeds were placed in ... BAP, 10 % (v/v) coconut water (prepared according to Dixon 1985) and.
Plant Cell Reports
Plant Cell Reports (1993) 13:103-106
9 Springer-Verlag1993
Plant regeneration from protoplast cultures of Passiflora edulis var. flavicarpa Deg., P. amethystina Mikan. and P. cincinnata Mast. Mareelo Carnier Dornelas and Maria Lficia Carneiro Vieira Departamento de Gen6tica, Escola Superior de Agricultura "Luiz de Queiroz", Universidade de Silo Paulo. Caixa Postal 83, 13418-900 Piracicaba~ SP, Brasil Received 12 December 1992/Revised version received 16 August 1993 - Communicated by J.J. Finer
Summary. Protoplasts isolated from seedling cotyledons of yellow passionfruit (Passiflora edulis var. flavicarpa Deg.) and two related wild species, P. amethystina Mikan. and P. cincinnata Mast., divided in culture and produced calli. Shoot regeneration was obtained in MS medium (Murashige and Skoog 1962) containing 2.0 mg/l 6-benzylaminopurine (BAP). Regenerated plants produced roots in half-strength hormone-free MS medium and could be transferred to soil after being acclimatized. Key words: Passionfruit - Passiflora - Protoplast culture - Passiflora wild species Abbreviations: B A P - 6-benzylaminopurine, MES 2-(N-Morpholino) ethanesulfonic acid, MS - Murashige and Skoog (1962) medium, NAA - 1-naphthaleneacetic acid.
Introduction The Passifloraceae family consists of 12 genera. The genus Passiflora is the widest spread (Sacco 1980), including 400 species (Cronquist 1981) of which more than 350 are found in tropical regions of South America (Killip 1938). While a large number of species have edible fruits, many Passiflora species are also of medicinal and ornamental value (Martin and Nakasone 1970). Passiflora edulis var. flavicarpa Deg., the yellow passionfruit, is an economically important species in America, Australia and Africa. Fruits are consumed fresh or processed for juice industries (Suzuki 1987).
Corresvondence to: M.L. Carnier Dornelas
P. amethystina Mikan. is a wild passionfruit species. It has purple-blue flowers which are very aromatic and is found in the rain forest of the Brazilian south-eastern coast (Fouqu6 1972). P. cincinnata Mast. (Figure 1.a.) is found from east of Brazil to Paraguay and Argentina (Fouqu6, 1972), and it is tolerant to Xanthomonas campestris f. passiflorae, a limiting disease in passionfruit orchards (Yamashiro 1991). Interspecific sexual hybridisation using the germplasm of wild species has being attempted in Passiflora breeding in order to promote disease resistance gene introgression and to transfer desirable traits into cultivated species (Torres and Martin 1974; Payanand Martin 1975). Interspecific hybrids are difficult to produce and have fertility problems (Payan and Martin 1975; Oliveira and Ferreira 1991). Tissue culture techniques, such as somatic hybridisation, offer additional methods for achieving gene flow between Passiflora species. A prerequisite for the production of somatic hybrid plants is the development of protoplast-to-plant technology. To date, plant regeneration from Passiflora protoplasts has been reported by Manders et al. (1991) and D'Ultra Vaz et al. (1993), where shoot regeneration was obtained from mesophyll cells of Passiflora edulis var. flavicarpa Deg. The present paper describes plant regeneration from protoplasts isolated from seedling cotyledons of Passiflora edulis var. flavicarpa Deg. and two related wild species, P. amethystina Mikan. and P. cincinnata Mast., using a protocol that may be also applicable to other species of Passiflora.
104 Materials and methods
(Manders et al
1991), plus 3.0% (w/v) sucrose and solidified with
1.5% (w/v) Phytagel (Sigma). After 20
Seed germination. Seeds of P. edulis var. flavicarpa and P. cincinnata were kindly provided by J C de Oliveira (FCAV/UNESP, Jaboticabal, Brazil). P. amethystina seeds were collected in Munhoz, Minas Gerais State, Brazil. Seeds were surface sterilised by immersion for 40 see in 70% (v/v) ethanol and for 20 min in 2% (v/v) NaOCI solution. The seeds were rinsed four times in sterile distilled water, and 5 seeds were placed in each 300 mi glass jars, containing 30 ml half-strength MS medium, 1.5% (w/v) sucrose and 0.8% agar (common purified agar, Sealgas, S~o Paulo, Brazil). The jars were sealed with parafilm (Rolopae, Silo Paulo, Brazil)and cultures were maintained at 2 5 _ 2°C in the dark until seed germination. Seedlings were then exposed to 22 #Em-2s 1 o f light intensity, provided by cool white fluorescent tubes, under 16 h light regime.
Protoplast isolation. Fifteen to 20 d after seed sowing, approximately 500 mg (ca.7 pairs) o f seedling cotyledons were excised and cut transversely into l m m thick slices. The tissues were plasmolysed for 20 min in a CPW solution (Frearson et al. 1973) containing 5mM MES and 9.0 or 13.0% (w/v) marmitol (CPW9M and CPW13M, respectively), at pH 5.8. CPW was then removed and the tissues incubated for 16 h in 5.0 ml of enzyme solution, in the dark. The enzyme mixture consisted of 2.0% (w/v) Cellulase R10, 0.2 or 0.4% (w/v) Macerozyme R10 (Kinki Yakult Manufacturing Co. Ltd., Nishinomiya, Japan) and 5.0 mM MES dissolved in CPW solution. Protoplast size was determined by using a micrometrie occular under optical microscopy.
Protoplast culture. Protoplasts were cultured in 5.0 cm Petri dishes, at densities of 5.0 x ItY , 1.0 x 105 and 2.0 x 105 protopasts/ml, in 4.0 ml of liquid KM8P medium (Kao 1977, modified by Gilmour et al. 1989), or were embedded in 0.6% (w/v) agarose (low gelling temperature, Sigma) as described by Power and Chapman (1985). Agarose beds were produced by mixing equal volumes of agarose with liquid KM8P medium containing twice the final protoplast densities. Four ml of this mixture were poured into 5.0 cm Petri dishes. After setting, the agarose was cut into four pieces, two of those pieces were transferred to another Petri dish. Droplets of 100 p.l of the protoplast-agarose mixture were also placed on Petri dishes. 3.0 ml of liquid KM8P was used to bathe the embedded protoplasts. The osmoticum o f the culture medium was progressively lowered, by replacing 1.0 ml of the medium (or bathing medium) with an equal volume of the KM8P:KM8 mixture every 7 d. The KM8 component was also modified according to Oilmour et al. (1989) from the formulation o f K a o (1977). KM8P and KM8 media were mixed in the ratios 2:1, 1:i and 1:2 (v:v), before use. Protoplast viability was determined according to Power and Chapman (1985)after 24h o f culture. The frequency of division (number o f dividing cells/living cells x 100) and the plating efficiency (number o f colonies obtained/protoplasts plated x 100) were also evaluated after 7 and 14 d of culture, respectively. All cultures were maintained at 25___'2°C in the dark until microcalli were formed. Experiments were repeated at least four times. Culture o f protoplast-derived microcalli and plant regeneration. After 28d of culture, yellowish colonies (ca. 2ram diameter) were transferred to MS medium supplemented with 5.0 mg/l NAA, 0.25 mg/l BAP
transferred to 22 #Em-2s -1 of
light
d,
the microcalli were
intensity
and placed on
Phytagel-solidified MS basal medium, supplemented with 2.0 mg/1 BAP, 10 % (v/v) coconut water (prepared according to Dixon 1985) and 3.0 % (w/v) sucrose to promote shoot regeneration. Shoots 2-3 em height were excised from the caUi and transfered to half-strength MS medium (1.5 % w/v of sucrose) to induce root formation.
Cytology. Regenerants of Passiflora edulis var. flavicarpa chromosome counts were made in the Feulgen-stained methaphase plates prepared from root meristems pretreated for 2:45 h in a solution of 0.03% (w/v) 8-hydroxyquinolineat 25 ___ 2 °C.
Results and Discussion When CPW9 was used to prepare the enzimatic solutions, the protoplasts lysed and produced much debris. The enzymatic treatment containing macerozyme at 0.2% gave poor yield of protoplasts that still showed residual cell wall. Better results were obtained using cellulaseat2.0% plus macerozyme at 0.4% dissolved in CPW13 solution. Protoplasts ofP. amethystina (Figure 1.b.) (54.9 ___9.4 /zm) and P. cincinnata (53.9 ___ 10.0 ~m) were larger than P.edulis var. flavicarpa (32.8 + 6.5 #m) protoplasts. After 24h of culture, the chloroplasts migrated to one pole of the protoplast-derived cells. Divisions were first observed in protoplast-derived cells cultured in droplets or layers (Figure 1.c.) after 3 days of culture. Protoplasts, that were embedded in droplets or quarters of agarose showed high division frequencies (Table I.) and plating efficiencies (Table II). The positive effect of embedded cultures of protoplasts has also been reported by Lorz et al. (1983), Shillito et al. (1983), Colijn-Hooymans et al. (1988) and Vieira et al. (1990). Binding et al. (1988) have reported that the droplet system may allow better nutrient exchange between solid and liquid phases. In liquid cultures the protoplasts aggregated and a brownish precipitate was observed. Under these conditions, the division frequency was very low. An effect of protoplast density on cell division has been reported for many plant species (Bhojwani et al. 1984, Pupilli et al. 1989, Vieira et al. 1990) and was also observed in the present study (Table I.). The density of lxl05 protoplasts/ml of medium was the most suitable for all the species studied. Protoplast-derived colonies grew fast in embedded
105
Figure 1. a. A p p e a ~ n e e of a Passiflora cincinnata plant in the field (Bar= 5 cm). b. Freshly isolated protoplasts of P. amethystina (Bar= 100 #m). e. First cellular division in a P. amethystina protoplast culture (Bar= 20/~m). d.-f. microcolonies (Bar = 100 #m) shoot regeneration (Scale= 1 em) and a potted plant (Bar= 5cm) derived from cultures of P.
Table I. Percentage of division (mean
cincinnata protoplasts.
__. SE) in protoplast-derived
Table II. Comparison o f yield, viability, plating efficiency and
cultures of Passiflora edulis var. flavicarpa, P. amethystlna and P.
regeneration ability of cotyledon protoplasts derived from Passiflora
cincinnata at different densities after 7d of culture. P. edulis P. amethystina Plating densities vat. flavicarpa 5.2_+ 1.2(LC) 3.5 _ 1.1 (LC) 5.0 x 10~ 27.9 + 3.2 (DC) 22.6 + 7.9 (DC) 21.3 + 5.5 (QC) 18.8 + 9.6 (QC)
edulis var. flavicarpa, P. amethystina and P. cincinnata (mean _ SE) P. edulis P. amethystina P. cincinnata var. flavicarpa Yield a 6.9x106+7.5x103 5x106_2.5x105 3.5xl0S_5.0xllY
1.0 x 1@
3.0_0.9(LC) 1.5_+0.8(LC) 40.1 _+ 8.5 (DC) 27.2 _+ 6.3 (DC) 35.0 _ 3.2 (QC) 30.7 _ 6.8 (QC)
~cmcmnam 3.6 _ 0.9 (LC) 14.1 _ 6.3 (DC)
10.3 + 5.7 (QC) 3.3 _ 1.2 (LC) 15.7 _+ 5.1 (DC) 12.8 _+ 3.4 (QC)
0.9 _ 0.1 (LC) 0.7 _+ 0.3 (LC) 1.1 __ 0.6 (LC) 36.2 _+ 7.3 (DC) 26.6 _ 9.2 (DC) 13.1 _+ 7.7 (DC) 27.6 + 8.2 (QC) 28.1 + 8.5 (QC) 11.9 _+ 5.5 (QC) LC: Liquid culture; DC: Droplets o f agarose; QC: Quarters of agarose 2.0 x 1@
protoplast Viability (%)
83.4 q- 4.4
93.8 + 0.9
Plating 2.7 + 0.9 (LC) 0.9 + 0.3 (LC) Efficiency (%)b 25.5 ___ 21.1(DC) 16.3 + 1.7(DC) 24.1 + 2.8 (DC) 19.1 _ 2.3(DC)
94.8 + 0.8
0.6 +_0.4(LC) 18.2 ___ 2.4(DC) 17.6 +_ 2.5(DC)
Regeneration Frequency (%)~ 48 _ 3 75 _ 6 93 _+4 a Number of protoplasts released per 500 mg of cotyledon tissue b Number of colonies obtained/total number of protoplasts plated x 100 c Number o f calli with shoot regeneration/total number of calli plated in MS medium with 2.0 mg/l BAP and 10% (v/v) coconut water LC: Liquid culture; DC: Droplets of agarose; QC: Quarters of agarose
106 cultures ( F i g u r e 1.d.) and could be transferred to solid m e d i u m after 28 d o f culture. produced became
friable
that medium, they
white-yellowish calli.
green when
These
calli
transferred to the regeneration
medium, under 22 /~Em'2s1 regeneration
On
occurred
o f light intensity. Shoot
through organogenesis with the
development o f adventitious buds on the surface o f the calli.
P. cincinnata showed the highest frequency o f shoot regeneration (Table II., F i g u r e I.e.). Despite the use of
coconut
water, recommended to improve cell
proliferation and shoot cultured
tissues
from Passiflora
production
(Mourad-Agha
and
1979, Kantharajah and D o d d 1989), protoplast-derived calli o f
P.
Dexheimer
only 48 % o f the
edulis vat. flavicarpa edulis vat.
regenerated shoots. H o w e v e r , callus o f P.
flavicarpa (on
average
12.5 _ 2.3
shoots/callus)
p r o d u c e d a large n u m b e r o f regenerated plants. Plants elongated and p r o d u c e d roots when transferred to h o r m o n e - f r e e
half-strength MS medium and could
then b e transferred to soil (Figure 1.f.). W e have examined 300 metaphases (30 plates per 10 rooted shoots) from which 40% ( 4 n = 3 6 ) . A l t h o u g h the reasons variations
are
still
showed polyploidy
for these chromosome
unknown,
they
might
have
originated due to the presence o f a callus phase and m e d i a components (Bayliss 1980, Lee and Phillips 1988). P o l y p l o i d y is particularly prevalent in in vitro cultures (D'Amato
1985)
and attempts must be made to obtain
or select Passiflora adventitious shoots cytogenetically normal and stable. Our results demonstrated that it is
possible
to
regenerate plants from protoplast cultures o f two wild and one cultivated species o f Passiflora. This protocol should be useful in future
research
for the application o f somatic techniques to Passiflora species. Refinements are still required to reduce the callus phase,
p o s s i b l y lowering
the
level
of
regenerated
polyploids. Somatic hybrids and genetically modified
Passiflora
plants
could
References
be valuable in yellow
passionfruit breeding programmes.
Acknowledgements. The authors acknowledgethe following Brazilian Institutions: FCAV/UNESP Jaboticabal, for providing the Passiflora seeds and FAPESP, for financial support. We also acknowledge Dr C.A.I.abate for reading the manuscript and Mr C. A. de Oliveira for technical assistance.
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