Scientia Horticulturae 99 (2004) 395–400

Short communication
Rapid micropropagation of passion fruit
(Passiflora edulis Sims.) varieties
D.K. Isutsa∗
Department of Horticulture, Egerton University, P.O. Box 536, Njoro, Kenya
Accepted 4 August 2003

Abstract

Lack of adequate, healthy plants can hamper production of passion fruits (Passiflora edulis Sims.).
Seed propagation results in undesirable variability, inadequate and seasonal supply. This research
aimed at rapid generation of P. edulis plants through modified ex vitro rooting techniques. Yellow
(P. edulis var. flavicarpa) and purple (P. edulis var. edulis) passion fruit shoot tops were proliferated
in vitro to stage II shoots, half of which were rooted ex vitro and the other half were conventionally
rooted in vitro. The design was completely randomized for each variety. Plantlets were assessed after
30 days. Data were analyzed using the MSTAT programme. After proliferating yellow passion fruit
on a medium containing 22.2 ␮M 6-benzylaminopurine (BAP), its rooting ex vitro was significantly
better (96% rooting, three roots per shoot, 92% survival) than rooting in vitro (62% rooting, one root
per shoot on 24.5 ␮M indole-3-butyric acid medium, 50% survival). Purple passion fruit proliferated
satisfactorily only on a medium containing both 22.2 ␮M BAP and 11.6 ␮M gibberellic acid 3. Like
difficult proliferation, and compared to yellow passion fruit, its rooting and survival also proved
difficult and poor (47% rooting, one root per shoot on 21.5 ␮M naphthalene acetic acid medium in
vitro, 32% survival) and (66% rooting, two roots per shoot ex vitro, 60% survival). Thus, the various
passion fruit varieties have different requirements for micropropagation. The key finding was that ex
vitro rooting is possible and significantly better than in vitro rooting of passion fruit shoots.
© 2003 Elsevier B.V. All rights reserved.

Keywords: Tissue culture; Passion fruit; Micropropagation; Passiflora; Rooting

1. Introduction

Passion fruit is an important fruit crop in many tropical and sub tropical countries. It
grows rapidly to replace low value cash crops, as well as those ravaged by diseases. Among

∗ Tel.: +254-51-42251; fax: +254-51-62527.

E-mail address: dki1@africaonline.co.ke (D.K. Isutsa).

0304-4238/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.scienta.2003.08.002
396 D.K. Isutsa / Scientia Horticulturae 99 (2004) 395–400

the cultivated varieties, purple passion fruit is the most popular in the juice industry. Its
fruits can also be exported or consumed locally. Yellow passion fruit is used as a rootstock
to control soilborne diseases such as fusarium wilt (F. oxysporum f. passiflorae) (Amugune
et al., 1993; Nakasone and Paull, 1998).
Most commercial passion fruit producers worldwide use seedlings to establish plan-
tations, because they do not spread the woodiness virus (Nakasone and Paull, 1998).
Propagation using cuttings and grafting is occasionally practiced, but these methods risk
spreading the woodiness virus (Nakasone and Paull, 1998). Micropropagation confers many
advantages such as being rapid and producing uniform, disease-free plants (Dornelas and
Vieira, 1994; Kawata et al., 1995; Faria and Segura, 1997). Poor quality seeds and lack
of disease-free plants can prevent adequate production and supply of passion fruits. Thus,
increasing the availability of healthy planting materials to growers could sustain production
and benefits of passion fruits. The major objective of this research was to develop a rapid
micropropagation protocol for generating healthy, uniform, and inexpensive passion fruit
planting materials.

2. Materials and methods

2.1. Seed germination, shoot proliferation, and in vitro rooting

Seeds of yellow and purple passion fruits were obtained from the National Horticultural
Research Centre at Thika, Kenya. For effective dormancy breaking and germination, dry
seeds were soaked in a solution containing 0.14 ␮M gibberellic acid 3 (GA3 ) for 7 days,
and then germinated in sterilized sand in a growth room maintained at 25 ± 2 ◦ C and 16 h
of 90 ␮mol m−2 s−1 .
Seedlings were excised after 1–2 months, surface-sterilized in 5% sodium hypochlorite
(3:1 v/v) for 10 min, and rinsed four times, using 2 l of sterilized distilled water. Shoot
tips were established on (Murashige and Skoog, 1962) basal salts medium supplemented
with 30 g/l sucrose, 8 g/l agar, and 22.2 ␮M 6-benzylaminopurine (BAP) alone for yellow
passion fruit (Kantharajah and Dodd, 1990), but in combination with 11.6 ␮M GA3 for
purple passion fruit. The pH was adjusted to 5.7, before 25 ml of the medium was poured
into each 100 ml glass jar, sealed with a cap, and autoclaved for 18 min at 121 ◦ C and
100 kPa. Shoot tip and internodal segments, measuring 1 cm long, were sub-cultured on
media similar to those used for initiation. Both explants and subcultures were maintained
at 25 ± 2 ◦ C and 16 h of 90 ␮mol m−2 s−1 (Faria and Segura, 1997). The resulting stage II
shoots were used in rooting experiments.
Preliminary tests were conducted to determine the medium that could effectively induce
in vitro rooting. Thus, 2 cm long shoots were excised and cultured on (Murashige and Skoog,
1962) basal salts media supplemented with 8 g/l agar, 20 or 30 g/l sucrose in combination
with 0, 9.8 or 24.5 ␮M indole-3-butyric acid. In further preliminary tests, media were
supplemented with 8 g/l agar, 30 g/l sucrose and 10.8 or 21.5 ␮M naphthalene acetic acid.
The pH was adjusted to 5.7 before 25 ml of the medium was poured into each 100 ml glass
jar and autoclaved for 18 min at 121 ◦ C and 100 kPa. Five shoots were rooted in each jar.
Cultures were maintained at 25 ± 2 ◦ C and 16 h of 90 ␮mol m−2 s−1 .
 D.K. Isutsa / Scientia Horticulturae 99 (2004) 395–400 397

2.2. Ex vitro versus in vitro rooting

Stage II shoots were divided into two groups for in vitro and ex vitro rooting. Both in vitro
and ex vitro rooting tests were conducted concurrently in time. The experimental design
was completely randomized for each variety. The design tested the effect of rooting method
on rooting and survival of plantlets. Twenty shoots, replicated five times, were rooted per
treatment. The experiment was repeated once with similar results.
In vitro rooting was performed on (Murashige and Skoog, 1962) basal salts media sup-
plemented with 8 g/l agar plus 24.5 ␮M IBA for yellow passion fruit and 21.5 ␮M NAA
for purple passion fruit. Environmental conditions were maintained at 25 ± 2 ◦ C, 16 h of
90 ␮mol m−2 s−1 , and 100% relative humidity in the jars. Data of rooted shoots and number
of roots per shoot were recorded after 30 days.
Ex vitro rooting was performed in sterilized sand:soil (2:1 v/v) mixture in the same
greenhouse as that used for acclimatizing plants. The sand:soil mixture was filled in prop-
agation boxes, in which shoots were inserted and covered with a clear, light polyethylene
sheet. Conditions were maintained at 25 ± 2 ◦ C, 16 h of 90 ␮mol m−2 s−1 and 98% relative
humidity in the boxes. After 30 days, plantlets were lifted, rinsed to remove the sand:soil
mixture, and counted to establish rooted shoots and number of roots.
After recording rooting data, both in vitro- and ex vitro-rooted plantlets were transferred
to sterilized sand:soil (2:1 v/v) mixture filled in perforated potting bags and acclimatized in
a shaded greenhouse maintained at 25 ± 2 ◦ C/18 ± 2 ◦ C and natural day length. To prevent
wilting, each plantlet was covered with a clear, light polyethylene bag for 1 week (Dornelas
and Vieira, 1994), and light was gradually increased. Plantlet survival was assessed after
30 days ex vitro.

3. Results

3.1. Shoot proliferation

During explant initiation, visual observations revealed that multiple shoot proliferation
and elongation on a medium supplemented with 8.8 ␮M BAP was either slow or inad-
equate, although Drew (1991) and Dornelas and Vieira (1994) recommended this level.
Subsequently, the level of BAP was increased to 22.2 ␮M, which successfully stimulated
multiple shoot proliferation and elongation in the yellow passion fruit explants, but only
proliferated numerous short shoots in the purple passion fruit explants. Thereafter, 22.2 ␮M
BAP and 11.6 ␮M GA3 were added to the medium used to initiate and proliferate the purple
passion fruit explants. After 4 weeks, previously short shoots of the purple passion fruit
variety elongated to generate long shoots that could easily be sub-cultured and rooted.

3.2. In vitro rooting

Yellow passion fruit shoots rooted on all media augmented with either 20 or 30 g/l sucrose
in combination with 0, 9.8 or 24.5 ␮M IBA, although 24.5 ␮M IBA resulted in significantly
higher rooting percentage (100%) than the other IBA levels (Table 1). The effects of sucrose
and its interaction with IBA were not significant. Visual comparison revealed that plantlets
398 D.K. Isutsa / Scientia Horticulturae 99 (2004) 395–400

Table 1
Effects of sucrose and IBA on percent rooting and number of roots of yellow passion fruit shoots rooted in vitroa
Sucrose (g/l) Percent rooting Number of roots per shoot
0 ␮M IBA 9.8 ␮M IBA 24.5 ␮M IBA 0 ␮M IBA 9.8 ␮M IBA 24.5 ␮M IBA
20 40 20 100 0 0 3
30 60 60 100 1 4 2
a
Values not followed by a letter within each variable are not significantly different, according to the F-test at
P = 0.05.

Table 2
Average percent rooting, roots per shoot and percent survival during ex vitro rooting without auxins for both
yellow and purple passion fruits, and during in vitro rooting with 24.5 ␮M IBA for yellow and 21.5 ␮M NAA for
purple passion fruitsa
Rooting Yellow passion fruit Purple passion fruit
method
Percent Roots per Percent Percent Roots per Percent
rooting shoot survival rooting shoot survival
Ex vitro 96 a 3a 92 a 66 a 2a 59 a
In vitro 62 b 1b 50 b 47 b 1a 32 b
a
Values followed by the same letter within each column are not significantly different, according to the F-test
at P = 0.05.

rooted on media supplemented with 20 g/l sucrose were thinner than those rooted using
30 g/l sucrose, which was then used throughout the in vitro rooting experiments. Purple
passion fruit shoots did not initiate roots on all IBA-augmented media. They initiated roots
only on 21.5 ␮M NAA-augmented medium, which was then used to root them in vitro.

3.3. In vitro versus ex vitro rooting

Results of the first trial and the second trial were statistically the same and hence averaged.
During rooting of yellow passion fruit, 96% of the shoots rooted ex vitro, whereas 62%
rooted in vitro (Table 2). The difference in percent rooting ex vitro and in vitro was significant
at P = 0.05. The number of roots per ex vitro-rooted shoot of yellow passion fruit was
significantly higher (3) than the one root per in vitro-rooted shoot (Table 2). Similarly,
plantlets surviving acclimatization after ex vitro rooting were significantly higher (92%)
than those surviving after in vitro rooting (50%).
The results for purple passion fruit revealed 66% rooting ex vitro, which was significantly
higher than the 47% rooting in vitro (Table 2). There was no significant difference in the
number of roots per shoot rooted ex vitro and in vitro (Table 2). The percentage of plantlets
surviving acclimatization after ex vitro rooting was significantly higher (59%) than the 32%
for those surviving after in vitro rooting (Table 2).

4. Discussion

During micropropagation Amugune et al. (1993) also found purple passion variety re-
calcitrant in tissue culture. In the current research, GA3 stimulated elongation of purple
 D.K. Isutsa / Scientia Horticulturae 99 (2004) 395–400 399

passion fruit shoots. There is no published literature, reporting the use of GA3 in tissue
culture media for passion fruit varieties. The current study, therefore, found a solution to
the problem of short shoots, which are often difficult to handle.
The easy rooting of yellow passion fruit in vitro agreed with findings of Kawata et al.
(1995). In the current study, 30 g/l sucrose produced robust plantlets, thereby increasing their
chances of survival ex vitro. Several researchers have recommended media augmented with
20 g/l sucrose, or without hormones to root passion fruit shoots (Dornelas and Vieira, 1994;
Kawata et al., 1995; Faria and Segura, 1997). The current result implied that genotypes
tested by the other researchers were different from the ones tested in this study (Amugune
et al., 1993; Nakasone and Paull, 1998). The purple passion fruit variety, on the other hand,
proved to be difficult-to-root in vitro, agreeing with the findings of Amugune et al. (1993).
In the present research, ex vitro rooting was consistently better than in vitro rooting in all
the assessed growth variables. The high number of well-developed roots on ex vitro-rooted
plantlets most likely enhanced the survival of plantlets during acclimatization (Kramer,
1983; Clemente et al., 1991). In vitro-grown plants usually show rapid wilting when trans-
ferred ex vitro if care is not taken to maintain high humidity in their new environment.
Susceptibility to wilting has been attributed to the severe impairment of water maintenance
mechanisms (Fila et al., 1998) and poor regulation of leaf transpiration—due to thin cuticles,
lack of leaf epicuticular waxes (Al-Ahmad et al., 1998) and poor stomatal regulation.
Based on visual assessment, the quality of ex vitro-developed roots of passion fruit was
better, as evidenced by branching and lateral root development, than that of in vitro-developed
roots, which remained branchless. Root hairs play an important role in the rhizosphere, ef-
fectively increasing the root surface area. Similarly water uptake is made more effective by
the presence of root hairs (Kramer, 1983). The short length or absence of root hairs may
render tissue culture-developed roots less functional, exacerbating the stress experienced
during and after acclimatization.
Hartmann et al. (1997) have recommended brief exposure to auxins for root induction and
not for prolonged growth. This fact could explain why most in vitro roots did not branch
when left for 30 days on rooting media supplemented with auxins. Thus in the present
research, ex vitro rooting proved to be more effective than in vitro rooting, agreeing with
results for Artemisia granatensis (Clemente et al., 1991), blueberry (Vaccinium spp.) and
apple (Malus domestica Borkh.) (Isutsa et al., 1994, 1998).

5. Conclusion

In all previous reports for passion fruit micropropagation, shoots spent three stages,
including rooting, under sterile conditions in vitro (Moran-Robles, 1978; Kantharajah and
Dodd, 1990; Drew, 1991; Dornelas and Vieira, 1994; Amugune et al., 1993; Kawata et al.,
1995; Faria and Segura, 1997). Thus, our findings represent an improvement in previously
published protocols for micropropagating passion fruit varieties. Ex vitro rooting eliminates
chemicals, agar, vessels, and time needed for in vitro rooting, and uses the same facilities
with the acclimatization stage. It, therefore, reduces micropropagation costs and minimizes
chances of developing somaclonal variants by shortening the in vitro period (Moran-Robles,
1978; Drew, 1991; Kawata et al., 1995; Faria and Segura, 1997; Rodrigues et al., 1998). Use
400 D.K. Isutsa / Scientia Horticulturae 99 (2004) 395–400

of ex vitro rooting is, therefore, recommended for rapid micropropagation of elite breeding
selections and the yellow variety that serves as a rootstock, protecting the purple variety
against soilborne pathogens.

Acknowledgements

This research was supported by a grant from the Rockefeller Foundation, and useful
advice from M/s F.B. Mumera of the National Plant Breeding Research Centre, Njoro,
Kenya.

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