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Rapid micropropagation of passion fruit (Passiflora edulis Sims.) varieties
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: [email protected] (D.K. Isutsa). 0304-4238/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2003.08.002
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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 plantations, 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
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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 supplemented 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 propagation 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 inadequate, 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
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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)
20 30
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
40 60
20 60
100 100
0 1
0 4
3 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 method
Ex vitro In vitro
Yellow passion fruit
Purple passion fruit
Percent rooting
Roots per shoot
Percent survival
Percent rooting
Roots per shoot
Percent survival
96 a 62 b
3a 1b
92 a 50 b
66 a 47 b
2a 1a
59 a 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 recalcitrant in tissue culture. In the current research, GA3 stimulated elongation of purple
D.K. Isutsa / Scientia Horticulturae 99 (2004) 395–400
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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 transferred 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, effectively 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
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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.
References Al-Ahmad, M., Hughes, H., Safadi, F., 1998. Studies on stomatal function, epicuticular wax and stem root transition region of polyethylene glycol-treated and non-treated in vitro grape plants. In Vitro Cell. Dev. Biol. 34, 1–7. Amugune, N.O., Gopalan, H.N., Bytebier, B., 1993. Leaf disc regeneration of passion fruit. Afr. Crop Sci. J. 1 (2), 99–104. Clemente, M., Pilar, C., Juana, S., Pliego-Alfaro, F., 1991. Micropropagation of Artemisia granatensis. HortScience 26 (4), 420. Dornelas, M.C., Vieira, M.L.C., 1994. Tissue culture studies on species of Passiflora. Plant Cell Tiss. Org. Cult. 36, 211–217. Drew, R.A., 1991. In vitro culture of adult and juvenile bud explants of Passiflora species. Plant Cell Tiss. Org. Cult. 26, 23–27. Faria, J.L.C., Segura, J., 1997. Micropropagation of yellow passion fruit by axillary bud proliferation. HortScience 32 (7), 1276–1277. Fila, G., Ghashghaie, J., Hoarau, J., Cornic, G., 1998. Photosynthesis, leaf conductance and water relations of in vitro cultured grapevine rootstock in relation to acclimatization. Physiol. Plant. 102, 411–418. Hartmann, H.J., Kester, D.E., Davies, F.T., Geneve, R.T., 1997. Plant Propagation: Principles and Practices, 6th ed. Prentice-Hall, Englewood Cliffs, NJ. Isutsa, D.K., Pritts, M.P., Mudge, K.W., 1994. Rapid propagation of blueberry plants using ex vitro rooting and controlled acclimatization of micropropagules. HortScience 29, 1124–1126. Isutsa, D.K., Pritts, M.P., Mudge, K.W., 1998. A protocol for rooting and growing apple rootstock shoots. Fruit Varieties J. 52 (2), 107–116. Kantharajah, A.S., Dodd, W.A., 1990. In vitro micropropagation of Passiflora edulis (purple passion fruit). Ann. Bot. 65, 337–339. Kawata, K., Ushida, C., Kawai, F., Kanamori, M., Kuriyama, A., 1995. Micropropagation of passion fruit from subcultured multiple shoot primordia. J. Plant Physiol. 147, 281–284. Kramer, P.J., 1983. Water Relations of Plants. Academic Press, New York. Moran-Robles, M.J., 1978. In vitro vegetative multiplication of axillary buds of P. edulis var. flavicarpa Degener. and P. mollissima Bailey. Fruits 33, 701–715. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497. Nakasone, H.Y., Paull, R.E., 1998. Tropical Fruits. CAB International, Wallingford, UK. Rodrigues, V., Neto, A.T., Neto, P.C., Mendes, B., 1998. Influence of the number of subcultures on somaclonal variation in micropropagated ‘Nanicao’ (Musa species, AAA group). Acta Horticulturae 490, 469–473.
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: [email protected] (D.K. Isutsa). 0304-4238/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2003.08.002
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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 plantations, 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 .
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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 supplemented 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 propagation 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 inadequate, 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
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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)
20 30
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
40 60
20 60
100 100
0 1
0 4
3 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 method
Ex vitro In vitro
Yellow passion fruit
Purple passion fruit
Percent rooting
Roots per shoot
Percent survival
Percent rooting
Roots per shoot
Percent survival
96 a 62 b
3a 1b
92 a 50 b
66 a 47 b
2a 1a
59 a 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 recalcitrant in tissue culture. In the current research, GA3 stimulated elongation of purple
D.K. Isutsa / Scientia Horticulturae 99 (2004) 395–400
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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 transferred 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, effectively 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
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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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