Research note. Plant regeneration of sweet orange (Citrus sinensis) from thin sections of .... In order to obtain larger adventitious buds, the use for 3 weeks and ...
Plant Cell, Tissue and Organ Culture 74: 99–102, 2003. 2003 Kluwer Academic Publishers. Printed in the Netherlands.
99
Research note
Plant regeneration of sweet orange (Citrus sinensis) from thin sections of mature stem segments A.K. Kobayashi
1,2
1 1 1, , J.C. Bespalhok , L.F.P. Pereira & L.G.E. Vieira *
1
ˆ ´ do Parana´ ( IAPAR), C.P. 481, CEP 86001 -970, Laboratorio de Biotecnologia Vegetal, Instituto Agronomico Londrina, Parana´ , Brazil ( 2 present address: EMBRAPA Mandioca e Fruticultura, C.P. 007, CEP 44380 -000, Cruz das Almas, Bahia, Brazil; * requests for offprints; Fax: 155 -43 -3376 -2101; E-mail: lvieira@ pr.gov.br) Received 3 January 2002; accepted in revised form 15 November 2002
Key words: adventitious buds, Citrus sinensis, mature tissue, sweet orange
Abstract An efficient system for in vitro plant regeneration from thin transversal stem sections explants (1–2 mm) using mature tissues of sweet orange cv. Pera was developed. Explants were cultured in different media to evaluate the frequency of regeneration and size of buds. A high percentage of explants (54% with 3.1 buds / explant) producing large buds (1–4 mm) was observed when the explants were cultivated for 2 weeks on Murashige and Skoog medium and then transferred to Woody Plant medium (WPM). Both media were supplemented with 1.8 m M 6-benzylaminopurine and 0.7 m M gibberellic acid. Adventitious buds were regenerated into whole plants by in vitro shoot-tip grafting. Regenerated plants started to flower after 12 months in the greenhouse, confirming their mature nature. Abbreviations: BA – 6-benzylaminopurine; GA 3 – gibberellic acid; MS – Murashige and Skoog; WPM – Woody Plant Medium
An initial requirement for successful gene transfer to plants is the establishment of a suitable tissue culture system which is compatible with a range of transformation techniques, mainly Agrobacterium mediated transformation and particle bombardment. Most reports on citrus transformation have used juvenile tissue as the source of explants (Moore et al., 1992; Gutierrez et al., 1997; Bond and Roose, 1998). Due to the juvenile characteristics of plants regenerated from this type of explant, several years are necessary before horticultural and commercial traits of the transgenic plants can be evaluated. Development of transformation procedures that could bypass the juvenile phase could greatly reduce the time needed to evaluate transgenic citrus trees (Cervera et al., 1998). Cervera et al. (1998) have used mature tissue as the primary explant for sweet orange transformation. However, the cultivar used in their research – Pine-
apple orange – is not largely planted in the main sweet orange producing countries of the world. Although there is little genetic variability among sweet orange cultivars, it appears that there are enough differences to affect in vitro regeneration and transformation efficiency (Bond and Roose, 1998). Reproducible and efficient protocols for in vitro regeneration using mature tissues of the most widely planted sweet orange cultivars have yet to be designed. In a previous paper, we have described a protocol for shoot regeneration of sweet orange cvs. Pera, Valencia and Folha Murcha from thin sections excised from juvenile explants (Bespalhok et al., 2001). Here, we report the development of an efficient system for shoot regeneration using thin sections of mature explants of sweet orange cv. Pera, one of the most widely planted cultivars in the world. The regeneration protocol described in this study can be easily used
100 for the effective selection and regeneration of genetically modified plants. One-year-old Pera sweet orange nursery trees (Citrus sinensis L. Osbeck) maintained under greenhouse conditions were used as source of mature explants for in vitro regeneration. These plants were pruned to induce new growth and elongated shoots (20 cm long) were used as the source of mature tissue (Figure 1A). After removing the leaves, the shoots were surface sterilized for 15 min in 1% sodium hypoclorite solu-
tion followed by five rinses in sterile distilled water. Internodal stem explants were cut transversally into thin segments (1–2 mm) and placed horizontally with the basal end of the explant in contact with medium. The thin transversal stem segments were cultured on MS medium (Murashige and Skoog, 1962) and / or WPM medium (Lloyd and McCown, 1980), both supplemented with 1.8 m M BA, 0.7 m M GA 3 , 3% (w / v) sucrose and 0.6% (w / v) Bacto agar. The pH was adjusted to 5.7 before autoclaving. For testing the
Figure 1. In vitro regeneration of Pera sweet orange using mature thin stem explants. (A) Newly developed shoots used as source of mature explants. (B) Adventitious buds on WPM medium after 5 weeks, bar51 mm. (C) Micro grafted shoots on Carrizo citrange, bar55 mm. (D) Flowering regenerated plant after 12 month on greenhouse.
101 influence of the media on bud induction, four treatments were used: – culture on MS medium; – culture on WPM medium; – culture on MS medium for 1 week and then transfer to WPM medium; and – culture on MS medium for 2 weeks and then transfer to WPM medium. Cultures were maintained in the dark at 2661 8C for 3 weeks and then exposed to 16 / 8 h photoperiod under approximately 45 m mol m 22 s 21 at the same temperature for 2 weeks. Twenty explants were inoculated per Petri dish (90320 mm) and five dishes were used for each treatment. After 5 weeks in the culture, the number of adventitious buds was recorded using a stereomicroscope. The experiment was repeated twice. Adventitious buds were regenerated into whole plants by in vitro shoot-tip grafting (Navarro, 1992). Briefly, Carrizo citrange (C. sinensis L. Osbeck3 Poncirus trifoliate L. Raf.) seeds were peeled by removing both seed coats, disinfected for 15 min in 1% sodium hypoclorite solution and rinsed five times with sterile distilled water. Seeds were sown on MS basal salts with 0.3% (w / v) Phytagel (pH 5.7) and grown in the dark at 2661 8C for 2 weeks. The seedlings were decapitated leaving 3 cm of the epicotyl and used as rootstocks. Adventitious buds of sweet orange larger than 1 mm were removed from the explant and inserted into an incision along the epicotyl of Carrizo rootstock. Grafted plants were cultured in liquid MS medium with 75 g l 21 sucrose. Grafted plants with at least three expanded leaves were removed from jars and individually placed in pots (18 l) containing a mixture of soil, sand and manure for acclimatization. In preliminary experiments, thin stem segments were cultured in MS medium supplemented with different concentrations of BA (0–28.4 m M). Adventitious buds were obtained at BA concentrations ranging from 0 to 7.1 m M (data not shown). Maximum
bud induction was achieved at 1.8 m M BA. Adventitious buds were regenerated from callus of the cambium tissue. This is the same region where regeneration occurs when juvenile explants are used (Ghorbel et al., 1999). However, the regenerated adventitious buds were very small (less than 1 mm) and not amenable to regeneration of whole plants even using micro grafting. In order to obtain larger adventitious buds, the use of WPM medium was evaluated. When stem segments were inoculated directly on WPM medium, the proportion of explants with buds was considerably lower compared to MS medium (Table 1). However, adventitious buds regenerated on WPM medium were larger (1–4 mm, Figure 1B) than the ones cultivated only on MS medium (less than 1 mm). The best results (highest percentage of explants producing larger buds) occurred when the explants were cultivated for 2 weeks on MS medium and then transferred to WPM (Table 1). WPM medium has been developed for tissue culture of woody species with recalcitrant regeneration problems (Lloyd and McCown, 1980). In our experiments we observed that explants cultured on WPM medium produced larger buds than those cultured on MS medium. One possible explanation for this result is the lower concentration of nitrogen and potassium in WPM medium compared to MS medium. Improvement of shoot growth / elongation by reducing the nitrogen and potassium concentration in the culture medium was also reported in other species (Bespalhok et al., 1992). Shoots started to grow 2 weeks after micro grafting (Figure 1C). Around 90% of micro grafted buds developed into whole plants. However, micro grafted plants transferred from test tubes directly to the soil mixture developed very slowly. Alternatively, the in vitro micro grafted plants were grafted again on 3month-old seedlings of Rangpur lime (C. limonia) by the cleft graft method in the greenhouse. The graft union was wrapped with Parafilm and the entire
Table 1. Effect of culture media on the bud regeneration frequency of mature thin internodal explants of Pera sweet orange Treatment
n
n with buds (%)
Buds total
Buds per explant a
MS WPM MS(1)-WPM b MS(2)-WPM c
180 200 200 200
71 (39.4) 29 (14.5) 98 (49.0) 108 (54.0)
355 109 544 622
2.060.2 0.560.1 2.760.3 3.160.3
a
Values are mean6standard error. Culture on MS medium for 1 week and then transferred to WPM medium. c Culture on MS medium for 2 weeks and then transferred to WPM medium. b
102 plant covered with a polyethylene bag which was gradually open after 2 weeks. Using this procedure, rapid acclimatization and development of the plants were observed under greenhouse conditions. The established plants showed to be mature based on morphological characters such as thorniness, growth habit, internode length and leaf size. After 12 months in the greenhouse, the micro grafted citrus plants started to flower (Figure 1), confirming their mature nature. Cervera et al. (1998) have obtained regeneration and transgenic plants from mature tissue of sweet orange cv. Pineapple using what was called an invigorating method. Explants were obtained from internode segments (1 cm) from stems developed from grafted buds. This protocol is quite tedious and requires large number of trees due to the size of the explants. Besides, regeneration frequency was low (23%) with 2.1 buds per explant. In comparison, the protocol reported here uses thin stem sections (1–2 mm) from stems of 1 m tall nursery plants, which results in more explants per plant. Regeneration frequency of 54% and 3.1 buds per explant was achieved in the best treatment (Table 1). Besides the higher regeneration frequency, another advantage of using thin stem explants is that fewer citrus plants are necessary as explants donors, requiring less space on greenhouse facilities. In this study we have described an efficient protocol for in vitro regeneration of sweet orange cv. Pera from mature tissue using thin internode stem sections as explants. This protocol can be used for transformation of sweet orange either by Agrobacterium or particle bombardment. Also, the use of thin explants may reduce the number of escapes, a great problem in citrus transformation, as the whole explant is effectively exposed to the selective agent in the culture medium. Transformed plants of Pera sweet orange via Agrobacterium tumefaciens have already been obtained in our lab using this regeneration protocol (in preparation).
Acknowledgements The authors thank Ms. S.A. Kudo and Ms. L.M. da Silva for their excellent technical assistance. A.K. Kobayashi and J.C. Bespalhok F. were supported by a RHAE / CNPq fellowship.
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