Acceso a plazas de formación sanitaria especializada

Assay

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4 Notes

1. The EDTA disodium salt will not go into solution until the pH of the solution is adjusted to ~8.0 by the addition of NaOH.

2. Ethidium bromide is a strong mutagen and a possible carcino-gen or teratocarcino-gen. Its hazardous properties require the use gloves for handling and safety disposal.

3. Rifampicin (25 mg/L) and kanamycin (50 mg/L) are added to grow Agrobacterium strain AGL1 transformed with pBI121.

4. To calculate the fi nal dilution vo lume of Agrobacterium ( V 1 ), use the equation C 1 V 1 = C 2 V 2 , where C 1 and C 2 are respectively the initial and fi nal OD and V 2 the fi nal volume. Use AIM to dilute the Agrobacterium cells. Diluted Agrobacterium culture has to be used immediately to avoid aggregation.

5. Shoots regenerated from the two cut ends of the explants can be considered independent events. It is important to cut off the calli at the base from the shoot.

publications/pdf/005449.pdf

3. Xu X, Pan S, Cheng S, Zhang B, Mu D, Ni P, Visser RG (2011) Genome sequence and anal-ysis of the tuber crop potato. Nature 475:189–195

4. Bradeen JM, Carputo D, Douches D (2009) Part 7 Transgenic sugar, tuber and fi ber crops.

doi: 10.1002/9781405181099 . k0704 in compendium of transgenic crop plants

5. De Block M (1998) Genotype-independent leaf disc transformation of potato ( Solanum tuberosum ) using Agrobacterium tumefaciens . Theor Appl Genet 76:767–774

6. Stiekema WJ, Heidekamp F, Louwerse JD, Verhoeven HA, Dijkhuis P (1998) Introduction of foreign genes into potato cultivars Bintje and Desiree using an Agrobacterium tumefaciens binary vector.

Plant Cell Rep 7:47–50

7. Sheerman S, Bevan MW (1988) A rapid trans-formation method for Solanum tuberosum using binary Agrobacterium tumefaciens vec-tors. Plant Cell Rep 7:13–15

8. Tavazza R, Tavazza M, Ordas RJ, Ancora G, Benvenuto E (1988) Genetic transformation

of potato ( Solanum tuberosum ): an effi cient method to obtain transgenic plants. Plant Sci 59:175–181

9. Wenzler H, Mignery G, May G, Park A (1989) A rapid and effi cient transformation method for the production of large numbers of trans-genic potato plants. Plant Sci 63:79–85 10. Mitten DH, Horn M, Burrel MM, Blundy KS

(1990) Strategies for potato transformation and regeneration. In: Vayda ME, Park WD (eds) The molecular and cellular biology of the potato.

CAB International, Wallingford, pp 181–191 11. Beaujean A, Sangwan RS, Lecardonnel A,

Sangwan-Norreel BS (1998) Agrobacterium - mediated transformation of three economi-cally important potato cultivars using sliced internodal explants: an effi cient protocol of transformation. J Exp Bot 49:1589–1595 12. Millam S (2006) Potato ( Solanum tuberosum

L.). In: Wang K (ed) Agrobacterium protocols, vol 2, 2nd edn. Humana, Totowa, NJ, pp 25–35 13. Chakravarty B, Wang-Pruski G (2010) Rapid regeneration of stable transformants in cul-tures of potato by improving factors infl uenc-ing Agrobacterium -mediated transformation.

Adv Biosci Biotechnol 1:409–416

14. Trujillo C, Rodriguez-Arango E, Jaramillo S, Hoyos R, Orduz S, Arango R (2001) One step transformation of two Andean potato cultivars

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( Solanum tuberosum L. subsp. andigena ).

Plant Cell Rep 20:637–641

15. Banerjee AK, Prat S, Hannapel DJ (2006) Effi cient production of transgenic potato ( S.

tuberosum L. ssp. andigena ) plants via Agrobacterium tumefaciens -mediated transfor-mation. Plant Sci 170:732–738

16. Narváez-Vásquez J, Ryan AC (2002) The sys-temin precursor gene regulates both defensive and developmental genes in Solanum tuberosum . Proc Natl Acad Sci U S A 99:15818–15821 17. Jefferson RA, Kavanagh TA, Bevan MW

(1987) GUS fusion: glucuronidase as a sensi-tive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

18. Po-Yen C, Chen-Kuen W, Shaw-Ching S, Kin- Ying T (2003) Complete sequence of the binary vector pBI121 and its application in

cloning T-DNA insertion from transgenic plants. Mol Breeding 11:287–293

19. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:

151–158

20. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15:

473–497

21. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual (3rd ed), CSHL Press, New York, NY 3:1–1453

22. Hoagland DR, Arnon DI (1950) The water culture method for growing plants without soil. University of California. Agric. Exp.

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Kan Wang (ed.), Agrobacterium Protocols: Volume 2, Methods in Molecular Biology, vol. 1224, DOI 10.1007/978-1-4939-1658-0_9, © Springer Science+Business Media New York 2015

Chapter 9

Taro ( Colocasia esculenta (L.) Schott)

Xiaoling He , Susan C. Miyasaka , Maureen M. M. Fitch , and Yun J. Zhu

Abstract

Genetic engineering of taro is an effective method to improve taro quality and the resistance to various diseases of taro. Agrobacterium tumefaciens -mediated transformation of taro is more effi cient than the particle bombardment transformation method based on current research. The development of a regenera-tion system starting from taro shoot tip explants could produce dasheen mosaic virus (DsMV)-free plantlets.

Highly regenerative calluses could be developed from DsMV-free, in vitro plantlets on the Murashige and Skoog (MS) medium with 2 mg/L BA and 1 mg/L NAA (M5 medium). The Agrobacterium tumefaciens - mediated transformation method is reported in this chapter. The highly regenerative calluses were selected and cocultivated with the Agrobacterium strain EHA105 harboring the binary vector PBI121 with either a rice chitinase gene chi11 or a wheat oxalate oxidase gene gf2.8 . After cocultivation for 3–4 days, these calluses were transferred to selection medium (M5 medium) containing 50 mg/L Geneticin G418 and grown for 3 months in the dark. Transgenic shoot lines could be induced and selected on the MS medium containing 4 mg/L BA (M15 medium) and 50 mg/L Geneticin G418 for 3 months further in the light.

Molecular analyses are used to confi rm the stable transformation and expression of the disease resistance gene chi11 or gf2.8 . Pathologic bioassays could be used to demonstrate whether the transgenic plants had increased disease resistance to taro pathogens Sclerotium rolfsii or Phytophthora colocasiae .

Key words Agrobacterium tumefaciens , Colocasia esculenta , Dasheen mosaic-free plantlets , Genetic engineering , Shoot tip explants , Taro , Transformation

1 Introduction

Taro ( Colocasia esculenta (L.) Schott) is an important tropical root crop which is cultivated worldwide especially in Southeast Asia and the Pacifi c Islands [ 1 , 2 ]. Traditionally, taro is propa-gated vegetatively using underground stems from sucker plants (i.e., cormels) [ 3 ]. Approximately 10 % of the previous crops’

cormels need to be used for propagation [ 3 ]. Several tissue cul-ture protocols for various taro cultivars have been developed. The development of an effi cient taro tissue culture system could reduce the usage of the cormels for propagating materials and increase the speed and production of the taro propagation. For example, Chand et al. in 1999 [ 4 ], Fukino et al. in 2000 [ 5 ],

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Hartman in 1974 [ 6 ], and He et al. in 2008, 2010, and 2013 [ 7 – 9 ] have reported that effi cient tissue culture systems could be developed using taro shoot tips as explants. Murakami et al. in 1995 [ 10 ] developed a regeneration system via protoplasts. In addition, Deo et al. in 2009 [ 11 ] reported a protocol of the somatic embryogenesis, organogenesis, and plant regeneration in taro.

In this chapter, we describe a protocol of establishment of in vitro stock culture using taro shoot tips as explants and a pro-tocol of inducing calluses and multiple shoots using in vitro shoot tips [ 7 – 9 ]. The protocols we used are easy to conduct and only need 1–2 cormels as propagating materials. Also, the regenera-tion effi ciency is relatively high with an average of 12 multiple shoots developed from each callus within 1 month [ 8 ]. In addi-tion, this regeneration method using shoot tip explants could eliminate the taro pathogen dasheen mosaic virus (DsMV) from infected taro [ 8 ].

Major limiting factors in taro production are various taro diseases that can severely decrease taro yields [ 12 ]. These taro dis-eases include: (1) taro leaf blight (TLB) caused by the oomycete pathogen Phytophthora colocasiae , (2) taro pocket rot (TPR) caused by a new species of Phytophthora , and (3) and southern blight caused by the fungal pathogen Sclerotium rolfsii [ 12 ].

Conventional breeding is ongoing to increase resistance to these diseases. However, one commercial taro cultivar that origi-nated in China (cv. Bun Long) rarely fl owers under natural environmental conditions in Hawaii. Cultivar Bun Long as well as several other cultivars does not respond to gibberellic acid (GA) which is applied to induce fl owering [ 13 ]. Lack of fl owering makes it impossible to improve taro quality and disease resistance by con-ventional breeding.

Genetic engineering of taro is an alternative to conventional breeding to improve disease resistance. To date, four journal arti-cles have reported transformation protocols of taro by either particle bombardment method or Agrobacterium -mediated method [ 5 , 7 – 9 ]. Fukino et al. in 2000 [ 5 ] fi rst reported transfor-mation of taro cultivar Eguimo using a marker gene glucuronidase (gus) gene. They used a particle bombardment method with a very low effi ciency (less than 0.5 %). He et al. in 2010 [ 8 ] also reported a successful particle bombardment transformation method to insert a disease resistance gene, rice chitinase gene chi11 with the same low effi ciency (less than 0.5 %). In addition, the Southern blot anal-ysis showed a high-copy insertion of the transgene (13 copies) that indicated a high risk of transgene silencing and rearrangement [ 8 ].

He et al. in 2008 [ 7 ] transformed a commercial taro cultivar Bun Long with the rice chitinase gene chi11 via Agrobacterium -mediated transformation, and transgenic plants showed increased disease resistance to the pathogen Sclerotium rolfsii . Also, He et al. in 2013 [ 9 ] Xiaoling He et al.

99

used the same Agrobacterium -mediated transformation protocol to insert a wheat oxalate oxidase gene gf2.8 into taro cv. Bun Long, and the transgenic plants showed increased disease resistance to the pathogen Phytophthora colocasiae. Agrobacterium -mediated trans-formation method had a higher transtrans-formation effi ciency (1–3 %) and lower-copy insertion of transgene (single copy or 2–3 copies) than the particle bombardment transformation method [ 5 , 7 – 9 ].

In this chapter, we describe this Agrobacterium -mediated transfor-mation protocol in step-by-step laboratory procedures.

2 Materials

Taro cv. Bun Long cormels were obtained from the University of Hawaii’s Waiakea Experiment Station. In vitro plantlets in culture were established from primary shoot apices and axillary buds on taro cormels. Highly regenerative calluses were induced from the DsMV-free in vitro plantlets. These calluses were the targets of Agrobacterium tumefaciens -mediated transformation.

1. α-Naphthalene acetic acid (NAA): 100 ml 0.5 mg/mL stock is prepared by dissolving 50 mg of NAA in 2 mL 1 N NaOH and making up to volume with 98 mL of sterile distilled water.

Stock solution can be stored at 4 °C for 6 months.

2. 6-Benzylaminopurine (BA): 100 ml 0.5 mg/mL stock is pre-pared by dissolving 50 mg of BA in 2 mL 1 N NaOH and mak-ing up to volume with 98 mL of sterile distilled water. Stock solution can be stored at 4 °C for 6 months.

1. Cefotaxime sodium salt: Prepare as 250 mg/mL stock solution in sterile distilled water, fi lter-sterilize through 0.2 μm mem-brane, aliquot 1 mL into 1.5 mL sterile Eppendorf tubes, and store at −20 °C for 6 months.

2. G418 Sulfate: Prepare as 50 mg/mL stock solution in sterile distilled water, fi lter-sterilize through 0.2 μm membrane, ali-quot 1 mL into 1.5 mL sterile Eppendorf tubes, and store at

−20 °C for 6 months.

3. Kanamycin monosulfate: Prepare as 50 mg/mL stock solution in sterile distilled water, fi lter-sterilize through 0.2 μm mem-brane, aliquot 1 mL into 1.5 mL sterile Eppendorf tubes, and store at −20 °C for 6 months.

4. Rifampicin: Prepare as 25 mg/mL stock solution in dimethyl sulfoxide (DMSO), fi lter-sterilize through 0.2 μm membrane, aliquot 1 mL into 1.5 mL sterile Eppendorf tubes, wrap Eppendorf tubes using foil paper to protect from light, and store at −20 °C for 1 year.

2.1 Plant Material

2.2 Growth