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Micropropagation of Prickly Pear by Axillary Shoot Proliferation

LG Luisa Gutiérrez-Quintana
CZ Carlos Zúñiga-Rizo
AB Asdrúbal Burgos
LP Liberato Portillo

Published: Jul 5, 2018 DOI: 10.21769/BioProtoc.2923 Views: 5738

Edited by: Adam Idoine Reviewed by: Jinping Zhao

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Abstract

A protocol for the axillary bud proliferation of prickly pear (Opuntia; Cactaceae) is presented. This genus is widely used as a crop in the arid and semi-arid areas of the globe worldwide, providing numerous benefits for human and animal consumption. In vitro culture for axillary bud proliferation is of great use to obtain a large quantity of plants in a short period of time, with potential uses in production and for the preservation of endangered species of the Opuntia genus.

The optimal medium for Opuntia in vitro culture consists of Murashige and Skoog medium (MS) and L2 vitamins. To increase the yield of the axillary bud proliferation, we recommend the addition of plant growth regulators (PGRs). This work suggests a 15 d incubation in the medium with 2.2 mg/L of benzyl aminopurine (BA) after which the explants are transferred to the medium without PGRs. We explain as well how to adapt the plant to ex vitro conditions.

Keywords: Prickly pear search In vitro culture search Axillary bud proliferation search Ex vitro adaptation search

Background

The genus Opuntia (prickly pear) is one of the members of the Cactaceae family (Bravo-Hollis, 1978). Although it is native to the Americas, it currently grows in the wild and commercial plantations of the South of Europe, North of Africa, Australia, Middle East, West Asia and other regions of the world (Ochoa and Barbera, 1995; Kiesling and Metzing, 2017). Prickly pear has profound effects on the arid and semi-arid environments as well as the human communities that live in those areas because of its high biomass yields despite growing in drylands (Acevedo et al., 1983). In many of such areas, the genus Opuntia is exploited for a number of things. The young cladodes of Opuntia can be consumed as a vegetable; the fruit is eaten directly or processed into jelly, juice or sweets (Barba et al., 2017). The whole plant can be used for animal forage, living fence (Las Casas et al., 2017) and recently it started to be used commercially to produce biofuel (Aké Madera, 2014). In addition, the Opuntia genus has a number of bioactive and nutritionally valuable compounds (Betancourt et al., 2017; Melgar et al., 2017).

The advantages of in vitro culture include high proliferation rates and the production of pathogen-free plants (Shedbalkar et al., 2010). In their native environments, some Opuntia species are suffering losses in their populations, due to exploitation without replacement that has been carried out traditionally (Rocha-Flores et al., 2017). In vitro culture thus brings the possibility of recovering the populations of endangered plants (Torres-Silva et al., 2018), as well as a large scale production for agriculture. Here we present a protocol for plant in vitro culture of the Opuntia genus that includes the cytokinin benzyl aminopurine (BA).

Materials and Reagents

  1. 1,000 glass culture containers (100 ml) (Sigma-Aldrich, catalog number: B8648 )
  2. Parafilm
  3. Scapel
  4. Petri dish
  5. Nursery trays (International GreenHouse, catalog number: CN-PLG )
  6. Humidity dome (International GreenHouse, catalog number: PR-DOME7 )
  7. An Opuntia sp. young tender cladode
  8. Dishwashing liquid
  9. Deionized water
  10. 96% alcohol
  11. 60% sodium hypochlorite (NaClO) (household bleach)
  12. 1 N NaOH
  13. 1 N HCl
  14. Tween 20 detergent
  15. Benzyl aminopurine (6-benzyl aminopurine) (Sigma-Aldrich, catalog number: B3408 )
  16. Activated charcoal (Sigma-Aldrich, catalog number: C9157 )
  17. Agar-agar (Sigma-Aldrich, catalog number: A1296 )
  18. Sucrose (C12H22O11) (Sigma-Aldrich, catalog number: S5391 )
  19. Ammonium nitrate (NH4NO3) (Sigma-Aldrich, catalog number: A3795 )
  20. Potassium nitrate (KNO3) (Sigma-Aldrich, catalog number: NIST193 )
  21. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: 223506 )
  22. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: 1374361 )
  23. Potassium dihydrogen phosphate (KH2PO4) (Sigma-Aldrich, catalog number: PHR1330 )
  24. EDTA (Na2EDTA) (Sigma-Aldrich, catalog number: PHR1068 )
  25. Ferrous sulfate heptahydrate (FeSO4·7H2O) (Sigma-Aldrich, catalog number: 1270355 )
  26. Manganese sulfate tetrahydrate (MnSO4·4H2O) (Sigma-Aldrich, catalog number: 229784 )
  27. Zinc sulfate (ZnSO4·7H2O) (Sigma-Aldrich, catalog number: Z0251 )
  28. Boric acid (H3BO3) (Sigma-Aldrich, catalog number: B6768 )
  29. Potassium iodide (KI) (Sigma-Aldrich, catalog number: 746428 )
  30. Sodium molybdate dihydrate (Na2MoO4·2H2O) (Sigma-Aldrich, catalog number: M1003 )
  31. Cupric sulfate pentahydrate (CuSO4·5H2O) (Sigma-Aldrich, catalog number: C3036 )
  32. Cobalt chloride hexahydrate (CoCl2·6H2O) (Sigma-Aldrich, catalog number: C2911 )
  33. Myo-inositol (C6H12O6) (Sigma-Aldrich, catalog number: I7508 )
  34. Thiamine hydrochloride (C12H17ClN4OS·HCl) (Sigma-Aldrich, catalog number: T1270 )
  35. Pyridoxine (C8H11NO3) (Sigma-Aldrich, catalog number: P5669 )
  36. MS + L2 vitamins salts (see Recipes)
  37. Disinfection solution (see Recipes)
  38. Maintenance medium (see Recipes)
  39. Stimulation medium (see Recipes)
  40. Rooting medium (see Recipes)
  41. Substrate for ex vitro adaptation (see Recipes)
Note: All reagents are stored at room temperature and have a long shelf life (~10 years). Maintenance, stimulation and rooting media can be stored up to 15 d at 4 °C. Disinfection solution should be prepared and used the same day.

Equipment

  1. Tweezers
  2. Microwave oven
  3. pH meter
  4. Weighing balance
  5. Autoclave
  6. Laminar flow hood
  7. Greenhouse
  8. Growth chamber

Procedure

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.

Category

Plant Science > Plant cell biology > Tissue isolation and culture

Plant Science > Plant breeding > Micropropagation

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