souza2018

Chapter 18
Cryopreservation of Pineapple Shoot Tips by the Droplet
Vitrification Technique
Fernanda Vidigal Duarte Souza, Everton Hilo de Souza, Ergun Kaya,
Lívia de Jesus Vieira, and Ronilze Leite da Silva
Abstract
Cryopreservation is a technique that allows the conservation of many species for long periods. Among the
protocols used for cryopreservation, droplet vitrification has shown efficient results in preserving shoot tips
of various wild and cultivated pineapple genotypes. The method consists of extraction of shoot tips from
plants grown in vitro, dehydration for a period of 48 h in a preculture medium supplemented with a high
concentration of sucrose, treatment in a plant vitrification solution (PVS2), and immersion in liquid nitrogen. The method described in this chapter has produced survival and regeneration indices of around 70%,
depending on the genotype and physiological conditions of the initial explants. The objective of this chapter is to describe in detail a droplet vitrification protocol for shoot tips that is easy to perform for cryopreservation of pineapple germplasm.
Key words Ananas comosus, Conservation, Plant vitrification solution
1
Introduction
Pineapple (Ananas comiosus L. Merrill) is one of the world’s most
popular tropical fruits [1] and Brazil is one of the centers of origin
and genetic diversity of this species [2, 3]. However, anthropization and the use of only a few varieties for commercial cultivation
have caused serious genetic erosion of the genus, requiring urgent
actions to conserve the species. Among the main conservation
strategies employed are conservation in field conditions or greenhouses/covered areas, slow growth in vitro conservation, and
cryopreservation [3–5].
In recent decades, due to the advances achieved with cryopreservation techniques, it has become an extremely interesting
alternative, since it can maintain species for long periods at ultralow
temperatures (−196 °C) or in the nitrogen vapor phase (−150 °C)
[5–14]. The use of this preservation strategy allows maintaining
large collections of biological material without the need of periodic
Víctor M. Loyola-Vargas and Neftalí Ochoa-Alejo (eds.), Plant Cell Culture Protocols, Methods in Molecular Biology, vol. 1815,
https://doi.org/10.1007/978-1-4939-8594-4_18, © Springer Science+Business Media, LLC, part of Springer Nature 2018
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Fernanda Vidigal Duarte Souza et al.
interventions, as occurring with in vitro conservation, since at
ultralow temperatures, the cell metabolism is so slow that biological deterioration is virtually halted [15]. The cessation of the plant
metabolism while maintaining the cellular integrity makes this
form of preservation attractive, as does the relatively low cost [16].
The most expensive aspect of this technique is the cost of establishing the basic storage structure, namely, the cryogenic tanks and
nitrogen flow system [17, 18]. However, the widespread use of
this technique still requires research and adjustments of factors
considered to be limiting. One of these limiting aspects is the high
level of specificity in the behavior of species when submitted to
ultra-frozen conditions, creating a genotype dependence on the
development of protocols [19]. Hence there is a need for studies
aimed at species of interest. To be successful, cryopreservation procedures demand precision and meticulous attention to the details
of each step. In the case of pineapple, studies in this respect began
in the 1990s [6]. More recently, Souza et al. [5] established an
efficient droplet vitrification protocol for 16 genotypes and 4 varieties, both wild and cultivated, achieving regeneration rates of
around 70%. This protocol has been since then applied to other
pineapple genotypes with good results and is presented in this with
sufficient detail to allow its replication.
The same authors have demonstrated the efficiency of this
technique and the causes of tissue injuries by means of light and
scanning electron microscopy.
2
Materials
Prepare all the solutions using ultrapure water and reagents with
high analytical purity grade. The solutions should be prepared and
stored at a temperature of 4 °C. The culture media should be
stored at room temperature in a dry and clean place, protected
from light. The residues generated should be treated and discarded
according to the applicable national or institutional regulations.
2.1 Culture Medium
for Multiplication
of Explants
1. MS culture medium [20] (basal salts), according to the manufacturer’s instructions, 0.05 μM of naphthalene acetic acid
(NAA) + 0.09 μM of benzyladenine (BA) and 0.09 M of
sucrose, 2.4 g L−1 of Phytagel® or 7 g L−1 of agar as solidifier,
pH 5.8 (see Note 1).
2.2 Preculture
Medium
1. MS culture medium [20] (basal salts) according to the manufacturer’s instructions, 0.3 M of sucrose and 2.4 g L−1 of
Phytagel® or 7 g L−1 of agar as solidifier, pH 5.8 (see Note 2).
2.3
1. Solution of MS [20] (basal salts) according to the manufacturer’s instructions, 30% (w/v) glycerol, 15% (w/v) ethylene
glycol, 15% (w/v) DMSO, and 0.4 M of sucrose (see Note 3).
PVS2 Solution
Cryopreservation of Pineapple
2.4
Washing Solution
271
1. Solution of MS [20] (basal salts) according to the manufacturer’s instructions, 1.2 M of sucrose (see Note 4).
2.5 Regeneration
Medium
1. MS culture medium [20] (basal salts) according to the manufacturer’s instructions, 0.22 μM of BAP, 0.09 M of sucrose,
2.4 g L−1 of Phytagel®, or 7 g L−1 of agar as solidifier, pH 5.8
(see Note 5).
2.6 Metal Supports
for the Shoot Tips
1. Sheets of aluminum foil with thickness of 0.25 mm,
2.5 cm × 0.5 cm (see Note 6).
3
Methods
Perform all procedures under standard laboratory conditions.
Some must be aseptic, in a laminar flow chamber.
3.1 Type of Explant
to Use
for Cryopreservation
1. Buds from pineapple plants grown in vitro that have been isolated and subcultured for 45 days in a multiplication medium.
3.2 Obtaining
and Multiplying
the Explants
1. Plants previously established in vitro [21] should be subcultured in a laminar flow chamber every 45 days in the multiplication culture medium (Fig. 1a).
2. The explants generated should be transferred to fresh multiplication medium and incubated in a growth chamber (27 ± 1 °C;
photoperiod of 16 h) for 45 days to standardize all the starting
material (Fig. 2b) (see Note 7).
3.3 Excision
and Preculturing
of Shoot Tips
1. The shoot tips with maximum length of 0.5 mm should be
excised in an aseptic environment (laminar flow chamber) with
the aid of tweezers, scalpel (sterilized), and a stereoscopic
microscope (Fig. 2a), leaving a single leaf primordium (see
Note 8).
2. The shoot tips should be distributed in a Petri dish (Fig. 2b)
containing preculture medium (see Note 9) and then incubated in an incubation chamber for 48 h at 26 ± 1 °C, photoperiod 16 h, and light intensity of 22 μmol m−2 s−1 to favor the
dehydration of the meristems.
3.4 Droplet
Vitrification of Shoot
Tips in PVS2 Solution
1. The precultured shoot tips should be deposited on aluminum
foil strips containing 4 μL droplets of the PVS2 vitrification
solution (Fig. 3) (see Note 10), remaining in contact with the
PVS2 solution for 45 min.
3.5 Immersion
of Shoot Tips in Liquid
Nitrogen
1. The foil strips containing the shoot tips should be inserted in
the sterile cryotubes and rapidly immersed in a bowl with liquid nitrogen (see Note 11).
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Fernanda Vidigal Duarte Souza et al.
Fig. 1 (a) Multiplication procedure for generation of buds. (b) Standardized buds after cultivation in multiplication culture medium
Fig. 2 (a) Excised pineapple shoots with 1 mm length. (b) Shoot tips distributed in Petri dishes. Bars: 1 mm
2. After being quickly closed, the cryotubes should be attached to
the cannulas and immersed in the cryogenic tank at
-196 °C. This closure should preferably be done with part of
the cryotubes still immersed in the liquid nitrogen, to prevent
any possibility of variation of the internal temperature. The
immersion in the LN2 should be immediate.
Cryopreservation of Pineapple
273
Fig. 3 (a–b) Shoot tips being transferred and deposited on droplets of PVS2 on aluminum foil strips
3. The samples should remain in liquid nitrogen for the time
necessary.
3.6 Washing
and Culturing
the Cryopreserved
Shoot Tips
1. The aluminum foil strips should be removed from the cryotube with the help of tweezers, and the face containing the
shoot tips should be placed in direct contact with the washing
solution so that they come loose and remain immersed in the
solution at room temperature for 20 min. This entire procedure should be performed in a laminar flow chamber.
2. The shoot tips should be cultured in Petri dishes containing
regeneration medium (see Note 12).
3. The dishes should be kept in a growth chamber with partial
absence of light in the first 48 h.
4. Cover the plates with white paper to reduce the incidence of
light on the tips recently removed from the LN2.
5. After this period, the dishes should remain in the growth
chamber (27 ± 1 °C; photoperiod of 16 h).
4
Notes
1. To prepare 1000 mL of the multiplication medium, follow
these steps: in a 1000 mL beaker, place 30 g of ultrapure
sucrose. Then add MS (basal salts) according to the manufacturer’s instructions and the BA and NAA regulators. Next add
a small volume of sterile deionized water until the beaker is
filled almost to 500 mL and homogenize the solution by swirling the beaker. Adjust the pH to 5.8 using a digital pH meter.
In another 1000 mL beaker, place about 500 mL of sterile
deionized water and 2.4 g of Phytagel®. Completely melt the
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Fernanda Vidigal Duarte Souza et al.
Phytagel® in a microwave oven and add the first solution. Top
up the volume to 1000 mL with sterile deionized water using
a test tube and distribute 80 mL aliquots of the culture medium
in a series of glass flasks with capacity of 800 mL, closing them
with lids. Autoclave the flasks at 120 °C for 20 min. Use the
culture medium only after it completely cools and solidifies
and within 1 month after its preparation.
2. To prepare 250 mL of culture medium, follow these steps: in a
250 mL beaker, place 25.675 g of ultrapure sucrose and 1.11 g
of MS (basal salts) according to the manufacturer’s instructions. Add a small volume of sterile deionized water until the
beaker is filled almost to 250 mL and homogenize the solution
by swirling the beaker. Adjust the pH to 5.8 using a digital pH
meter. Top up the volume with sterile deionized water to
250 mL using a test tube. Place 0.6 g of Phytagel® in a 500 mL
Erlenmeyer flask and add 0.6 g of the first solution. Seal the
Erlenmeyer flask and sterilize the solution by autoclaving at
121 °C for 20 min. In a laminar flow chamber, distribute the
culture medium in Petri dishes. Use the culture medium only
after it completely cools and solidifies and within 1 month after
its preparation.
3. To prepare 250 mL of the PVS2 solution, follow these steps.
In a 250 mL beaker, place 75 g of glycerol (purity > 99.5%),
34.1 mL of dimethyl sulfoxide (DMSO) (purity > 99.5%), and
33.8 mL of ethylene glycol (purity > 99.5%). To this mixture
add 34.25 g of ultrapure sucrose and 1.11 g of MS (basal salts)
according to the manufacturer’s instructions. Add a small volume of sterile deionized water until the beaker is filled almost
to 250 mL and homogenize the solution for several minutes by
swirling the beaker. Adjust the pH to 5.8 using a digital pH
meter. Top up the volume with sterile deionized water to
exactly 250 mL using a test tube. Take the solution to the laminar flow chamber and carry out ultrafiltration using a syringe
and filter with pore diameter smaller than 0.22 μm. The recipient flask must be sterile, and the volume can be divided into
two or more Erlenmeyer flasks, which should be protected
from light and can be stored at 4 °C for up to 1 month. The
PVS2 solution cannot be sterilized by autoclaving, because it
contains volatile substances that can be lost during the
process.
4. To prepare 250 mL of the washing solution, follow these steps:
in a 250 mL beaker, place 102.69 g of ultrapure sucrose. Then
add 1.11 g of MS (basal salts) according to the manufacturer’s
instructions. Next, add a small volume of sterile deionized
water until the beaker is filled almost to 250 mL and homogenize the solution by swirling the beaker. Adjust the pH to 5.8
using a digital pH meter, and top up the volume with sterile
Cryopreservation of Pineapple
275
deionized water to exactly 250 mL utilizing a test tube. Take
the solution to the laminar flow chamber, and carry out ultrafiltration using a syringe and filter with pore diameter smaller
than 0.22 μm. The recipient flask must be sterile, and the volume can be divided into two or more Erlenmeyer flasks, which
should be protected from light and can be stored at 4 °C for
up to 1 month. The washing solution also should be sterilized
by autoclaving. However, the solution can become darker in
color due to the start of caramelization of the sucrose.
5. To prepare 250 mL of the regeneration medium, follow these
steps: in a 250 mL beaker, place 7.5 g of ultrapure sucrose.
Then add MS (basal salts) according to the manufacturer’s
instructions. Next, add 12.5 μg of BA followed by a small volume of sterile deionized water until the beaker is filled almost
to 250 mL, and homogenize the solution by swirling the beaker. Adjust the pH to 5.8 using a digital pH meter, and top up
the volume with sterile deionized water to exactly 250 mL
using a test tube. In a 500 mL Erlenmeyer flask, place 0.6 g of
Phytagel® and add the first solution. Seal the Erlenmeyer flask,
and sterilize the solution by autoclaving at 120 °C for 20 min,
and distribute the medium in Petri dishes with diameter of
90 mm. Use the culture medium only after it completely cools
and solidifies and within 1 month after its preparation.
6. Open the aluminum foil on a totally smooth countertop.
Moisten a cotton swab with acetone (purity > 99.5%) and pass
it over the aluminum foil several times, until the surface is
smooth and homogeneous. Using a ruler, fold the foil into two
parts and form a double strip with width of 1 cm. Cut into narrower strips with width of 0.5 cm and length of 2 cm. Deposit
the strips in glass flasks and autoclave them at 120 °C for
20 min.
7. The number of buds should be twice the number of shoot tips
intended for extraction, because losses during retrieval are frequent. At the moment of excising the shoot tips, the buds
should have a standard size, obtained by subculturing for
45 days, as mentioned in Subheading 3.2. If there is any sign
of etiolation of the plants, they should not be used to dissect
shoot tips. To prevent this, the incubation conditions should
be constant and precisely monitored.
8. Instruments (tweezers and scalpels) with small points should
be used to excise the shoot tips with the size necessary for
cryopreservation, since they facilitate removal of leaves around
the tips without causing injury to the surrounding tissue. This
is a process that requires great skill of the operator but can be
achieved with sufficient training and practice. To confirm that
the tips have length of 1 mm, a strip of ruled millimeter graph
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Fernanda Vidigal Duarte Souza et al.
paper (sterile) can be used as a reference. The removal of the
shoot tips is a crucial step for the technique’s success, because
the regeneration will only occur if the vegetative structures
have been preserved.
9. The shoot tips should be distributed with spaced 1 cm apart,
directed upward, with the base partially immersed in the
medium.
10. Aluminum foil strips should be distributed in the Petri dishes
over small blocks of sterile ice, and one of the ends of each strip
should be folded to facilitate handling. Sterile eyedroppers
should be used to distribute the droplets of the PVS2
solution.
11. The whole process of immersion in the liquid nitrogen and
transfer to the cryogenic tank should be carried out as fast as
possible to avoid sudden temperature variations. It is recommended first to cool the cryotubes and to introduce the aluminum foil strips with the shoot tips within the LN2 in the bowl.
12. The excess solution should be removed using sterilized filter
paper. The tips should be placed on this paper for a few minutes and then distributed in the Petri dishes with the regeneration medium, spaced 1 cm apart, directed upward, with the
base partially immersed in the medium.
Acknowledgments
The authors acknowledge Fundação de Amparo à Pesquisa do
Estado da Bahia (FAPESB), Conselho Nacional de Desenvolvimento
Científico
e
Tecnológico
(CNPq),
Coordenação
de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES)/
EMBRAPA program, and Embrapa Mandioca e Fruticultura for
financial support and Helder Lima Carvalho, technician of the
Laboratório de Cultura de Tecido de Plantas, for his valuable collaboration in preparing this chapter.
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