 |
Ipomea
(sweet
potato)
|
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Work
in Progress
~~~~~~~~~~~~~~~~~~~~~~~~~~
Cryopreservation
of sweet potato has been an ongoing project at NCGRP. Published protocols have resulted in variable survivability results
(0-60%) on accessions stored on site.
Various modifications to cryopreservation techniques have been employed
to increase the survivability of this ‘hard to freeze’ more tropical plant
and are presented here as a work in progress, however, consistent viability
results have eliminated placing accessions into long-term LN2
storage at this time.
Sweet
Potato Vitrification
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
- In
vitro plants are initiated
from nodal segments and placed in sweet potato medium (modification of Murashige
and Skoog, 1962). Plantlets are
cultured at 25°C with a 8hr dark and16hr light cycle (55 µm/m-2/s-1).
Shoot tips are generally ready for excision in 4-13 weeks, (accession
dependent).
- Plants are
subjected to a 8 hr dark period just prior to shoot tip excision.
- Shoot tips (0.5mm-1.0mm)
consisting of 2-3 leaf primordia and apical dome are dissected with a hypodermic
needle under a dissecting microscope and placed in 2% sucrose* at 25°C
for 24 hr. Then shoot tips are placed in 0.3M sucrose* at 25°C for 24hr.
- Shoot tips are then placed
in 0.4M Sucrose + 2M glycerol* for 60 minutes at 22°C. Shoot tips are next placed into PVS2* for 16
minutes. Just prior to the 16 minutes
PVS2 incubation, transfer the shoot tips + approximately 2-3 µl of
PVS2 onto heat sterilized aluminum foil strips.
Fold foil strips in half.
- At exactly
16 minutes, immerse shoot tips into LN2-slush, place into LN2 cooled cryovials
and store in LN2.
Retrieval
- Cryovials are quickly removed
from LN2 and immersed into a 40°C water bath for 3-5 seconds.
Caps are removed and aluminum foil strips with shoot tips are quickly
placed into 3ml of 1.2M sucrose* at 22°C for 20 minutes.
- Shoot tips are placed on modified
recovery medium* for 5 days under reduced light conditions (first 2 days in
dark, remaining 3 days in dim light (27 µm/m-2/s-1)),
followed by 9 days under normal lighting (55 µm/m-2/s-1).
- Shoot tips are transferred to
sweet potato medium* and observed for plantlet development.
Further experimental
trials have included the following modifications to the protocol described above:
- Smaller (0.8mm) or larger shoot
tips (1.2mm+).
- Alteration of removal of plant
growth regulator concentrations in the preloading, cryoprotectants and recovery
medium.
- Variations in exposure times
to preloading and cryoprotectant (PVS2) solutions
- Various shoot tip post-freezing
washing techniques
- LN2 exposure instead of LN2
slush exposure
- Alteration of recovery medium
agar concentrations
Sweet
potato encapsulation/vitrification
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Sweet potatoes are grown and shoot
tips are excised as described in steps 1-3 in the Vitrification Procedure.
The encapsulation/vitrification protocol followed is described in Hirai
and Sakai, 1999 and Reed, 2001. All solutions (2M Glycerol+0.4M sucrose, PVS2
and 1.2M sucrose) used within the protocol were modified using the same solutions
as described in the vitrification sweet potato solutions*.
*All
osmotica, Cryoprotectant, and medium recipes are specific for sweet potatoes.
References
~~~~~~~~~~~~~~~~~~~~
Hirai, Dai and
Akira Sakai. 1999. Cryopreservation of in vitro-grown meristems of potato
(Solanum tuberosum L.) by encapsulation-vitrification. Potato Research
42: 153-160.
Murashige, T.
and F. Skoog. 1962. A revised medium for rapid growth and bioassays
with tobacco tissue culture. Physiology Plantarum 15:473-497.
Pennycooke, J.C.
and L.E. Towill. 2000. Cryopreservation of shoot tips from in vitro
plants of sweet potato [Ipomoea batatas (L.) Lam.] by Vitrification.
Plant Cell Reports 19:733-737.
Reed, B.M.
2001. The basics of in vitro storage and cryopreservation. National
Clonal Germplasm Repository-Corvallis. Corvallis, OR, USA. Pp. 30.
Towill,
L.E. and R.L. Jarret. 1992. Cryopreservation of sweet potato (Ipomoea
batatas [L.] Lam.) shoot tips by vitrification. Plant Cell Reports
11:175-178.
Recipes
~~~~~~~~~~~~~
All
nutrient medium is based on medium of Murashige and Skoog (1962).
Sweet potatoes have been observed as sensitive to NH4.
Therefore, the macroelement NHNO3 is omitted from modified
sweet potato recovery media and vitrification solutions. All recipes are for 500ml total volume and
based on stock solutions.
Sweet
potato medium
|
Macroelements
NHNO3 5 ml
KNO3
5 ml
MgSO4 5 ml
CaCl2 5 ml
KH2PO4 5 ml
Iron 5 ml
|
Micronutrients
5 ml: MS vitamins 5ml
Disaccharides
Sucrose (5% use table sugar) 25g
pH 5.7
Gelling
Agent
Agar ( .7%) 3.5 g
|
Sweet
Potato Vitrification
All
Vitrification solutions are the same as noted on Medium
Recipes with exception of omitted NHNO3.
2% sucrose
0.3M sucrose
0.4M
Sucrose + 2M glycerol
PVS2
1.2M
sucrose
Modified
Sweet Potato Recovery Medium
This medium is
the same as the Sweet Potato Medium with the following additions and omission
of NHNO3:
Add
the following growth regulators:
Kinetin (.0001 mg/ml) .232 ml
BAP (.0005 mg/ml) 5 ml
IBA (.0001 mg/ml) .25 ml
Sweet
Potato Encapsulation/Vitrification
Sweet
Potato CaCl2 Medium
This medium is
the same as the Sweet Potato Medium with the following substitutions:
Replace 25g table
sugar with Sucrose (.35M Grade II) 30g
Add 3.65g of CaCl2 instead of .5mls of macroelement stock
Sweet
Potato 0.4M sucrose and 3% Alginate Beads Medium
This medium is
the same as the Sweet Potato Medium with the following substitutions and omissions:
Omit CaCl2 macroelement
from medium
Replace 25g table sugar with Sucrose (.4M Grade II) 34.23g
Before heating
very, very slowly add Alginic acid
Alginic Acid (3%) 7.5g
Heat after it
is all dispersed in the liquid. If it is heated first, it will form an
insoluble ball. The ball will not melt in the autoclave unless it is well
dispersed first.