USE OF CRYOPRESERVATION IN ORNAMENTALS.

1. PRESENTED BY:
SHEEBA BELWAL
I.D No: 44440
Department of Horticulture
Floriculture and Landscaping
G.B.P.U.A&T Pantnagar, Uttarakhand

2.  Use of very low temperatures to preserve
structurally intact living cells and tissues.
 Cryopreservation (Greek, krayos-frost) means
preservation in the frozen state.
 Principle:
To bring the plant cell and tissue cultures to a zero
metabolism or non-dividing state by reducing the
temperature in the presence of cryoprotectants.

3.  James Lovelock was the first theoritician of
cryopreservation.
 The first information on cryopreservation of
ornamental species was reported by Fukai
(1989) and regarded Dianthus hybrida.

4.  It is ideal method for long term conservation of
material.
 Disease free plants can be conserved and
propagated.
 Recalcitrant seeds can be maintained for long time.
 Endangered species can be maintained.
 Pollens can be maintained to increase longivity.
Rare germplasm and other genetic manipulations
can be stored.

5.  i. Over solid carbon dioxide (at -79°C)
 ii. Low temperature deep freezers (at -80°C)
 iii. In vapour phase nitrogen (at -150°C)
 iv. In liquid nitrogen (at -196°C)

6.  Morphological and physiological conditions of
plant material influence the ability of
explants to survive during cryopreservation.
Different types of tissues can be used for
cryopreservation such as:
 Ovules
 Anther/pollen
 Embryos
 Endosperm
 Protoplast, etc.

7.  Tissue must be selected from healthy plants.
 Small and Young
 Rich in cytoplasm
 Meristematic cells can survive better than
the larger
 Highly vacuolated cells.

8.  Pregrowth treatment protect the plant tissues
against exposure to liquid nitrogen.
 Pregrowth involves the application of additives
known to enhance plant stress tolerance. Eg.
abscisic acid, proline, trehalose.
 E.g. C. roseus cells are precultured in medium
containing 1M sorbitol before freezing. (Chen et
al., 1984) Digitalis cells were precultured on
6% Mannitol medium for 3 days before freezing.
(Seitz et al., 1983) Nicotiana sylvestris with 6%
sorbitol for 2-5 days before freezing. (Maddox et
al., 1983)

9. The cryopreservation technique followed by
the regeneration of plants involves following
steps :
 1. Selection of material: meristem, embryo,
ovules, seeds etc.
 2. Addition of cryoprotectant:These are
sucrose, alcohols, glycols, some amino acid
(proline), DMSO (dimethyl sulfoxide)
 3.Freezing:The sensitivity of the cells to low
temperature is variable and largely depends
on the plant species.

10.  1. Slow-freezing method:
0.5-5°C/min from 0°C to -100°C, and then transferred to liquid
nitrogen. used for the cryopreservation of suspension cultures.
 2. Rapid freezing method:
-300° to -1000°C/min. Used for the cryopreservation of shoot tips
and somatic embryos.
 3. Stepwise freezing method:
The plant material is first cooled to an intermediate temperature
and maintained there for about 30 minutes and then rapidly
cooled by plunging it into liquid nitrogenused for
cryopreservation of suspension cultures, shoot apices and buds.
 4. Dry freezing method: dehydrated cells are found to have a
better survival rate after cryopreservation.

11.  4. Storage in liquid nitrogen: 70 to -196 degree. Prolong storage is
done at temperature of -196 degree in liquid nitrogen.
 5. Thawing: Thawing is usually carried out by plunging the frozen
samples in ampoules into a warm water (temperature 37-45°C) bath
with vigorous swirling.
 6. Washing and reculturing: The preserved material is washed few
times to remove the cryoprotectant. This material is then recultured
in a fresh medium.
 7. Measurement of viability: It is calculated by formula : No of
cells growing / no of cells thawed * 100.
 8. Regeneration of plants: Addition of certain growth promoting
substances, besides maintenance of appropriate environmental
conditions is often necessary for successful plant regeneration.

13.  Arabidopsis shoot tips revealed that cryoprotectant
treatments induce gene expression and critical
pathways may include those involved in lipid
transport and osmoregulation.
 The vitrification method has been used in the
cryopreservation of pollen from two different
cultivars of the genus Dendrobium: D. 'Sena Red
Thailand' and D. 'Mini W/RL'. Chrysanthemum
grandiflora (Halmagyi et al., 2004), Humulus spp.
(Reed et al., 2003), Acer mono (Park et al., 2005),
Gentian spp. (Tanaka et al., 2004), Dianthus
caryophyllus (Halmagyi and Deliu, 2007), and
Ribes spp. (Johnson et al., 2007).

14.  Camellia spp. (Ballester et al., 1997), Humulus spp. (Reed et
al., 2003), Nerium oleander and Photinia fraseri
(OzdenTokatli et al., 2008), Splachnum ampullaceum
(Mallón et al., 2007), and Rosa (Previati et al., 2008) deal
with the in vitro conservation of ornamental plants, all based
on the cold storage approach for generating Slow growth
storage.
 Cryotherapy is being successfully applied to many species of
commercial and ornamental use. Em Chrysanthemum
morifolium, Jeon et al. (2016) demonstrated that
cryopreservation was determinant in the efficacy of
elimination of the virus complex chrysanthemum stunt viroid
(CSVd).
 Beneficial for pyrethrin biosynthesis by Chrysanthemum
cinerariaefolium cell cultures.

15.  It was shown that employment of slow dehydration
method for preparation of plant material for
cryopreservation allowed to obtain a viable callus
tissue of Rosa ‘New Dawn’ and propagating
Phlebodium aureum gametophytes.
 In case of chrysanthemum, there is only one report
of the application of synseed technology.
 Among the cryopreservation protocols for orchids,
vitrification is an effective, simple, safe, inexpensive
technique that is applicable to a wide range of orchid
explants.