CRYOPRESERVATION IN ORNAMENTAL CROPS

USE OF CRYOPRESERVATION IN ORNAMENTALS.

1. Cryopreservation as a method to conserve genetic resources in ornamental crops PRESENTED BY: SHEEBA BELWAL I.D No: 44440 Department of Horticulture Floriculture and Landscaping G.B.P.U.A&T Pantnagar , Uttarakhand

2. Cryopreservation 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. HISTORY 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. Application 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. Storage of germplasm at very low temperatures: 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. SELECTION OF PLANT MATERIAL 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. FACTORS: 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 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. Mechanism of cryopreservation 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. Four different types of freezing methods 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.

12. Application of cryopreservation to herbaceous species 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).

13. 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.

14. 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.

15. THANK YOU