FIELD OF INVENTION

The present invention relates to methods for the cryopreservation of plant cells and to methods for the recovery of plants cells which have been cryopreserved. The invention also relates to plants, viable plant cells and plant cells cultures which have been successfully recovered from cryopreservation.

BACKGROUND OF THE INVENTION

Long term preservation of plant germplasm is possible by storage of plant tissues under liquid Nitrogen (-196°C) such a technology can be effective in storage clonally propagated germplasm as well as seeds. This presumed to be remains without any genetic changes for very long duration. Cryopreservation of rare plants, clones, plant varieties also routine use of plant germplasm are in practice. The technology also useful in elimination viruses. The technique of storing plant tissues in liquid nitrogen temperatures requires development of protocol which includes slow freezing, cold acclimatization and vitrification procedures as also protocols are optimized in combination of two or more of these techniques. The present invention provides a method to preserve the Antarctic vascular plant D. antarctica can survive or tolerate freezing temperature below sub zero temperature. Cold acclimatized cells can survive by production of dehydrins/antifreeze like proteins. The procedure is devised to accumulation of these proteins by incubating the plants under low temperature before cryopreservation of the plants by vitrification protocol, which protects the plants under ultra low temperature.

OBJECTIVES OF THE INVENTION

1. Germplasm conservation of the Deschampsia Antarctica.

2. Achieve Cryopreservation of Deschampsia antarctica by combination of cold acclimatization and vitrification techniques.

3. Assess the plant ability to survive under sub-zero temperature -20°C and -196°C and subsequent recovery.

4. Design optimum recovery medium for recovery of the plant from cryogenic storage

BRIEF SUMMARY OF INVENTION

The Deschampsia antarctica plant inhabits the maritime antarctica and survives in sub zero temperatures. The plants remain dormant during winter and new growth is observed in summer.

The plants survives by adapting to several mechanism including reduced photosynthesis, accumulation of higher levels sucrose and encoding several antifreeze proteins which protects plants in the sub zero temperature (Bravo and Griffith 2005, Gidekel et. al. 2003, Zuniga-Feest 2005). The protocol was devised based on the cold acclimatization of Deschampsia antarctica by exposing the plants to freezing temperature for accumulation of protective substance, high level of sucrose antifreeze proteins that protect plant cell by super freezing the water in the cell, which is needed for cellular activity. Further to cold acclimatization, the plants are exposed to the cryoprotective solution, which mcludes sucrose, dimethyl sulfoxide (DMSO), glycerol, and ethylene glycol, which desiccates the cells, by vitrification under liquid nitrogen temperatures (-196°C). The recovery of the plants from cryogenic condition after exposure to freezing temperature and ultra low temperatures was achieved in these plants by combination of cold acclimatization and subsequent vitrification. Plants are recovered by preliminary viability assessment by use of viability staining (Fluorescein diacetate) and visualization under confocal microscopy. Recovery of cryopreserved plant was assessed by growing dormant buds retrieved from cryopreservation on a recovery medium designed. Recovered plants are later transferred to multiplication medium, rooting medium and than to the hardening in soilrite/coirpeat.

DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1: Cold acclimatization at +4,-10 and -20°C

Figure 2: Exposure to PVS2

Figure 3: Viability of shoot apical meristematic tissue after cryopreservation assessed by FDA staining

Figure 4: Recovered plants after cryopreservation

Figure 5: Rooting of cryopreserved D. antarctica in a Activated charcoal medium

Figure 6: Cryopreserved D. antarctica plants transferred to coir-peat

DETAILED DESCRIPTION OF THE INVENTION

Collection of plant material:

Deschampsia antarctica culture plates have been received from Chile and 2 samples were deposited in NBPGR for germplasm storage.

Procedure

The tissue culture plant of D. antarctica are sleeted for the cryopreservation by vitrification,

Deschampsia antarctica cultures were screened for dormant side buds which were separated from the cultures.

The buds were dried in laminar airflow bench for 3-5 minutes to remove excess moisture
The plantlets were exposed to two different cold treatments (4 to -20°C and -196°C)
Sixteen sets of cultures were exposed to 4°C for two weeks and later to-10°c and -20°C for same duration.(Figure 1)

Cold acclimatized buds were transferred to cryovials 1.8ml and modified PVS2 and glycerol sucrose solution 1:1 was added separately (ca. 1ml) vials were shaken for 2-3 minutes. (Figure 2)

The vials were incubated in 4°C for infiltration of cryoprotectant solution for 20 minutes.
Later the vials were directly immersed in liquid nitrogen and transferred to liquid nitrogen storage container.

Vials are retrieved by thawing in water bath at 35**C-40°C one minute.

The viability of the shoot apical meristem was assessed by means of FDA staining and visualized under con-focal microscopy Shoot apical meristem was recovered from ultra low temperature before testing the cell viability using FDA method (Yamori et. al., 2005).

Shoot apical meristem was recovered from ultra low temperature and was transferred to liquid nitrogen container before testing the cell viability using FDA method (Yamori et. al., 2005)

Plant sample was examined under stereomicroscope and shoot apical meristematic tissue was dissected.

Apical meristem was transferred to buffer saline in cryovials and 20ul of FDA in acetone and water mixture stain solution was added. The vials were incubated in 4°C in a refrigerator for 20 to 30 min. The plant parts were transferred to microslide and cover slip was mounted, the specimen was observed through Olympus Flow-View 1000 confocal microscopy under 480nm excitation filter. The bright green fluorescence was observed showing good viability of cells (Figure 3)

Recovery and growth of cryopreserved plants of D. autartica plants

The plants cryopreserved in liquid nitrogen were assessed for recovery percent in the medium designed. The shoots buds cold acclimatized at +4*0 followed by -10 and -20''C

The shoot buds not acclimatized and were growing at 22"'C (control)

These experimental sets, which were cryopreserved, were retrieved from the cryocans. The 16 plates from these experiments were transferred to recovery medium and incubated for growth of multiple shoots. 50 plant lets are used as a control without cold acclimatization and exposure to liquid nitrogen. The plants were also transferred to the medium with reduced sucrose concentration of 0.5 per cent to induce phototropic condition. The medium was designed to high recovery of D. antarctica plants from cryogenic storage and induction of quick root and multiple shoots

MS basal medium supplemented with

Adenine sulphate -160mg/l
Tyrosine -200mg/l
Casein hydrolysate -1000 mg/1
Thiamine HC1 -1 mg/1
Sucrose -3 per cent
Benzyl adenine -5 mg/1
Indole Acetic Acid -2 mg/1
pH- -5.6

The plants exposed to Liquid nitrogen were found to be affected by chlorosis and stunted growth. The plants transferred showed good recovery from cold treatment after 2-3 days of culture. (Figure 4)Total of 16 plates with 115 plants were cultured on the recovery medium. Total of 78 plants were found recovered with in eight days of culture and multiple shoot development observed after 75 days of culture full development of multiple shoots were observed after the duration of 90 days. Plants recovered from cryopreservation with the induction of multiple shoots was transferred to regular (MS medium with BAP-1.0 mg/1, Kinitin O.lmg/1, CuSo4 -0.75 mg/1, Sucrose -30 g/1, pH-5.7) medium for further growth and multiple shoots development.

Rooting of cryopreserved D. antarctica Plants

The plants recovered from cryopreservation experiment are fiirther transferred to rooting medium for induction of roots and further hardening of the plants. Cryopreserved D. antarctica plantlets with multiple shoots are transferred to rooting medium with the following combination

Basal MS medium salts
MS Vitamins modified 100 rag/1
lAA 1.0 mg/1
Kinetin 0.lmg/1
Sucrose 30 g/1
Activated charcoal Ig/L
PPM 3ml/L
CUSO4 0.75mg/L
pH 5.7

The profuse rooting was observed after 20-25 days after transferring. (Figure 5)

Hardening of/), antartica cryopreserved plants in soilrite

The rooted plants of D. antarctica were tested for ability to grow on soilrite medium (perlite and vermicompost, 50:50). The 1-1.5cm rooted plantlet are washed in distilled water and immediately transferred to soiWte enriched with MS basal salts and GA3 50ppm for better root growth in a polycarbonate box with the closing lid to maintain 90 per cent humidity. The plants are placed 3cm apart and kept for day/night photoperiod 16/8h and soilrite temperature of 26°C±2*'C. After one week of observation with watering once in two days, the all plants are recovered and growing healthy with new crown development.

Transfer of Cryopreserved D. antarctica plants for hardening in Coir-peat medium

The coir-peat was used for hardening of the Cryopreserved D. antarctica plants. The additional nutrient supplements GA3 20 PPM were added to the coir peat. The rooted plants of D. antarctica was washed gently in distilled water to remove adhering agar medium and transferred to the coir-peat medium in the poly box. Totally three-culture bottle were transferred (Figure 6). Initially pre-hardening was carried out at Day/Night photoperiod of 16/8h and humidity of 90 percent and plants are maintained under green house condition with 22°C and 90 percent humidity.

Table- 1 Recovery of 2). Antarctica

We Claim

1. A process for preservation of plant tissues comprising the steps of dehydrating, pre-freezing and cryo-freezing plant derived tissue, wherein the plant tissue to be cryo-preserved is a primary explant that has been subjected to an induction treatment for regeneration.

2. The process of claim 1, wherein the plant tissue is derived from a orthodox species.

3. The process for the cryo-preservation according to any of the claims 1 and 2 wherein the plant tissue is a primary explant capable to regenerate buds.

4. The process for the cryo-preservation according to any of the claims 1 and 2, wherein the plant tissue is a primary explant capable to regenerate after exposure to 4°C to -196°C.

5. The process for the cryo-preservation according to any of the claims 1 to 4, wherein the plant tissue utilised is derived from a plant belonging to the species Deschampsia antarctica.

6. The process for the cryo-preservation according to above claims wherein the plant tissue utilized is derived from Dischampsia antarctica and use of combination of cryopreservation methods cold acclimatization and vitrification.