Long term conservation through dormant bud cryopreservation
Authors: Vartika Srivastava
Scientist, Tissue Culture and Cryopreservation Unit
ICAR-NBPGR, Pusa Campus, New Delhi



Dormant bud cryopreservation

Advances in biotechnology have generated new opportunities for genetic resources conservation and utilization and maintenance of plant materials at cryogenic temperatures (cryopreservation) is now a suitable option for long-term storage. Dormant vegetative buds of diverse species can be preserved using cryopreservation. Since long, these have been used to propagate identical clones of fruit crops as a routine procedure. The dormancy of these buds can be utilized for cryopreservation. Cryopreserved dormant buds can be regenerated either in vivo or in vitro and propagated further. Cryopreservation of dormant buds offers distinct advantages viz., genetic integrity is maintained, long term storage, simple process and space efficient and low cost of maintenance. Sakai (1960) provided one of the first studies showing that winter twigs of popular and willow can survive ultra low temperatures of liquid nitrogen (LN), when slowly cooled prior to immersion in LN. Later a study demonstrated that this simple technique is also applicable to twigs of several fruit species (Sakai and Nishiyama, 1978).

The method of cryopreservation of dormant buds includes different techniques viz., controlled rate cooling, vitrification and encapsulation dehydration. The selection of the method depends on the species as well as its cold hardiness. Eg. Many apple species are cold hardy and nodal sections can be directly used for cryopreservation using controlled rate cooling (Forsline et al. 1998; Towill and Bonnart, 2005). Persimmon is not as cold hardy as apple and thus its shoot tips excised from dormant buds can be cryopreserved through vitrification (Matsumoto et al. 2001).

General Methodology of dormant bud cryopreservation:

Two-step freezing

Nodal explants (2.0-2.5cm long, one dormant bud attached on nodal segment) are isolated and kept in steel container in charged silica gel at -10˚C for desiccation till the moisture content reaches around 23-25%. These desiccated buds were then used for cryopreservation in liquid nitrogen at -196˚C using two-step freezing protocol. The two-step freezing was achieved by sequentially lowering the temperature at -5˚C per day up to terminal temperature of -30˚C before plunging in liquid nitrogen at -196˚C. The desiccated buds were then sealed in heat shrinkable tubes with plugs in both ends. These tubes were shifted sequentially at 5˚C, 5˚C, -10˚C, -15˚C, -20˚C and -25˚C, and -30˚C keeping at each of the temperature for a minimum of 48 hrs. The tubes were held at -30˚C for 72 hrs and then directly plunged in the liquid nitrogen at -196˚C.



Recovery method

Cryopreserved nodal sections are thawed by slow thawing. The nodal sections were placed at 5˚C for 48 hour for thawing. These are rehydrated in sterile moist peat vermiculite for 7 days at 5˚C. The viability of fresh and cryopreserved dormant buds can be tested through TTC and in vitro culturing method or by direct grafting on to desired root stock.

Viability by TTC method

The longitudinal sections of the fresh and cryostored buds (with scales) are cut and transferred in 1% (w/v) TTC solution. These sections were incubated at room temperature in dark for overnight and observation can be taken under the stereo microscope.

Viability by in vitro method

The buds excised from the rehydrated nodal sections were taken and 4 to 5 outer scales were removed using microscope. The explants were washed with Tween 20 for 15 minutes. Tween 20 was rinsed off with running tap water. These buds were then surface sterilized with 0.1% mercuric chloride for 9 minutes, rinsed three times with autoclaved distilled water (5 min each). The sterilized buds were cultured on the suitable multiplication medium standardized for that species. The sprouted buds were then sub-cultured on the fresh medium for further multiplication and root induction.

Critical factors for successful cryopreservation:

Most important factors for successful cryopreservation of dormant buds include extent of cold acclimation, extent of desiccation obtained before cooling and cryopreservation method used.


References:

Forsline PL, Towill LE, Wddell JW, Stushnoff C, Lamboy WF, McFerson JR. (1998). Recovery and longevity of cryopreserved dormant apple buds. J Am Soc Hortic Sci 123:365-370.

Matsumoto T, Mochida K, Itamura H, Sakai A (2001). Cryopreservatio of persimmon (Diospyros kaki Thumb.) by vitrification of dormant shoot tips. Plant Cell Rep 20:398-402.

Sakai A (1960). Survival of the twig of woody plants at -196˚C. Nature. 185:392-394.

Towill LE, Bonnart R (2005) Cryopreservation of apple using non-desiccated sections from winter-collected scions. Cryoletters 26:323-332.


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