FIELD OF THE INVENTION

Deschampsia antarctica is an endemic grass of peninsular Antarctica. This plant is luxuriantly grown in the arctic regions where the temperature is between 15-20 °C. This plant shows great UV protection activity. An effort is made to grow this plant in Indian conditions where temperature is generally varies between 20-30 °C.

Vegetative propagation is slow and unable to generate enough plant material for laboratory requirements. Micro propagation of D. antarctica that can solve the problem of availability and prevent excessive collection from its natural habitat.

Deschampsia antartica normally reproduces both by seed and self-fertilization and also vegetatively from tillers (Holderegger et al. 2003). This vegetative propagation is very difficult in Indian climatic conditions to propagate and also to generate enough plant material for laboratory requirements and also for large-scale production. We have made an attempt to develop in vitro method for the production of large scale in short periods. This is the first report for shoot initiation, root initiation from shoot tip with different hormonal treatments and hardening of this plant. In present invention maximum numbers of shoots (23.2±2) were noticed from shoot tip. In earlier reports by Mareley et al. (2005) shooting (25.4±1.3) and rooting response from callus was reported. In our investigation rooting was achieved 100% in the medium containing Vz MS +lmg/l IAA+ O.lmg/1 Kinetin + 0.75 mg/1 CuS04 with 2% sucrose, there were no previous literature available for the rooting and hardening of this plant.

BACKGROUND OF THE INVENTION

The present invention pertains to micropropagation and multiplication studies on Deschampsia antarctica - a frost resistant plant from arctic region. The present invention further relates to root and shoot initiation using tissue culture method. Thus grown Deschampsia antarctica in controlled condition, may give enhanced quantities of secondary metabolites further, which will be used as medicine or medicament.

OBJECTIVES OF THE INVENTION

One objective of the invention is to provide a highly efficient in vitro system for micro-propagation of Deschampsia antarctica.

Yet another objective of the invention is to provide a process for induction of multiple shoots and roots of Deschampsia anractica.

Yet another objective of the invention is to identify the explants, media and culture condition for producing large number of Deschampsia prazeri.

Yet another objective of the present invention is to harden the plant material, which was developed in in-vitro conditions.

SUMMARY OF THE INVENTION

Deschampsia antarctica is one amongst two angiosperms available in Peninsular Antarctic region. The plant is generally grown in cold, saline and moist windy climates. The slow growth of this plant by vegetative and by seeds prompted us to carry out in-vitro propagation studies of this plant. The plant has got UV radiation protecting activity, which may lead to the use of this plant as a medicine or medicament. We have made an attempt to develop in vitro method for the production of large scale in short periods.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Culture plates received from Chile

Figure 2: Cultures after incubation in controlled condition

Figure 3: Cultures after incubation in controlled condition (After 5 days)

Figure 4: Cultures transferred to the bottles after 5 days

Figure 5: Cultures transferred to the bottles after 10 days

Figure 6: Cultures transferred to the bottles after 30 days

Figure 7: Multiple shoots of after 45 days

Figure 8: Multiple shoots of after 60 days

Figure 9: Plant showing roots in MS+1.0mg/l IB A (40 days)

Figure 10: Plant showing roots in 1/2 MS+1.0mg/l IAA+2% Sucrose (60 days old)

Figure 11: Close up view roots in 1/2 MS+1.0mg/l IAA+2% Sucrose (60 days old)

Figure 12: Plant showing roots in MS+1.0mg/l IAA+3% Sucrose (60 days old)

Figure 13:Plant showing roots in MS+1.0mg/l NAA+ 3% Sucrose (60 days old)

Figure 14: Plant showing roots in l/2MS+1.0mg/l NAA+ 3% Sucrose (60 days old)

Figure 15: Plant showing roots in l/2MS+1.0mg/l ffiA+3% Sucrose (60 days old)

Figure 16: Primary hardening (After 30 days)

Figure 17: Secondary hardening (After 60 days)

Figure 18: Secondary hardening (After 90 days)

DETAILED DESCRIPTION OF THE INVENTION

Collection of plant material:

The Deschampsia antarctica plant material (Starting material) was brought from Chile and was maintained in controlled condition and these plants were incubated at 22°C at 16/8 h light/dark as a mother plants for further invention. Before bringing it to the lab germplasm was deposited in NBPGR, India. Preparation of the media:

Stock solutions of the media ingredients were prepared in distilled water. Appropriate aliquots of these solutions were mixed to prepare the media. Concentrations of the macro- and micro-element salts and organic constituents of the MS (Murashige and Skoog, 1962) basal medium. Stock solutions of growth regulators (PGR) were prepared and stored. For media preparation a calculated amount of aliquots were added from these stock solutions. Carbohydrate was weighed and added in required quantity and allowed to dissolve.

Unless mentioned, pH of all the media was adjusted to 5.7 using IN NaOH or IN HC1 after mixing all the constituents except the gelling agent. The volume was made up with distilled water. Gelling agent (agar agar or phytagel) was then added. Culture media was autoclaved for 20min. at 121°C and 15 lbs/(inch) 2. Autoclaved media was poured in sterile petri dishes before gelling, as and when required. Auxins and 3ml/l of PPM™ used for rooting media were filter sterilized through Millipore membranes (0.22 mM) and added aseptically to the autoclaved liquid medium or in the autoclaved semisolid medium before it gels.

Procedure:

Inoculation of the plant material is carried out in laminar hood; Cultures were initiated from the explants in laminar airflow cabinets. All the dissections were carried out on sterile filter papers. The instruments used for aseptic dissection or transfers of tissues were pre-autoclaved. During

aseptic operations these were flamed intermittently after dipping in rectified spirit. After transferring the tissues, the petri dishes were sealed with cling wrap. All cultures were labeled appropriately prior to incubation.

For multiplication of plantlets shoot clumps were obtained (2-3 shoots) they were separated into smaller clumps (single shoot) and sub cultured into fresh MS medium supplemented with 1 mg/1 BAP, Kinetin 0.1 mg/1, CuSo4 0.75 mg/1, 3% Sucrose.
Results:

After 30 days of incubation shoots were 2.0 cm length these shoots were sub cultured in the same MS medium with the same hormones under 16/8 h light/dark. On first subculture of plantlets 4-5 multiple shoots observed after 30 days from the basal region. After 45 days of incubation of these plants, plants lets showing 5-7multiple shoots.6-8 multiple shoots are arising from the basal region, vigorous growth has been observed after 60 days with shoot length recorded 6-7 cm.

In some of the cultures rooting were observed after 45 days and 60 days of cultures. Latent contamination is particularly dangerous because it can easily be transferred among cultures. Initially our cultures are affected with bacterial/yeast contamination in primary subculture, for this contamination the cultures are controlled by adding the 3 ml of PPM (Plant Preservative mixture) to the media, now the cultures are in good condition. The plants that are contaminated recovered by transferring into PPM™ containing medium.
The primary shoots that obtained from BAP (1 mg/1) and kinetin (0.1 mg/1) containing medium upon sub-culturing into the same medium show significant shoot elongation was noticed from the base of shoot, of the explants within 8-10 weeks. When an explant has grown into a plantlet with two to three leaves, transfer it to fresh medium every 2 months.

Elongated shoots (4-5 cm) were transferred to rooting medium supplemented with NAA (1 mg/1) for rooting, roots were observed after 15 to 20 days. These obtained roots were failed to elongate, for the vigorous root initiation the media composition has been modified with different concentrations auxins (IAA, IBA and NAA). The maximum roots (were observed in Vi MS +lmg/l IAA with 2% sucrose, as compared with other combinations tried. 12-15 roots were noticed with root length of 1.5-2.0cm. These proliferated plantlets were shifted to cavity pots containing 9:l(Soilrite: perlite) for the primary hardening in laboratory condition. For the secondary hardening these plantlets were shifted to pots containing Soilrite: Sand: Redearth: FYM (5:1:1:3) in green house condition.

These plants well acclimatized to the green house condition (20±2 °C with 70% humidity) showing luxuriant growth.

The best root response (12-15 roots/plant) was observed in the presence of V2 MS +lmg/l IAA+ Kinetin 0.1mg/l + CuSo4 0.75 mg/1 with 2% sucrose was found to be significant, higher than roots induced from the base in other concentration of auxins (IAA, IBA and NAA in either MS or half strength MS basal medium) used in the present study.

The present study describes a well-documented and reliable Micro-propagation protocol of D. antarctica form basal portion (tillers) with much higher rate of multiplication. This protocol can be used as a basic tool to commercialize cultivation of the frost resistant plant.

Its effects would be studied in Ultraviolet radiations (in Different wavelengths). Further, studies on tolerance to salinity stress, leading to cell dehydration would also be carried out. Main objective is to mass multiply this plant so as to make it commercially feasible and equally effective as compared to native Antarctic Deshampsia antarctica plant. DNA profiling of mother plant with in-vitro grown Deschampsia antarctica by AFLP method would also be conducted and the germplasm would be conserved through cryo-preservation. References:

1. Leon A. Bravoand Marilyn Griffith, Characterization of antifreeze activity in Antarctic plants, Journal of Experimental Botany, Vol. 56, No. 414, pp. 1189-1196, April 2005 and Advance Access publication 21 February, 2005.

2. Holderegger, R., Stehlik i., Lewis Smith R.I. & Abbott R.J. 2003,Populations of antarctic hairgrass (Deschampsia antarctica) show low genetic diversity. Arctic, Antarctic and Alpine Research, 35, 214-217.

3. Marely Cuba, Ana Gutierrez-Moraga, Barbara Butendieck And Manuel Gidekel, Micropropagation of Deschampsia antarctica - a frost-resistant Antarctic plant, Antarctic Science 17(1): 69-70 (2005)

4. Murashige T and Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15(3): 473-497.

5. Katarzyna J. Chwedorzewska Preliminary genetic study on species from genus Deschampsia from Antarctic (King George I.) and Arctic (Spitsbergen), Polar Bioscience, 19,142-147,2006

1) A method for in-vitro micro propagation of Deschampsia antarctica , said method comprising step of: inoculating explants into modified murashige and skoog medium supplemented with combination of growth hormones comprising Benzyl amino purine at a concentration ranging from 0.25 mgL'1 to 3.0 mgL"1, preferably at a concentration of about 0.5 mgl"1 and Napthalene acetic acid at a concentration ranging from 0.01 to 0.05 mgL"1, preferably at a concentration of about 0.01 mgl"1, optionally along with Gibberlic acid at a concentration ranging from 0.01 0 mgL"1 to 0.05 mgL"1 , preferably at a concentration of about 0.01 mgl"1 followed by incubation to obtain micro propagated Deschampsia Antarctica.

2) The method as claimed in claim 1, wherein said explants are surface sterilized sequentially with Tween 20, cetrimide, bavistin and mercuric chloride.

3) The method as claimed in claim 2, wherein said sterilization with Tween 20 is performed for a time period ranging from 10 to 15 minutes followed by rinsing with water for about 15 minutes.

4) The method as claimed in claim 2, wherein said sterilization with cetrimide is performed for a time period ranging from 8 to 10 minutes followed by rinsing with water for about 15 minutes.

5) The method as claimed in claim 2, wherein said sterilization with bavistin is performed for a time period ranging from 15 to 20 minutes followed by rinsing with water for about 15 minutes.

6) The method as claimed in claim 2, wherein said sterilization with mercuric chloride is performed for a time period of about 5 minutes followed by rinsing with water for about 15 minutes.

7) The method as claimed in claim 1, various explants is selected comprising leaf, leaf petiole, stem internodal segment, stem nodal segment, apical bud and auxiliary bud.

8) The method as claimed in claim 1, wherein said incubation is carried out for a time period ranging from 14 to 16 hours of day time and 6 to 8 hours in dark at temperature of about 25± 2°C.

9) The method as claimed in claim 1, wherein said harvested multiple shoots are washed and transferred to coco peat mix with red soil for hardening. MwV^ftVvv. 7