Picrorhiza Kurroa

Different populations of P. kurroa were studied in their natural habitat (>2000 m msl) to understand the plant for domestication. Following observations were recorded on morphological features.

Habitat: The plant is naturally distributed in alpine and temperate regions of Himalaya from 2500 to 3500 m. It is a perennial creeping herb, which spreads by stolons. A whorl of radical leaves arise from rhizome tip. The flowering scape attains an average height of 16.0 to 17.5 cm.

Root morphology:  Adventitious roots arise from the rhizome. Maximum average root length (38 cm) was observed in case of PK-6. Maximum average primary roots (5) were recorded for PK6 and PK7. PK5 recorded highest number of secondary roots (52) and maximum fresh weight (230 g) of the roots. 

Root characteristics of different accessions

Shoot morphology:  Stem is represented by stolon and underground rhizome which bear leaves and flowering scape. Maximum fresh weight (375.19g) of the shoot was recorded in PK4

Flowering: Flowering occurs in one or two phases depending upon altitude of the growing site.  Under relatively lower elevation of 2500-3500 m msl, the fist phase starts in 1st week of May and continues up to 3rd week of June and the second flowering begins in August and continues up to end of September.  In alpine regions (>4000 m), flowering occurs only once in July-August and seeds develop in September. Flowers are borne on a scape in an indeterminate spike forming more or less a triangular head. Flowers are purple coloured, bisexual and having convex thalamus.

Maximum number of inflorescences (15.67) was recorded for PK2 and longest inflorescences (33.92 mm) in PK1.  Maximum number of flowers (11.83) was recorded in PK3. Maximum length of bract (5.17 mm) was recorded in case of PK7 while maximum breadth was recorded for PK6.

Fruit: Fruit is an ovoid capsule which dehisces by means of lateral splits.  The average dimension of fruits was 9 x 5 mm.

Seed: The extremely small sized seed is 1.3 x 1 mm in size.  Embryo is enclosed in the large bladdery loose hyaline reticulate testa. The seeds test weight was about 57.5 mg.

Cultivation in field polyhouse: Cultivation was undertaken in two different seasons using fresh rhizomes as propagule.  Crop performance was better in polyhouse than in open field in terms of plant spread, height, leaf- size and growth of stolons.  Autumn season (mid September to mid October) was favourable for establishment of the transplants and production of viable seeds over summer season (April-May). Crop remained dormant during January-February in field but it grew profusely in polyhouse.  The crop in open field was also adversely affected due to heavy rains during July-August.  The crop in polyhouse was infested with white fly.  Black spot fungal disease was noticed in both the conditions of cultivation.

Seed production: A breakthrough was achieved with regard to viable seed production at relatively lower elevation (1300 m msl) at Palampur in polyhouse. Time of planting was standardized so that seed development took place before onset of summer which is not favourable for seed development under the subtropical conditions. 

Seed biology: Freshly harvested seeds showed 100% germination when inoculated on basal MS medium supplemented with 3% sucrose (Table 2.11).  However, with storage, the germination percentage declined gradually and seeds became non viable beyond 9 months.

Table Seed germination (%) of P. kurroa

In order to understand the biology of seeds, plants of eight accessions were maintained in polyhouse and their flower initiation, fertilization/anthesis, fruit set and stage of fruit/seed development were recorded (Table 2.12; Fig. 2.13).

Table Calendar for reproductive phase in P. kurroa

RH was maintained at 65-70% in the poly tunnel

About 36% germination was observed up to 6 weeks of inoculation and MS with 3% sucrose was necessary for germination. Since germination was low even on sucrose supplemented MS medium, the seeds were subjected to different treatments like hot water treatment (35, 40, 45, 50, 60^oC) for 30, 60 seconds and 30 minutes respectively. Treatment of seeds with water at 40-45^oC for 30-60 seconds yielded 48 % germination even on filter paper moistened with distilled water.  However, the seedlings were weaker as compared to those germinated on MS medium. Hence it is recommended that the seeds be first treated with water at 40-45^oC for 30-60 sec prior to their inoculation on MS medium.

Micropropagation: In P. kurroa, nodal segments (Table 2.13) from stem and rhizome were used for initiation of shoot cultures using MS medium containing kinetin (5.0 mM) and NAA (0.5 mM). Callus induction took place from leaf explants (Table 2.14) on MS medium supplemented with TDZ (5-20 mM), 2,4-D (5-20 mM) and BAP (5-20 mM). When such calli were transferred to PGR free medium or in the presence of KN (1.0 mM), regeneration via shoot bud formation occurred. Multiple shoots were formed within 2 weeks.

Table  Effect of PGR’s on shoot proliferation from nodal segments on MS medium

Table  Callus induction from leaf explant in P. kurroa

Scale-up of cultivation under suitable agro-climatic conditions: A suitable site was selected adjoining to natural habitat of P. kurroa for its large-scale cultivation in district Chamba (3000 m msl), Himachal Pradesh. About 400 sq.m area was brought under its cultivation in June 2001. Presently, the crop is in second year of its growth. 

Chemical evaluation: Wild samples of P. kurroa collected from different altitudes were evaluated for the estimation of Picroside-I and II (Table 2.15)

 Picroside-I and II in different accessions of P. kurroa

Among the wild growing population analysed Picroside-I was maximum in PK-13 and Picroside-II was maximum in PK4.  A new analytical technique was standardized where only few milligrams of material is required for qualitative evaluation.

Domestication and Cultivation

Eight genotypes collected from wild were evaluated for growth performance under polyhouse. Significant differences were observed for all the traits under study. High values of coefficients of variability, heritability and genetic gain for number of inflorescence, length of the stem and number of branches suggested the effectiveness of straight selection for these three traits.

Comparative growth performance of P. kurroa  genotypes collected from different locations 

Effect of growth regulators on  performance of P. kurroa under polyhouse, was studied. The nodal cuttings taken from rhizomatous stolons were treated with different concentrations of BAP/KN/Thiadiazuron (TDZ), with water as control. Mean numbers of sprouting shoots/cuttings, rooting percentage, number of roots/ cutting, root length/cutting and survival percentage were recorded after 16 weeks which was significantly higher for TDZ treatment.

Effect of rooting media on nursery raising

Different rooting media were evaluated for its propagation. One hundred cuttings were planted in each treatment with 4 replications. Early rooting (10 Days After Planting, DAP) was observed when sand + soil + vermiculite was used, whereas maximum rooting (89.52%) and survival (62.81%) was recorded in soil + vermicompost + peat.

Chemical characterization

Eleven accessions collected from wild were established under polyhouse conditions.  These were tested for picroside-I and II content. Acc.10 was distinctly rich in picroside-I (3.63%) and Acc.8 in picroside-II (1.50%).  Nursery raising through seeds and vegetative propagation was carried out.

Picroside content of different accessions of P. kurroa collected from wild

Among the wild high altitude populations, P-I was maximum from 3000 m altitutde and P-II was maximum from 3200 m altitude.

The plant was subjected to different abiotic stress conditions. After giving treatment, these were analysed for P-I and P-II. In sample PK-13, P-I was maximum (0.12%) while P-II was maximum in PK-4 (0.29%).           

Picroside content after stress treatment of P. kurroa  accession

Determination of picrosides in hepatoprotective herbal preparations

HPLC-PDA method was developed and validated for determination of picrosides in herbal preparation containing P. Kurrooa

Ultra-perofrmance LC electrospray ionization quadrupole time-of -flight mass spectrometry for characterization of picroside

Molecular cloning and characterization of regulatory genes involved in picrosides metabolism in Picrorhiza kurrooa Royle ex Benth plants (Funded by Department of Biotechnology, New Delhi)

Genes involved in picroside biosynthesis, the upstream sequences of  1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), 3-hydroxy-3-methylglutaryl Co-A reductase (HMGR), 1-deoxy-D-xylulose-5-phosphate synthase (DXS), 4-(CDP)-2-C-methyl-D-erythritol kinase (IspE), 1-hydroxy-2-methyl-2-(E)-butenyl-4-PP reductase (IspH) and acetoacetyl Co-A thiolase (ACTH) were cloned and analysed. Full length cDNA of WRKY (1,825bp) and bZIP (1,440bp) transcription factors were also cloned. A Regeneration protocol was standardized for the evaluation of promoters. Leaves obtained from in vitro shoot cultures were transformed using HMGR promoter cloned upstream to GUS gene in a promoter-less pBI101. Gene expression was observed at low temperature (16ºC) and light .