Cellular differentiation, regeneration, and secondary ... - Springer Link

Plant Cell Tiss Organ Cult (2013) 112:143–158 DOI 10.1007/s11240-012-0223-9

REVIEW

Cellular differentiation, regeneration, and secondary metabolite production in medicinal Picrorhiza spp. Tapan K. Mondal • Pranay Bantawa • Bipasa Sarkar • Parthodev Ghosh • Pradeep K. Chand

Received: 3 December 2011 / Accepted: 17 August 2012 / Published online: 29 August 2012 Ó Springer Science+Business Media B.V. 2012

Abstract Picrorhiza kurroa and P. scrophulariiflora are two important endangered medicinal plant species of the Indo-China Himalayan region. These species contain several bioactive compounds that have therapeutic properties. In vitro culture studies have been conducted for developing protocols for shoot proliferation via apical/axillary meristem culture, rhizogenesis, acclimatization of plantlets, and nursery establishment. Moreover, successful efforts have been made to induce somatic embryogenesis from callus cultures as well as synchronous maturation of somatic embryos and plantlet conversion. In addition, regeneration has also been achieved via de novo shoot organogenesis, callus-mediated organogenesis, and from synthetic seeds following nutrient-alginate encapsulation. Factors impeding T. K. Mondal (&) P. Bantawa Biotechnology Laboratory, Faculty of Horticulture, Uttar Banga Krishi Viswavidyalaya, Cooch Behar 785165, West Bengal, India e-mail: [email protected] Present Address: T. K. Mondal National Research Center on DNA Fingerprinting, National Bureau of Plant Genetic Resources, Pusa, New Delhi 110012, India B. Sarkar Department of Chemistry, Faculty of Science, University of North Bengal, Darjeeling 734013, West Bengal, India P. Ghosh Department of Botany, Faculty of Science, University of Kalyani, Nadia 741235, West Bengal, India P. K. Chand Plant Cell and Tissue Culture Facility, Post-Graduate Department of Botany, Utkal University, Vani Vihar, Bhubaneswar 751004, Odisha, India

successful in vitro micropropagation have also been investigated. Clonal fidelity of micropropagated plants have been assessed using DNA markers. More recently, genetic transformation of P. kurroa has been reported via Agrobacterium tumefaciens or A. rhizogenes. Hairy root cultures (rhizoclones) containing higher levels of the bioactive compounds kutkoside and picroside I have also been identified. Two genes involved in picroside biosynthesis in P. kurroa have been identified, and these are found to be up-regulated under illumination and low temperature. High throughput de novo transcriptome sequencing has revealed abundance of trinucleotide simple sequence repeat markers associated with temperature-dependent biosynthesis of picrosides. Progress made in developing regeneration, transformation, as well as biochemical and molecular analysis of valuable bioactive compounds present in Picrorhiza species will be reviewed. Keywords Genetic improvement In vitro regeneration Phytochemistry Picrorhiza kurroa Picrorhiza scrophulariiflora Picrosides

An overview Picrorhiza scrophulariiflora Pennell. and P. kurroa Royle ex. Benth. are two highly valued endangered medicinal plant species of the Himalayan region. The former species is restricted to Central through Eastern Himalayas at an altitude of 4,300–5,200 m, while the latter is distributed in Western through Central Himalayas at an altitude of 3,000–4,300 m (Smit 2000). Both the species have various pharmaceutical utilities. Traditionally, native communities of India use these plants to treat several diseases and disorders. Indian pharmaceutical manufacturers such as

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‘Ayush’, ‘Dabur’, and ‘Himalayan Drugs’, among others, have been exploiting both species as important ingredients while preparing medicinal compounds according to formulations of ‘Ayurveda’, a traditional system of medicine in India. Plant populations of both species have been dwindling in their natural habitats due to factors such as over-exploitation (as raw material for medicinal use), harvesting of rhizomes, over-grazing, landslides, tourist inflow, and anthropogenic activities (Bantawa et al. 2009a). Both species are now on the endangered list of plant species.

Botanical characterization The generic name of Picrorhiza is derived from its bitter root, which is used as a native medicine (Royle 1835). In Greek, ‘‘picros’’ means bitter, while ‘‘rhiza’’ means root. The specific name is derived from ‘‘Karu’’, the Punjabi name of the plant, which means bitter (Coventry 1927). Smith and Cave (1911) collected a Picrorhiza species at the base of the Zemu glacier in Sikkim at a height of 4,300 m, and identified it as P. kurroa. There is a specimen at the Herbarium of the Academy of Natural Sciences of Philadelphia, which was identified by Pennell as the shortstamened form described by Hooker (Smit 2000). Furthermore, all collections from the Western Himalayas also had long stamens, while those from the Eastern Himalayas and Yunnan had short stamens. Therefore, the two species were characterized as follows: P. kurroa is the species located in the dry Western Himalayas, while P. scrophulariiflora is the species endemic in the moist Eastern Himalayas (Pennell 1943). P. scrophulariiflora and P. kurroa (Scrophulariaceae) are better known as ‘Nepalese Kutki’ and ‘Kutki’, respectively, in Indian languages. Both species are also known by the same vernacular name due to their morphological similarities. Their vernacular names are ‘Kutki’ in Nepali and Hindi, ‘Katuki’ in Bengali, ‘Karu’ in Punjabi, ‘Putising’ in Dzongkha; ‘Kutki’ or ‘Kutaki’ in Lhotshampkha. In English, it is called ‘Nepalese Kutki’. P. scrophulariiflora It is an herbaceous perennial stout creeping plant with jointed and zigzag growing underground rhizomes (Fig. 1a). It yields off-shoots of 8–12 cm in length from joints of rhizomes. Flowering stems and stout rootstocks are covered with old leaf blades. Leaf blades are 10–14 cm in length. Flowers are dark blue-purple in color (Fig. 1b), and they appear as a dense cylindrical head borne on a stout stem arising from a rosette of conspicuously serrated leaves. Corolla (ca. 1.5 cm) possess a long three-lobed

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upper-lip and a short lower-lip. The calyx is hairy, nearly as long as the floral tube. The fruiting stem measures 5–10 cm; and capsules are 6–10 mm in length of deeppurple in color (Fig. 1c) (Polunin and Stainton 1990) (Fig. 2). P. kurroa Morphologically it is much similar to P. scrophulariiflora except that mature P. kurroa is smaller in height (5–9 cm) with smaller leaves (7–11 cm) (Fig. 1d) (www.ihbt.res.in/ picro.htm). Its leaves are almost all basal, spathulate to narrow elliptic, coarsely saw-toothed, 5 narrowed to a winged stalk. The flowers are pale or purplish blue in colour and its corolla is much smaller, (ca. 0.8 cm), 5-lobed to the middle and with very much longer stamens. The pedicels are usually longer than petioles (Polunin and Stainton 1990).

Medicinal and ethnomedicinal value Both species of Picrorhiza have long been used in the preparation of a large number of medicinal compounds. Rhizomes of Picrorhiza have been used in many Ayurvedic preparations. Of a total of 444 classical preparations investigated, 37 were found to contain Picrorhiza rhizome powder (Bantawa et al. 2011a). Most of these preparations are used in the treatment of fever, skin disease, or liver disease; however, the contents of Picrorhiza in these are low. One of the best known Ayurvedic preparations is Arogyavardhini, containing 50 % Picrorhiza, used for treatment of lingering fever, obesity, diabetes, skin disease, and liver disease. Several folk medicines, used among tribal and rural communities in India, are also known to contain Picrorhiza rhizome powder. In Nepal, Picrorhiza is routinely used in preparations prescribed for liver diseases (Anon 1993). The dried rhizome is reported to contribute to alleviating fever, malnutrition caused by digestive problems, jaundice, diarrhoea, and dysentery. The rhizome of this species is also used as an adulterant of, or as a substitute for, Gentiana kurroa.

Phytochemistry Most reports on biological activities and constituents of Picrorhiza deal with P. kurroa, while P. scrophulariiflora is rarely mentioned. This is due to the fact that both species share almost similar chemical composition, and tillnow, the only known difference is that P. scrophulariiflora contains additional compounds such as phenylethanoids, glycosides,


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Fig. 1 Picrorhiza spp. a Mature plants of P. scrophulariiflora Pennell, b P. scrophulariiflora plant bearing flowers, as well as c Fruit d Mature plants of P. kurroa. (The photograph of mature

plants of P. kurroa was kindly provided by Dr Bikram Singh, Principle Scientist, IHBT, Palampur, India)

and plantamajoside, which are all absent in P. kurroa (Li et al. 1998; Smit 2000). Rhizomes of P. scrophulariiflora are widely used in pharmaceuticals (Olsen 1998) due to presence of active constituents such as picroside III (Weinges and Kunstler 1977), picroside, kutkoside (Aswal et al. 1984), picroside V (Simons et al. 1989), veronicoside (Stuppner and Wagner 1989), picroside II (Wang et al. 1993), D-mannitol, vanillic acid (Yang 1996), phenylethanoid glycosides, along with iridoid 8 glucoside (Li et al. 1998) and pikuroside (Jia et al. 1999). Recently, Bantawa et al. (2010) found that the contents of picroside I and picroside II are significantly higher in P. scrophulariiflora than in P. kurroa.

important biological and physiological functions (Sacchettini and Poulter 1997). These iridoid glycosides are derived from geranyl diphosphate (GDP), which is produced by sequential head-to-tail condensation of dimethylallyl diphosphate and its isomer isopentenyl pyrophosphate (Wise and Croteau 1998). Synthesis of isopentenyl pyrophosphate and dimethylallyl diphosphate is achieved via cytosolic mevalonate (MVA) and plastid methylerythritol phosphate (MEP) pathways (Mahmoud and Croteau 2002; Fig. 4). A cross-talk between these two pathways has also been reported (Hampel et al. 2006). Extensive research has been devoted to standardizing iridoid fractions of P. kurroa; e.g, kutkin and picroliv (Smit 2000). The main iridoid glycoside reported from P. kurroa is ‘kutkin’, a mixture of picroside I and picroside II, which is responsible for the hepatoprotective activity (Shukla et al. 1991). Several groups have used high-performance liquid chromatography (HPLC) to quantify picrosides (Dwivedi et al. 1997; Sturm and Stuppner 2000; Sturm and Stuppner 2001; Vaidya et al. 1996; Sharma et al., 2012; Kumar et al. 2012). Later, Singh et al. (2005) developed a new method for simultaneous quantification of picroside I and picroside II, respectively, using high-

Bioactive compounds of therapeutic value Iridoid glycosides Several iridoid glycosides, medicinally important hepatoprotectants, have been isolated and characterized from Picrorhiza (Fig. 3). These belong to the terpenoids family, which has more than 30,000 members possessing

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Leaf with venation Inflorescence Calyx with stamens

Calyx with gynoecium

Upper leaf Corolla with stamens

Fruit Basal leaf

Placentation

Rhizome

Fig. 2 A diagram of P. scrophulariiflora Pennell. showing different plant parts

performance thin layer chromatography (HPTLC). A sensitive and selective HPLC method has been reported by Lv et al. (2007) using UV at wavelengths of 262 and 277 nm for simultaneous determination of picroside I and picroside II. Linear calibration curves were obtained in ranges of 0–50 lg/ml for picroside I and 0.25–200 lg/ml for picroside II. Accuracy and precision of this validated method were both within the acceptable limits of \15 % at three quality control concentrations. Bantawa et al. (2010) utilized different compositions of mobile phases and the desired resolution of picroside I and II with symmetrical and reproducible peaks was achieved using mobile phases water: acetonitrile (70:30) (Fig. 5a). Identifying these compounds was confirmed by TLC (Fig. 5b), and was later carried out by comparison of retention

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times, UV spectrum with standard compounds, and by spiking samples with a standard stock solution. Although different techniques such as spectrophotometry (Narayanan and Akamanchi 2003) and HPTLC (Singh et al. 2005) have been standardized to quantify the picroside content, HPLC remains the most preferred (Dwivedi et al. 1997; Sturm and Stuppner 2000; Sturm and Stuppner 2001). Mean values of total picrosides (i.e., picroside I and picroside II) varied from 6.35 % (dry weight) to 7.2 % (dry weight) in P. scrophulariiflora collected from Ha in Bhutan. Similar high picroside contents were reported earlier by Smit et al. (2000). However, picroside contents, on dry weight basis, varied from 0.021 to 3.1 % in P. kurroa collected from Western Himalayas, which was much lower than that of P. scrophulariiflora. Such


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Fig. 3 Different iridoid glucosides isolated from P. kurroa and P. scrophulariiflora

R3= OH, R2= OH, R1=

R3= OH, R2= OH, R1=

R1= OH, R2= OH, R3= OH Catalpol (Wang et al. 1993)

O

O

O

O

Veronicoside (Stuppner and Wanger 1989)

Veronicoside (Stuppner and Wanger 1989)

R3= OH, R2= OH, R1=

OH OMe

O O

R2= OH, R3= OH, R1=

OH

Picroside II (Weinges et al. 1972; Wang et al. 1993)

R1

O O

Specioside (Li et al. 1998)

O

O R3 R2= OH, R3= OH, R1=

R2 OH

HO HO

O

R2= OH, R3= OH, R1=

O O

OH O

Verminoside (Li et al. 1998)

OH

OMe

OH

O

R2= OH, R3= OH, R1= O O OH

O 6-feruloylcatalpol (Simons 1989; Stuppner and Wagner 1989)

OMe Picroside V (Simons 1989)

R1= OH, R2= OH, R3=

R1= OH, R2= OH, R3=

O R1= OH, R2= OH, R3=

O Picroside I (Kitagawa et al. 1971)

OH

O

OH

O

O OMe

Picroside IV (Li et al. 1998)

O Picroside III (Weinges and Künstler 1977) O

OH

MeO

HO

I R= H

H

O

O

H

HO

OH HO HO

HO

OH

HO

OH

O

HO HO

O

O O

HO

2 R= HO

O

HO HO

MeO

H O

O

OH Aucubin (Wang et al.1993)

OH Pikuroside (Jia et al. 1999)

O O

RO O

O

O O

HO

O

3 R=

OH

(Kim et al. 2006)

H

O O

H

H

H

OR

H O

HO

O

OH

HO OH H H

1 R = Vanilloyl 2 R = Trans-p-coumaroyl

(Huang et al. 2006) O

R1= HO HO

OH

R1= H

O

O OH

R2= OH

O

HO HO

O

R2= H OH

HO

O R2O HO

Picrogetioside B

O

HO

HO HO

O HO

O

OR1

Picrogetioside A

O

OH

OH

O

O

(Zou et al. 2008)

O

C

O

CH3

O O

O O OH

OH HO HO

O O OH

Picrogetioside C

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MEP pathway

MVA pathway

Pyruvate + Glyceraldehyde-3-phosphate

Acetyl coenzyme A

1-deoxy-D-xylulose-5phosphate synthase

Acetoacetyl CoA thiolase

1-Deoxy-D-xylulose-5-P

Acetoacetyl coenzyme A

1-deoxy- D-xylulose-5Phosphate reductoisomerase

3-hydroxy-3-methylglutaryl coenzyme A synthase

2-C-Methyl-D-erythritol-4-P 2-C-methylerythritol 4-phosphate cytidyl transferase 4-(CDP)-2-C-methyl-D-erythritol 4-(cytidine-5 -diphospho)-2Cmethylerythritol kinase

3-Hydroxy-3-methylglutaryl coenzyme A 3-hydroxy-3-methylglutaryl coenzyme A reductase Mevalonate

4-(CDP)-2-C-methyl-D-erythritol-2-P

Mevalonate kinase 12 phosphomevalonate kinase

2-C-methylerythritol-2,4cyclophosphate synthase

Mevalonate phosphate

2-C-methyl-D-erythritol-2,4-cyclo-PP

Mevalonate-5-pyrophosphate decarboxylase

1-hydroxy-2-methyl-2-(E)-butenyl -4-diphosphate synthase

Mevalonate pyrophosphate

1-Hydroxy-2-methyl-2-(E)-butenyl-4-PP

Mevalonate-5-pyrophosphate decarboxylase Isopentenyl pyrophosphate isomerase

1-hydroxy-2-methyl-2-(E)-butenyl -4-diphosphate reductase

Isopentenyl pyrophosphate

Dimethylallyl pyrophosphate

Geranyldiphosphate synthase Geranyldiphosphate Cinnamate/Vanillate (from phenylpropanoid pathway)

Glucose Iridoid moiety

Picrosides Fig. 4 Schematic pathway for picrosides biosynthesis (adapted from Kawoosa et al. 2010 and Mahmoud and Croteau 2002). Solid arrows indicate which has already been known steps, whereas dotted arrows indicate unknown steps

P-I

P-I

P-II

P-II

a b Fig. 5 Representative chromatogram profile of picroside I (P–I) and picroside II (P–II) from the rhizome of P. scrophulariiflora a HPLC and b TLC (Bantawa et al. 2012; Bantawa 2010)

variability of chemical content has already been reported in a number of other medicinal plants (Drasar and Moravcova 2004; Hisiger and Jolicoeur 2007), and in particular in Picrorhiza (Singh et al. 2005).

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Phenylethanoids Yet another group of compounds has been isolated from Picrorhiza, consisting of the phenylethanoid glycosides

Objective

Micropropagation

Micropropagation

Micropropagation via synthetic seeds

Plant regeneration via callusmediated organogenesis

Micropropagation

Micropropagation

Biological hardening of micropropagated plants


Caulogenesis

Micropropagation

Picrorhiza species

P. kurroa

P. kurroa

P. kurroa

P. kurroa

P. kurroa

P. kurroa

P. kurroa

P. kurroa

P. kurroa

P. kurroa

Nodal segments

Leaf disc, nodal segment and root segments

Leaf discs, nodal segments and root segments

INA

Nodal segments

Nodal segments

Leaf and nodal cutting from mature plant

Shoot tips

Terminal and nodal cuttings

Shoot tips

Explant type

Table 1 In vitro propagation of Picrorhiza spp.

Shoot proliferation

MS ? NAA (0.6)

MS ? 2,4 D (2) ? IBA (0.5)

MS ? NAA (0.6)

MS ? BA (2) ? Kin (3) ? activated charcoal (8)

MS ? IAA (2) ? Kin (3) ? table sugar (3 %)

INA

INA

MS ? BA (0.23)

MS0

MS ? BA (0.23)

MS ? BA (0.25)

MS ? BA (0.23)

MS ? 2,4 D (0.5–2) ?NAA (4) ? Kin (1)

MS0

MS ? BA (0.69)

MS ? Kin (5) ? IAA (1)

Shoot initiation

Nutrient medium

MS ? NAA (0.4) ? IAA (0.1) ?IBA (0.5)

MS ? IBA (3) ? NAA (2)

MS ? IBA (3) ? table sugar (3 %)

INA

MS ? IBA (3)

MS ? IBA (0.2)

MS ? NAA (0.2)

MS ? NAA (0.2)

MS ? NAA (1)

Rooting

Among the 3 different bacteria, Bacillus sabtalis was most effective which registered 92.5 % survival in vitro plants

100 % rooting of in vitro raised plants were achieved

INA

Four-week-old plantlets were successfully established in soil

INA

INA

Rooted plantlets were transferred to clay pot containing sterile sand, soil 86.7 % survival

Hardening and field transfer

Rooting after 15–25 days

Root initials started in 28 days

The survival percentage was 81.5 % in hardening chamber

Hardening mixture was sand: soil: vermiculite(1:1:1) and the plantlets were covered with glass beaker for 9–10 days for root establishment

Low cost in production due to use of table sugar

Root establishment in 60 d

Complete protocol of vegetative propagation is reported

Shoot proliferation occurred within 4 weeks

Half-strength nitrogen was best for conversion of bud primordia into shoots

Encapsulated shoot tips could be stored up to 24 weeks on nutrient-free from 4 to 10 °C in dark

Multiple shoots sprouted in 28 days and rooting was initiated in 18–20 days

Shoot initiation occurred in 28 days; root initiation in 7–9 days

Remarks

Jan et al. (2009)

Sood and Chauhan (2009b)

Sood and Chauhan (2009a)

Trivedi and Pandey 2007

Chandra et al.(2006)

Chandra et al. (2004)

Lal and Ahuja (1996)

Lal and Ahuja (1995)

Upadhyay et al. (1989).

Lal et al. (1988)

References

Plant Cell Tiss Organ Cult (2013) 112:143–158 149

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Objective

Micropropagation

Micropropagation and somatic embryogenesis

Callus culture for picroside production

Micropropagation via synthetic seeds

Micropropagation

Micropropagation


Picrorhiza species

P. kurroa

P. kurroa

P. kurroa

P. kurroa

P. scrophulariiflora



Table 1 continued

123 In vitro leaf

Shoot tips, nodal segments

In vitro shoot tips, nodal segments

In vitro derived leaf disc, nodal segment, root In vitro derived leaf,shoot tips, nodal segments

Nodal segment Leaf tissue and of in vitro plantlets

Nodal segments

Explant type

MS ? bavistin (100) and adenine sulphate (100)

MS ? Kin (0.5)

WPM ? BA (0.05)

WPM ? BA (0.05)

WPM ? BA (0.1)

INA

INA

WPM ? NAA (0.1) ? Kin (0.05)

INA

MS ? Kin (2) ? IBA (0.5)

MS ? NAA (0.2) for callus induction; MS ? NAA (0.6) for shoot bud initiation

Shoot proliferation

INA

MS ?Kin (2) ? IBA (0.5)

MS ? 2,4- D (0.25) for callus induction; MS ? BA (0.25) for shoot bud initiation

Shoot initiation

Nutrient medium

Rooted on WPM ? NAA (1)

MS ? NAA (1)

MS ? NAA (1)

MS ? NAA (1)

INA

IBA (1)

MS ? NAA (0.4)

Rooting

Multiple shoots formed within 28 days and root initiation started in 15–18 days In vitro root initiation was noticed in 14 days

Bavistin along with adenine sulphate gave better in vitro multiplication rate

Rooting was initiated after 28 days

Callus induction took place in MS ? 2,4-D (2) in 6–7 days

Callus induction took place 20 days after inoculation from leaf explants

INA

Remarks

Survival rate of plants subsequently transferred for hardening was 90 %. After another 10 months the well hardened plants were distributed to the local farmers for planting

Rooted plantlets were transferred to plastic cups containing sterile virgin soil and sand (9:1)

The regenerated plantlets were hardened in plastic cups (6 9 8 cm) containing 9 : 1 virgin soil and soil with 97 % survival

INA

Picroside content was determined at different stages of callus culture

Nodal segments plated on the MS ? TDZ (0.11) and IBA (0.5) formed somatic embryos

Rooted plantlets were hardened in polycups containing sand:soil:vermiculite (1:1:1) with a survival percentage of 81.5 %


Bantawa et al. (2011a, 2011b)

Bantawa et al. (2010)

Bantawa et al. (2009b)

Mishra et al. (2011)

Sood and Chauhan (2010)

Sharma et al. (2010)

Jan et al. (2010)

References

150 Plant Cell Tiss Organ Cult (2013) 112:143–158

Bantawa (2010)

Bhat et al. (2012)

Survival rate after 5 months was ca. 82 %

Plantlets hardened in Styrofoam cups containing mixture of soil:sand:vermiculite:: 1:3:2. After 3 weeks, hardened plants were directly transplanted to trays containing a sand:soil:: 1:1 mixture exhibiting 93 % survival

scrodide A-C and plantamajoside (Li et al. 1998). Presence of plantamajoside was also reported from P. scrophulariiflora (Zhou et al. 1998), which is otherwise found in Plantago major (Plantaginaceae) (Ravn et al. 1990). Recently, new phenyl glycosides, including scrophenoside D, phenylethyl glycoside, scrodide F together with three known phenylethyl glycosides, scrodide A, plantainoside D, and plantamajoside were identified in stems of P. scrophulariiflora (Zou et al. 2007). Cucubitacins These constitute a group of triterpenic compounds that are known to be both bitter and toxic. Cucurbitacins are normally present in plants in the form of b-glycosides which have a wide range of biological activities (Smit 2000). A total of 23 different cucurbitacin glycosides and a single aglucone have been isolated from Picrorhiza (Fuller et al. 1994). Various cytotoxic effects of this group of compound have been reviewed previously (Miro 1995; Smit 2000; Whitehouse and Doskotch 1969; Witkowski and Konopa 1981). For example, cucurbitacins D, I, E, and 2- ethoxycucurbicin E are responsible for the induction of morphological changes to Ehrlich ascites tumor cells at low concentrations (Gitter et al. 1961). Furthermore, lymphocytes of chronic lymphatic leukemia are very sensitive to cucurbitacin D (also known as elatericin A) (Shohat et al. 1967). Similar activities of cucurbitacin E on cytoskeleton are also reported (Duncan et al. 1996; Duncan and Duncan 1997). Cucurbitacins have shown inhibitory activities in vivo in several carcinoma, sarcoma, and leukemia models (Fang et al. 1984; Gitter et al. 1961; Konopa et al. 1974; Kupchan et al. 1972; Lavie and Glotter 1971; Reddy et al. 1988).

INA information not available

Phenolics

Figures in parenthesis denote concentrations in mg/l

B5 ? Kin (3) ? IBA (1) Plant regeneration via adventitious (de novo) shoot organogenesis P. kurroa

Leaf explants from in vitro shoot cultures

B5 ? Kin (3) ? IBA (1)

B5 ? Kin (3) ? IBA (1) ? activated charcoal (10)

INA MS ? Kin (0.5) ? GA3 (0.5) for germination MS ? ABA (0.1–1.0) for 2 weeks for maturation Plant regeneration via somatic embryogenesis P. scrophulariiflora

In vitro leaf derived callus

MS ? BA (0.1–2.0)

Rooting Shoot proliferation Shoot initiation

Nutrient medium

Explant type Objective Picrorhiza species

Table 1 continued

151

High frequency (94 %) multiple shoot bud regeneration occurred in 21–28 days which proliferated to shoots (10–12 shoots/explant) in 42–56 days. Shoots rooted in 21–28 days

References Remarks



These are very common in the plant kingdom. They are precursors and degradation products of lignin, which provides sturdiness to the plant and a physical defense barrier against parasites (Steinegger and Hansel 1988). Furthermore, they usually act as antifungal agents, providing direct protection to the plants (Kokubun and Harborne 1995). Picrorhiza is a good source of phenols. Various phenolics such as vanillic acid (Rastogi et al. 1949), apovynin (Basu et al. 1971), androsin (Wang et al. 1993) and picein (Stuppner and Wagner 1989) have been isolated from Picrorhiza. These phenolics are associated with antioxidative activities (Muller et al. 1999), and anti-inflammatory activities (Lafeber et al. 1999). Moreover, they act against sepsis (Wang et al. 1994), atherosclerosis (Chen et al. 1998), and asthma (Iwamae et al. 1998).

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Shoot proliferation for clonal mass propagation In vitro shoot proliferation offers a useful tool for conservation of germplasm and mass propagation of threatened plant species (Coste et al. 2012; Rabba’a et al. 2012). Further, in vitro clonal propagation of medicinal plants allow for large-scale production of therapeutically highvalued taxa for commercialization and sustainable utilization in the industrial sector (Xu et al. 2012). De novo regeneration has also been reported for a growing list of medicinal and aromatic plants (Pant et al. 2010). A summary of in vitro regeneration efforts of this genus is presented in Table 1. Although several parts of the plant have been used as explants, nodal segments of mature plants have been widely used (Table 1). Lal et al. (1988) are the first to attempt in vitro proliferation of P. kurroa using shoot-tips. Later, runners, axillary shoots (Chandra et al. 2006), terminal buds, as well as single node stem cuttings (Upadhyay et al. 1989) have also been used for shoot proliferation of P. kurroa. Recently, our group has exploited shoot-tips and nodal explants for proliferation of P. scrophulariiflora (Bantawa et al. 2009b; Bantawa et al. 2010). While Murashige and Skoog (1962) (MS) is the main basal media (Table 1), yet different growth regulators, such as 6-benzyladenine (BA) thidiazuron (TDZ), and

b

a

e Fig. 6 Different stages of micropropagation of P. scrophulariiflora. a Bud break and axillary shoot proliferation on WPM ? 0.1 mg/l BAP, b, c Shoot proliferation on WPM ? 0.5 mg/l Kin, d Root

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kinetin (Kin), either alone or in combination with indole3-acetic acid (IAA), a-napthaleneacetic acid (NAA), or indole-3-butyric acid (IBA) have been used mostly for inducing bud-break and axillary shoot proliferation, as well as for subsequent shoot elongation. Lal et al. (1988) used MS medium supplemented with varying concentrations of Kin (3–5 mg/l) for rapid proliferation of multiple shoots of P. kurroa. Moreover, adding 1 mg/l IAA to the Kin-containing medium showed a marked improvement in growth of regenerated shoots. However, Chandra et al. (2006) used BA at a lower concentration (0.23 mg/l) for production of multiple shoots of P. kurroa. Upadhyay et al. (1989) utilized BA at a range of concentrations (0.11–2.25 mg/l) either alone or in combination with either IAA (0.02–0.2 mg/l) or gibberellic acid (GA3) (0.03–0.35 mg/l) for P. kurroa. Presence of 0.2 mg/l BA alone was found to be the best, inducing the highest frequency of shoot proliferation as well as promoting shoot growth. Sood and Chauhan (2009a) developed a low-cost micropropagation protocol for P. kurroa. For shoot proliferation of P. scrophulariiflora, lower concentrations of Kin (0.5 mg/l) alone were preferable (Fig. 6 a–c). Addition of IAA or NAA along with Kin did not improve the frequency of shoot proliferation, but instead increased vitrification and induced callusing of shoot cultures (Bantawa et al. 2009b).

c

f

d

g

h

induction on WPM ? 1.0 mg/l NAA, e, f Acclimatization of plants in soil, g, h Wellestablished plants after 6 months of acclimatization displaying well rooted condition (Bantawa et al. 2010)


Rooting and acclimatization of shoots Lal et al. (1988) were successful in inducing about 14 roots per microshoot of P. kurroa within 7 days on MS medium supplemented with 1 mg/l NAA. Among various treatments, 0.22 mg/l IBA resulted in 100 % rooting with 3.2 roots per plantlet of P. kurroa (Trivedi and Pandey 2007). For P. scrophulariiflora, the use of 1.0 mg/l IBA also proved to be the best for rooting of regenerated microshoots (Bantawa et al. 2009b) (Fig. 6d). As with other medicinal plants, acclimatization of plantlets is an important step for micropropagation of Picrorhiza species. Sterilized sand, soil, and manure (1:1:1) were used for transfer of regenerated plantlets of P. kurroa into soil mix during acclimatization (Lal et al. 1988). Recently, biological acclimatization of micropropagated plants has been attempted (Lincy and Sasikumar 2010). Hardening of P. kurroa micropropagated shoots with Bacillus megaterium resulted in 94 % survival, compared to 38.5 % for control (Trivedi and Pandey 2007). Transferred plantlets are maintained in a plastic house, under high humidity (80 % or above) for 8 weeks, and then transferred to larger plastic pots (8 cm 9 10 cm) containing the same potting mixture (Fig. 6 e–h).

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incubated on MS medium fortified with 2 mg/l 2,4dichlorophenoxyacetic acid (2,4-D), 0.5 mg/l IBA, and solidified with 0.8 % (w/v) agar (Jan et al. 2010). Callus cultures transferred to MS medium containing different concentrations and combinations of BA, Kin, and IBA underwent organogenesis and differentiated into multiple shoots. Highest frequency of regeneration (76.7 %) was observed from root-derived callus grown on solidified medium containing 2 mg/l BA and 3 mg/l Kin along with 3 % (w/v) sucrose. Most calli derived from nodal segment underwent rhizogenesis in stead of caulogenesis. Recently, our group developed a reproducible in vitro regeneration system of P. scrophulariiflora from leafderived callus. Induction of more than seven shoot buds per explant was achieved on a Woody Plant Medium (WPM) (Lloyd and McCown 1981) supplemented with 0.1 mg/l NAA and 0.05 mg/l Kin. Shoots elongated when transferred to WPM supplemented with 0.1 mg/l BA, and after transfer to WPM supplemented with 0.1 mg/l NAA, they developed within 2 weeks. Genetic uniformity of these plantlets was confirmed following analysis of leaf tissues using random amplified polymorphic DNA (RAPD) markers analysis (Bantawa et al. 2011b).

Analysis of picroside contents in regenerated plants Direct adventitious (de novo) shoot organogenesis Recently, Bhat et al. (2012) have reported on direct regeneration via shoot organogenesis from leaf explants derived from in vitro-grown shoot cultures. These leaf explants were incubated on a regeneration medium (RM) consisting of Gamborg’s B5 medium (Gamborg et al. 1968) supplemented with 3 mg/l Kin and 1 mg/l IBA. Within 3–4 weeks, induction of multiple shoot buds, at a high frequency (94 %), was observed. Subsequently, shoot buds grew into well-developed shoots (9–12 shoots/explant) within 6–8 weeks. Shoots were rooted on RM augmented with 1 % activated charcoal within 3–4 weeks. Plantlets were successfully acclimatized in Styrofoam cups containing mixture of soil:sand:vermiculite :: 1:3:2. After 3 weeks, hardened plants were directly transplanted to trays containing a sand:soil (1:1) mixture in a controlled environment chamber with 93 % survival.

Indirect shoot organgensis via callus Callus cultures were established from different explants, such as leaf sections as well as nodal and root segments of P. kurroa (Jan et al. 2010). Callus induction was highest (70 %) in root segments, followed by leaf sections (56.3 %) and nodal segments (38.3 %) when explants were

Although cell cultures offer a suitable biological system in a controlled environment wherein the morphogenic events can be maintained and regulated by manipulating levels of growth regulators in the nutrient medium, thereby resulting in a rapid production of plant metabolites of pharmaceutical value (Ray and Jha 2001). However, there is little information pertaining to biosynthesis and accumulation of picroside-I and picroside-II in cultures of P. kurroa. Sood and Chauhan (2010) attempted to detect the picroside accumulation in the cell culture of P. kurroa. The picroside-I content was 1.9, 1.5, and 0.04 mg/g dry weight in leaf sections, stem, and root segments, respectively. The picroside-I content declined to almost non-detectable levels in callus cultures derived from leaf sections, stem segments with no change in picroside-I content in root segments or calli derived thereof. Biosynthesis and accumulation of picroside-I started in callus cultures during differentiation of shoot primordia, reaching levels comparable to those of donor explants; contents of 2.0 mg/g dry weight and 1.5 mg/g dry weight for plantlets derived from leaf sections and stem segments, respectively. Shoots developed from root-derived callus cultures were relatively slow in growth and the amount of picroside-I content was low (1.0 mg/g, dry weight) compared with shoots derived from callus cultures derived from leaf and stem segments. Thus, biosynthesis and accumulation of picroside-I in P. kurroa are

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developmentally regulated during different stages of differentiation.


90 % survival rate was recorded after 8 weeks. Well-rooted plantlets, following acclimatization, were successfully established in the field (Bantawa 2010).

Somatic embryogenesis Synthetic seeds Somatic embryogenesis has been reported in both these species of Picrorhiza (Table 1). For P. scrophulariiflora, Bantawa (2010) used different explants such as in vitroderived roots, leaves, and nodal segments incubated on WPM basal medium supplemented with auxins (0.1–2.0 mg/l of each of 2,4-D and NAA) either alone or in combination with cytokinins (0.05–0.1 mg/l of each of Kin and BA) for embryogenic callus induction. Somatic embryo production differed significantly with respect to the type of explant. Leaf explants developed friable callus along the base of leaf blades. Upon transfer of this friable callus to WPM medium with either Kin or BA (0.1–2.0 mg/l), embryogenic callus was observed, and subsequent transfer of somatic embryos to WPM medium supplemented with ABA (0.1–1.0 mg/l) for 2 weeks, these embryos achieved maturation (92 %). Approximately, 73 % of mature somatic embryos germinated in medium containing WPM with 0.5 mg/l Kin and 0.5 mg/l GA3. Sharma et al. (2010) reported that in vitro-derived nodal explants of P. kurroa cultured on MS medium supplemented with 0.11 mg/l thidiazuron (TDZ) along with 0.5 mg/l IBA developed somatic embryos. However, these somatic embryos failed to regenerate into whole plantlets. Synchronization of maturation of somatic embryos is a necessary prelude for developing whole plants. For P. scrophulariiflora, Bantawa (2010) achieved synchronous maturation of somatic embryos from leaf-derived callus by transferring these somatic embryos onto a solidified MS medium containing 0.5 mg/l ABA for 2 weeks, followed by transfer to a fresh MS medium containing 0.5 mg/l Kin for another 4 weeks. Frequency of maturation of somatic embryo differed considerably based on the plant growth regulator (PGR) composition, incubation time on ABAcontaining medium, and explant source. Conversion of somatic embryos into plantlets often constitutes a limiting step in somatic embryogenesis due to the inability of somatic embryos in certain plant systems to mature (Quoirin 2003). In our study on P. scrophulariifolra, somatic embryos did not germinate in PGR free medium. Matured somatic embryos were formed in clusters, each comprising more than ten embryos. These embryos of cotyledonary stage, when separated individually and transferred to medium containing Kin (0.5 mg/l) along with GA3 (0.5 mg/l) germinated at a high frequency (72.84 %) within 4 weeks (Bantawa 2010). Well-rooted plantlets were transferred from culture tubes into plastic cups containing virgin soil mixed with sand (9:1). Over

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Synthetic seed technology involves the use of suitable propagules such as somatic embryos derived from tissue culture or from non-embryogenic unipolar meristematic explants, such as shoot apices, axillary buds, or nodes encased in a protective coating by nutrient-alginate encapsulation (Rihan et al. 2011). Somatic embryos of P. scrophulariiflora were encapsulated in calcium alginate gel matrix with full liquid strength MS basal medium augmented with a combination of 0.5 mg/l Kin and 0.5 mg/l GA3 (Kinoshita and Saito 1992). A range of sodium alginate levels (2–6 %) was used, and 100 % survival was achieved when beads were stored up to 15 days at 4 °C. Interestingly, when the alginate beads were prepared in MS medium containing 0.5 mg/l Kin alone and subsequently stored for 15–45 days, they produced multiple shoots within 10–16 days. Longer storage at lower temperatures resulted in the production of a single shoot per bead. The time required to germinate also increased from 10 to 12 days (at 0-day storage time) to 25 days (at 105-day storage period). Sprouting frequency did not vary between 0 and 105 days storage time, where 100 % germination was recorded (Bantawa 2010). The rate of germination of encapsulated somatic embryos declined with longer storage time. In mulberries and pomegranate, there was a decline in germination of synthetic seeds, derived from nutrient-alginate encapsulation of nodal explants, when they had been stored at a low temperature for longer period of time (Pattnaik and Chand 2000; Naik and Chand 2005). Pandey and Chand (2005) reported similar observations for Hyoscyamus muticus, and concluded that failure of germination of alginate beads prepared in distilled water might be due to poor nutrition. In addition to somatic embryos, in vitro grown microshoots of P. kurroa were also encapsulated in alginate beads (Mishra et al. 2011). Re-growth of encapsulated microshoots, using alginate encapsulation, reached 89.33 % following storage for 3 months. Amongst 21 developing plantlets, 42.66 % exhibited formation of multiple shoots at the onset of regrowth and 21.43 % demonstrated simultaneous formation of shoots and roots. Healthy root formation was observed in plantlets following 2 weeks of their transfer to 1/2 MS containing 0.2 mg/l NAA. Plants were transferred in three batches to the greenhouse where they survived at a frequency of 95 %. Further the genetic fidelity of P. kurroa plants growing out after storage at 4 °C in encapsulated form was ascertained


by RAPD analysis (Mishra et al. 2011). In vitro plantlets derived from encapsulated somatic embryos of P. scrophulariiflora were transferred from the nutrient medium to plastic cups (6 cm 9 8 cm) containing a mixture of autoclaved virgin soil and sand (9:1). After 8 weeks, 80 % survival was observed. These plantlets were re-potted in polyethylene bags each containing the same potting mixture and were transferred to nursery beds. Following 8 weeks of field transplantation, survival rate was scored to be 78 % (Bantawa 2010). The effects of gradual acclimatization of in vitro propagated plantlets on glasshouse or field cultivation was investigated. It was observed that the plants covered with polythene bags for 4 weeks showed an average survival rate of 91 % plant where as those uncovered after 7, 14 and 21 days exhibited a relatively low frequency survival of 7, 24 and 46 % respectively. Other hardening treatments such as application of anti-transpirants or liquid paraffin failed to yield any significant improvements. The synthetic seed system may be useful for developing ex situ conservation strategies for P. kurroa and their large-scale plantation to replenish degraded habitats (Sharma et al. 2010).

Bottlenecks of micropropagation Establishment of in vitro culture of tissues of adult origin is comparatively more difficult than that of the juvenile donor (Drew and Smith 1986). Establishing the field collected explants of Picrorhiza species in tissue culture has proved difficult due to browning perhaps because of the fact that the species are rich in phenolic compounds. Transparent jelly like exudates are often noted during in vitro culture of P. scrophulariiflora (Bantawa et al. 2010). Those cultures turned necrotic despite frequent subculturing onto either the same or on basal WPM. It might be due to the deposition of some stress-induced phyto-chemicals, which block the uptake of the nutrients (Bantawa et al. 2010). Besides, high rates of microbial contamination during initial establishment of in vitro culture are also observed in Picrorhiza spp. (Bantawa et al. 2010).

Genetic transformation A protocol for induction of Agrobacterium rhizogenesmediated transformed hairy root cultures of P. kurroa was established (Verma et al. 2007). Cultivation of leaf explants with A. rhizogenes strain LBA9402 strain resulted in development of hairy roots at 66.7 % relative transformation frequency. Nine independent, opine and TL-positive hairy root somaclones (rhizoclones), were analyzed to assess their growth and ability to produce specific

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glycosides (i.e., kutkoside and picroside I) during different phases of growth. Bioactive compounds were detected in all rhizoclones, although distinct inter-clonal variations in contents of these compounds were observed. Yield potential, both of biomass as well as individual glycoside contents, including kutkoside and picroside I, of the hairy root somaclone 14-P, was higher than all other hairy root clones. Recently, Bhat et al. (2012) have reported on successful A. tumefaciens-mediated transformation of P. kurroa. A. tumefaciens strain GV3101 harboring the binary vector pCAMBIA1302 carrying the green fluorescent protein and hygromycin phosphotransferase encoding genes was used. Leaf explants from in vitro-grown shoots were the most responsive when pre-cultured (2 days) on the regeneration medium prior to co-cultivation with Agrobacterium suspension culture grown with 200 lM acetosyringone. A high frequency of transformation (presumably reported to be 56 %) was recorded with an average of 3.4 ± 0.4 transgenic plantlets per explant. Putative transformants were selected on media stressed with 15 mg/l hygromycin and analyzed using by PCR and fluorescence microscopy. Therefore, this protocol claims efficient transformation and plant regeneration that may pave the avenues towards metabolic engineering in transgenic P. kurroa.

Functional genomics The emerging field of functional genomics offers tremendous opportunities to discover novel genes and assign functions to those genes. Thus far, there are a few and limited genomic resources available for Picrorhiza (Bantawa et al. 2012; Hussain et al. 2009). A total of 728 ESTs of P. kurroa and 27 of P. scrophulariflora have been deposited at NCBI (www.ncbi.nlm.nih.gov). Kawoosa et al. (2010) have identified two regulatory genes of terpenoid metabolism namely, 3-hydroxy-3-methylglutaryl coenzyme A reductase (pkhmgr) and 1-deoxy-D-xylulose5-25 phosphate synthase (pkdxs) from P. kurroa. Full length of these genes along with their up-stream sequences have been cloned by by rapid amplification of cDNA ends (RACE). This revealed presence of core sequences for light and temperature responsiveness. Electrophoretic mobility shift assay confirmed binding of protein to these motifs. Results have demonstrated that illumination and temperature would play a key role in regulating the expression of pkhmgr and pkdxs, and associated picrosides level in Picrorhiza. Expression of pkhmgr and pkdxs is up-regulated at a low temperature (15 °C) and under illumination as compared to a relatively high temperature (25 °C) and dark conditions. Picroside contents have exhibited similar trends to levels of gene expression. To rule out the possible

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limitation of the carbon pool under dark growth conditions, plantlets of Picrorhiza were grown in vitro on MS medium supplemented with 3 % sucrose. Similar findings have been observed, wherein up-regulation of both genes accompanying higher picroside contents in the presence of light have been detected in these axenic plantlets. Next generation sequencing is a high throughput sequencing technology due to which generation of genomic resources become rapid and cost-effective. It is also a superior technique for identifying the differentially expressed gene. Accumulation of secondary metabolites is highly influenced by environmental factors such as light and temperature (Szopa et al. 2012). As picroside accumulation depends on ambient temperature, a highthroughput de novo transcriptome sequencing was carried out using plants grown at 15 and 25 °C. Using Illumina sequencing technology, a total of 20,593,412 and 44,229,272 reads were obtained after quality filtering for 15 and 25 °C respectively. A total of 74,336 reads were assembled, with an average coverage of 76.6 % and average length of 439.5 bp. The guanine and cytosine (GC) content was observed to be 44.6 % and the transcriptome exhibited an abundance of trinucleotide simple sequence repeat (45.63 %) markers (Gahlan et al. 2012). Acknowledgments Major funding support to our work was provided by the Department of Science and Technology (DST) and the Department of Biotechnology (DBT) under the Ministry of Science & Technology, Government of India, New Delhi through the award of competitive research grants. The authors acknowledge Dr. Swapan Kumar Ghosh, Uttar Banga Krishi Viswavidalaya, West Bengal, India and Dr. Paramvir Singh Ahuja, Institute of Himalayan Bioresource Technology, Himachal Pradesh, India for their encouragement to carry out some of the experiments. We wish to thank Dr. Jatindra K. Nayak, Professor of English, Utkal University, Bhubaneswar, India for having critically read the manuscript and made useful corrections in the English language of the text.

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