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IntroductionPicrorhiza kurroa Royle ex. Benth.(Family:Scrophulariaceae), native to Western Himalayanregion, between 3000-5000 m elevation (Hooker1885; Chopra & Ghosh 1934; Blatter 1984; Jain 1996;Agrawal 2003) is valued as hepato-protective, anti-periodic, cholagouge, stomachic, anti-amoebic,anti-oxidant, anthelmintic, anti-inammatory,cardio-tonic, laxative, carminative, expectorant,etc. (Chopra & Ghosh 1934; Uphof 1959; Kapoor1990; Kapahi et al. 1993; Singh et al. 2006; Bhatt &Bhatt 1996: Gaddipati et al. 1999; Prajapati, 2003).

Picroside I and II are the two important chemicalconstituents present in its rootstock which havetherapeutical importance(Dutt et al. 2004). In orderto full the increasing national and internationaldemand, the raw drug is largely wild extractedsince the species has very limited cultivation thathas made it critically endangered (Nayer andSastry 1987; Rai et al. 2000). The species has alsobeen put in the negative list of exports by Ministryof Commerce, GOI [Vide Notication No. 03 (RE-2003)/2002-2007 (Appendix II) dated 31st March,2003 issued by Director General of Foreign Trade,Govt. of India].

Despite considerable therapeutical importance, noinformation regarding its reproductive biology,ploidy status and breeding systems is availablein literature. The present study was undertakento understand its breeding behaviour that isfundamental to undertake any genetic improvementfor higher yield and quality attributes.

Material and methods

Plant Material

Wild and eld grown plants of the species atRahla, Himachla Pradesh, India (2800m amsl)were used for the study.

MethodsMale meiotic studies were conducted by xingunopened ower buds in Carnoys xative IIconsisting of absolute alcohol: glacial aceticacid: chloroform (1:1:1) for 24 hours. Onepercent propionocarmine was used for stainingchromosomes by the usual squash method and aminimum of 20 good cells at proper stage werestudied for taking observations. Pollen stainabilitywas determined by staining freshly dehiscedpollens with 1% propionocarmine.

Pollination studies were conducted with three

treatments viz., (i) open pollination, (ii) selng bybagging of single spikes and (iii) selng 2-4 spikes/plant with seven replications in randomized blockdesign. Minimum 10 oral spikes were used ineach replication.

Results and Discussion

Morphology

Plants of P. kurroa are perennial, herbaceous withcreeping rootstock and aerial parts represented bybasal leaves and owering scape only (Fig.1). Suchfeatures make P. kurroa highly adaptable to harshenvironmental conditions of the areas it inhabits.The plants spread through stolons and apparentlyseparate looking plants at above ground levelare actually joined together by stolons beneath.However, when these stolons get detached fromthe mother stock, the plants become independent.The simple leaves arising from the tip of theupturned stolons are present in rosettes orwhorls. Cauline leaves are present only during theowering phase.

The inorescence is indeterminate terminal spikeforming a more or less triangular head. The owersare sessile, zygomorphic, bilipped, bisexual andpurple (Fig. 2) borne in the axils of small green

Reproductive Biology of Picrorhiza kurroaa CriticallyEndangered High Value Temperate Medicinal Plant

R. Raina*, Tara Singh Mehra, Romesh Chand and Yash Pal Sharma

Department of Forest Products, Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni- Solan (HP) 173 230*Corresponding author: raviraina4@yahoo.co.in

Abstract: Picrorhiza kurroa Royle ex. Benth.(Family: Scrophulariaceae), an important endangered temperatemedicinal plant used as hepatic stimulant is a perennial herb propagated mostly by stolon segments.Chromosome number and its reproductive behaviour are reported for the rst time. Meiosis is normalin the species with 2n=34 chromosomes. P. kurroa mostly prefers out-crossing as evinced by pollinationexperiments and oral architecture.

Key Words: Picrorhiza kurroa, endangered medicinal plant, reproductive biology

Manuscript received: 5 February, 2010 Manuscript accepted: 3 June, 2010

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coloured bracts opening during June to September.Sepals persist up to capsule development stageand are of unequal in size.

Filaments, which are colourless in bud stage,become purplish after anthesis. This is probably

to add colour to the ower for attracting insectsfor affecting pollination. The four free stamensare epipetalous and alternating with the petallobes. Stamens are didynamous with 2 long (15.50mm) and 2 short (13.25 mm) stamens. Althoughthe stamens are exerted, the anthers are borneon incurved laments, which make their positionintrorse. Simultaneous with anthesis the stamensbecome exerted with anthers continuing in theintrorse position. The stye also keeps pace withthe laments and at the stage of maximum lengthboth were of almost equal length. The anthers

start shedding pollen once they come out of thecorolla cavity till the maximum growth of thelament. Bombus spp. and Apis orae were observedvisiting the owers. The ower architecturemostly favours cross-pollination. Although exertedstamens are a step towards cross pollination,introrse anthers ensure that some pollen may fallon the owering spike itself so as to affect self-pollination. As is known (Kaul & Kaul 1996) andas well observed during the present studies, thisspecies occurs at high altitudes characterized byshort growing seasons. In environments, as in

high alpine or arctic region and deserts those arepoor in pollinators because of the unfavourableconditions; autogamy is often the only possiblemeans of sexual reproduction (Hagerup 1932 &1951). However, in Picrorhiza kurroa, the exertedstamens as well as introrse anthers ensure bothallogamy and autogamy. This was also observedin pollination studies (discussed later).

The ovoid, turgid and acute dry dehiscentcapsules of this species dehisce septicidally and/or loculicidally shedding extremely small sized(1.30 x. 1.0 mm) and light seeds (1000 seeds

weigh approximately 60 mg) (Fig.4). The seed iscovered by hyaline reticulate loose sac like seedcoat enclosing the minute embryo. This ensures an

efcient seed dispersal mechanism as the plants ofthis species inhabit high wind zones and the windcarries its seeds far away from the mother plant. Italso avoids overcrowding around the seed- settingparent. Approximately 41% seeds germinated

under laboratory conditions as compared to 35%under nursery conditions (Fig.5).

One can have a reasonable clue to the evolutionaryprimitiveness and/or advancement of a speciesbased on certain morphological features (Cronquist1968). Based on these (Cronquist 1968), theprimitive characters observed in P.kurroa are long-

Table 1. Effect of different pollination methods on fruit set in P. kurroa.

Sr.No.

Treatments Opened owers/spike (no.)

Fruits /spike(no.)**

Fruit set(%)*

Fruit size(mm)**

Seed set/spike(mg)**

1. Open pollination 22.29 17.43(4.17)

77.56(61.83)

7.73(2.72)

24.57(4.95)

2. Selng (by bagging of singleinorescence)

21.14 0(0.71)

0(0)

0(0.71)

0(0.71)

3. Selng (by caging of 2-4inorescences per plant)

21.43 2.00(1.49)

9.02(17.46)

6.84(2.61)

1.57(1.25)

CD (P=0.05) N S 0.227 1.933 0.101 0.117

(values in parenthesis are *arc sign transformed values; **sq. root transformed values)

Figs. 1-9: Picrorhiza kurroa

1 Flowering plant, 2 Floral parts (a) single fower (b) calyx

(c) corolla with four stamens (d) pistil, 3 Propionocarmine

stained pollen, 4 Seeds, 5 Seed germination in nursery,

6 Effect of open and self pollination on spike length, 7Diplotene (2n=34), 8 Diakinesis(2n=34), 9 Metaphase-I

(2n=34)

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lived habit, simple chlorophyllous leaves, perfectowers, hypogynous ovary forming dehiscentfruit and epigeal germination; where as temperatehabitat, herbaceous and deciduous habit, cyclicoral parts with few of each kind, perianthdifferentiated into calyx and corolla, petals united,

stamens differentiated into laments and anthersproducing triaperturate pollen (Fig.3), carpel fullyclosed and united with axile placentation andterminal stigma, pollination by insects other thanbeetles (Bombus spp. and Apis orae) are the majoradvanced characters.

Cytological studies

In all the pollen mother cells studied, 17 bivalentswere regularly observed at diplotene, diakinesisand metaphase-I followed by regular anaphase -Iwith chromosomes segregation of 17-17 univalentsat each pole (Fig.7, 8&9). In none of the pollenmother cells could any abnormality like laggards,micronuclei, etc be observed. Pollen stainabilitywas found to be approximately 95%.

Despite extensive literature survey, no publishedrecord of cytogenetical details including thechromosome number of P. kurroa could be found.With 2n=34 chromosomes observed in the presentstudy, the presumed base number would be17. Base numbers of above 12 are consideredsecondary base numbers having evolved as aresult of ascending or descending dysploidy and a

cross between forms with different base numbersfollowed by chromosome doubling (Stebbins 1971).It is presumed that this base number (17) hasarisen as a result of a cross between progenitorswith different base numbers. One possibility couldbe of a cross between progenitors with x=8 and 9followed by chromosome doubling.

The primary base numbers reported in familyScrophulariaceae form a series from x=6 up to 12and taxa with 2n=12 to 168 are reported in literature

(Federov 1969) and 2n= 34 is not unknown in thefamily. Twenty nine species belonging to this

family are with 2n=34 chromosomes (Federov1969). Out of these 29 species, 23 species belongto genus Veronica, 2 species to genus Celsia, 3species to Verbascum and 1 species to Veronicum.It is interesting to note that the genus Veronicaand Picrorhiza have been placed in the samesubtribe (Veroniceae), tribe (Digitaleae) and series(Rhinanthideae) of family Scrophulariaceae. Due to theabsence of any literature regarding chromosomenumber of the species, the present observations of2n=34 in P. kurroa appears to be the rst report ofchromosome number in this species.

Pollination studiesApproximately 77 per cent fruit set was observedin open pollinated spikes whereas no fruit set

was observed when single spikes were bagged.However, approximately 9 per cent fruit set wasobserved when 2-4 spikes of the same plantwere bagged together. The capsule size of openpollinated spikes was 7.73 mm as compared to 6.44mm when 2-4 spikes/plant were bagged together.

Seed yield per spike was also signicantly higher(24.57 mg) under open pollination as comparedto 1.57 mg recorded when 2-4 spikes/plant werebagged together (Table-1). The length of openpollinated spikes increased considerably whereasself pollinated ones did not show any lengthincrease and subsequently dried with dry oralparts remaining attached (Fig.6). No fruit set underselfed (bagging) conditions and signicant fruit setunder open conditions were indications towardscross compatible behaviour of this species. Thisview is also supported by oral morphology of

this species (exerted stamens and free hangingstigma; compacted inorescences and bluishcoloured owers).

Development of fruit set, when more numberof spikes of a single plant was bagged together,indicates some degree of self pollination. Itappears that as an insurance against failed cross-pollination, the introrse anthers ensure setting ofsome seed through self- pollination. This indicatesthat although the species favours cross-pollination,some extent of self pollination is also possible andself incompatibility barriers are not completelyexpressed. It also suggests that the species iscross pollinated and in the absence of the agents/conditions, pollinations wont occur.

References

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Chopra, R. N. and Ghosh, S. 1934. Some commonindigenous remedies. Indian Journal of MedicalResearch 22: 263-264.

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