P. N. Krishnan

Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Tiruvananantapuram, Kerala, India

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Publications (17)18.59 Total impact

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    ABSTRACT: A rapid in vitro propagation protocol for large-scale cultivation of Lagerstroemia speciosa L., an important medicinal and ornamental tree was established using nodal explants in Schenk and Hildebrandt (SH) medium optimized with various concentration and combinations of auxins and cytokinins. The study revealed, for the first time, accumulation of corosolic acid (CRA), an anti-diabetic and anti-obese pentacyclic triterpene in the in vitro plantlets. Multiple shoot induction was maximum in SH medium supplemented with 4.44 µM Benzyl Amino Purine (BAP) and shoot initials, when transferred to SH medium supplemented with 2.22 µM BAP, resulted in shoot multiplication and elongation. Rooted plantlets, after 2 weeks of hardening in mist-house and 3 months of rearing under shade-net, transferred to forest segments showed successful establishment (100 %). In a short duration of 24 weeks, ~2,450 hardened plantlets were obtained from a single nodal explant. The ISSR banding profiles of the regenerants and the mother plant were highly monomorphic and similarity matrix (0.96–0.99) confirmed genetic fidelity of the clones. Chemical analysis using HPLC showed that CRA content is more in mature (0.012–0.062 % DW) than young leaves (0.004–0.007 % DW) of the in vitro derived plantlets. The differential synthesis of CRA in micropropagated and mother plant was studied using gene expression profiles. The expression of upstream rate limiting genes in terpenoid biosynthesis was relatively high in young leaves while downstream Cytochrome P450 hydroxylase (CYP450H), catalyzing the final step(s) in CRA synthesis, was more in mature leaf, suggesting a strong correlation between observed CRA variation and gene expression. The study signifies the role of in vitro methodology for true-to-type regeneration and its possible utility for biosynthesis of CRA, throughout the year.
    Plant Cell Tissue and Organ Culture 12/2014; · 2.61 Impact Factor
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    ABSTRACT: A rapid in vitro propagation protocol for large-scale cultivation of Lagerstroemia speciosa L., an important medicinal and ornamental tree was established using nodal explants in Schenk and Hildebrandt (SH) medium optimized with various concentration and combinations of auxins and cytokinins. The study revealed, for the first time, accumulation of Corosolic acid (CRA), an anti-diabetic and anti-obese pentacyclic triterpene in the in vitro plantlets. Multiple shoot induction was maximum in SH medium supplemented with 4.44 µM Benzyl Amino Purine (BAP) and shoot initials, when transferred to SH medium supplemented with 2.22 µM BAP, resulted in shoot multiplication and elongation. Rooted plantlets, after 2 weeks of hardening in mist-house and 3 months of rearing under shade-net, transferred to forest segments showed successful establishment (100%). In a short duration of 24 weeks, ~2450 hardened plantlets were obtained from a single nodal explant. The ISSR banding profiles of the regenerants and the mother plant were highly monomorphic and similarity matrix (0.96-0.99) confirmed genetic fidelity of the clones. Chemical analysis using HPLC showed that CRA content is more in mature (0.012 to 0.062% DW) than young leaves (0.004 to 0.007% DW) of the in vitro derived plantlets. The differential synthesis of CRA in micropropagated and mother plant was studied using gene expression profiles. The expression of upstream rate limiting genes in terpenoids biosynthesis was relatively high in young leaves while downstream Cytochrome P450 hydroxylase (CYP450H), catalyzing the final step(s) in CRA synthesis, was more in mature leaf, suggesting a strong correlation between observed CRA variation and gene expression. The study signifies the role of in vitro methodology for true-to-type regeneration and its possible utility for biosynthesis of CRA, throughout the year.
    Plant Cell Tissue and Organ Culture 12/2014; · 2.61 Impact Factor
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    ABSTRACT: An efficient in vitro propagation protocol was standardized in K. galanga, wherein shoot cultures were raised from rhizome with axillary bud explants in Murashige and Skoog (MS) medium supplemented with different hormonal regimes. Maximum 10.6±0.83 multiple shoots per explants were obtained in MS medium supplemented with 4.0 mgl-1 6-benzyl adenine (BA) along with 1.0 mgl-1 each of α-naphthaleneacetic acid (NAA) and kinetin. Subsequent 2-3 subculture passages enhanced the shoot multiplication rate to 3-4 times. The multiple shoots thus produced were having individual root system and elongated substantially when kept in the same medium for 4-6 weeks. There was no need of elongation and root induction phase and this two-step multiplication procedure reduced the period of plantlet production compared to the earlier protocols. The regenerated plants after a short hardening phase got established in the field at 80-90% efficiency and their genetic uniformity was confirmed through ISSR analysis. The protocol thus standardized is an efficient method for rapid propagation of this high value medicinal species by which at least 30 shoots per explants can be produced within 12 weeks duration. It offers the possibility of raising physiologically uniform plants for easy dissection of shoot tip meristems which can be efficiently utilized for subsequent long-term conservation through cryopreservation.
    IOSR Journal of Pharmacy. 12/2014; 4(12):16-23.
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    ABSTRACT: An efficient protocol has been devised for the propagation and field establishment of Eulophia cullenii (Wight) Bl., a terrestrial orchid having ornamental potentialities, and is critically endangered in Western Ghats, India. Seeds extracted from 60–90-d-old capsules germinated in ½ MS, ¼ MS, Knudson C, or Mitra liquid medium developed into 1.4–2.5-mm-diameter protocorms in 60 d. Supplementation of organic additives like coconut water, peptone, yeast extract, and casein acid hydrolysate (CH) significantly enhanced protocorm growth. Upon subculture onto agar-gelled Mitra medium fortified with 0.05% CH, 56% of protocorms regenerated into shoots through the formation of linear mini-rhizomes. The regenerated shoots grew vigorously in ½ MS, producing new rhizomes. Mature rhizomes from axenic seedlings produced maximum (13 ± 1.4) shoots/whole rhizome in ½ MS fortified with 44.4 μM 6-benzylaminopurine (BAP), in 120–150 d. Horizontal and longitudinal halves of the rhizome also gave multiple shoots (6–8.5) in the presence of 44.4 μM BAP. Shoots or shoot clumps sub-cultured onto ½ MS basal medium produced roots followed by rhizomes in 60–150 d. Seedlings with mature rhizomes showed 70% establishment in the nursery and added a new rhizome at the end of one growth cycle. An average of 70.6% of the rhizomes originating from seedlings during the second growth cycle sprouted to produce new shoots, when planted in the native localities. Asymbiotic germination and cloning through rhizomes thus can provide a large number of vigorous plants of E. cullenii for ornamental exploitation as well as eco-restoration, if rhizome as storage organ is ensured in the propagule.
    In Vitro Cellular & Developmental Biology - Plant 11/2013; · 1.14 Impact Factor
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    ABSTRACT: Zygotic embryos excised from immature green fruits of the rattan palm, Calamus thwaitesii and cultured for 16 weeks under optimum culture conditions in Murashige and Skoog (MS) medium supplemented with 31.67 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 35.23 μM 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) produced mixed (compact and friable) calli at 70 and 92 % rates. The semi-friable part of the callus (~500 mg) separated and subcultured in medium containing 2.22 μM 6-benzyladenine and 1.07 μM α-naphthalene acetic acid produced groups of 10.37 ± 0.60–21.52 ± 0.48 discrete globular embryoids of varied size in 6–8 weeks. Calli raised in presence of 2,4,5-T were relatively more prolific, friable and embryogenic than those induced by 2,4-D. Embryoids (2.0–3.0 mm) isolated and cultured in basal medium germinated into plantlets at 65 % efficiency while the immature (0.5–2.0 mm) ones produced calloid structures. Approximately 15 % of the in vitro plantlets raised from the 2,4-D-induced embryogenic calli produced secondary immature embryoids on the sheath and lamina parts of leaves which were isolated and cultured in basal medium developed into rooted plantlets at 62 % rate in 12–16 weeks. The continued growth of the embryo-derived callus through successive subcultures together with differentiation of embryoids into plantlets, and the formation of immature embryoids on in vitro plantlets in MS basal nutrient medium reports for the first time a reliable method of producing at least 116 plants from a single embryo in a year. Rooted plantlets treated with 50 % glycerin survived at 78 % rate after hardening and 82.7 % of the hardened plants reintroduced into forest segments showed uniform growth free of morphological abnormalities after 3 years of observation. In addition to embryogenesis, cryopreservation of the zygotic embryos through simple drying and encapsulation–dehydration methods resulting 60–70 % recovery rates also offers another option for long-term conservation and sustainable utilization of this plant genetic resource.
    3 Biotech. 06/2012; 3(3).
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    ABSTRACT: Musa acuminata ssp. burmannica, one of the wild progenitors contributing 'A genome' to the present-day dessert bananas, has a long evolutionary history intervened by human activities. In this study, ISSR markers were used to analyze the pattern of genetic variation and differentiation in 32 individuals along with two reference samples (viz., Musa acuminata ssp. burmannicoides, var. Calcutta 4 and Musa balbisiana) of wild Musa, which corresponded to three populations across the biodiversity-rich hot spot of southern Western Ghats of India. High levels of genetic diversity were revealed both at the species and population levels, using Nei's diversity indices. The hierarchical analysis of molecular variance showed pronounced genetic differentiation, as 96% of the total variance was fixed within population and only 4% among populations. Nei's genetic differentiation coefficient (GST=0.1823) and low gene flow (Nm=1.18) further confirmed this. The positive correlation (Mantel test) between geographic distance and genetic distance (r=0.338 P<0.001) indicates geographic isolation as one of the key factors in shaping the population genetic structure. Grouping of individuals was largely in conformity with their spatial distribution, which was confirmed by UPGMA cluster analysis and PCA scatter plot clustering all 32 individuals into three major groups along a geographical gradient. The discontinuous distribution and dwindling population due to habitat fragmentation are serious threats to prevailing genetic diversity in this species. Conservation measures based on diversity pattern are suggested for long-term preservation and sustainable utilization of this precious genetic resource. KEY MESSAGE: A diverse germplasm of Musa acuminata ssp. burmannica exists in southern Western Ghats as a possible repository of useful resistant traits, which can be effectively utilized for crop improvement.
    Plant Cell Reports 05/2012; 31(9):1591-601. · 2.94 Impact Factor
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    R.K. Radha, William S. Decruse, P.N. Krishnan
    Current Frontiers in Cryopreservation, 03/2012; , ISBN: 978-953-51-0302-8
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    ABSTRACT: In vitro propagation of diploid land race of Musa cv. ‘Ambalakadali’ was achieved through organogenesis both from shoot primordial meristems and male floral apices taken out from mature disease free field grown plants. Healthy cultures were successfully initiated from shoot primordial meristems when independent dosage of BAP at 4.0-mg/l was given in MS nutrient media. The inflorescence apices responded the most to the in vitro culture conditions at 6.0-mg l-1 BAP in MS media itself. In both the cases, culture proliferation requires comparatively lower dosages of BAP. Subsequent cultures displayed enhancement in rate of shoot initiation and multiplication in the inflorescence derived cultures. Transfer of shoots (4-5 cm) into MS basal medium containing 0.1% activated charcoal (AC) favoured generation of healthy shootlets and formation of profuse roots during a period of 4 weeks. The plantlets (15 cm) after acclimatization in green house were distributed to local farmers for field evaluation. Plants flowered normally within 12 months and produced uniform characters as that of the mother plants.
    INTERNATIONAL JOURNAL Of ACADEMIC RESEARCH. 03/2011; 3(2):1088-1095.
  • Peringattulli Narayanan Krishnan, S. W. Decruse, R. K. Radha
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    ABSTRACT: Climate change, alien species, and use of land for intensive farming and development are causing severe threat to the plant genetic diversity worldwide. Hence, conservation of biodiversity is considered fundamental and also provides the livelihoods to millions of people worldwide. Medicinal plants play a key role in the treatment of a number of diseases, and they are only the source of medicine for majority of people in the developing world. The tropical regions of the world supply the bulk of current global demand for “natural medicine,” albeit with increasing threat to populations in the world and its genetic diversity. India is a major center of origin and diversity of crop and medicinal plants. India poses out 20,000 species of higher plants, one third of it being endemic and 500 species are categorized to have medicinal value. The Western Ghats is one of the major repositories of medicinal plants. It harbors around 4,000 species of higher plants of which 450 species are threatened. Currently, the number of species added to the red list category in this region is increasing, and the valuable genetic resources are being lost at a rapid rate. Demand for medicinal plants is increasing, and this leads to unscrupulous collection from the wild and adulteration of supplies. Providing high-quality planting material for sustainable use and thereby saving the genetic diversity of plants in the wild is important. During the last 25years of intensive research, Tropical Botanic Garden and Research Institute has developed in vitro protocol for rapid regeneration and establishment of about 40 medicinally important rare and threatened plants of Western Ghats. In situ conservation alone would not be effective in safeguarding these important species. Thus, utilizing the biotechnoligical approach to complement ex situ conservation program is becoming vital. Propagating biotechnology tools in plant conservation program is a prerequisite to succeed in sustainable use and to complement the existing ex situ measures. In addition to propagation, storage of these valuable genetic resources is equally important. In vitro slow growth of 35 species and cryopreservation using embryo/meristem/seed in 20 different species of rare medicinal plants of this region is accomplished. Plants developed in vitro of ten medicinal plants, which have restricted distribution, were reintroduced in the natural habitat as well. KeywordsConservation–Medicinal plants–Western ghats– In vitro conservation–Cryopreservation–Micropropagation
    In Vitro Cellular & Developmental Biology - Plant 02/2011; 47(1):110-122. · 1.14 Impact Factor
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    ABSTRACT: An efficient micropropagation protocol was developed for direct shoot induction from the seedlings of Rubia cordifolia , a species sparsely distributed in the Western Ghats of India. Plant regeneration was achieved using shoot tip, nodal and split nodal half explants on Murashige and Skoog medium. Effect of plant growth regulators like 6-Benzyladenine, Kinetin, 6-Benzyladenine + Indole-3-acetic acid and 6-Benzyladenine + α-Naphthalene acetic acid on shoot multiplication and Indole-3-acetic acid, Indole-3-butyric acid and α-Naphthalene acetic acid on rooting was studied. R . cordifolia shoots showed abundant proliferation and rooting capacity, both of which are significantly influenced by the varying concentrations of the different plant growth regulators. The optimum number of shoot obtained were 5.9 and 5.2 per explants in 2 weeks on the medium supplemented with 1 mg L<SUP>-1</SUP> Benzyladenine and 0.02 mg L<SUP>-1</SUP> Indole-3-acetic acid in nodes and split vertical halves of the node respectively. The results suggest that the nodal explants (intact and split vertical halves) are better sources of shoot formation than the shoot tip explant. Shoot multiplication was rapid and consistent for 4 subcultures with 0.5 mg L<SUP>-1</SUP> Benzyladenine. The best root induction (98%) and survival was achieved on 1 mg L<SUP>-1</SUP> Indole-3-butyric acid followed by 1 mg L<SUP>-1</SUP> Indole-3-acetic acid. Rooted plantlets were successfully transferred to green house conditions with 89% survival. Micropropagated plants displayed normal phenotypes in ex situ conditions. These plantlets can be used to replenish declining populations in the wild, for the extraction of bioactive compounds and reducing pressure on wild stocks.
    International Journal of Botany 01/2011;
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    S.Mukunthakumar, G.Praveen, P.N. Krishnan, S.Seeni
    National conference on Banana; 10/2007
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    National Conference on Banana; 10/2007
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    ABSTRACT: This study reports a protocol for successful micropropagation of Decalepis arayalpathra (Joseph and Chandras) Venter. (Janakia arayalpathra Joseph and Chandrasekhran; Periplocaceae), a critically endangered and endemic ethnomedicinal plant in the southern forests of the Western Ghats which is overexploited for its tuberous medicinal roots by the local Kani tribes. Natural regeneration is rare and conventional propagation is difficult. Conservation of the species through micropropagation was attempted. The nodal explants of greenhouse-raised plants, were more desirable than cotyledonary nodal explants of aseptic seedlings. The basal nodes (73%) of 12–16-wk-old greenhouse-grown plants cultured in Murashige and Skoog (MS) medium containing 12.96 μM 6-benzyladenine (BA), 2.48 μM 2-isopentenyladenine (2-ip) and 2.68 μM α-naphthaleneacetic acid (NAA) formed 16–17 cm long unbranched robust solitary shoots in 8 wk. Cotyledonary nodal explants cultured in the same medium showed multiple shoot formation and axillary branching. But the shoots were thin, fragile and not suitable for mass propagation. Single nodes of a solitary shoot subcultured on MS medium containing 2.22 μM BA and 0.24 μM 2-ip together produced 9.8±0.3 nodes from 18.0±0.6 cm long shoots within 5–6 wk. The basal nodes of the shoots so formed were repeatedly subcultured to increase the stock of propagules while the 2.5–3.0 cm terminal cuttings were used for rooting. The best root induction (68%) and survival (86%) was achieved on half-strength MS medium supplemented with 1.07 μM NAA. Field-established plants showed uniform growth and phenotypic similarity to parental stock.
    In Vitro Cellular & Developmental Biology - Plant 01/2005; 41(5):648-654. · 1.14 Impact Factor
  • C. G. Sudha, P. N. Krishnan, P. Pushpangadan
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    ABSTRACT: A rapid micropropagation system was established forHolostemma annulare (Roxb.) K. Schum., (H. ada-kodien R. Br. ex Schult; Asclepiadaceae), a rare medicinal plant. Shoot tips (0.5–0.8 cm) and terminal and basal nodes (1.0–1.5 cm) harvested from actively growing shoots of conventionally raised plants were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA). Multiple shoot formation (3.8) was observed in 68% of basal nodes cultured on medium with optimum concentration of 4.43 μM BA and 0.54 μM NAA after 8 wk. Terminal nodes were not suitable for inducing multiple shoots. Irrespective of the orientation (vertical/horizontal), all shoot tip explants responded with a single shoot in all the combinations of plant growth regulators tried. Effects of other cytokinins (kinetin and 2-isopentenyladenine) and auxins [indole-3-acetic acid and indole-3-butyric acid (IBA)] to enhance the regeneration potential of basal nodes were analyzed. Shoots were multiplied by subculture of basal nodes and stumps (the original explant tissue free of shoots, but with remnant axillary, meristem and two or three protruding buds) in a reduced concentration of BA (2.21 μM) and NAA (0.27 μM). Liquid medium for multiplication was found to be ineffective due to a high degree of hyperhydricity. To make the multiplication process cost effective, culture bottles with polypropylene, caps were used for multiplication. The best root induction (75%) and survival (80%) was achieved on 0.5 strength MS medium supplemented with 1.48 μM IBA. Field-established plants had uniform growth habit traits in terms of height of plants and number, length, and weight of the tuberous roots.
    In Vitro Cellular & Developmental Biology - Plant 12/1997; 34(1):57-63. · 1.14 Impact Factor
  • P N Krishnan, C G Sudha, S Seeni
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    ABSTRACT: Rapid micropropagation of Trichopus zeylanicus Gaertn. subsp. travancoricus Burkil ex Narayanan, a rare ethnomedicinal herb endemic to the Western Ghats of southern India, was achieved by culturing shoot tips (0.3-0.5 cm) of 2-month-old axenic seedlings on Woody Plant Medium. Among the cytokinins tested, only BAP induced callus-free multiple shoot bud formation, with a maximum of 8.5±0.4 buds per explant being obtained with 2.0 mg.l(-1) BAP after 8 weeks of culture. Shoot tips containing proliferated buds were divided and subcultured on medium containing 0.2 mg.l(-1) BAP to produce 12.0±1.0 shoots per explant in 6 weeks. Excision of buds after culture initiation, with subculture of the debudded basal tissue in 2 successive passages yielded 20.0±1.0 and 13.5±0.5 buds per explant respectively. Each bud cultured in turn for 4 weeks on WPM with 1.0 mg.l(-1) BAP formed 3.8±0.4 secondary buds which were repeatedly recultured to increase bud production. Altogether this method enabled an estimated harvest of 7848 buds from a single shoot tip in 28 months. Shoots (3-5 cm) developed from bud cultures were rooted in half-strength WPM medium with 0.5 mg.l(-1) each of NAA and IBA, and 90-100% of the rooted plants were established in the field after hardening. Micropropagated plants were grown to maturity free of defects in growth, morphological, flowering and seed set characteristics.
    Plant Cell Reports 08/1995; 14(11):708-11. · 2.94 Impact Factor
  • P N Krishnan, S Seeni
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    ABSTRACT: A rapid propagation method comprising initiation of in vitro shoot tip culture from field-grown flowering plants and reculture of the nodal segments of regenerated shoots in Schenk and Hildebrandt (1972) medium was developed for Woodfordia fruticosa (L.) Kurz., a rare medicinal shrub. A medium supplement of 6-benzylaminopurine (0.2 mg.l(-1)) induced high frequency (88%) development of axillary shoot buds (3.2) in 4-5 weeks. Subculture of the explants with multiple new shoots in fresh medium for 30 days yielded an even larger number (9.7) of shoots. Highest multiplication (26-35 shoots) was recorded when using culture initiation media with 0.5 mg.l(-1) each of BAP and NAA followed by subculture in 0.2 mg.l(-1) BAP. The shoot multiplication rate was further accelerated by reculturing 0.4-0.6 cm nodal segments of regenerated shoots in media with 1.0 mg.l(-1) BAP. Shoot cuttings (3.5-7.0 cm) were rooted in 0.2 mg.l(-1) IAA. Regenerated plants displayed uniform morphological, growth and flowering characteristics.
    Plant Cell Reports 11/1994; 14(1):55-8. · 2.94 Impact Factor
  • R K Radha, S William Decruse, P N Krishnan
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    ABSTRACT: Desiccation sensitivity and freezing tolerance of excised embryonic axes from the seeds of Nothapodytes nimmoniana (Graham) Mebberly and their cryopreservation were examined. Zygotic embryos with cotyledons excised from fully ripened fruits possessed 55.7% moisture content (MC) and 86.67% of them germinated in Murashige and Skoog (MS) medium devoid of plant growth regulators (PGR). Dehydration under laminar airflow for 120 min reduced the MC of the embryos to 19.6% and germination to 68%. Subsequent reduction in MC to 15.4, 14.2 and 12.1% also reduced the germination. Embryos dehydrated for 120 min after 1-wk-storage in Liquid nitrogen (LN) showed 60% germination, which was the optimum condition obtained in the study. The LN treated embryos developed into healthy seedlings after 30-60 d of inoculation in MS basal medium similar to desiccated control. Prolonged desiccation damaged the plumule where only root formation was observed. The study reveals the possibility of long term ex situ conservation of N. nimmoniana, which produces large and intermediate type of seeds, through zygotic embryo cryopreservation. Nothapodytes nimmoniana (Graham) Mebberly, a member of family Icacinacea, is a vulnerable tree species of the Western Ghats. It is valued for cytotoxic quinoline alkaloid, antitumor drug 1 , camptothecin (CPT) and also has immense medicinal applications against retrovirus and human immuno-deficiency virus. Conservation efforts are much more required for this species as their population is becoming narrow due to habitat destruction and over exploitation 2 . Seeds of N. nimmoniana are large and intermediate type, and showed 86.67% germination under controlled conditions. Embryonic axes with cotyledons, having moisture content (MC) of 55.7% and presumed to be intermediate in nature, lose their viability within a short period after maturity. Cryopreservaton of zygotic embryos is recognized as an effective tool for the long-term preservation of such plant species those produce recalcitrant/large seeds 3 . The present communication describes the feasibility of long-term conservation of N. nimmoniana germplasm through cryopreservation of embryonic axes. N. nimmoniana seeds (Figs 1a & b) were collected in October-November from the mature trees of Pallivasal (Idukki District, Kerala) in the Southern Western Ghats. The seeds were separated from the fruits (drupe), rinsed in running tap water for 1 h to remove the mucilage and washed in 1% Teepol detergent (Godrej India Ltd., Mumbai), followed by continuous washing in running tap water for 10-20 min. Seeds were then surface decontami-nated by immersion in 0.01% (w/v) HgCl 2 for 5-10 min, followed by 3-5 rinses in sterile distilled water. The embryos with cotyledons were dissected out after breaking seed coat and endosperm in a laminar air flow cabinet. The embryos were subjected to desiccation for a period of 0, ½, 1, 1½, 2, 2½, 3 and 3½ h; and for each treatment, 25 embryonic axes (total 200 embryonic axes) were desiccated in open Petridishes in the laminar air flow chamber. After respective desiccation period, one lot of the embryos were inoculated into MS 4 medium devoid of plant growth regulators as control and another lot was transferred into 2 mL cryo-vials and plunged into liquid nitrogen (LN) at –196°C. After 24 h storage, the vials were retrieved from LN and rewarmed in a water bath at 40°C for 1-2 min. The rewarmed embryos were then transferred to MS medium and maintained under 12 h light (25-50 µE m –2 s -1) at 25±2°C for 8 wk for recovery. Moisture content of the embryos was determined by constant temperature oven method (at 103°C); samples were kept in the oven for 17h. Observation on the germination of embryos was made after 8 wk and results were analyzed statistically in a completely randomized model. Survival rate was assessed as percentage of embryonic axes that exhibited any kind of growth including seedling development, shoot growth and root growth.