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Oldenlandia Umbellata L.: a Medicinal Forest Plant

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Oldenlandia Umbellata L.: a Medicinal Forest Plant Regular Article pISSN: 2288-9744, eISSN: 2288-9752 J F E S Journal of Forest and Environmental Science Journal of Forest and Vol. 35, No. 1, pp. 54-60, March, 2019 Environmental Science https://doi.org/10.7747/JFES.2019.35.1.54 Foliar Micromorphological Response of In Vitro Regenerated and Field Transferred Plants of Oldenlandia umbellata L.: A Medicinal Forest Plant Revathi Jayabal, Latha Rasangam, Manokari Mani and Mahipal Singh Shekhawat* Department of Botany, Kanchi Mamunivar Center for Postgraduate Studies, Puducherry 605008, India Abstract Plant tissue culture techniques offer quick methods of regeneration of plants of medicinal importance but the survival chances of such plants are always questionable when shifted to the in vivo conditions. The present study enumerates the micromorphological developments in the leaves of in vitro regenerated and field transferred plantlets of Oldenlandia umbellata. The leaves developed in vitro after 4th subcultures of multiplication phase and after 6 weeks of field transferred plants were used. Statistically significant differences in the number of stomata, veins, raphides, crystals and trichome density per square mm were observed. The improvements in stomatal apparatus and density (decreased from 41.85 to 32.20), developments in leaf architectural parameters and emergence of defense mechanism through increased numbers of raphides (8 to 15), crystals and trichomes (13.5 to 18.2) proved acclimation of tissue culture raised plantlets from in vitro to the in vivo environments lead to 100 % success in field establishment of the plantlets. The in vitro induced foliar abnormalities (changes in stomata, venation pattern, vein density, trichomes, crystals etc.) were repaired while hardening of plantlets in the greenhouse and finally in the field. The observed micromorphological response of leaves under altered environmental conditions could help in determination of proper stage of field transfer and prediction of survival percentage of in vitro regenerated O. umbellata plantlets. Key Words: acclimatization, foliar micromorphological analysis, Oldenlandia umbellata, survival Introduction printing and coloring of wool and silk fabrics for centuries (Yoganarasimhan and Chelladurai 2000). Oldenlandia umbellata L. (Rubiaceae) is a natural dye All parts of O. umbellata are traditionally used by the yielding plant. It is a small, prostrate, profusely branched tribal people and ethnic communities of India and China perennial herb. The plant is native to Indian subcontinent for the treatment of asthma, bronchitis and bronchial ca- but distributed in the forests of Myanmar, Sri Lanka, tarrh (Yoganarasimhan and Chelladurai 2000; Samy et al. Cambodia, Indonesia, India, Pakistan and Africa (Siva 2008). This plant produces some important bioactive com- 2007). It is commonly known as Madder plant or Chay pounds including anthraquinone derivatives, saponins, tan- root and the red dye extracted from the roots used in calico nins, terpenoids, ursolic acid, kaempferol-3-O-rutinoside, Received: January 10, 2019. Revised: January 30, 2019. Accepted: February 18, 2019. Corresponding author: Mahipal Singh Shekhawat Department of Botany, Kanchi Mamunivar Center for Postgraduate Studies, Puducherry 605008, India Tel: +91-8943157187, Fax: +91-04132251687, E-mail: [email protected] 54 Journal of Forest and Environmental Science http://jofs.or.kr Jayabal et al. oledicoumarin, hedyotiscone, cedrelopsin, pheophorbide East-coast of the Union Territory of Puducherry, south etc. (Ramamoorthy et al. 2009). India (11.9416°N, 79.8083°E). Seedlings were transported The natural population of this plant has been depleted to the laboratory and maintained in the botanical garden due to over exploitation of O. umbellata for its roots (for red and greenhouse of the K. M. Centre for Postgraduate dye) and medicinal values (Siva et al. 2009; Siva et al. Studies, Puducherry, India to collect the fresh and healthy 2012). This plant is propagated by seeds only, but the plants explants from these mother plants. harvested before flowering and fruiting therefore, naturally In vitro propagation of O. umbellata grown plants cannot fulfill the ever increasing demand. The tissue culture protocols were developed by various research The tissue culture protocol developed for O. umbellata groups as alternate method of propagation to conserve this in our previous study (Shekhawat et al. 2012) is described important forest plant (Siva et al. 2009; Shekhawat et al. here in brief because the same procedure was used again to 2012; Siva et al. 2012; Kumar et al. 2014; Krishnan and establish the cultures. One year old O. umbellata plants Siril 2016; Krishnan and Siril 2017a, 2017b). Though, in were used as mother plants to collect the explants. The ex- vitro propagation techniques attained immense importance plants (nodal segments, bearing 2-3 nodes) were treated in recent years, the hidden drawbacks rely in the loss of tis- with 0.1% (w/v) broad spectrum antifungal agent Bavistin sue culture raised plants during field transfer. The presence (a systemic fungicide; BASF India Ltd., India) for 5-7 min of growth regulators, sucrose, constant temperature and and washed with autoclaved double distilled water for 4-6 low CO2 concentrations under in vitro conditions lead to times. The surface sterilization was achieved with 0.1% the formation of abnormal anatomical structures (variations (w/v) mercuric chloride (disinfectant, Hi-Media, India) in stomatal distribution and pattern, venation pattern and solution for 4-5 min and finally rinsed five times with steri- vein density, trichomes, raphides and crystals) affect surviv- lized distilled water under aseptic conditions. Murashige al of plantlets under harsh natural conditions. The foliar and Skoog’s (MS) medium (Murashige and Skoog 1962) micromorphological studies might be extremely useful in incorporated with 3.0 mg L-1 6-benzylaminopurine (BAP) acclimation research as the internal structures of the leaves with additives was effective for shoot bud induction. The are highly responsive to environmental changes which lead shoots were multiplied on full strength MS medium aug- to improve the survival percentage of tissue culture raised mented with 1.0 mg L-1 BAP and 0.5 mg L-1 indole-3 ace- plants under natural habitats (Revathi et al. 2018). tic acid (IAA). The adventitious roots were induced on half Understanding of foliar micromorphological develop- strength MS medium containing 2.5 mg L-1 indole-3 buty- ments of in vitro and field grown plantlets could help to ric acid. The in vitro produced shoots were also rooted by overcome the difficulties in successful establishment of ex vitro rooting when pulse treated with 200 mg L-1 of IBA plants under field conditions. Moreover, foliar micro- for 3.0 min. The rooted shoots were hardened in the green- morphological changes can also be served as one of poten- house (28±2°C temperature with 60-70% relative humid- tial marker to study the changes during acclimatization of ity) and successfully shifted to the natural conditions. micropropagated plants under natural conditions. This is Foliar micromorphological studies the foremost report exploring the comparative foliar micro- morphological investigations of tissue culture raised and Foliar micromorphological experiments were conducted field transferred plants (after 6 weeks) of O. umbellata to to investigate the quantitative and qualitative structural predict the proper stage of the plantlets to be shifted to the changes taking place in the foliar constants and leaf archi- natural habitats. tecture (i.e. stomatal distribution and pattern, venation pat- tern and vein density, trichomes, raphides and crystals) Materials and Methods which support gradual adaptation of O. umbellata plantlets from lab to land. Shoots and leaves were randomly selected Study area and collection of plants for the micromorphological evaluation from in vitro and Oldenlandia umbellata plants were collected from the field conditions. The leaves developed in vitro after 4th sub- J For Environ Sci 35(1), 54-60 55 Foliar Micromorphological Analysis of Oldenlandia umbellata culture in multiplication phase and after 6th weeks of field Absolute veinlet termination (V.T.) number per sq. mm= transferred plants were used in this study. The entire foliar Average veinlet termination number per mm2×Area of the apparatus at third to seventh leaves from the base of the leaflet in mm2 shoots were excised manually. The epidermal peels from the Trichome density (T.D.) per sq. mm=Average no. of tri- 2 2 leaves were separated manually by standard method to chomes per mm ×Area of the leaflet in mm study the developments in stomata (Johansen 1940). Fresh Raphide density (R.D.) per sq. mm=Average no. of ra- leaves were initially fixed in FAA solution (formalin, acetic phides per mm2×Area of the leaflet in mm2 acid and ethyl alcohol) in the ratio of 1:1:3 (v/v), cleared in The results were tested with the t-test in order to de- 70% ethanol (v/v) until the chlorophyll was removed termine statistically significant differences in the two differ- (12-24 h), bleached with 5% (w/v) NaOH for 24-48 h, and ent environments at p≤0.05. The t-test was performed by rinsed three times in distilled water for the study of venat- means of Microsoft Excel version 7. The results were ex- ion, trichomes and crystals. The leaves were then stained pressed as mean±standard error (SE) of 10 focal views and with 1% (v/v) safranine (Loba chemie, India) aqueous sol- the average values were
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