Foliar Micromorphological Response of In Vitro Regenerated and Field Transferred Plants of Oldenlandia umbellata L.: A Medicinal Forest Plant

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 $4^{th}$ 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.

Introduction

Oldenlandia umbellata L. (Rubiaceae) is a natural dye yielding plant. It is a small, prostrate, profusely branched perennial herb. The plant is native to Indian subcontinent but distributed in the forests of Myanmar, Sri Lanka, Cambodia, Indonesia, India, Pakistan and Africa (Siva 2007). It is commonly known as Madder plant or Chay root and the red dye extracted from the roots used in calico printing and coloring of wool and silk fabrics for centuries (Yoganarasimhan and Chelladurai 2000).

All parts of O. umbellata are traditionally used by the tribal people and ethnic communities of India and China for the treatment of asthma, bronchitis and bronchial catarrh (Yoganarasimhan and Chelladurai 2000; Samy et al. 2008). This plant produces some important bioactive compounds including anthraquinone derivatives, saponins, tannins, terpenoids, ursolic acid, kaempferol-3-O-rutinoside, oledicoumarin, hedyotiscone, cedrelopsin, pheophorbide etc. (Ramamoorthy et al. 2009).

The natural population of this plant has been depleted due to over exploitation of O. umbellata for its roots (for red dye) and medicinal values (Siva et al. 2009; Siva et al. 2012). This plant is propagated by seeds only, but the plants harvested before flowering and fruiting therefore, naturally grown plants cannot fulfill the ever increasing demand. The tissue culture protocols were developed by various research groups as alternate method of propagation to conserve this important forest plant (Siva et al. 2009; Shekhawat et al. 2012; Siva et al. 2012; Kumar et al. 2014; Krishnan and Siril 2016; Krishnan and Siril 2017a, 2017b). Though, in vitro propagation techniques attained immense importance in recent years, the hidden drawbacks rely in the loss of tissue culture raised plants during field transfer. The presence of growth regulators, sucrose, constant temperature and low CO2 concentrations under in vitro conditions lead to the formation of abnormal anatomical structures (variations in stomatal distribution and pattern, venation pattern and vein density, trichomes, raphides and crystals) affect survival of plantlets under harsh natural conditions. The foliar micromorphological studies might be extremely useful in acclimation research as the internal structures of the leaves are highly responsive to environmental changes which lead to improve the survival percentage of tissue culture raised plants under natural habitats (Revathi et al. 2018).

Understanding of foliar micromorphological developments of in vitro and field grown plantlets could help to overcome the difficulties in successful establishment of plants under field conditions. Moreover, foliar micromorphological changes can also be served as one of potential marker to study the changes during acclimatization of micropropagated plants under natural conditions. This is the foremost report exploring the comparative foliar micromorphological investigations of tissue culture raised and field transferred plants (after 6 weeks) of O. umbellata to predict the proper stage of the plantlets to be shifted to the natural habitats.

Materials and Methods

Study area and collection of plants

Oldenlandia umbellata plants were collected from the East-coast of the Union Territory of Puducherry, south India (11.9416°N, 79.8083°E). Seedlings were transported to the laboratory and maintained in the botanical garden and greenhouse of the K. M. Centre for Postgraduate Studies, Puducherry, India to collect the fresh and healthy explants from these mother plants.

In vitro propagation of O. umbellata

The tissue culture protocol developed for O. umbellata in our previous study (Shekhawat et al. 2012) is described here in brief because the same procedure was used again to establish the cultures. One year old O. umbellata plants were used as mother plants to collect the explants. The explants (nodal segments, bearing 2-3 nodes) were treated with 0.1% (w/v) broad spectrum antifungal agent Bavistin (a systemic fungicide; BASF India Ltd., India) for 5-7 min and washed with autoclaved double distilled water for 4-6 times. The surface sterilization was achieved with 0.1% (w/v) mercuric chloride (disinfectant, Hi-Media, India) solution for 4-5 min and finally rinsed five times with sterilized distilled water under aseptic conditions. Murashige and Skoog’s (MS) medium (Murashige and Skoog 1962) incorporated with 3.0 mg L-1 6-benzylaminopurine (BAP) with additives was effective for shoot bud induction. The shoots were multiplied on full strength MS medium augmented with 1.0 mg L-1 BAP and 0.5 mg L-1 indole-3 acetic acid (IAA). The adventitious roots were induced on half strength MS medium containing 2.5 mg L-1 indole-3 butyric acid. The in vitro produced shoots were also rooted by ex vitro rooting when pulse treated with 200 mg L-1 of IBA for 3.0 min. The rooted shoots were hardened in the greenhouse (28±2°C temperature with 60-70% relative humidity) and successfully shifted to the natural conditions.

Foliar micromorphological studies

Foliar micromorphological experiments were conducted to investigate the quantitative and qualitative structural changes taking place in the foliar constants and leaf architecture (i.e. stomatal distribution and pattern, venation pattern and vein density, trichomes, raphides and crystals) which support gradual adaptation of O. umbellata plantlets from lab to land. Shoots and leaves were randomly selected for the micromorphological evaluation from in vitro and field conditions. The leaves developed in vitro after 4th subculture in multiplication phase and after 6th weeks of field transferred plants were used in this study. The entire foliar apparatus at third to seventh leaves from the base of the shoots were excised manually. The epidermal peels from the leaves were separated manually by standard method to study the developments in stomata (Johansen 1940). Fresh leaves were initially fixed in FAA solution (formalin, acetic acid and ethyl alcohol) in the ratio of 1:1:3 (v/v), cleared in 70% ethanol (v/v) until the chlorophyll was removed (12-24 h), bleached with 5% (w/v) NaOH for 24-48 h, and rinsed three times in distilled water for the study of venation, trichomes and crystals. The leaves were then stained with 1% (v/v) safranine (Loba chemie, India) aqueous solution for 4-8 min and rinsed carefully in distilled water to remove excess stain, mounted in dilute glycerol, examined under Light microscope (Labomed iVu 3100, USA) and analyzed using Pixelpro software.

Experimental design, data collection and analysis

Microscopic fields were randomly focused from the leaves of 10 samples (in vitro and field transferred plantlets) to obtain the better mean number of stomata per square mm. The statistical analysis and terminology of stomatal types, density and venation pattern were of Salisbury (1932) and Hickey and Wolfe (1975).

Formulae used for calculation of stomatal density, frequency and index

Stomatal density (S.D.)=Number of stomata per square millimeter of leaf area

\(Stomatal frequency (S.F.) =\frac{\mathrm{S}}{\mathrm{E}} \times 100\)

\(\text { Stomatal index (S.L.) }=\frac{\mathrm{S}}{\mathrm{S}+\mathrm{E}} \times 100\)

Where, S=Average number of stomata per unit leaf area, and E=Average number of epidermal cells in the same unit area (Salisbury 1932).

Formulae used for calculation of vein-islet numbers, veinlet terminations, trichome density and raphide density

Absolute vein-islet number (V.I.) per sq. mm=Average vein-islet number per mm² ×Area of the leaflet in mm²

Absolute veinlet termination (V.T.) number per sq. mm= Average veinlet termination number per mm² ×Area of the leaflet in mm²

Trichome density (T.D.) per sq. mm=Average no. of trichomes per mm² ×Area of the leaflet in mm²

Raphide density (R.D.) per sq. mm=Average no. of raphides per mm² ×Area of the leaflet in mm²

The results were tested with the t-test in order to determine statistically significant differences in the two different environments at p≤0.05. The t-test was performed by means of Microsoft Excel version 7. The results were expressed as mean±standard error (SE) of 10 focal views and the average values were presented.

Results and Discussion

Establishment of cultures of O. umbellata

The cultures of O. umbellata were established using nodal shoot segments collected from the mature and healthy mother plants as described in the earlier report (Shekhawat et al. 2012). The buds were induced on MS medium augmented with 3.0 mg L-1 BAP. These shoots were further multiplied in liquid MS medium (without agar) contained BAP and IAA. The shoots were rooted by in vitro as well as ex vitro rooting methods using IAA and IBA. The rooted plantlets were successfully hardened in the greenhouse. The leaves of in vitro multiplied shoots and the field transferred plants were collected for the micromorphological analysis.

Foliar micromorphological studies

Significant improvements were observed in terms of stomatal frequency, veins and vein numbers, trichomes, raphides and crystals of the leaves from in vitro to the in vivo environments due to developmental adaptations in the foliar apparatus under natural conditions (habitats).

Morphologically, the in vitro raised shoots and leaves resembled the donor plants growing in nature with minor qualitative differences in size and thickness of the leaves. The leaves were sessile, opposite, verticillate, lanceolate, pubescent and the midrib was prominent on abaxial surface.

Leaves were amphistomatic and the type of stomata was rubiaceous/paracytic, typical to the family Rubiaceae. Epidermal cells were irregular in shape with undulating margins and arranged in regular connections with the stomatal guard cells. The epidermal cells of in vitro grown leaves possessed U-shaped sinuous walls but V-shaped walls were presented with the leaves of field transferred plants. The in vitro raised leaves possessed abundant (high frequency) open stomata with smooth cuticle and underdeveloped contiguous stomata (Figs. 1a, c), but the field transferred plant’s leaves were characterized by reduced stomatal frequency, well developed cuticle, compact epidermal cells with functional stomata (Figs. 1b, d). The stomatal density and stomatal index of the abaxial surface of in vitro leaves were 41.85±0.31 and 25.48±0.21, which were gradually reduced to 32.20±1.00 and 19.11±0.73 respectively after 6 weeks of field transfer (Table 1). The decreased stomatal density under field environments is reported to contribute the reduction of water loss in field grown plants (Chirinéa et al. 2012). Well developed cuticle and reduced stomatal index might help in physiological adaptation of micropropagated plantlets in the field. Bahadur et al. (1971) also reported mesogenous paracytic types of stomata in O. umbellata.

Table 1. Comparative foliar micromorphological studies of in vitro regenerated and field transferred plants of Oldenlandia umbellata

 

Fig. 1. Foliar micromorphological studies of O. umbellata. (a) Stomatal density in abaxial epidermis of in vitro developed leaves, (b) Stomatal density in the leaves of field established plants. (c) Magnified view of epidermis with stomata of in vitro leaves. (d) Magnified view of stomata of in vivo leaves. (e) Foliar vein architecture in the leaves under in vitro environment. (f) Developmental increase of vein-islets, veinlet terminations of field transferred plants (CS, contiguous stomata; PS, paracytic stomata; VI, vein islet; VT, veinlet termination).

Reticulate and unicostate types of venation patterns were observed in this species. The midrib was more prominent in the leaves of field established plants but less conspicuous midrib observed in the in vitro raised leaves. In general, the secondary veins were triplinerved in both the leaves. Vein islets were rhomboidal and polygonal in shape and presented 2-3 single as well as branched veinlet terminations. Vein islet numbers and veinlet termination were 14.1±0.39 and 29.0±0.10 per sq.mm with in vitro raised leaves which were gradually increased to 16.8±0.24 and 32.3±0.25 in the field established plants (Table 1; Figs. 1e, f). The gradual developments in the pattern of vein islet and veinlet terminations from rudimentary vascular tissues of in vitro leaves to the well developed vasculature in the natural conditions improve the field performance of the plants.

Calcium oxalate raphides, druces and crystal sands were present in the leaves of O. umbellata. The density and structures of these crystals varied in both the environmental conditions. The in vitro leaves possessed underdeveloped raphides with fewer numbers (8±0.00) gradually increased to 15±0.28 under field habitat (Table 1; Figs. 2a, b). The druses were varied in size and distributed all along with the major and minor veins in the leaves. Visibly more number of druse crystals were recorded in the field transferred leaves as compared to in vitro leaves (Figs. 2c, d). In general, the calcium oxalate crystals were reported to be toxic in plant cells and organs, and their formation in plants are determined genetically (Ilarslan et al. 2001). The development of crystals could also be influenced by light, temperature, pH and ion concentration in the surrounding environments (Kuo-Huang et al. 2007; Revathi et al. 2018). Calcium crystals in the cells were emerging as herbivory mechanism and also adopted for the intra genus classification (Tütüncü et al. 2014). Again, the location of the crystals in a species is a taxonomic feature and reported to be species specific (Lersten and Horner 2000).

 

Fig. 2. Raphides, crystals/druses and trichomes with the O. umbellata leaves. (a) Distribution of calcium oxalate raphides under in vitro condition. (b) Increased raphide density in the leaves of field transferred plants. (c, d) Calcium oxalate druses of in vitro and field transferred plants. (e) Density of trichomes developed under in vitro conditions. (f) Increased trichomes density observed with the leaves developed under field environment (URP, underdeveloped raphides; DRP, developed raphides; UTR, underdeveloped trichomes; DTR: developed trichomes).

The foliar trichomes were non glandular, unicellular, uniseriate and observed all over the lamina. The leaves raised under in vitro environments possessed underdeveloped trichomes with less density (13.5±0.17) (Fig. 2e) than the field transferred plants (18.2±0.33) (Table 1; Fig. 2f). Foliar trichomes help to reduce water loss, protect the tissues from UV radiation and participate in anti-herbivore/defense mechanism towards harsh environments and predators (Naidoo et al. 2009; Shekhawat et al. 2017). The increase in the densities and improvements in the structure of trichomes may be the indication of the development of defense mechanism against stressed environmental conditions and pathogens which ultimately improve the survival percentage of tissue culture raised plants under natural habitats.

Conclusion

The foliar micromorphological developments towards field adaptation of micropropagated plantlets of O. umbellata were studied for the first time. It can be concluded that the characters of epidermal cells, stomata, trichomes and accumulation of crystals in the leaves of in vitro developed and field transferred plantlets showed developmental repairs for successful establishment under the natural habitats. Foliar micromorphological evaluation at different stages of plant developments could reveal the most suitable stage of field transfer from the greenhouse. It was noted that four weeks of hardening of in vitro regenerated plants in the greenhouse could assist in effective acclimatization of plantlets of O. umbellata. The study could help to understand the foliar micromorphological response of plants under changed environmental conditions.

Conflict of Interest

The authors disclose that they have no conflict of interest in this publication.