The in Vitro Seed Germination Protocol for Ephedra Foliata

INTRODUCTION

Ephrdra foliata Boiss. (Ephedreace) commonly known as Somlata and unthphog in north western part of India. The plants are dioeciously woody shrubs with 3-5 m long trailing or scandent stem, having subverticillate or fascicled, thin, persistent branches with 2.5–10 cm long internodes. Leaves usually 3, linear setaceous, shortly connate at base, stem performs the function of assimilation and female plants bear semitransparent, nutritious edible berries (female cones) having sweet taste due to the fleshy bracts. These fleshy bracts are important during the food scarcity in arid regions (Meena B et al 2019). From the ancient time, decoction of whole plant is used to cure asthma, fever, snake bite, stomachic, blood purification, dropsy, worms and as cardio tonic (Silori et al. 2005; Quattrocchi 2012). It also retains antimicrobial and hepatoprotective properties (Alqasoumi and Abdel-Kader 2012; Bissa 2015) and high nutraceutical value (Kamboj 2000). But unsustainable exploitation, large scale habitat abolishing, disinclined growth rate, poor rejuvenation, foraging and other man made dilemmas have caused enormous reduction in its natural populations. Therefore, E. foliata has now become a rare or endangered species from a vulnerable category in its natural habitats (Kharin 2002; Joshi et al. 2013) Therefore conservation is primary need for existence of this species. Most of in vitro work carried on to Ephedra related to alkaloid content of callus (Khanna and Uddin 1976, 1977; Ramawat and arya 1977). Survey of literature reveals that no attempt has been made to develop in vitro seed germination protocol of Ephedra foliata. Present report deals with design the protocol for in vitro seed germination.

Ephedra foliata Boiss. seeds were collected from mature plant growing in Botanical garden of Department of Botany, Kurukshetra University, Kurukshetra. The seeds were washed under running tap water followed by 0.2 % solution of Tween twenty a detergent for five minutes. The surfaces of seeds were sterilized in 0.4% bavistin and 0.2% streptomycine solution for 25 minutes followed by three times rinsed with DDW. Prior to inoculation seeds were surface sterilized with 0.1% HgCl2 solution for two minutes and then repeatedly washed in DDW. Explants were cultured on Murashige and Skoog basal medium supplemented with 2% sucrose, different combinations of plant growth regulators and solidified with 0.7% Agar-agar was used. The medium free from PGR‘s acted as control. The pH of medium was adjusted to 5.7-5.8 with 0.1 N HCL and NaOH before autoclaving at 121° C for 15 minutes. The cultures were reserved under fluorescent light at 25° C ± 2 with a 16 hour photoperiod.

the seed germination of Ephedra foliata and next to no data is accessible for the generation of quality planting material by means of tissue culture. The best germination response of Ephedra foliata with complete development of root and shoots was seen as on MS medium with various combinations of growth hormones. The seeds of Ephedra were tested against their germination potential in field and lab conditions. In field only 4-5 % germination was observed in the month of mid-June to end of July. The germinated seedlings showed high mortality rate and only 1 % seedlings survived. While no seed germination observed on vermiculite throughout the year.

Collected Seeds From Plant Ephedra foliata in Botanical garden of Kurukshetra University On the other hand 30-55% germination of seeds observed in vitro conditions by influence of plant growth regulators. Various combinations and concentrations (0.25-2.5 mg/l) of PGRs were used for this experiment. The best results were found in half strength MS medium supplemented with 2 mg/l GA and 2mg/l BAP + 0.2 mg/l Kinetin. While 1.5 mg/l NAA, 0.5 mg/l BAP and Kinetin alone showed the Each experiment had 25 test tubes, five test tubes used as control with three replicates. Control did not show a single seed germination. Maximum germination was observed in half MS supplemented with 2 mg/l GA (55%) with mean seedling length 6.9 ± 0.02 cm, 30% germination was given by 2 mg/l BAP + 0.2 mg/l Kinetin with mean seedling length 6.7 ± 0.01 followed by 0.5 mg/l BAP (22%), Kinetin (18%) and 1.5 mg/l NAA (20%) with mean seedling length 2.1±0.5cm, 2.7±0.3cm and 1.6±0.4cm after the three weeks of inoculation.

Past in vitro investigations of Ephedra seeds which lack the key data for production of quality planting material. The seeds showed maximum germination percentage with GA but the higher concentration of GA inhibits the germination, the same influence of GA, BAP and IBA on seed germination of dragon fruit (Hylocereus undatus) studied (Vishnupriya J. et al 2019) and GA and IAA on seed germination of radish (Raphanus sativus L.) (Neelam D. and Abhishek B., 2019). The seed doesn‘t germinate below 20ºC and above 45ºC. The surface sterilizing chemicals also affect the germination percentage ie higher concentration of HgCl2 which might kills the embryo due to this seed unable to germinate. On the other hand some chemicals enhance the than in vivo plants. Ephedra is a source of different secondary metabolites and have great medicinal and pharmaceutical value, which may be useful for many industries.

We are thankful to the Plant tissue culture laboratory, Soenti, Kurukshetra, Haryana, India for providing infrastructure.

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Research Scholar, Department of Botany, Kurukshetra University, Kurukshetra, India mssonikuk@gmail.com