III. Materials and Methods

III. Materials and Methods

III.1 Materials:

Three medicinal plant species growing in North - West Himalayan region and facing varying degree of threat due to overexploitation constitute the material for present study. The distribution range and threat status of these species is provided in the table III. 1.

Table III. 1 Distribution range and threat status of species under study

III.2 Methods

Three species proposed for study were located in natural populations and individuals of V. wallichii and P. kurrooa were also transplanted in the Lead Botanic Garden of the University. Space coordinates were recorded using Mobile Mapper Global Positioning System (MAGELLAN, France). The methodology used in assessing magnitude of threat and studying different aspects of reproductive biology, cytology and analysing genetic diversity using DNA based molecular markers is summarized as under:

III.2a Assessment of threat status:

For assessment of the existing status, cause and magnitude of threat to the species in nature, natural populations of these species were surveyed repeatedly over a period of four years and data regarding population size, density, area of occupancy was recorded. Quadrate method was used to study the number of individuals of the species under study at any site along with the other plant species growing in the same habitat. In case of Picrorhiza kurrooa, quadrate of the size 1 m x 1 m was used but in case of Valeriana wallichii and Ferula jaeschkeana quadrate size was increased to 10 m x 10 m keeping in view comparatively larger area covered under any population. Due to the stoloniferous habit, Picrorhiza kurrooa existed in small patches and as such each offshoot in a patch was counted as one individual plant. Information on parameters like soil type, moisture conditions, and availability of sunlight was also recorded. Data on ethnobotany, present and past distribution of the three species was also collected by interviewing the locals.

III.2b Reproductive biology:

Data on plant and floral morphology, phenology, pollen viability, pollen – ovule ratio, pollination mechanism, pollinator variability and their comparative efficiency, pollen – pistil interaction, genetic system, reproductive output, seed germination and seeding recruitment were collected for all the three species.

b (i) Floral – biology:

Data on anthesis was collected, under in situ as well as ex situ conditions. Time and pattern of anther-dehiscence was determined by periodic examination of anthers under the light microscope. For checking stigma receptivity, Stigmata were manually pollinated after different time intervals and examined for pollen germination either directly or after fixation in Carnoy’s fixative (3 parts absolute alcohol and 1 part glacial acetic acid) and then shifting them to 70% ethyl alcohol. For microscopic examination pistils were stained in Lewis’ stain ( mixture of 2 ml of 1% aq. acid fuchsin, 2 ml of 1% aq. light green and 40 ml lactic acid and 46 ml distilled water) (Lewis, 1979) and also in Aniline blue for fluorescence microscopy (Shivanna and Rangaswamy, 1993).

b (ii) Pollen- Ovule ratio:

Pollen output per flower was calculated by first counting the number of pollen grains per anther and multiplying this figure by the number of stamens per flower. The ovule count per flower was determined by putting the ovaries in 4N NaOH for12 – 14 h at 60° C in a hot air oven, washing them thoroughly in water to remove all traces of NaOH and then gently squashing the ovaries in a drop of Lewis stain and then observing under the microscope. The number of pollen divided by number of ovules per flower gave pollen – ovule ratio for the species.

b (iii) Pollen - stainability and viability:

Pollen – stainability and viability in each of three species was studied using stainability test in 1% acetocarmine and FCR test using a mixture of FDA in acetone and sucrose respectively. The concentration of sucrose optimized for all the three species was 50%. In stainability test only the fully turgid pollen with stained cytoplasm were considered viable while the shriveled and empty pollen were taken as non viable. In FCR test viable pollen were distinguished through their fluorescent cytoplasm in contrast to the non florescent non viable pollen.

b (iv) Pollen - pistil interaction:

This was studied right from the germination of pollen on stigma and tracing the path of pollen tubes through stylar tissue to the ovules. Pollen tubes entering the ovules were taken as an evidence of successful fertilization. Aniline blue fluorescence method (Shivanna and Rangaswamy, 1993) was used for such studies. Pistils were cleared by keeping them in 4 N NaOH at 60˚C for 12 – 16 hours in a hot air oven in case of Picrorhiza kurrooa and Valeriana wallichii. But in case of Ferula jaeschkeana, 8 N NaOH was used and the clearing time was also increased up to 20 – 24 h. Fluorescence microscopy of pistils was also performed to compare the rate of pollen tube growth in case of female and hermaphrodite flowers and also in case of self or cross pollination in V. wallichii by first manually pollinating the pistils and then subjecting these pistils to fluorescence microscopy after fixing them in Carnoy’s fixative at different time intervals of pollination. In case of P. kurrooa, presence or absence of pre – fertilization barriers in inter - morphoform crosses were also determined using the same procedure.

b (v) Pollination mechanism:

In order to understand the pollination mechanisms operating in the three species under study, different features of the floral architecture like type of corolla, position of the reproductive parts in a fully opened flower and kind of pollen and stigma were thoroughly studied. Further to check anemophily, slides smeared with Mayer’s albumin were suspended at varying distances from the populations except for P. kurrooa. These slides were examined after 24 h by staining in Lewis stain for the presence or absence of the pollen of the species under study. To check entomophilly as the possible mode of pollination, plants growing in nature as well as ex situ were observed to check whether or not the flowers were visited by some insects and also if any attractants or rewards were offered by the flowers to visiting insects. Behaviour of different insects visiting these flowers was monitored to classify them as pollinators or only robbers. These insects were collected and pollen load on their body parts was calculated to confirm their role in pollination process.

b (vi) Mating or Breeding system:

To understand the nature of mating system in these species, experiments to check the self and cross compatibility status by carrying out manual pollinations (manual cross and self) were undertaken. Inflorescences were also subjected to forced selfing by bagging. Fruit set or fertilization of ovules was taken as a mark of confirmation of compatibility in any particular pollination experiment. Results of these experiments were compared and correlated with the type of pollination, floral architecture, pattern of blooming of flowers within a plant and inflorescence and also time and sequence of maturation of reproductive parts in each flower to confirm the breeding system in each of these species.

b (vii) Meiotic system:

Pollen mother cell (pmc) meiosis was studied in order to understand the level of genetic variation generated through male sex tract. This was done by calculating the chiasmata frequency by counting both interstitial and terminal chiasmata per bivalent in a PMC. Chiasmata frequency was then used to calculate the recombination index of a species using the following formula:

Recombination index = Total no. of chiasmata per cell + Haploid chromosome no.

of the species.

To study the pmc meiosis, young floral buds were fixed in Carnoy’s fixative at a suitable time of the day standardized for each species. After keeping the buds in this fixative for about 3 - 3.30 h, they were thoroughly washed under running tap water and then transferred to 70% ethanol in refrigerator. Anthers were squashed in a drop of 1% acetocarmine and slides were scanned for appropriate stages of meiosis after gentle heating and tapping in the folds of filter paper. The unambiguous cells were marked and photographed using Nikon eclipse 80i microscope.

b (viii) Reproductive output:

For determining reproductive output, percent fruit set per plant and percent seed set per fruit were calculated. Both these parameters were studied for open pollination in nature and under controlled pollination experiments involving manual self and cross pollinations (msp and mcp). To calculate the percent fruit set for a species, first the average number of flowers per plant and then average number of fruits formed per plant was calculated. Percentage fruit set was calculated using the following formula:

Average no. of fruits formed per plant

Percentage fruit set = ______x 100

Average no. of flowers produced per plant

In case of P. kurrooa, outcome of pollination experiments was followed through the fluorescence microscopy to check whether or not the pollen tubes traverse the stylar tissue and enter the ovules. In case of F. jaeschkeana, results of mcp and msp were followed by observing an enlargement in the size of pollinated ovaries more than 4 times the original size to confirm the fruit set.

Percentage seed set was calculated by first taking the average number of ovules per ovary and then the average number of seeds per fruit in case of each species and then applying the following formula:

Average no. of seeds per fruit

Percentage seed set = ______x 100

Average no. of ovules per ovary

III. 2c Seed germination:

The final outcome and success of sexual reproduction can be measured through the germination of seeds and survival and maturation of seedlings up to reproductive stage. To check this, seeds of these species were put for germination under lab conditions and in different media like moist Whatman paper, moist sand and mixture of sand, manure and garden soil in the ratio 1:1:1. Total number of seeds germinated divided by the total seeds put for germination gave the percentage seed germination. Percent seed germination was compared during different times of year and also in seeds produced as a result of different pollination treatments. Picrorhiza and Ferula seeds were also subjected to subfreezing temperature in order to simulate the conditions required for their germination in their natural habitat.

III. 2d Analysis of genetic diversity:

For studying the level and pattern of genetic diversity distributed in natural populations of these species, two DNA based molecular markers, Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) were used. It involved DNA isolation from fresh or preserved leaf material, purification and quantitation of the crude DNA, optimization of PCR program for each species and also for each marker system, screening of both kinds of primers to select those producing the best results and then final amplification using the selected primers. The results were scored and analyzed using different softwares to interpret the level of genetic diversity in these species. The protocol followed and modifications done in different species is given below:

d (i) DNA isolation:

Total genomic DNA was extracted following the CTAB method of Doyle and Doyle (1990) with minor modifications (Sharma et al., 2013). Fresh leaf samples were used in case of Valeriana wallichii and Picrorhiza kurrooa whereas in Ferula jaeschkeana, leaf samples collected from natural populations were stored in liquid nitrogen before transportation to lab and were then used for DNA isolation. Samples were ground to fine powder using liquid nitrogen. A homogenate of 3 - 5 g leaf tissue was prepared using 10 ml of extraction buffer (2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris HCl, pH 8.0). Added 2 - 2% mercaptoethanol to buffer at the time of Isolation and also added 1ml of PVP (3%) in the suspension. Vortexed and incubated the sample at 60o C for 45 min. Samples were allowed to cool down at room temperature. Then the mixture was treated with equal volume of chloroform: isoamyl alcohol (24:1) and mixed by gentle inversions for 5 min followed by centrifugation at 12,857 g for 15 min at 25o C. The upper aqueous layer was transferred to a fresh microcentrifuge tube to which equal volume of pre - chilled isopropanol was added. The tubes were left at -20o C for 3 - 5 h after which they were centrifuged at 15,557 g for 20 min at 4oC. The DNA pellet was washed in 70% alcohol and air dried at room temperature for 4 - 6 h and resuspended in 2 ml of TE buffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0).

d (ii) Purification of DNA:

The crude DNA, extracted by above method was not found suitable for amplification. DNA in such cases was light to dark brown. Such samples were then purified. For DNA purification, equal volume of phenol chloroform (1:1) was added to the dissolved DNA. After centrifugation at 8,228 g for 15 min at 25oC, supernatant was collected and added equal volume chloroform: isoamyl alcohol (24:1). Again after centrifugation at 12,857 g for 15 min at 25o C equal volume of absolute ethanol and 1/10th volume of 3 M sodium acetate was added to the aqueous layer. Left the samples at -20o C for 6 h and centrifuged at 12,000 rpm for 20 minutes. The DNA pellet was washed with 70% alcohol and air dried at room temperature. Finally dissolved the DNA pellet in 1ml of TE buffer to obtain the form of DNA ready to be used in PCR amplification. This purification step was essentially used in case of Ferula jaeschkeana and in some samples of Picrorhiza and Valeriana which were slightly brown in colour and were not getting amplified during PCR.