Evaluation of Ferula assa-foetida accessions for germination parameters 1 under cold stratification to overcome seed dormancy and effect of media 2 mixtures on seedling growth

Ferula assa-foetida L. is a perennial of Apiaceae family having seed dormancy which inhibits 12 the germination. Oleo-gum resin obtained from the rhizomes of Ferula assa-foetida plant has 13 several medicinal properties and used for the treatment of various diseases, pharmaceutical 14 industries and in cooking of food in some countries. In present study, three different temperature 15 treatments (5°, 15° and 25°C) were used to break seed dormancy in six Ferula assa-foetida 16 accessions repeatedly for two years. Also the seedling survival rate with other morphological 17 parameters like plant height, no. of leaves, leaf width, leaf length, root length and root diameter 18 were observed on 3 month old seedlings in six different media mixtures during year 2019-20. 19 The germination ranges from 3.63 (EC968466 at 25°C) to 81.88 percent (EC966538 at 5°C) with 20 an average of 31.14 percent over all temperatures and genotypes. The mean germination time 21 was ranged from 19.69 to 42.71 days with an average of 30.14 days. The highest germination 22 (66.9%) and least mean germination time was observed at 5ºC (20.85) which showed that this 23 crop required a 5ºC best performer for overall germination as well as seedling survival parameters and could be used 28 as base population in future selection and improvement breeding programs. 29

best performer for overall germination as well as seedling survival parameters and could be used 28 as base population in future selection and improvement breeding programs. 29 Keywords: Seed dormancy, chilling, germination, genotypes, Apiaceae and Ferula assa-foetida. 30 Ferula assa-foetida L. is a small perennial monocarpic herb belonging to family Apiaceae 31 (Umbelliferae) and commonly known as "Heeng" in Hindi 1 . The plant height is about 1-1.5 32 meter in length, large size compound leaves and a large size thick taproot with root hairs is 33 present and has a pungent smell. This plant is native to Iran and Afghanistan 2,3 . About 170 34 species are found all over the world and 60 species of Ferula assa-foetida are geographically 35 distributed around North Africa, Central Asia and Europe 4 . This species is distributed at an 36 elevation between 2000-4000m above mean sea level, with an annual rainfall of 250-350mm 5 . 37 Two species of genus Ferula i.e. Ferula alliacea and Ferula jaeschkeana are found in India 6 . The 38 oleo-gum resin is present in the fleshy tap roots and this exudate extracted from the roots is 39 called asafoetida. F. assa-foetida is one of the most significant plant of Iran and Afghanistan 7 40 because of high export demand for its oleo-gum resin. It has medicinal use in traditional 41 medicines 8 . Asafoetida plays an important role in cooking food as a condiment and in medicine 42 EC966538 accession at 5°C, while EC968469 accession showed lowest germination (55.50%) at 113 the same (5°C) level of temperature. In case of adverse high temperature of 25°C, the accession 114 EC966538 showed maximum germination (8.13%), while EC968466 had very poor germination 115 (3.63%) at this adverse temperature (Table 5) and (Fig. 2). The results of genotypic effect on 116 germination showed that the accession EC966538 has consistently higher germination 117 percentage among all the accessions in all temperature treatments. Seed dormancy is the major 118 challenge in F. assa-foetida and other Ferula species 52,53,54,5 and genetic background of this 119 accession (EC966538) could be used in future germination improvement breeding programs. 120

Other germination parameters 121
Seeds of F. assa-foetida are not germinated in a single flush, and involvement of multi-level seed 122 dormancy causes continuous germination up to several days even with some favorable 123 environment. Hence, germination percentage should not be only the single parameter to access 124 the germination capability of this crop. Thus, in present study we have observed several other 125 parameters to identify best accession and environment for good germination. These parameters 126 are germination index (GI), mean germination time (MGT), mean daily germination (MDG), 127 coefficient of velocity of germination (CVG), peak value (PV), germination value (GV), days to 128 25% germination (DG25%), days to 50% germination (DG50%), days to 75% germination 129 (DG75%), radical length (RDL) and seed vigor index (SVI). 130 The pooled and individual analysis of variance (ANOVA) for both the years 2018 and 2019 131 showed significant difference (p≤0.01) for temperature treatments, genotypes and their 132 interaction for all the other germination parameters studied (Table 4). Mean germination time 133 (MGT) measures mean time required by any seed sample to initiate and terminate germination. 134 Lower is the value of MGT, faster a seed lot has germinated 55 . The results pertaining to mean 135 germination time (MGT) showed that germination was achieved very fast (Table 3) and (Fig. 3 Germination index (GI) is a measure of both germination percentage and speed of germination. It 147 gives maximum weightage to early geminating seeds and less weightage to late germination 55 . 148 The GI ranged from 0.09 (EC968466 at 25°C) to 3.94 (EC966538 at 5°C) with an average of 149 1.44 over all temperatures and genotypes. The average germination index for all the genotypes 150 was highest (Table 5) at 5°C (3.47) than 15°C (0.73) and 25°C (0.14). 151 Coefficient of velocity of germination (CVG) denotes the rapidity of germination and increases 152 with germination of seeds and time required for their germination is reduced 55 . CVG ranged 153 from 2.35 (EC968466 at 25°C) to 5.08 (EC968469 and EC968470 at 5°C) with an average of 154 3.59 over all temperatures and genotypes. Recently, the mean germination time, germination 155 index and coefficient variation of germination were studied for Magnolia grandiflora plant after 156 cold stratification 40 . The results pertaining to PV, GV and SVI were also in accordance to results 157 of mean germination time and per day germination rate i.e. the temperature treatment of 5°C and 158 accession EC966538 followed by EC968470 were found best performer (Table 3). Also, the 159 radical length ( Fig. 4 and Fig. 5) and seed vigor index was highest at 5°C (2.55 cm and 1.70, 160 respectively) followed by 15°C (1.71 cm and 0.36,). However, in overall genotypic effects, the 161 highest radical length and seed vigor was found for EC968470 (1.87 cm and 0.84, respectively). 162 These seed germination parameters also studied in some Himalayan leguminous and actinorhizal 163 plants 57,58 . 164

Seedling survival rate 165
To check the seedling survival rate, all newly germinated plants were examined in the year 2019-166 20 under six different media mixtures (Table 1) for various morphological traits viz. plant height 167 (cm), number of leaves, leaf length (cm), leaf width (cm), root length (cm) and root diameter 168 (mm). The results of analysis of variance for seedling survival traits showed that all the 169 genotypes and media mixtures were highly significant (p<0.01) for all traits studied (Table 6). 7 and root diameter. It showed that the studied accessions were genetically diverse for seedling 172 survivals and could be utilized as base population for further selection and breeding programs. 173 The mean performance of different media mixture and genotypes showed that the accession 174 EC968466 in media M4 and EC968467 in M5 have highest seedling survival (91.50%) ( Table 5)  175 and ( Fig. 6 and Fig. 7). Over all media mixtures, M5 has the highest seedling survival rate 176 (87.66%), followed by M4 (87.08%). Media M4 has highest plant height (21.49 cm), number of 177 leaves (5.27), leaf length (9.22 cm), leaf width (6.58 cm), root length (11.93 cm) and root 178 diameter (5.36 mm). It showed that the combination of soil, sand, FYM and cocopeat in 1:2:2:1 179 ratio was the best mixture to attained maximum survival of F. assa-foetida seedlings. While, 180 media M6, which was only soil has lowest seedling survival rate (46.50) and also found lowest 181 for all other seedling survival parameters, which indicated that this crop needs a survival media 182 at initial stage to get good seedling establishment. 183 Genotypic effects on seedling establishment showed that accession EC968467 has highest 184 survival rate (75.94%) and number of leaves (5.17) but lowest root diameter (4.16 mm). 185 Accession EC968470 has highest plant height (15.15 cm) and root length (9.67 cm), while 186 accession EC966538 was best for other leaf parameters i.e. number of leaves, leaf length and leaf 187 width But maximum germination of F. asafoetida through seed is very difficult and almost very low. 203 Media mixture of sand, soil, FYM and cocopeat (1:2:2:1 and 1:1:1:1 ratio) gave higher seedling 204 survival. It was also observed that the accession EC966538 and EC968470 were the best 205 performer for overall germination as well as seedling survival parameters. 206

Seed viability test 220
Seeds of F. assa-foetida were examined for seed viability with the help of Tetrazolium test 221 (2,3,5-tri-phenyltetrazolium chloride). For that, 1% Tetrazolium solution was prepared by adding 222 1g 2,3,5-tri-phenyl-2H-tetrazolium chloride (TTC) in 100 ml of doubled distilled water in a 223 brown bottle, mixed well and confirmed the pH of the solution at 7. Further, four replicates of 25 224 seeds from each accessions were dissected using a magnifying lens. Then the dissected seeds 225 with embryo were kept in 1% tetrazolium solution and incubate at room temperature for 24 hours 226 in dark 19 . Seeds were evaluated on their staining pattern and colour intensity as the bright red 227 color stained seeds considered as viable while partially or light stained seeds were considered as 9 non-viable 25,26 . The seed viability test was repeated three times in every 6 months during the 229 study period i.e. in 2018, 2019-I and 2019-II. 230

Surface sterilization 231
Healthy seeds of F. assa-foetida selected on the basis of their shape and size were surface 232 sterilized by pre-washing with tap water for 1h and then soaking the seeds in 1% sodium 233 hypochlorite solution (NaOCl) with tween-20 (2 drops /100 ml) for 25 min and then washed with 234 sterilized double distilled water to remove traces of sterilizing agents before putting in petri-235 dishes. 236 Where, ni is the number of germinated seeds on fi th day of observation during germination time 260 (from 0 to 45 days) and N is the total number of germinated seeds. 261

Coefficient of velocity of germination 262
Coefficient of velocity of germination (CVG) was computed using following formula 35 . 263 Where Ni is the number of seeds germinated on Ti th day of observation from seeding. 265

Germination index 266
Germination index (GI) was calculated by using formula 36

Seed vigor index 282
The seed vigor index (SVI) was computed using following formula 41 . 283

Seed vigor index (V i ) = × Pg 100 284
Where Vi is the vigor index, Ls is the length of seedling and Pg is germination percentage. 285 Days to 25%, 50% and 75% germination 286 Days to 25%, 50% and 75% germination were calculated when 25, 50 and 75 percent 287 germination of Ferula assa-foetida seeds was achieved. 288

Radical length 289
Radical length was measured with the help of meter scale in centimeter (cm). 290

Media Mixtures 291
After cold stratification treatment germinating seeds of all the six accessions were transferred to 292 different media mixtures under glasshouse conditions to study the survival and growth of the 293 germinating seedlings Table 1