In vitro shoot regeneration of hypocotyl explants of Foeniculum vulgare-a culinary and medicinal herb

A reproducible protocol for in vitro shoot regeneration and plantlet establishment was developed from hypocotyl-derived callus explants of Foeniculum vulgare. Callus induction frequency was the highest (92%) on SH (Schenk & Hildebrandt) medium supplemented with 4.44 μM BA (N 6 -benzyladenine) and 1.34 μM NAA (α-naphthaleneacetic acid) followed by 86% and 80% on MS (Murashige & Skoog) and NN (Nitsch & Nitsch) medium respectively after 40 d of culture. The organogenic callus induced better shoot regeneration frequency (92%) on SH medium containing 6.66 μM BA as compared to 87% and 75% on MS and NN medium respectively. The highest number of 9.2 shoots per callus explant was achieved with the mean shoot length of 5.48 cm on SH medium supplemented with 6.66 μM BA followed by decreasing shoot production of 8.2 shoots and 7.3 shoots per callus explant on MS and NN medium respectively. The in vitro regenerated shoots produced the maximum root induction frequency of 94% on SH medium with 1.34 μM NAA, while on MS and NN media,frequencies of root induction were 86% and 77% respectively. There were 6.6 roots per shoot with an average root length of 6.4 cm on SH medium with a level of1.34 μM NAA, while MS and NN media with the same level of auxin (1.34 μM NAA) induced an average of 5.8 and 5.3 roots per shoot respectively after 60 d of culture. In vitro derived plantlets acclimated successfully with 100 % survival.


Special Issue of First International Conference on Advancements in Management, Engineering and Technology (ICAMET 2020)
In vitro shoot regeneration of hypocotyl explants of Foeniculum vulgare-a culinary and medicinal herb 1

. Introduction
The species F. vulgare (Fennel) is an aromatic and flavor rich perennialherb with culinary and medicinal uses. The species Foeniculum vulgare is with soft feathery, almost hair-like foliage found in northern temperate regions and in the tropical parts favor's warm, sunny localities. Besides, it is well established for its pharmacological properties and thus much valued as carminative, digestive and diuretic agents (4).The development of an in vitro culture system would deal exceptional opportunities for producing pharmaceuticals in the laboratory without having to depend on field cultivation (20).
. To our best knowledge, there have been no reports on successful acclimatization of this species prior to this present investigation underscoring the need for a complete reproducible protocol for rapid regeneration and plantlet recovery of this species. Thus, in this study an efficient and reproducible protocol was established from hypocotyl explants of 30 d old in vitro seedlings of Foeniculum vulgare.

Materials and Methods 2.1Expalnt source for callus induction
Seedlings ofFoeniculum vulgare were established as per the seed sterilization procedure followed in the earlier report of Carum copticum (10) after obtaining seeds from source organization namely Central Institute of Agriculture Engineering, Bhopal, India. Seeds were exposed to the treatment solution consisting of 0.1% (w/v) HgCl 2 for the duration of 3 min followed by washing seeds three times with sterile double distilled water ensuring removal of traces of sterilant. For germination growth, these seeds were inoculated on three culture media consisting of MS (12), NN (13) and SH (17) each containing different concentrations of gibberellic acid (GA 3 : 0.75, 1.50, 3.0, 3.75 µM). In vitro seedling culture was continuously maintained for the constant source of hypocotyl explants for the present study. Percentage of shoot producing roots, average number of roots per shoot and root length were recorded after 60 d of culture.

Aacclimatization of rooted shoots
Regenerated rooted shoots were removed from culture vessels and washed under slowly running tap water to make the roots free from adhering medium traces prior to transfer to pots containing a mixture of 1:1 ratio of autoclaved vermicompost and soil. Pots containing plants were surrounded with polyethylene bags for duration of fourteen days for keeping humidity percentage high. Thereafter, the hardened plants were shifted to bigger pots where plants were growing healthy at medicinal garden of the institute.

Statistical Analysis
Each experiment was repeated thrice with 10-12 replicates and was completely randomized design. Data analysis was carried out by one way analysis of variance (ANOVA) and the means were scored using Tukey's test on statistical package of SPSS (ver. 20).Treatments were significantly different at P< 0.05.

Seed germination
Successful seed germination (100%) was observed on SH medium with 0.75 µM GA 3 after 30 d while MS and NN media with higher concentration of GA 3 (1.50 µM) stimulated 100% germination (data not shown). Germinated seedlings were used as source for explants (hypocotyls) for callus induction (see Figure 3 a-c).

Callus initiation
The hypocotyl explants of F.vulgare were successfully induced on MS, NN and SH basal media with PGR for callus formation. The highest frequency of callus induction from hypocotyl explants (92%) on SH medium with 4.44 μM BA and 1.34 μM NAA was significant in contrast to 86% and 80% on MS and NN medium respectively and produced friable green callus after 40 d of culture (see Table 1 and Figure 3 a-c). Callus induction frequency of 52% was observed on SH medium with 2.22 μM BA and 2.69 μM NAA in contrast to 42% on MS and 44% on NN medium containing 2.22 μM BA and 2.69 μM NAA. However, the frequency of callus induction decreased to 26% on SH medium with 6.66 μM BA and 1.34 μM NAA while callus induction frequencies on MS and NN medium with the same plant growth regulators were 22% and 16% respectively. The next best callus frequency was (80%) on SH medium with 1.16 μM Kn and 1.34 μM NAA in contrast to 64% and 56% on MS and NN medium respectively ( see Table 1 and Figure  3 a-c).  Figure 1 a-c, Figure 3

Rooting of shoots
In vitro regenerated healthy shoots of F.vulgare did not root either on MS or NN or SH medium without auxins but rooted readily when these media were supplemented with different concentrations of auxins such as IAA or IBA or NAA. On 1.42 µM IAA supplemented SH medium with the root induction response was 50% as compared to 46% and 44% on MS and NN medium respectively whileSH medium with 1.23 µM IBA induced 40% root induction as compared to 32% and24% on MS and NN medium respectively. However, the highest root induction frequency (94%) was observed on SH medium with 1.34 µM NAA, whereas, on MS and NN medium induced 86% and 77% respectively after 60 d of culture.The highest number of 6.6 roots per shoot with an average root length of 6.4 cm were observed on SH medium with 1.34 µM NAA, while, MS and NN media with NAA (1.34 µM) induced 5.8 and 5.3 roots per shoot respectively. Moreover, NAA at 9µM on the MS, NN and SH media resulted decreased number of roots per shoot. On the other hand 1.42 µM IAA in the SH medium produced 4.8 roots per shoot with a mean root length of 4.7 cm in contrast to 3.2 roots and 2.7 roots per shoot on MS and NN medium respectively (see Table 2, Figure 3 a-c). Rooted shoots of F.vulgare were washed under tap water to remove adhering media from the surface of roots and then transferred to plastic pots (7x5 cm) containing a mixture of sterilized soil and vermicompost (1:1) and sheltered with polythene bag to maintain high humidity necessary for efficient acclimatization. NAA at the level of 1.34 µM was used for regular spraying to stimulate rooting and maintaining congenial environment of high humidity and kept in the growth room prior to field transfer where plants showed 100% survival rate (see Figure 3 a-c).Healthy in vitro regenerated plantlets had noapparent phenotypic variations.

Discussions
The hypocotyl explants of F.vulgare were successfully induced on MS, NN and SH basal media with PGR for callus formation. These observations conforms to the earlier observations on callus induction from hypocotyl and radical explants of Asparagus densiflorus on MS medium (21), whereas in vitro cultures of fennel reported 100% callus formation on MS medium with IAA, Kn and 2-iP each at 0.5 mg/l (3). Similarly, hypocotyl and radical explants of Asparagus densiflorus were induced to maximum callus induction and proliferations (95%) on MS medium with 0.5 mg/l BA and 0.3 mg/l NAA (21), while in Ammi majus cotyledonary leaves produced callus on MS medium supplemented with 2 mg/l IAA, 0.5 mg/l Kn and 40 ml Adenine (14). In accord with the shoot regeneration from callus cultures, previous reports indicated that the most effective shoot regeneration of 88.8% from hypocotyl and cotyledon-derived callus of Brassica oleracea var. capitata seedling on MS medium with 8.88 µM BAP and 0.53 µM NAA producing the highest nuber of 7.5 shoots/buds per callus explant (5). In line with our observation it was reported that hypocotyl derived callus explants of Foeniculum vulgare (16) seedlings produced 100% shoot regenerating frequency with 8 shoots per explant on MS medium containing BA (0.1 mg/l) and NAA (0.1 mg/l) whilein Ruta graveolens (2)leaf segment-derived callus produced 70% shoot bud frequency with maximum production of 32.2 shoots per unit callus on MS medium with 7.5 μM BA but obtained enhanced shoot bud regeneration frequency of 92% with the highest number of 92.4 shoots per unit callus having a shoot length of 4.1 mm on MS medium containing 7.5 μM BA plus1.0 μM NAA. Also similar observations have been recorded in Helicteres isora (19) where shoot regeneration frequency of 67% occurred from nodal explant-derived callus producing 3.2 shoots per 0.5g callus on MS medium with 2.22 µM BA and 2.32 µM Kn within 35 d of culture, inIpomoea mauritiana(7) optimum shoot regeneration frequency (30%) with 2-3 shoots per callus were obtained from internodal segment-derived callus on MS medium containing 1.0 mg/l BAP and 0.2 mg/l IAA while plants were successfully regenerated (over 65%) from immature leaf derived-callus in Bambusa ventricosa on 13.3 µM BA and 2.7 µM NAA (22). Similar findings of root initiation were reported in many medicinal plants including Ipomoea mauritiana in which 6.2 roots per shoot with a root length of 4.42 cm were obtained on MS medium with 0.5 mg/l NAA (7) and 7.5 roots with an average root length of 4.16 cm produced in Curcuma longa on MS medium with 3.0 mg/l NAA (9). In Brassica oleracea var. capitata in vitro regenerated shoots rooted on MS medium with 5.37 µM NAA (5), while in vitro raised shoots of Carum copticum were induced to root formation on MS medium with 0.5 mg/L NAA (18). Similarly, in Glycine max rooting of in vitro regenerated shoots occurred on B5 medium containing 14.7 µM IBA (15) while in Primula vulgaris shoots rooted on WPM (11)medium with 0.5 mg/L IBA (6). Plantlets were acclimatized successfully using natural soil or vermiculite and soil (1:1) in many other plant species such as in Primula vulgaris (6), Momordica charantia (1), Ipomoea maurtiana (7), and Salvia officinalis (8).

Conclusion
An efficient and reproducible protocol was established from hypocotyl explants of in vitro seedlings through indirect organogenesis of Foeniculum vulgare. This regeneration system showed large scale shoot regeneration from hypocotyl-derived callus and high rooting ability. These plantlets survived acclimatization and grew healthy in the medicinal garden. Moreover, hypocotyl explant-derived callus proliferation may serve as a source of medicinally important secondary metabolites. Thus, this reproducible regeneration protocol holds promise for sustainable supply of sterile plant materials to the pharmaceutical industries besides substantial contribution towards conservation of germplasm.