**2. Propagation**

#### **2.1 Propagation through seeds**

Stevia is mainly reproduced through seeds in its wild habitat. Stevia seeds are small in size, and approximately 40-gram seed is required for a one-hectare stevia plantation. Furthermore, 1 ha may produce approximately 8.1 kg seed, which is sufficient for a plantation of 200 ha area [44]. Nursery plants are typically cultivated from January to March, although in polyhouse conditions, they may be raised all year. Seedlings with 5–7 leaves that are two months old are put in the field. However, in temperate locations, seeds are frequently ineffectual in germination [62]. In the short growing season of the northern region, farmers use glasshouses or greenhouses for better establishment of stevia crop. The seeds should be sown up to a depth of 1–2 cm in soil and Irrigation should be necessary at regular intervals. Germination studies have revealed the presence of two types of seeds i.e. tan and black colored. Black seeds show more viability and germination in light as compared to tan seeds [38].

#### **2.2 Vegetative propagation**

Stem cuttings are used for vegetative propagation, which is an excellent strategy for multiplying stevia plants [30]. Because some plants do not produce viable seeds for germination, vegetative propagation is sometimes the only method of replication. During February, a hardy branch cutting from a fresh stem or shoot with three to four nodes is ideal for planting in the soil [30, 63–65]. A one-year plant's leaf axials exhibit 98–100 percent rooting [64] however cutting from other areas of the plant affects its roots and growth. In February, axial stem cutting with two pairs of leaves produces the best rooting results, while three pairs of leaves cutting produce outstanding results in April [66]. In comparison to other seasons, late winter is the greatest period for stevia rooting [67]. Growth regulators can sometimes encourage roots and sprouting. Several growth regulators raise the content of stevioside in leaves [68, 69].

## **2.3 Micro-propagation**

Propagation through tissue is a technique in which a single tissue (or explant), such as seeds, auxiliary shoots, leaves, sprouts, inter-nodal explant, and shoot primordia, is used for successful development of a new plant [39, 70–75]. Micro-propagation via tissue culture is a quick procedure of multiplying disease-free plants of a selected or bred clone, and it has also been recorded in the literature [39, 43, 74, 76–78]. Maximum shoot development and biomass growth occurred in Murashige and Skoog (MS) media improved with 0.25 mg/l kinetin and 0.5 mg/l BAP (6-benzylamino purine) solution, while roots developed well in MS (Murashige and Skoog) media supplied with 1.0 mg/l IBA (Indole-3-butyric acid) solution [79]. The cultures were always incubated at 24 ± 2°C with 60–80 percent relative humidity and photoperiods of 16 hours with a light intensity of 3000 lux [80].

## **2.4 Conservation of plant genetic resources**

The plant genetic resources include genetic stocks, active collection, general germplasm, pre-breeding material, base collection, breeder's collection, interspecific derivatives, etc. Synthetic seed technology with added osmotic agents was used for germplasm conservation of *Stevia rebaudiana* Bert [81]. Synthetic seed production in *Stevia rebaudiana* with micropropagation may solve many problems [82]. In vitro propagation protocol of *S. rebaudiana* has been also established to meet the demand [83, 84]. Shoot tips obtained from in vitro shoot cultures of *Stevia rebaudiana* Bertoni encapsulated in 4% calcium alginate used as synthetic seeds. Synthetic seeds capsulated with 0.05 M mannitol after 6 weeks are the most suitable for conversion [85].

Due to the presence of self-incompatibility, seeds produced from the individual plant would be representative of half-sib progeny [10]. Lack of homozygous populations due to self-incompatibility can be overcome by double haploid production which can be used for the further breeding program. The crucial restraint in stevia cultivation is the lack of evergreen plant cultivars. Germplasm for delayed flowering to attain a prolonged vegetative phase has been developed through mutation breeding and can be transferred to high Rab-A consisting varieties or any other desirable genotype. Diverse lines from various breeding institutions can be shared for hybrid production. Recovery of phytochemicals through processing technology precisely, green technology should be boosted. The development of tetraploid and triploid germplasm can be used for hybridization and commercial exploitation.
