**9.** *In vitro* **propagation**

490 Genetic Diversity in Plants

al*.*, (1997). Evaluating diversity and genetic relationships among the progenitor *A. longicuspis* and 27 garlic varieties collected from different regions of the world, these authors found a dissimilarity of 0 between two samples, and a highest dissimilarity of 0.82 between two large groups. Through the RAPD technique used in this work, the two 'Perla' cultivars were grouped with the best production varieties, where the two selections presented a dissimilarity of 0.1. However, the 'Perla' C-3-1/25 cultivar showed a band of 2100 bp, and could thus be identified as a possible molecular marker. Our results allowed to identify highly related garlic varieties ('Sonora'-'Napuri', 'Criollo Aguascalientes'-'Español', 'California'-'Chileno' and 'Pata de Perro'-'Guatemala'), and separate them from varieties that are characterized by a lower yield (i.e., 'Pata de Perro' and 'Napuri'), and from mixed

garlic that has been generated from introduced commercial varieties ('Criollo

Fig. 6. Dendrogram obtained by the RAPD technique and general description according to physiological, morphological and genetic characteristics in garlic (*Allium sativum* L.)

varieties cultivated in the Central Region of Mexico. Arms or branches of the dendrogram: 1.

'Perla' C-3-1/25, 2. 'Perla' C-37-1/8, 3. 'Coreano', 4. 'California', 5. 'Chino', 6. 'Criollo Aguascalientes', 7. 'Español', 8. 'Cortazar', 9. 'Positas', 10. 'Pepita', 11. 'Massone', 12. 'Durango', 13. 'Chileno', 14. 'Hermosillo', 15. 'Sonora', 16. 'Napuri', 17. 'Nicaragua', 18.

'Ixmiquilpan', 19. 'Pata de Perro' 20. 'Guatemala'.

Most Mexican garlic cultivars are somewhat susceptible to pests and diseases. Furthermore, garlic is a seedless plant that could carry diseases to the next generation through vegetative propagation. "Seed cloves" per hectare range from 200,000 to 250,000 for typical plant density; therefore, totally clean vegetative material may be too difficult to be generated. The solution for this kind of problems is considered under biotechnological view such as *in vitro* culture so that we can obtain homogeneous healthy plants. *In vitro* bulbils after four years may produce, depending on the cultivar, from 1,400 to 10,700 bulbils ready to be used as "seed" (Burba, 1993). A recent report mentioned plants produced from cloves cultivated *in vitro* with 1 mg L-1 TDZ-1 (Thidiazuron), 1 mg L-1 GA3 (Gibberelic acid) and coconut milk (Lagunes, 2009). *In vitro* garlic plants were also obtained on MS supplemented with 2.0 mg L-1 2iP (2-isopentenyl adenine), 0.1 mg L-1 NAA (Naphthalene acetic acid) and 30 g L-1 sucrose (Mujica et al., 2008). Basal plate from cloves has the highest callus production as compared to leaves, stem segments, pedicels and aerial bulbils (Rabinowitch & Brewster, 1990). Another report mentioned that callus formation from 'Rojo de Cuenca' was the best on media having BA and NAA; furthermore, high BA concentration promoted adventitious shoot formation, but did not show influence on callus formation (Barandiaran et al., 1999). Callus was also obtained from leaves exposed to 0.3 a 0.5 mg L-1 2,4-D (Fereol et al., 2002).

Our own results showed that explants about 5 mm2 of clove sections were enough to regenerate *in vitro* whole plants from 'C-3-1/8' and 'C-37-1/25', 'Chino' and 'Coreano' garlic genotypes. The first step began with explants for root production, that were placed into MS medium supplemented with combinations of 0.15 mg L-1 2,4-D, 5 mg L-1 adenine, 1.4 mg L-1 4-amino-3,5,6-trichloropicolinic acid (Pichloram) and 1 mg L-1 6-(γ,γ-dimetilamino) purine (2iP) (Table 4). MS medium also contained 30-50 g sucrose and 3.5 g L-1 Phytagel. Cloves were soaked in 70% ethanol 1min; after that, were transferred to 20% commercial bleach for 20 min. Then, cloves were rinsed thrice with dH2O, placed back to 70% ethanol 1 min and rinsed again. Incubation conditions were: 28 ±2 °C and 16/8 h photoperiod. Adventitious roots were used to produce protocorms and protocorm-like bodies. These protocorms were cultured for 30 weeks on MS supplemented with 1 mg L-1 IAA and 5 mg L-1 adenine.

#### **9.1 Protocorm formation**

Protocorms 0.5-1 cm merged from root tips after 8 weeks on MS supplemented with 1.5 mg L-1 2,4-D and 5 mg L-1 adenine (Table 4). Then they were placed into basal MS during 3 weeks, before protocorms were inoculated into four regeneration media at 18 ±4 °C and 16/8 h photoperiod during six weeks (Capote et al., 2000; Robledo-Paz et al., 2000; Quintana-Sierra et al., 2005). Protocorm and protocorm-like structures were both light and dark-green colored, compact and easily detachable (Fig. 7a, b and c), somehow similar to organogenic callus from *A. cepa* (Van der Valk et al., 1992; Zheng et al., 1998). 'Chino' and 'Coreano' protocorms were even more easily detached, dark-green colored as compared to 'Perla' derived genotypes; Novák et al., (1986) also found differential pigmentation among genotypes. Media supplemented with Pi (1.4 Mg/L-1) and 2iP (1 Mg/L-1) only promoted long roots.

#### **9.2 Microbulbil formation**

The treatment supplemented with 2,4-D showed the highest number of protocorm formation (Table 4); when the treatment included IAA and adenine, 'Chino' and 'Coreano' doubled to genotypes 'C-3-1/8' 'C-37-1/25'. Other treatments induced root formation. This different varietal response was found by Capote et al., (2000) and Quintana-Sierra et al., (2005) for *Allium cepa* and would be related to differences in sensibility to growth regulators (Fehér et al., 2003). Microbulbils placed, during three weeks, on basal MS (no regulators) increased their size (Fig. 7d), but after 30 weeks they developed into bulbils 1 cm diameter (Fig. 7e and 7f) and finally grew into whole plants.


Table 4. Effect of growth regulators on protocorm and microbulbil formation of four garlic 12 genotypes. IAA: Indole acetic acid, NAA: Naphthalene acetic acid, Kin: Kinetin, 2,4-D: 2,4-13 Dichlorophenoxyacetic acid, Pic: Pichloram, 2iP: 2-isopentenyl adenine.

Fig. 7. *In vitro* regeneration of garlic plants (*A. sativum* L.). A) Protocorms regenerated from root tips, B) 'Chino' protocorms, C) 'Perla' protocorms, D) Microbulbil, E) and F) 'Chino' whole plant.
