Agronomic and Biotechnological Strategies for Breeding Cultivated Garlic in Mexico

Garlic is an apomyctic diploid species (2n=2x=16) with vegetative reproduction that belongs to the Allium genus (Alliaceae), which includes onion (Allium cepa), leek (A. ampeloprasum) and shallot (A. ascalonicum) (Mc-Collum, 1987; Figliuolo et al., 2001; Ipek et al., 2003; 2005). The importance of garlic was recognized by humans at bronze era about 5000 years ago, and since these early times, has been used as food, condiment and medicine by Asians and Mediterranean (Ipek et al., 2005). World production of garlic is ranked 14th among vegetables with a total of 14.5 million ton (Trejo, 2006). In Mexico, its consumption is about 400 g per capita (Chavez, 2008), and the national production is considered low as compared to other countries such as China (80 % world production), India, Korea and the rest of the world (20 %) (FAOSTAT, 2011). Still, Mexico has a place as exporter of garlic produced mainly from the states: Zacatecas, Guanajuato, Aguascalientes, Baja California, Puebla and Sonora (Trejo, 2006). The areas in Mexico during winter 2008, dedicated to garlic were 5,085 ha with a total yield of 49,968 ton (SIAP, 2011).


Introduction
Garlic is an apomyctic diploid species (2n=2x=16) with vegetative reproduction that belongs to the Allium genus (Alliaceae), which includes onion (Allium cepa), leek (A.ampeloprasum) and shallot (A.ascalonicum) (Mc-Collum, 1987;Figliuolo et al., 2001;Ipek et al., 2003;2005).The importance of garlic was recognized by humans at bronze era about 5000 years ago, and since these early times, has been used as food, condiment and medicine by Asians and Mediterranean (Ipek et al., 2005).World production of garlic is ranked 14 th among vegetables with a total of 14.5 million ton (Trejo, 2006).In Mexico, its consumption is about 400 g per capita (Chávez, 2008), and the national production is considered low as compared to other countries such as China (80 % world production), India, Korea and the rest of the world (20 %) (FAOSTAT, 2011).Still, Mexico has a place as exporter of garlic produced mainly from the states: Zacatecas, Guanajuato, Aguascalientes, Baja California, Puebla and Sonora (Trejo, 2006).The areas in Mexico during winter 2008, dedicated to garlic were 5,085 ha with a total yield of 49,968 ton (SIAP, 2011).
Among other problems, Mexican garlic has a limited spectrum of high yielding cultivars for different environments and, at the same time, have good market qualities.Keeping in mind that kind of problems, this chapter was mainly focused on agriculture and biotechnology research done at four institutions.The first two sections include morphological, physiological and cytogenetic characterizations of the most common cultivars and related germplasm; the third section describes some advances on garlic micropropagation.The last canopy.Experimental yields from this genotype usually range from 16 to 18 ton/ha.Physiological disorders are common, such as brush-like plant growth with excessive number of thinner leaves; the more severe this problem, the more the plant opens its canopy of leaves with reduced sheath.Bulbs of brush-like plants lose their covering layers, producing naked cloves.This disorder is high temperature-dependent, having the highest temperature influence on March and April; therefore, it varies in severity from year to year.Experimental observation indicates that some other factors alone or combined may be related to the induction of brush-like plants such as: early planting, excessive nitrogen fertilization, and planting density.This disorder worsens when these factors appear combined.

Field performance of promissory genotypes
Besides the previously described genotypes, some more garlic accessions from the germplasm bank of INIFAP-CAEPAB (Fig. 1) were tested for their performance on the field (Aguascalientes, Mexico).These accessions have features suitable for breeding, as described below.

Postharvest photosynthesis and respiratory activity of stored cloves
In order to understand some physiological events of stored garlic, analyses of six genotypes under storage were focused on the respiratory process and photosynthesis.To accomplish that goal, photosynthetic activity of stored cloves during 0, 30, 60 and 90 d were measured on three cloves selected at random from the container of each genotype.Measurements included: evaporation rate mM/s/m2/s (E), stomatal conductance mM/m2/s (G), net photosynthesis assimilation µM/m2/s (A) and CO 2 internal concentration ppm (CI).From these measurements, it was found that for some genotypes like 'C-CN-9/2' evapotranspiration was the highest at 30 d, as opposed to 'Criollo Aguascalientes' and 'Chino Jaspeado' with the lowest value at 90 d.Stomatal conductance was high 'C-CN-9/25', mainly after 90 d.Most genotypes showed negative photosynthesis rate, and internal CO 2 showed no clear tendency within genotypes.Weight remained stable during the first 60 d, but after that period, it decreased about 1/3 of the initial values.The bulbs behavior at the final of postharvest period is show in the Fig. 2.
Unfortunately, whole plant regeneration remained elusive.

Karyotyping Mexican garlic genotypes
Karyotypes of C-CN-95/2, C-37 1/8, C-3 1/25 (all these 'Perla' type genotype), 'Chino', 'Coreano' y 'Criollo' were obtained from root tips.Cloves of these genotypes were placed inside petri dishes containing wet cotton wool in order to induce roots 1-2 cm long.Roots were removed and soaked with 0.05% w/v colchicine and placed in the darkness for 3:30 h at 25°C.These roots were fixed with Farmer´s solution (ethanol and glacial acetic acid, 3:1 v/v); then, they were hydrolyzed with 1N HCl at 60 C for 10 min.Feulgen stain was applied to fixed roots before maceration with an enzymatic solution (2% pectinase, 5% celulase and citrate buffer pH 4.5) for 30 to 60 min.Roots tips were placed on microscope glass slides with a drop of 2% propionic orcein, and sandwiched with a cover glass.The slides were heated for few seconds with an alcohol burner with a very soft press so that single cells could be freed from the tissue.Then the cover glasses were gently tapped with a pencil in order to squeeze single cells for releasing and spreading the chromosomes.Observation of microphotography 100x25" allowed the following counts and measurements: chromosome number, short arms (p), long arms (q) total length, relative size of the chromosome and arm relationship (García, 1990).All of these observations were useful to classify each garlic genotype according to karyotype nomenclature and formule from Levan et al., (1964).

Genetic profile of Mexican garlic
Genetic markers are efficient tools for genetic analysis of populations and individuals.
According to this concept, molecular characterization of garlic around the world has been performed either through RAPDs (Bradley & Collins, 1996;Eom & Lee, 1999;Shasany et al., A total of 20 µl of mineral oil was placed over the reaction mixture.Amplifications were carried out in a DNA thermocycler (Model FPR0G02Y Techne Progene, England), under the following conditions: an initial denaturalization step of 2 min at 94 °C, followed by 35 cycles of 1 min at 94 °C, 1 min at 35 °C, and 2 min at 72 °C, with a final extension step of 7 min at 72 °C.Amplification products were analyzed by electrophoresis in a 1.2% agarose gel.It was run at 100 V for 4 h, and detected by staining the gel with ethidium bromide (10 ng/100 ml of agarose solution in TBE).All visible and unambiguous fragments amplified by the chosen primers were entered under the heading of total visible fragments.Fragment data were entered on a spreadsheet to form a binary matrix, where (1) represented fragment presence and (0) fragment absence for each fragment accession combination.Cluster analysis was conducted by converting the data matrix into a similarity matrix using a simple matching coefficient.This coefficient was calculated by dividing the number of matches (0-0 and 1-1) by the total number of comparisons (Nei & Li, 1979).A cluster analysis was then conducted using the unweighted pair group method, with arithmetical averages (UPGMA) process using the S- distinct polymorphic fingerprint were selected to reveal the genetic variation among the garlic samples.In almost all varieties, it was possible to identify around 10 bands.

In vitro propagation
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 GA 3 (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).

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.

Individual selection for breeding Mexican garlic cultivars
Garlic in some cases may produce inflorescences but infertile seed; hence, crosses are not possible.Sometimes, bulbs o bulbils develop onto inflorescences (Brewster, 1994).Individual selection on best plants (yield or quality) has been used for breeding (Heredia and Heredia, 2000;González, 2006;Con, 1997).The CAEPAB group worked with individual selection from 'Perla' clones and 'Chileno' having heads with fewer cloves than the average for the original cultivar taking into account also: head size, vigor (hardiness) and plant healthiness.This initial work led to obtain two garlic cultivars: 'San Marqueño' from line 'C-37-1/8' (Macías et al., 2009) and 'Diamante' from line 'CAL-RN-11-1-1-2-4' derived from an Aguascalientes-Zacatecas collection (Macías & Maciel, 2003).In brief methodology for garlic breeding: 1. Bulb collection of promissory plants from the fields of outstanding growers of Aguascalientes and Zacatecas (May 1999).2. Bulbs were planted in the experimental fields at CAEPAB, in order to check all of the collected material under the same growing conditions.3. Evaluation and selection of garlic plants during 6-8 years (Table 6).4. Storage of best clones at the germplasm bank (CAEPAB).5. "Seed" production, enough to be transferred to growers for commercial validation.6.After validation, best genotypes, having consistent yield results through time, are released to farmers.
Clones obtained through this kind of breeding are grown by farmers from Aguascalientes and Zacatecas (Macías et al., 2009).Nowadays, 'San Marqueño' and 'Diamante' garlic are demanded in Europe because of their high quality that makes them suitable to be exported (Fig. 8).These clones have their optimal conditions at 2000 meters over the sea level, on loamy soils, well drained, without salinity or pedregosity.Lab test are encouraged to check for soil pathogens that may reduce yield.

Conclusion
Horticulturists around the world look for answers from experimental stations to problems such as low yields, pests, diseases and quality defects.Similarly, garlic growers from Central Mexico have been in contact with institutions such as CAEPAB, PRODUCE-Ags and ITEL, in order to agree on agronomic and biotechnological research that may be applied to their fields.Original garlic genotypes from these growers and some other introduced to Mexico were the source for new cultivars and promissory genotypes developed by CAEPAB.Some of these that were analyzed showed a good correlation between bulb sizes and clove weight against bulb weight.It was also found that Mexican genotypes have a wide variety of on clove size and number that reflects a good genetic pool for breeding through individual selection for this seedless plant.Some other characteristics are qualitative that may have positive impact for worldwide market demands.For example, it was found for late cultivars the longest storage life.
Garlic biotechnology was also directed to characterize of Mexican cultivars.Molecular and cytogenetic characterizations for these cultivars may help to identify and to register, unambiguously, cultivated varieties.Molecular analysis such as AFLP´s or RAPDs is required in order to establish genetic uniqueness or relatedness.So far, only RAPDs have been performed but this kind of work is not concluded yet.Another biotechnological application is to produce in vitro pathogen-free vegetative material for massive propagation that may be released to growers.Protocorm and protocorm-like bodies were produced in vitro before they grew into bulbils and whole plant.Nevertheless, massive propagation of garlic has not been achieved as desired.Therefore, garlic biotechnology is going at slow pace.Finally, breeding through individual selection allowed releasing cultivars appropriate for national and international demands.

Fig. 2 .
Fig. 2. View of stored garlic at 90 d.Sprouted heads are from short-shelf life cultivars.Heads with low number of sprouting bulbs are from 'Perla' (bottom left corner).

Fig. 4 .
Fig.4.Chromosomes from somatic cells (n=8x=16) from garlic genotypes: 'C-CN-95/2', 'C-37-1/8', C-3 1/25, 'Chino','Coreano' and 'Criollo'.2000; Peiwen et al., 2001;Ipek et al., 2003;Paredes et al., 2008;Pardo et al., 2009) or through AFLP(Rosales & Molina, 2007).Our version of this kind of analysis with 20 Mexican genotypes was as follows: Twenty garlic genotypes were subjected to RAPDs in order to construct a distance tree using clustering with the Unweighted Pair Group Method with Erithmetic Mean (UPGMA).DNA was extracted according toDoyle and Doyle (1990); DNA samples were run on agarose gel 0.8% and DNA concentration was measured with a spectrometer (model GBC Cintra 10e UV-visible).RAPD reactions were performed in a 25 ml volume, consisting of 10x buffer solution [10 mM Tris-HCl buffer (pH 8.0), 50 mM KCl 2 ], 2.5 mM MgCl 2 , 2.5 units of Taq DNA polymerase (Promega), 100 µM dNTP, 50 ng genomic DNA and 0.4 µM OPB series (OPB-8, OPB-9, OPB-10, OPB-11, OPB-15 and OPB 17) primer (Operon Technologies, Alameda, CA, USA).A total of 20 µl of mineral oil was placed over the reaction mixture.Amplifications were carried out in a DNA thermocycler (Model FPR0G02Y Techne Progene, England), under the following conditions: an initial denaturalization step of 2 min at 94 °C, followed by 35 cycles of 1 min at 94 °C, 1 min at 35 °C, and 2 min at 72 °C, with a final extension step of 7 min at 72 °C.Amplification products were analyzed by electrophoresis in a 1.2% agarose gel.It was run at 100 V for 4 h, and detected by staining the gel with ethidium bromide (10 ng/100 ml of agarose solution in TBE).All visible and unambiguous fragments amplified by the chosen primers were entered under the heading of total visible fragments.Fragment data were entered on a spreadsheet to form a binary matrix, where (1) represented fragment presence and (0) fragment absence for each fragment accession combination.Cluster analysis was conducted by converting the data matrix into a similarity matrix using a simple matching coefficient.This coefficient was calculated by dividing the number of matches (0-0 and 1-1) by the total number of comparisons(Nei & Li, 1979).A cluster analysis was then conducted using the unweighted pair group method, with arithmetical averages (UPGMA) process using the S-Professional Plus 2000 program.The results obtained were compared with others studies realized by different authors, and were discusses as following: Six decamer OPB primers showing

Table 2 .
Photosynthetic and respiration rate of cloves from six garlic genotypes after 90 d storage.

Table 3 .
Professional Plus 2000 program.The results obtained were compared with others studies realized by different authors, and were discusses as following: Six decamer OPB primers showing Chromosome morphological description from Mexican garlic genotypes.LBL=Long arm, LBC=Short arm, LT=Total length, LR=Relative length, r=Arm relationship and N=Centromere nomenclature.