Micropropagation in Agave Species: Comparison
Please note this is a comparison between Version 2 by Jason Zhu and Version 3 by Jason Zhu.

The Agave genus is composed of approximately 210 species distributed from south United States to Colombia and Venezuela. Numerous Agave species have been used for the preparation of alcoholic beverages and have attracted interest in the pharmaceutical and food industry. Despite their economic importance, there are few initiatives for the improvement and selection of characteristics of interest. This is mainly due to its morphology, long lifecycles, and monocarpic nature. Micropropagation is a feasible alternative to the improvement of Agave species. It has been used for multiple purposes, including massive propagation, induction of somaclonal variation to enhance agronomic characteristics of interest, maintenance of specific genotypes, and genetic transformation using molecular techniques.

  • Agave genus
  • bulbils
  • co-cultivation

1. Introduction

An alternative to conventional propagation in Agave is the use of plant tissue and cell cultures, in order to easily obtain new plantlets in a short time and on a large scale. Micropropagation presents several advantages which are directly applicable for commercial Agave production, including mass production of plantlets within a short timescale; micropropagated plants, which are free from pathogens thus reducing the spread of diseases between plantations; and the production of plants that are uniform in age and size, leading to homogeneous plantations which facilitate and optimize the process of harvesting. Consequently, several of the main tequila companies are currently testing the wide-scale use of micropropagated A. tequilana germplasm in comparison to the traditional practice of planting offsets [1].
Since the 1980s, several species have been propagated using micropropagation for multiple purposes, including massive propagation of endangered species such as A. arizonica and A. victoria-reginae; induction of somaclonal variation to enhance agronomic characteristics of interest; maintenance of specific genotypes, as in the case of the tequila industry; and genetic transformation using molecular techniques [2][3][4][5][6].
Regeneration responses have been achieved through various approaches including axillary bud proliferation, as reported by Ramírez-Malagón, for A. tequilana, A. salmiana ssp. Crassispina, A. duranguensis, A. oscura, A. pigmaea, and A. victoria-reginae. For A. tequilana, regeneration was obtained through temporary pulse treatments with different concentrations of 2,4-D, yielding 12 shoots per explant with 6.8 mM 2,4-D and three days of exposure to the hormone. Regarding the other species, the pulse system did not achieve any shoot formation since explants became necrotic. Nevertheless, IBA and BA were also tested at different concentrations, in the other species leading to axillary shoot formation as expected [7][8].
Direct organogenesis is another response exploited for micropropagation and there are several reports where new shoots have been induced. For instance, in A. sisalana, A. fourcroydes, and A. cantala, several combinations of growth regulators were evaluated, where 0.40 µM NAA + 0.49 µM IBA + 2.32 µM KIN proved to be the best combination to promote shoot formation and avoid callus production during the process [8].
On the other hand, indirect organogenesis is also a way to induce shoots. In this case it is necessary to first pass through a callus induction phase and then promote shoot proliferation. It has been observed that undifferentiated tissues, such as meristem cells, are an appropriate choice to induce new buds, even for direct somatic embryogenesis [9]. Indirect organogenesis in A. tequilana was reported, obtaining a suitable response when meristems were exposed to different zeatin and 2,4-D combinations to produce callus, which was grown on in 5.2 µM NAA in order to maintain indefinitely the growth of callus tissue. For promoting shoot formation, the treatment that showed the best results was 0.11 µM 2,4-D in combination with 44 µM BA, with a bud forming capacity (BFC) index of 14.5. This BFC index represents the relation between the mean number of buds per explant and the percentage of explants forming buds [10].
Other systems of regeneration widely used in many plant species are direct and indirect somatic embryogenesis, considered a viable alternative for genetic improvement, since cultures initiate from a single cell or, in some cases, a group of cells. Rodríguez-Garay reported for the first time the production of somatic embryos in the genus Agave, using 2,4-D as a growth regulator. Somatic embryos were produced on leaf blades of in vitro A. victoria-reginae plantlets when the medium was supplemented with 1.4 µM 2,4-D and germination was achieved on half-strength MS medium without growth regulators [3].
An example of indirect somatic embryogenesis is that reported by Tejavathi. The response was obtained in A. vera-cruz Mill, the main source of natural fiber in India. 2,4-D and NAA were shown to successfully produce embryogenic callus, in comparison to IAA and IBA, which produced non-embryogenic callus. The addition of 5.37 µM NAA + 0.91 µM Zeatin + 40 g/L sucrose to the medium was the best combination for somatic embryogenesis in this species [11].
Independently of the regeneration response, micropropagation may also be combined with various methodologies for mass propagation. The most common system is semisolid culture, although there are additional techniques, such as temporal immersion systems and thin cell suspension layers, that are suitable options for enhancing the number and quality of shoots/somatic embryos obtained [12][13].

2. Medium, Growth Regulators and Response Comparison of Agave Agave Species Micropropagation

Depending on the intended use, micropropagation protocol development for Agave species is focused on specific regenerative pathways, as mentioned above, and the use of specific growth regulators also corresponds to the expected response. Moreover, it is important consider that results may vary within the same genus under similar cultivation conditions [7] and even different genotypes of the same species may influence the responses obtained. Therefore, it is fundamental to establish a general micropropagation protocol that will serve for the majority of Agave species.
In many reports for Agave species, researchers used MS salts, supplemented with L2 vitamins, and in some cases, modified the ammonia concentration. A common growth regulator used to produce an indirect response, as in the case of organogenesis or somatic embryogenesis, is 2,4-D at a concentration ranging from 0.1 to 9.05 µM. However, it has also been reported to directly promote shoot formation in combination with cytokinins such as BA or KIN, with concentrations ranging from 4.44 to 38.2 µM and 2.32 to 27.84 µM, respectively.
Another example is the case of A. cantala, A. fourcroydes, A. sisalana, and A. peacockii. Contrasting concentrations of KIN are required to induce direct organogenesis: 27.84 µM for A. peacockii, and 2.32 µM KIN for the others. In addition, A. peacockii also requires the supplement of the cytokinin BA, whereas the other species require the addition of the auxins NAA and IBA. The reported numbers of shoots generated under these conditions vary extensively from 87 to 4, respectively [8][14]
Regarding regeneration efficiency, the system with the highest regeneration rate for the Agave genus is that of somatic embryogenesis, both direct and indirect.

3. Genetic Transformation in Agave Species

The development of micropropagation methods represents a significant opportunity to develop genetic transformation protocols. Exploitation of this technology may be a feasible strategy to introduce genes and improve certain traits, such as tolerance to diseases, and increase the production of probiotic compounds.
During the last two decades, protocols to transform Agave species have been tested with promising, although variable, results. For example, a patent for genetic transformation of the Agave genus by particle bombardment involving A. tumefaciens and A. rhizogenes has been filed. In the bioballistic method, embryogenic calli were bombarded with tungsten particles covered with plasmid DNA containing marker genes. On the other hand, embryogenic calli were placed in co-culture with Agrobacterium for 48 h and then transferred to a selective medium to obtain transformed cells. PPT/Bar and hpt genes (which confer resistance to phosphinothricin and hygromycin respectively) were used as selectable markers and the uidA gene (β-glucuronidase) was used as a reporter gene. Herbicide- or antibiotic-resistant plants were obtained using these protocols [15].
In a subsequent report, A. salmiana was transformed using co-cultivation with A. tumefaciens and particle bombardment. The uidA gene was used as a reporter gene in both cases, and nptII (neomycin phosphotransferase II) and bar genes were used as selectable markers for A. tumefaciens or bioballistic mediated transformation methods, respectively. The conditions for both shoot regeneration and rooting were optimized using leaves and embryogenic calli. Agrobacterium co-cultivation was the most effective method, obtaining 32 rooted transgenic plants regenerated from calli, with a transformation efficiency of 2.7%. The transgenes were detected in 11-month-old plants. Alternatively, the particle bombardment protocol produced transgenic calli that tested positive with the GUS assay after 14 months on a selective medium [6].
Likewise, root regeneration was induced in leaves, stems, and roots of A. salmiana mediated by A. rhizogenes A4. In vitro plantlets were inoculated with different concentrations of bacteria and acetosyringone. Leaf tissue showed the best response, producing 63% of transformed roots when 1 × 109 bacteria mL−1 and 200 µM acetosyringone were used. The nptII and uidA genes were used as a selectable marker and a reporter gene, respectively. A rate of transformation of 80% of the tissues was determined for the reporter gene and 60% for the selectable marker [16].
A successful example of the use of transgenic agave plants is in the case of zebra disease, which is caused by Phytophtora nicotianae and attacks Hybrid 11648 in all regions where it is cultivated. Conventional plant breeding could be a strategy for obtaining plants tolerant to zebra disease. However, this method is difficult to achieve due to the long lifecycle of the hybrid, which takes around 10 years to bloom. Hence, a transgenic strategy could be an alternative to produce enhanced tolerance to P. nicotianae in Hybrid 11648 plants in a short period. Therefore, a transgenic strategy to express hevein-like peptides in calli of Hybrid 11648 was reported. The optimum culture media for callus induction were SH, 13.2 µM BA, 2.68 µM NAA, and 0.45 µM 2,4-D. The shoot regeneration media were SH, 6.66 µM BA, and 2.68 µM NAA. Several factors influencing transformation efficiency were also tested. The effective time for infection was 10 min and acetosyringone was used at a concentration of 200 µM. The optimum time for pre-culture of callus was three days, and the optimum co-culture time was four days. Thirty-seven lines from 150 explants were obtained and the hevein-like gene was expressed in seven lines [17].
In spite of these successful reports of transformation of Agave species, the process is laborious and time consuming and development of a rapid and easy transformation protocol would be a great advantage. A method for the transformation of A. tequilana and A. desmettiana mediated by A. tumefaciens was therefore developed based on direct organogenesis. Bulbil meristems were used as explants and co-cultivated with A. tumefaciens strains LBA4404 and GV2260 using phosphinothricin (PPT/Bar) as the selective agent. A. desmettiana produced a much higher number of shoots per explant in comparison with A. tequilana (2–20 shoots and 1–2 shoots respectively) [18].


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