La propagación de plantas, a través de la embriogénesis somática, Tesis presentada en opción al grado científico de Master en Biotecnología Vegetal. UCLV. Embriogénesis somática en papaya cultivar ‘Maradol Roja’: una alternativa para either to produce a high number of plants (high quality vegetal material to be. La regeneración de plantas de cacao mediante embriogénesis somática se ha El manejo del material vegetal se llevó a cabo en cámara de flujo laminar.
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Here we report the establishment of a methodology for plant regeneration via somatic embryogenesis SE from immature zygotic embryos, what supports high survival and high genetic homogeneity of the regenerated plants as determined somarica AFLP. We also report the first studies in papaya under photoautotrophic conditions during the rooting phase, critical for the in vitro culture of this crop.
These are novel and promising findings which make more efficient the in embriogenexis regeneration system. Somatic embryos, rooting, Floroglucinol, photoautotrophism, in vitro acclimatization, zeolite.
Topicos de La Embriogenesis Somatica Vegetal : Florio De Real Sunshine :
Vegetl high nutritional value of embriogenesid is the basis for a variety of food and medicinal applications .
In Cuba, in sonatica crop sommatica yields of tons, with a cultivated surface of ha . This veegtal is essentially obtained by cross-polynization and propagated by seeds. However, previous treatment of seeds is required for its direct use, since they are not able to keep some of the characteristics of starting genetic material for instance, genetic segregation properties.
Such fegetal generate variations in the quality and size of fruits, influencing crop yields. Another problem comes from the difficulties for determining the sex of seeds, since papaya is a trioecious plant and sex cannot be determined until flowering, having to wait for up to two or three months since planting.
There are three types of plants according to the sex of the inflorescence: In this somatida, two or three seeds have to be planted per planting spot in order to generate a full hermaphrodite plantation, also having to eliminate the other two undesired phenotypes at flowering.
Such practice normally raises production costs by increasing the number of planted seedlings and their cultivation until removal . One strategy effective to circumvent these problems is the use of in vitro culture techniques, either to produce a high number of plants high quality vegetal material to be planted or to support embriogenessi improvement programs and the commercial introduction of new or hybrid cultivars.
In fact, the massive propagation by organogenesis or somatic embryogenesis SE is justified for this crop due to its superior quality and uniformity of the plants obtained, despite its associated costs in comparison to seed propagation techniques .
Another technical difficulty comes from the relative nature of papaya as refractive to tissue culture, with low percentages of in vitro establishment, low multiplication rates and the presence of endogenous bacteria when propagated vevetal cultures under controlled conditions. This is further amplified by difficulties in rooting due to the lack of a basal callus, what hamper or limit the direct connection of roots with the stem, ultimately causing high losses during acclimatization by limited ex vitro rooting.
All this has led to the preferential use of SE for papaya propagation and its genetic improvement. SE has been mainly started from zygotic embryos as the most widely used type of explant for all the cultivars.
Nevertheless, a high number of works report callus formation prior to the generation of the somatic embryos, even in the case of using tissues with soamtica cells zygotic embryosthereby increasing the risks of genetic variability in the plants obtained . There are also few reports on the obtainment of secondary multiplication from primary somatic embryos, as a key phase to generate higher number of plants in SE. Besides, somatic embryo germination is affected by hyperhydricity and callus formation at the base of the explant, leading to incomplete germination , and having the in vitro shoots to be passed to a culture medium for rooting.
All this constitutes the main problem of tissue culture for this species .
Other difficulties come from the influence of environmental factors on the resulting plants, which influence on the growth, development and morphogenesis of shoots and plants generated in vitro mainly during the trophic phase. According to Kozai , in vitro photoautotrophism can be induced by excluding carbohydrates from the culture medium and by increasing gas exchange in the culture vessel. Photoautotrophic culture provides several advantages, including growth and photosynthesis stimulation, high percentage of survival during the in vitro to ex vitro transition, the correction of physiological and morphological disorders in the cultured plants, avoids callus formation at the base of the explant what increases rooting, and reduces the losses due to microbial contamination.
All these could provide an alternative for papaya cultivation, in order to circumvent the plant losses during the ex vegeetal acclimatization phase. So far, there was found a single report on the papaya tissue culture in vitro by using photoautotrophic conditions , but no details were referred about the most critical phase: Specifically for SE, there were no reports on the use of zeolite as substrate for shoots or in vitro plants, or by using the culture flask with increase aeration.
Another alternative considers the potential introduction of biologically active substances of national production and available for crop improvement in the in vitro regeneration process of papaya, in order to improve rooting in vitrowhile substituting the import of equivalent foreign products.
It is produced veegetal the enzymatic hydrolysis of pectic acid extracted from Persian lime Citrus latifolia Tanaka .
It is able to induce and develop rooting and to notably increase the development and vigor of plants in vitro in several crops. Another product to be considered is phloroglucinol PGa phenolic compound obtained by degrading floridcin and a precursor in the synthetic pathway of lignin.
It has been demonstrated to display vegetal growth promotion activity, with positive effects in the lignification of in vitro plantsthe elimination of callus formation at the base of explants obtained from somatic embryos, and in the rooting phase . In addition, current morphologic, cytological, isoenzymatic and phenol contents analysis techniques are unable to determine plant sex in papaya seeds or seedlings .
Most embroigenesis do not distinguish hermaphrodite plants and are not suitable for screening a high number somaticca samples. This has led to the implementation of strategies using molecular markers for sex determination in this species, particularly techniques vgetal on PCR amplification of molecular markers to determine the sex at early stages of development under field conditions .
Consistent with an strategy aimed to vegetl the high mortality rates in vitro during the ex vitro acclimatization phase due to difficulties for root development, a high ex vegetak survival rate was obtained, and regenerated plants displayed a high genetic homology. Somaica work report callus formation for other papaya cultivars prior to the formation of somatic embryos, despite of starting from zygotic embryo tissues with embryogenic predetermined cells which ultimately lead to an increase in genetic variability risks.
Conversely, few reports are available on the secondary multiplication of primary somatic embryos, a phase considered crucial for high multiplication rates in SE. The methodology developed was supported by histology and electron microscopy studies, and provides a consistent tool to increase the efficiency of the propagation process. The study also provided evidence on the role of auxin transporters at all stages of histological differentiation during the development of somatic embryos.
Photoautotrophic micropropagation for rooting and increased ex vitro survival rates The photoautotrophic culture has several advantages, such as stimulating plant growth and photosynthesis, together with an increase in survival rates during the in vitro – ex vitro transition.
Our results of in vitro cultivation of papaya showed that the methodology developed was able to preserve somatic embryos from death during the ex vitro acclimatization phase. This was possible due to the increased ventilation of culture vessels, by capping them with metallic foil paper with two holes.
Also, the flow of photosynthetic photons estabilized in the range of This was also complemented with the use of zeolite as substrate for plant in vitro cultivation, resulting in higher photosynthesis rates, the correction of morpho-physiological disorders, the prevention of basal callus formation, which combined with indole butyric acid treatment in the culture medium supported high percentages of rooting.
This work also provided the first report, up to our knowledge, on the soatica of zeolite as substrate for the cultivation of shoots in vitro, in combination with increased ventilation as it was carried out. This procedure allowed us to improve the quality of the plants obtained with a better ex vitro acclimatization of in vitro obtained explants, and higher survival rates. These results also accounted for the pioneering studies on the photoautotrophic culture conditions in Cuba for this plant species .
Increased rooting to PG treatment also improved embriogenewis morpho-physiological parameters of the plants obtained. So far, these were the first reports on somarica these growth regulators for the improvements of papaya in vitro cultivation during the most critical phase rooting [18, 19]. The methodology established can be implemented in commercial laboratories biofactories in Cuba and abroad. It is expected fegetal these results could have a positive impact on the propagation of this commercially relevant crop in Cuba in the short time, also setting guidelines for future research on the genetic regulation during SE in papaya species.
Factors that influence the yield and viability of protoplasts from Carica papaya L. Ministerio de la Agricultura. Variation of plants derived from indirect somatic embryogenesis in cotyledon explants of papaya.
Improved protocol for high frequency plant regeneration through somatic embryogenesis in Carica papaya. Better rooting procedure to enhance survival of field growth Malaysian Eksotika papaya transformed with 1-aminocyclopropanecarboxylic acid oxidase gene. Photoautotrophic micropropagation-environmental control for promoting photosynthesis. Teixeira da Silva JA. Photoauto- ,Photohetero-and Photomixotrophic in vitro propagation of papaya Carica papaya L. Thammasat Int J Sci Technol.
Oficina Cubana de la Propiedad Industrial.
Revista Colombiana de Biotecnología
Phloroglucinol in plant tissue culture. Effects of different culture conditions photoautotrophic, photomixotrophic and the auxin indole-butyric acid on the in vitro acclimatization of papaya Carica papaya L. Red Maradol plants using zeolite as support. Effect of phloroglucinol on rooting and in vitro acclimatization of papaya Carica papaya L. Received in October, Accepted in March,