Patent Application
20160278315
The present invention relates to a process for the production of clonal plants of arboreal species, especially eucalyptus, in large-scale and high-volume, whereby the step of vegetative propagation, performed in vitro, occurs under conditions such that the obtaining of healthy and vigorous microcuttings of the arboreal species is maximized, while the rate of hyperhydricity of the micropropagation phase in the temporary immersion regime is minimized.
Micropropagation consists of a technique of plant tissue culture which is used as a basis for the formation of seedlings, being a practical, safe and fast method of mass propagation of plants in a reduced space. Conventionally, micropropagation is performed via the semi-solid system, typically in a gelling medium with agar. However, this is still a costly practice, allowing room for the use of equipment and new technologies with equipment called bioreactors that help in the production of plants in large scale operation and reduce the high cost of labor.
Bioreactors are devices for the cultivation of seedlings in a regime of temporary or permanent immersion to obtain plants. The bioreactor consists of a set of containers with compartments and accessories for optimal management of key growth factors to maximize the biological reactions. By means of this equipment, it is possible to obtain higher rates of multiplication of the culture by the use of a liquid nutrient medium, injection and exchange of gases, e.g. air enriched with carbon dioxide, inside the container, as well as better physiological control of cultivation conditions and process automation. There are various types of bioreactors, which may vary depending on the culture container and culture medium, the immersion-type (temporary or permanent), and their functionality. The temporary immersion systems best known in Brazil are RITA® (temporary automated immersion container—for example, created by CIRAD-France) and BIT® (temporary immersion bioreactor—for example, created by Embrapa-Cenargen).
Due to the potential of the micropropagation technique, there is growing interest in this technology as demonstrated by the prior art. For example, document WO 2012/061950 describes a method and bioreactor for the micropropagation of an Antarctic plant species, wherein the method is characterized by the use of a nutrient medium and appropriate conditions for the development of said plant species, and additionally employs a protocol for supplying UV radiation and temperature control for the production of valuable metabolites for medical and cosmetic use.
Document WO 2012/156440 describes a temporary immersion system and method of micropropagation of plant species characterized by the provision of appropriate conditions for the development of propagules, such as lighting, a temperature and immersion system with intervals ranging from 1 to 10 times per day, and immersion time of 1 to 2 minutes or 3 to 4 minutes depending upon the plant species being developed.
Watt (see Watt, M P “The status of temporary immersion system (TIS) technology for plant micropropagation.” African Journal of Biotechnology, Vol. 11 (76), p. 14025-14035, Sep. 20, 2012) provided a retrospective of the micropropagation of plants, particularly with respect to proposals to reduce hyperhydricity, citing as the main factors, the shortening of the immersion time and increasing periods of rest (time between two successive immersions).
González (see Gonzalez, R. et al. “Multiplication in vitro of Eucalyptus globulus by temporal immersion system.” Bosque 32(2): 147-154, 2011) reported the factors affecting the stage of in vitro multiplication of Eucalyptus globulus by temporary immersion and concludes that the rate of multiplication of microcuttings of this arboreal species increases significantly when a soak time of 1-2 min and a frequency of immersion (rest period) for 12 hours are employed. These authors also concluded that hyperhydricity increases with the concentration of nutrients and sucrose.
Oliveira (see Oliveira M L, “Micropropagation of clones of Eucalyptus grandis×E. urophylla in a temporary immersion bioreactor.” Master's Thesis Dissertation. Federal University of Viçosa. 2011) reports the study of in vitro micropropagation of two eucalyptus species, concluding that there is a high percentage of occurrence of hyperhydricity in the development of propagules when the technique of temporary immersion is employed. The author also concluded that “there is a need for adjustment in the crop management phases of multiplication and elongation to obtain shoots with greater vigor, able to root in ex vitro conditions in order to make this technique feasible on a large scale.”
The aforementioned prior art shows that although there has been great progress in the technique of in vitro micropropagation of plants, the problem of hyperhydricity remains to be solved and, at the same time, employing suitable conditions to obtain clonal seedlings of plants, particularly of the arboreal species, and more particularly of the eucalyptus species, enabling the production of said cloned seedlings on a large scale and high volume and to achieve commercial production of healthy and luxuriant plants.
The present invention refers to the obtaining of clonal plant seedlings, in particular of arboreal species, particularly the species of eucalyptus, using the technique of in vitro micropropagation of plant material, comprising the steps of multiplication, elongation, rooting, acclimatization, and growth and acclimatization of seedlings ready for commercialization.
In a first embodiment, the present invention relates to a process for in vitro micropropagation of plant material, especially of arboreal species, particularly from a species of eucalyptus, comprising the steps of multiplication and elongation of shoots developed from plant propagules by the temporary immersion method, as follows: (a) in the step of multiplication of the shoots, the material to be propagated is placed on a suitable support in a first container (plant propagation container) and is subjected to management of temporary immersion in a liquid nutrient medium from a second container (storage container of a liquid medium), said management comprising alternated periods of immersion and rest, with the immersion time being preferably at least 1 minute, preferably at most for 3 minutes, more preferably for 1 to 2 minutes, with the resting period preferably at least 2 hours, preferably for up to 4 hours, more preferably for 2 to 3 hours; (b) in the step of elongation of shoots, the propagation material is subjected to management of temporary immersion, preferably employing the same nutrient medium of step (a), said management comprising alternated periods of immersion and rest, the immersion time being preferably for at least 20 seconds, preferably for at most 60 seconds, more preferably for 20 to 30 seconds, with the rest period preferably being at least 4 hours, preferably for at most 6 hours, more preferably for 4 to 5 hours; said method being characterized by the fact that, in said elongation step, said immersion periods are significantly shorter and said rest periods are longer compared with the periods of immersion and rest, respectively, in the multiplication step.
In a second embodiment, the invention relates to a process for the production of clonal plant seedlings ready for field development comprising the steps of: (i) subjecting the plant propagules to vegetative propagation conditions of temporary immersion in vitro comprising the stages of: (a) multiplication of the shoots in which the material to be spread on suitable support is placed in a first container (micropropagation container) and said material is subjected to the management of temporary immersion in a liquid nutrient medium, from a second container (liquid medium storage container), with said management comprising alternating periods of immersion and rest, with the immersion time preferably at least 1 minute, preferably at for most 3 minutes, more preferably for 1 to 2 minutes, and with the period of rest being preferably at least 2 hours, preferably for at most 4 hours, more preferably for 2 to 3 hours; (b) elongation of shoots in which the material is subjected to propagation management of temporary immersion, preferably using the same nutrient medium of stage (a), said management comprising alternating periods of rest and immersion, with the immersion period being preferably at least 20 seconds, preferably for at most 60 seconds, more preferably for 20 to 30 seconds, and the rest period being preferably at least 4 hours, preferably for at most 6 hours, more preferably for 4 to 5 hours; (ii) rooting and acclimatization of the multi buds obtained in step (i) in containers and conditions suitable to maintain plant turgor and reduce evaporative demand; and (iii) growth and acclimatization of the microcuttings obtained in step (ii).
The production system of microcuttings of clonal arboreal species, such as eucalyptus, in high scale and high productivity in temporary immersion bioreactors has as its main objective the gradual replacement of traditional clonal gardens of operational forest nurseries by plant biofactories, aimed at producing industrial micropiles for subsequent rooting and acclimatization in specific greenhouses (cloches).
In bioreactors, it is possible to obtain higher rates of multiplication and crop growth by continuous use of a liquid nutrient medium, supplemented with air injection and carbon dioxide enrichment and photosynthetically active radiation with light-emitting diode lamps inside the plant containers. Thus, the management of production of shoots and biomass is best optimized via automated manipulation of key growth factors, enabling better physiological control of culture conditions.
What has been sought in this technique is the minimization of hyperhydricity, which causes disturbances and degeneration of the cloned seedlings obtained by the method of temporary immersion and, at the same time, retains the advantages of this technique in terms of production costs and health/vigor of the produced seedlings. This challenge was met by the inventors of this invention who have created a method in which alternating periods of immersion and rest of the material in vegetative propagation are differentiated in the stages of multiplication and elongation of shoots. This procedure has made it possible to reduce hyperhydricity and maximize the production of clonal plants seedlings, in particular arboreal species, and most preferably of the eucalyptus species. In
The process of the present invention has been customized to meet the physiological demands for the vegetative propagation of arboreal species, especially eucalyptus, allowing the intensive and precision development of plants, differentiated to result in the best quality standard and uniformity and vigor of shoots produced, providing greater rooting of microcuttings and subsequent better acclimatization and early yield of clonal seedlings for industrial use on a large-scale.
The process of the present invention allows the obtaining of productivity at least 30 times greater than the traditional method of producing seedlings from commercial nurseries, in addition to providing a 33% reduction in cycle time for production of a clonal change. Additionally, the operational deployment of the process of the invention in nurseries provides a reduction of manpower and a reduction in the construction cost of clonal gardens.
The process of the invention solves, among others, the following problems:
Production of clonal seedlings on a large scale and high volume, with uniformity in quality, for maximum efficiency and productivity of vegetative propagation of arboreal plants, for example, eucalyptus, with reduced operating costs and production cycles.
Reduction of physiological abnormalities and disorders related to hyperhydricity of the explants, a phenomenon commonly referred to as plant vitrification. The management of gas exchange is one of the advantages of the invention, which is preferably performed in a bioreactor of the BIT type, but may be performed in a bioreactor of another type, such as RITA, which practically eliminates these biological problems.
Reduction of the cost of production of eucalyptus clonal seedlings associated with reduced labor involved in clonal gardens in the operational process.
Uniformity of the pattern of physiological quality of plant matrices and micropiles in vegetative propagation, performed in a temporary immersion bioreactor, in contrast with the lack of uniformity of plants and cuttings obtained from plants grown in clonal gardens, where environmental conditions are not uniform.
Difficulties of the vegetative propagation of species and clones of arboreal species, including eucalyptus, recalcitrants are contoured with vegetative propagation in vitro in bioreactors, allowing the use of new and more productive clones that would not be produced by conventional means.
Clonal cleaning, by obtaining matrices of plants and crops free of pathogenic microorganisms and, also, cultures of high genetic fidelity of single clone without mixing with other clones.
Acceleration of breeding programs by multiplication of superior clones, aiming to produce seedlings in less time and in greater quantity and reduced space, in addition to the rejuvenation of selected clones and matrices.
It is important to note that the use of sprouts (or products of micropropagation) obtained according to the process of the invention, for direct seeding in specific containers (which can be tubes, plugs, Styrofoam trays or otherwise), replaces the supply of shoots to the market from known clonal gardens (either clonal mini-gardens or clonal micro-gardens). This is a great advantage of the technique employed in the micropropagation process of the present invention, and one that, with the production of eucalyptus, presents a return in terms of unmatched quantity and quality which is not comparable with the technique used in clonal gardens.
Throughout the process of growth and development of plants in the bioreactor, two basic culture media can be used, as shown below. These media are chemically modified according to the present invention to attend specific clones, according to the need and physiological stage of the culture. The media are supplemented with growth phytoregulators, fostering bud multiplication, elongation of shoots and pre-induction of ex-vitro rooting of microcuttings.
The types of concentrations of growth regulators, as well as their use in the management of the bioreactor process are those commonly employed in the art, with proper adaptation to the cultivation of the desired species for propagation, preferably including arboreal species, and more preferably eucalyptus.
As shown in
After proper allocation of the pellets in the trays, they must be placed with their bottom (base) in contact with a shallow layer of pure water so that the pellets in the trays are not covered (or float) in the water. This is the process of expansion of the pellets, which in this case “rehydrates” them by capillarity. It is important to note, that the base of the pellets should be kept in contact with this layer of water until fully expanded. If the water depth is reduced (due to absorption by the pellets), the water must be replaced. There must be no concern about “excess water.” This operation takes only a few minutes. If one wishes to speed up the expansion process, one can make use of heated water of approximately 45° C. In this case, it is essential to carry out the expansion of the pellets some minutes in advance, such that their temperature approaches room temperature, before being taken to receive the microcuttings.
In general, in a population of micropropagated eucalyptus plants, it is likely that a small subpopulation will present anatomical, morphological and physiological abnormalities due to imbalances of the conditions inside the bioreactor and other flasks in the micropropagation. All these changes are associated with unsuitable conditions for the process of rooting microcuttings and subsequent acclimatization of seedlings, and thus reduce survival rates and the quality of seedlings.
The application of acclimatization techniques aims to provide greater graduation in the transition between the in vitro (bioreactor) and the external (nursery) environment. Among these techniques, we highlight the use of mist and fog systems, which help to maintain the plant turgor and decrease evaporative demand. Shading helps to reduce high levels of brightness from the external environment and also helps to reduce the temperature and stress of the plants in acclimation.
Alternatively, other products and protected environments are used in the planting of microcuttings produced in bioreactors. Products used include substrates stabilized by biodegradable polymers for planting in wood fiber containers, and coconut fiber and Sphagnum peat. These containers function as pressed pellets and are expandable when hydrated.
Cultivation System with Fog and Cloche
This system generates a mist of extremely small water droplets, with diameters smaller than 15 micrometers, which are nebulized within the cloche environment using specific nozzles or an ultrasonic nebulizer, generating an ultrathin (<5 μm) dry fog. These droplets are so small that they remain suspended in the air until evaporated, contributing to the increase in humidity and temperature reduction and, at the same time, avoiding leaf wetness. These water droplets in suspension are able to maintain a very thin film of water vapor on both leaf surfaces, which keeps the seedlings turgid and temperature controlled.
The fog system is used in conjunction with the cloche structure, preferably a structure designed specifically for rooting and acclimatization of eucalyptus. The cloche consists of the assembly of a mini-greenhouse or over-tunnels within a normal greenhouse, kept suspended on rounded tables. During the growth and development of seedlings in the cloche, a sub-surface irrigation system (floating) is utilized, which provides temporary immersions of the base of the biodegradable plugs in water or nutrient solution which is used to supply nutrients to the plants without wetting the aerial part of the seedlings.
The planting system of the microcuttings produced in the bioreactor in biodegradable containers consists of selecting standard microcuttings of satisfactory quality and inserting the base of the microcuttings into bags or previously hydrated pressed pellets (plugs), as described above.
After placing the pellets in the trays, they must be positioned with their bottom (base) in contact with a shallow layer of pure water at room temperature so that the pellets in the trays are not covered (or float) in the water, until full expansion, before planting the microcuttings.
Rehydration of the pellets is accomplished only by capillarity and not by their submersion in water. It is important to note that the base of the pellets should be kept in contact with the layer of water until they are fully expanded. If the water level is reduced (due to absorption by the pellets), it must be replaced. There must be no concern with regard to excess water. This operation takes only a few minutes.
If one wishes to speed up the expansion process, one can make use of water heated to about 45° C. In this case, it is essential to perform the pellet expansion a few minutes early, so that their temperature approaches room temperature before being taken to receive the microcuttings during planting.
By way of non-limiting example, some information is provided about the techniques available to perform the planting of the micropiles produced in the bioreactor.
The application of the depth of irrigation water should be adjusted to achieve the field capacity of each substrate, especially at the stage of rooting (CV) and Acclimation (CA), where the Ellepot and Jiffy must not be saturated with water.
In the greenhouse, irrigation management should produce an irrigation interval of short duration and intervals between irrigations, for example, 6 seconds duration every 10-15 minutes between irrigations, to increase the level of relative humidity without irrigation of the substrate occurring.
In acclimatization, irrigation management should allow complete wetting of the substrate of the Ellepot and Jiffy, without causing excess and saturation of the same. The shading, for example, of the shading screen type, must remain extended only during the hotrest hours of the day, for example, from 11 am to 2 pm.
To the contrary, in the Stages of Growth (PC) and Rustification (PR), the Ellepot and Jiffy should be almost saturated, due to the need for the greater depth of water to compensate for evaporation losses from the container, root uptake and leaf transpiration.
Additionally, in the growth and rustification terrace, irrigation of the Ellepot and Jiffy is recommended with an irrigation rod or shower head for uniform wetting of the entire substrate.
During the process of rooting and seedling growth in the Ellepot and Jiffy container, it is necessary to monitor the moisture content of the substrate in order to recommend the irrigation shift (duration and interval) so that substrate moisture remains within the ideal range of field capacity.
These instant, real-time measurements of substrate moisture must be performed using a portable moisture meter+conductivity+temperature, for example, the Delta-T brand, model WET Sensor. This equipment must be calibrated in the laboratory for the substrate being used in the test.
The humidity sensor must be inserted in at least two positions (depths) within the Ellepot and Jiffy, because only the tip of the sensor is able to accurately record the values in the rhizosphere region. In the CV, this position should be shallow (2-3 cm), in the CA this position should be median (3-6 cm) and in the PC position it should be at the bottom (7-9 cm).
Between one measurement and the other it is necessary to wait for a period of time (10-15 seconds) for sensor stabilization in the moist substrate. In the same Ellepot or Jiffy, the 1st measurement must be processed at a point of lower humidity, and the 2nd measurement at a point of higher humidity.
We recommend the following ranges of moisture for the substrate used in the test, measured with the wet sensor, as shown below:
The process of the invention, applied to obtain eucalyptus clonal seedlings in a bioreactor by temporary immersion technique, comprises the following steps:
Step 1—Plant material remains at rest during the interval between immersion of the plant in culture media and gas exchange of the atmosphere of the internal compartment of the bioreactor. The medium is stored in the outer container (lower) and the explants in the inner compartment (above). The active photosynthetic radiation bioreactor is made of tubular fixtures with LED lamps in blue, red and white colors.
Step 2—The solenoid valve is activated by a timer in a range of pre-set time and duration. This allows entry of compressed air into the outer container, increasing pressure in the outer compartment (lower), causing the liquid culture medium to be transferred to the inner chamber (upper) of the plants, via the connection channels between the containers, and comes into contact with the base of the cultures. The management of temporary immersion varies with the type of eucalyptus clone and also with the stage of the culture. In the multiplication phase of shoots, 2-3 hours between temporary immersions and immersions lasting between 1-2 minutes are used. At the stage of shoot elongation, 4-5 hours between temporary immersions and immersions lasting between 20-30 seconds are used.
Step 3—After the immersion period, the solenoid valve is turned off and the medium returns into the outer housing through the drains by gravity. At this stage, the exchange with the injection of compressed air into the inner container where the plant material is allocated occurs. This gas exchange (air and carbon dioxide) is actuated by a solenoid valve controlled by a timer.
Step 4—The gas exchange occurs with the injection of specific concentrations of CO2 in the inner container at a flow rate of 1.3 liters per minute, with a flow of 500 micromole CO2 per second, in the concentration of 800-1000 micromole CO2 per mole of air, where the plant material is grown, to increase the photosynthetic and productive capacity of the crops. This gas exchange is also driven by a timer-controlled solenoid valve.
PAR Radiation—throughout the production cycle we use photosynthetically active radiation (PAR), which corresponds to the range from 400 to 700 nm for the growth and development of the crop. In this case, electric lamps with light emitting diodes (LED) are used instead of the traditional fluorescent lamps employed in biofactories. The plants mainly absorb spectra in the red range (600-700 nm) and in the blue (400-500 nm), therefore, the management of illumination is controlled to provide a ratio of 3/1 (red/blue) to 2/1 (red/blue), respectively for multiplication and elongation. The intensity of PAR also varies according to the physiological stage of the culture. Typically, in the multiplication phase 20-30 μmol/m2/s is used, and in the elongation phase 40-60 μmol/m2/s is used.
All publications and patent applications mentioned in this specification are indicative of the level of those skilled in the art to which the present utility model refers. All publications and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application were each specifically and individually indicated to be incorporated for ease of reference.
While certain embodiments have been described, they are only shown in an exemplary way, with no intention to limit the scope of the present utility model. The accompanying claims and their equivalents in this description are considered to cover such forms or modifications as they may be within the scope and spirit of the present utility model.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/BR2014/000391 | 10/30/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61897680 | Oct 2013 | US |
