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Peterson 1. The bamboo productive chain is still incipient in Brazil, and the low supply of plantlets due to low-efficient conventional propagation methods presents a significant bottleneck to its development. This study aimed to establish a micropropagation protocol for Guadua chacoensis.
Explants from donor plants cultivated under controlled environment showed less contamination, if compared to explants from plants grown in the field.
The obtained cultures were then multiplied using either in vitro -derived nodal segments or clump division in the presence of increasing contents 0 mM, 10 mM, 20 mM, 30 mM or 40 mM of 6-Benzylaminopurine BAP. The number of shoots increased with increasing BAP concentrations, but this also resulted in a reduced rooting rate and root length. Additionally, the shading improved the plant survival rates, if compared to those under non-shaded conditions, which presented photoinhibition and photodamage symptoms.
Peterson is a neo-tropical woody bamboo native from Argentina, Bolivia, Brazil and Paraguay Judziewicz et al. In Brazil, it occurs in the Atlantic Rainforest and Pantanal biomes, generally in riparian forests and lowlands Shirasuna According to Benton , G. In this way, G. Although Brazil presents the greatest bamboos diversity in the Americas Judziewicz et al.
Few native Brazilian bamboo species have been studied, despite their possible usefulness, an issue that grows in importance as agricultural frontiers expand, leading to biodiversity loss, genetic erosion and the possible permanent loss of valuable species.
Therefore, efficient plantlets production methods are essential for degraded areas and riparian forests reforestation, as well as to ensure a proper production for the developing industry.
However, the availability of seeds in American woody bamboos is irregular due to the long flowering cycles, often with intervals of 30 years Guerreiro Due to the seed scarcity, woody bamboos are conventionally propagated by asexual methods such as the division of clumps, rhizomes and section of culms. In many cases, such macropropagation techniques could present drawbacks, such as low efficiency, high costs, seasonality and problems with the transportation logistics of large-sized propagules Gielis et al.
There are several reports on bamboo tissue culture research, although many focused on Asian species Singh et al. A consensus between in vitro bamboo culture studies is that epiphytic and endophytic microorganisms may contaminate the medium when the bamboo cultures have already advanced into the multiplication stage Banerjee et al. In addition to the typical sanitation methods, there are two general ways of decreasing contamination rates: first by preventing the explant source plants from coming into contact with microorganisms, and second by controlling the proliferation of microorganisms that survive the initial disinfestations.
After the culture lines have been established, they must also be effectively multiplied. Cytokinins, such as 6-Benzylaminopurine BAP , may stimulate in vitro bamboo cultures to produce a greater number of culms, if compared to untreated ones.
However, individual genotypes may have different responses to BAP. Therefore, a range of concentrations must be tested to determine which ones will result in the highest multiplication rates with the least unwanted side effects Sandhu et al. The in vitro environment restricts the normal development of photosynthesis due to various physical factors, such as an exogenous supply of sucrose, high relative humidity and low luminous intensities Hazarika These conditions impair the acclimatization of plants to survive under greenhouse or field conditions.
Most studies on bamboo ex vitro acclimatization generally evaluated the plant survival rates, but not the factors limiting survival. Therefore, it is a suitable tool for monitoring the plant performance during the ex vitro acclimatization. Thus, this study aimed to establish a micropropagation protocol for the Guadua chacoensis bamboo, with a focus on developing a feasible large-scale production of healthy plantlets, as required for the consolidation of a bamboo productive chain in Brazil.
A total of nine plantlets were cultured using macroproliferation from a single plant. To determine if the growing conditions of the donor plant would affect the contamination or establishment rates of the in vitro cultures, the plantlets were cultivated for 6 months summer-autumn in one of the three environments: i indoors, in a commercial substrate Tropstrato Florestal - Vida Verde Ltda.
For hydroponic cultivation, the plants were grown on expanded clay, in a 20 L dripping bucket system. The pH hydroponic solution was weekly adjusted to 5. The plants in the commercial substrate were cultivated in 10 L pots and watered weekly for the indoor treatment and, when necessary, for the outdoor treatment. All treatments were fertilized every 15 days with the same nutrition solution as the hydroponic system. The pH was adjusted to 5. Nodal segments with mm in length and still protected by their sheaths were collected from primary branches and washed with a neutral detergent with a brush under running tap water.
Before the in vitro inoculation, nodal segments were reduced to 5 mm in length, by removing the tissues damaged by the disinfestation procedure. For multiplication, two experiments were performed using previously-established in vitro cultures maintained in MS basal medium free of plant growth regulators. The plantlets were multiplied via two approaches: i in vitro -derived nodal segments division; ii clump division.
After 40 days, the bud breakage and shoot number and heights were recorded. Each experimental unit consisted of one nodal segment per glass tube with 10 mL of medium, with a total of 30 nodal segments per treatment. Each experimental unit consisted of one clump with five culms per mL jar containing 40 mL of culture, with a total of 18 clumps per treatment.
To acclimatize the plantlets to ex vitro conditions, shoot clumps were first divided again into culms per clump, depending on the rhizome structure, and the roots were reduced to 2 cm in length, for standardization. These clumps were then planted in 72 multi cell plug trays and, after 60 days, transferred to cm 3 tube pots, in a greenhouse.
Survival was determined at 30 days after transfer to ex vitro conditions. After days of acclimatization, plants with culms were macro-proliferated into clumps of culms with rhizome and roots. The analyzed leaves were marked and tracked during the ex vitro acclimatization for 30 days. After 7 days, senesced leaves were substituted by the next young expanded leaf.
The parameters in the multiplication experiments were analyzed using regression analysis. All statistics were analyzed on the R environment R Core Team Due to the anatomy of nodal segments, contamination rates are usually quite high, because microorganisms may be shielded from disinfestation procedures by the space under bud prophylls, in the space between the bud prophyll and culm, or within vessels.
Though mercury-based solutions e. HgCl 2 are commonly reported for efficient explant sterilization Marulanda et al. The bud breaking occurred from days after the inoculation Figure 1A. The conditions in which the explant donor plants were kept greatly affected the contamination rate. Plants raised under hydroponic conditions had less contamination than those raised in commercial substrate indoors. The results suggest that the condition of donor plants is an important factor for increasing the likelihood that a given explant will survive disinfestation and establish a stable culture line.
A previous experiment showed a better bud breakage Figure 1C in well-developed nodal segments from the base of in vitro culms, especially the first and second nodes data not shown. As the BAP concentration increased, the number of shoots increased as well. However, the mean shoot height tended to decrease Figures 2B and 2C. Chlorosis and oxidation were observed in the shoots obtained from in vitro -derived nodal segments cultured in BAP-supplemented media.
At this phase, BAP could be used to improve the multiplication rates using the clump division method. The second multiplication approach, via division of clumps, is an efficient method commonly used for bamboo in vitro multiplication. For G. However, the spontaneous root formation reduced in response to increasing BAP concentrations Figure 3B.
Marulanda et al. Although the highest BAP concentration resulted in a 3. In comparison, Gutierrez et al. In the no shading treatment, even ex vitro -developed leaves senesced. A similar behavior was described by Matysiak , who found that ex vitro -developed leaves still presenting photodamage symptoms were not able to acclimatize. According to Harazika , the survival of leaves in the ex vitro condition depends on the environmental stress level.
These leaves have an important photosynthetic role in acclimatization, but non-persistent leaves function only as a nutritional reserve. These results are in accordance with those reported by Kumar et al. This result highlights that the direct transfer into full sunlight would likely be highly damaging to these plantlets, impairing their survival and the sprout of new leaves. On the other hand, shading improved the plantlets survival and quality. A higher survival rate obtained on the high shading treatment resulted as a consequence of the persistent-leaves plasticity under moderate light.
Similar results were also reported by Marulanda et al. These plantlets were further macroproliferated, leading to a increase in the number of plantlets, thus improving the protocol efficiency. A feasible micropropagation protocol was established for Guadua chacoensis. New clumps were regenerated from nodal segments of in vitro plants. Culture medium supplemented with BAP significantly increased the in vitro multiplication rate of G.
Moving plantlets onto medium without BAP reversed these detrimental effects. A two step method for accelerated mass propagation of Dendrocalamus asper and their field evaluation. Physiology and Molecular Biology of Plants , v. Priority species of bamboo. Bamboo : the plant and its use. Cham: Springer, Chlorophyll fluorescence as a probe of the photosynthetic competence of leaves in the field: a review of current instrumentation. Functional Ecology , v. Bambus nativos no Brasil: oportunidades e desafios para seu conhecimento.
Tissue culture strategies for genetic improvement of bamboo. Acta Horticulture , v. Flowering cycles of woody bamboos native to southern South America. Journal of Plant Research , v. African Journal of Biotechnology , v. Morpho-physiological disorders in in vitro culture of plants. Scientia Horticulturae , v. In vitro propagation of the neo-tropical giant bamboo, Guadua angustifolia Kunth, through axillary shoot proliferation.
Guadua chacoensis in Bolivia : - an investigation of mechanical properties of a bamboo species