In vitro germination and acclimatization of Hamatocactus setispinus

Seed propagation preserves the population genetic variability and helps selecting desirable features. This study evaluated the in vitro germination of Hamatocactus setispinus in six different culture media, 1MS basal medium full strength; 2halfstrength MS basal medium; 31.0 g L-1 of Peter’s CalMag® 15-05-15 formulation; 40.5 g L-1 of Peter’s CalMag® 15-05-15 formulation; 5MS basal medium supplemented with 10% coconut water and; 6water and agar, with and without activated charcoal, and the speed of germination index, the mean germination time and the germination rate, root length, shoot length and the number of roots were evaluate. The seedlings with superior development obtained from in vitro germination were acclimatized in two substrates: Biomix® Floreira; Biomix® Floreira + sand. Seedling survival, shoot length, shoot diameter, root length, root number, shoot fresh matter weight, root fresh matter weight, shoot dry matter weight and root dry matter weight were evaluated. Peter’s 1.0 g L-1 medium without activated charcoal led to the best results for root length (11.36 mm) and root number (3.84). There was 100% of seedling survival. Acclimatization substrates did not differ among themselves and, therefore, they did not affect seedling growth.


INTRODUCTION
Seed germination stands out among the methods for cacti propagation, because it allows the preservation of genetic diversity of populations (ROJAS-ARÉCHIGA and VÁSQUEZ-YANES, 2000), which might help in the selection of desirable features, such as biomass production, fruit quality, tolerance to stress-promoting factors, etc. (ALTARE et al., 2006).
Many cactus species have slow growth and low seed germination.Thus, in order to increase the production of these plants, in vitro propagation is an important tool (MEDEIROS et al., 2006), not only for enhancing increased growth rates, but also for producing plants free of pathogens (ROJAS-ARÉCHIGA and VÁSQUEZ-YANES, 2000).
For a successful in vitro cultivation, it is important to know the nutritional requirements of cells and tissues in culture.Therefore, one of the factors that affect the success of this technique is the culture medium in which the explants are grown.
Culture media are based on plant nutrient requirements and, in general, consist of essential components that comprise inorganic salts, carbon source and energy, water, vitamins and growth regulating substances.However, in order to comply with specific needs, some modifications can be made by adding optional components, such as amino acids and amides, organic acids and complex natural substances (THORPE, 1981).
After in vitro cultivation, plants must be acclimatized and this process should be carried out very carefully, due to differences between in vitro and greenhouse environmental conditions (HAZARIKA, 2003).
Thus, the objective was to evaluate the in vitro germination of Hamatocactus setispinus in different culture media, with and without the addition of activated charcoal, and seedling acclimatization in different substrates.

Plant material
Seeds were obtained from fruits of Hamatocactus setispinus, which were harvested in June 2009, in Maricá (22° 55' 10'' S, 42° 49' 07'' W), a town in Rio de Janeiro State, Brazil.The fruits were packed in paper bags and transported to the laboratory for tests.
Culture jars containing 30 mL of medium were autoclaved at 121° C and 1.5 atm for 15 minutes.Coconut water used to prepare Medium 5, which was extracted from a fresh green coconut, was initially filtered through cotton and, then, in filter paper to remove impurities.In a laminar flow chamber, the coconut water was filtered through a Millipore filter of 0.22 mm and then added to the respective culture medium that had already been autoclaved.
Six days after harvest, the fruits were opened and their seeds were carefully removed and spread on Kraft paper for the elimination of the mucilage.Subsequently, seeds were disinfested in a solution of 0.50% of sodium hypochlorite under constant shaking for 15 minutes in a laminar flow chamber environment.Seeds were then triple washed in deionized autoclaved water for 5, 10 and 10 minutes each.Then the seeds were inoculated in the culture jars containing 30 mL of the culture media described above, sealed with plastic caps and wrapped in PVC film.
The jars containing the seeds were kept in a culture room with fluorescent daylight-type lights of 40 W, light intensity of 25 mmol m -2 s -1 , at a temperature of 27 ± of 2° C and a photoperiod of 16 hours.
Germination was evaluated every three days during a period of 60 days.Seeds were considered germinated at radicule emission.The germination rate was evaluated and mean germination time (MT) calculated according to Edmond and Drapala (1958), whereas the speed germination index (SGI) was calculated by the formula suggested by Maguire (1962).
For each seedling, the number of roots (NR) was counted and root (RL) and shoot length (SL) were measured with a digital caliper.The experiment consisted of a 6x2 factorial scheme six different culture media and two concentrations of activated charcoal in a completely randomized design with ten replications.Each replication consisted of one culture jar containing 10 seeds, totalizing 120 jars and 1200 seeds.The germination rate data were transformed to arcsine (x/100) ½ .The data were subjected to variance analysis and means were compared by Tukey test at 5% probability with the help of Genes program (CRUZ, 2001).

Acclimatization experiment
Four acclimatization experiments were established with seedlings from in vitro germination, in a greenhouse covered with white polyethylene film (100 m) and a 70% shade cloth (Sombrite ® ).The experimental designs were in randomized blocks with two substrate treatments (S1-Biomix Floreira ® ; S2-Biomix Floreira ® + sand).
The first experiment was carried out with seedlings obtained from seed germination in Peter's ® CalMag 1.0 g L -1 medium (Medium 3) without activated charcoal, with three replications with two pots per plot, containing 11 seedlings per pot, totalizing 66 seedlings.The second one used seedlings from seed germination in Peter's ® CalMag 1.0 g L -1 medium (Medium 3) with activated charcoal, with three replications and two pots per plot, each containing 12 seedlings, in a total of 72 seedlings.The third one used seedlings from seed germination in Peter's ® 0.5 g L -1 medium (Medium 4) without activated charcoal with three replications and two pots per plot, each containing 12 seedlings, in a total of 72 seedlings.The fourth and last experiment used the seedlings obtained from seed germination in Peter's ® 0.5 g L -1 medium (Medium 4) with activated charcoal and consisted of three replications with two pots per plot, each containing 11 seedlings, totalizing 66 seedlings.
Seedlings were removed from the culture medium and the excess of medium was washed from the roots before transplanting to plastic pots (0.5 L) containing Biomix Floreira ® (S1) or Biomix Floreira ® + sand (S2) as substrate.The pots were kept in the greenhouse for three months, during which the average minimum and maximum temperatures registered were 24.3 + 1.3° C, and 29.5 + 1.2° C, respectively; the average air relative humidity was 63 + 2.3% with a photosynthetically active photon flow of 150.8 + 31µmol fotons m -2 s -1 .
At the end of the experimental period were evaluated: seedling survival (S%), shoot length (SL) and shoot diameter (SD), root length (RL), number of roots (NR), shoot fresh and dry weights (SFW, SDW) and root fresh and dry weights (RFW, RDW).Shoot length was measured from bottom to top of the stem; shoot diameter was measured in the middle portion of the stem (most uniform and juicy).Roots and shoots were separated for fresh weight measurements.After that, roots and shoots were dried at 70º C for 72 hours in a convection oven before dry weight measurements.
Data were subjected to joint variance analysis and means were compared by Tukey test at 5% of probability, with the help of the program SAEG (SAEG, 2007).

In vitro germination experiment
Seed germination began on the sixth day after in vitro sowing.It could be considered fast based on data for germination of eight cacti species of the genus Turbinicarpus, whose onset of in vitro germination was on the fourteenth day (DÁVILA-FIGUEROA et al., 2005) and on similar results observed for Pilosocereus robinii (QUIALA et al. 2009), whereas Arrojadoa spp (cactus foxtail) germination began on the eighth day after sowing (DIAS et al., 2008).
For speed germination index (SGI), the best results were obtained with Peter's 1.0 g L -1 (Medium 3) or water and agar (Medium 6) media, without the addition of activated charcoal, obtaining 0.91 and 0.96 seed germinated per day, respectively.The lowest values of SGI were obtained with MS medium (Medium 1) and MS supplemented with 10% coconut water (Medium 5), regardless of the addition of charcoal (Table 1).

SGI
For seed germination rate, no significant differences were observed among media or between activated charcoal treatments, except for Medium 1 that showed the lowest values either with or without charcoal (Table 1).Rosas-López and Collazo-Ortega (2004) evaluating three different culture media on the germination of Polaskia chichipe and Echinocactus platyacanthus (water and agar, half-strength MS basal medium and; full-strength MS basal medium) also observed the lowest germination rate in MS medium with 100% of minerals.Such results might be due to the concentration of salts decreasing the water potential in the medium, which would consequently reduce the water availability necessary for germination (ROSAS-LÓPEZ and COLLAZO-ORTEGA, 2004).According to the same authors, the medium consisting of water and agar not only had a higher germination rate, but also a higher SGI due to higher water potential of the medium when compared to the other media used.The same relationship between the concentration of salts in the medium and its water potential was discussed by Santos (2009).
The use of activated charcoal in the medium led to the highest values in shoot length (SL) (Table 2).A similar result was observed by Bellintani et al. (2007) for two species of bromeliads, where plants from the medium with charcoal had greater values of shoot length, which might be related to the adsorption of undesirable substances by the charcoal.According to Hartmann et al. (2002) one of the advantages of activated charcoal is its ability to adsorb substances secreted by explants, or present in the culture medium, which can inhibit growth.
Medium 3 led to the highest SL values, while the seedlings on Medium 6 had the lowest ones (Table 2), probably this medium did not provide enough nutrients for normal seedling growth.The presence of water just accelerated seed germination, but for seedling continued development nutrients from the culture medium would be required.
In a study with seeds of Cattleya loddigessi, Moraes et al. ( 2009) also obtained higher root length and number of roots on media supplemented with commercial soluble formulation of minerals.They observed highest values of seedling root length on culture medium supplemented with orange Kristalon fertilizer (NPK: 6-12-36) and best results for root number on culture medium supplemented with Hyponex fertilizer (NPK: 6.5-6-19), when compared to results obtained on mineral half strength MS medium.

Acclimatization experiment
The seedling survival was 100% in all four acclimatization experiments.There was no significant difference in SD (Table 3), RN (Table 4), SFW, RFW (Table 5), SDM and RDM (Table 6) of seedlings from either substrate on the four acclimatization experiments.
There was a significant interaction between substrate x in vitro culture medium for shoot (SL) (Table 3) and root length (RL) (Table 4).Seedlings from Peter's ® CalMag 1.0 g L -1 culture medium with activated charcoal had the highest SL, when grown in Biomix ® Floreira (S1), whereas those from Peter's ® 1.0 and 0.5 g L -1 culture media with activated charcoal had the highest SL (Table 3).Moreover, Peter's ® 0.5 g L -1 medium without activated charcoal led to the lowest values of SL for plants grown in both substrates.Seedlings grown in Biomix ® Floreira + sand (S2), originally obtained from Peter's ® 1.0 g L -1 without charcoal and Peter's ® 0.5 g L -1 with charcoal in vitro culture media, showed the highest values of SL.Seedlings, originally obtained from other in vitro culture media, did not differ in SL values regardless of the acclimatization substrate used.
Seedlings grown in S1, originally obtained from Peter's ® 1.0 and 0.5 g L -1 in vitro culture medium with charcoal, showed the highest SD values (15.33 and 14.65 mm, respectively), while those from Peter's ® 0.5 g L -1 medium without charcoal showed the lowest ones (12.00 mm) (Table 3).In acclimatization substrate S2, seedlings originally from Peter's ® 0.5 g L -1 medium without charcoal also had the lowest SD values, whereas seedlings from other culture media showed higher SD values and did not differ among themselves.
Seedling RL did not differ according to their original culture media, when plants were grown in acclimatization substrate S1.Nevertheless, when acclimatized in substrate S2, seedlings obtained from Peter's ® 1.0 g L -1 medium with charcoal had longer RL than those originally from Peter's ® 0.5 g L -1 medium without charcoal (Table 3).Seedlings originally from Peter's ® 1.0 g L -1 culture medium without charcoal showed higher values of RN in both acclimatization substrates (Table 4) compared to seedlings from the other culture media.
The SFW values of seedlings from Peter's ® 1 and 0.5 g L -1 media with charcoal grown in both acclimatization substrates did not differ significantly among themselves and were higher than those of seedlings from the other in vitro culture media, but SFW values of those from Peter's ® 0.5 g L -1 without charcoal were the lowest ones (Table 5).
There was no significant difference among the effects of the in vitro culture media on the seedling RFW, when they were grown in acclimatization substrate S1, whereas those grown in substrate S2, originally from Peter's ® 1.0 g L -1 medium (either with or without charcoal), showed the highest values for this trait (Table 5).
There was no significant difference between acclimatization substrate effects on SDM and RDM, irrespective of the seedling original culture medium (Table 6).
In the in vitro germination experiment, a detrimental effect of activated charcoal on germination was observed (Table 1).This result contrasts that observed in the acclimatization experiments in which the seedlings from media containing charcoal either did not differ or showed superior results, for some characteristics, than seedlings originally from media without charcoal.Thus, envisaging the large scale in vitro production of seedlings, the use of activated charcoal could be recommended for this cactus species.Nevertheless, considering the seedling results, it would be advisable to further investigate extended acclimatization periods for a conclusive recommendation based on the presence of activated charcoal in the germination culture medium and its effect on the seedling growth during acclimatization.Table 6.Shoot dry (SDW) and root dry (RDW) weights of Hamatocactus setispinus seedlings obtained from in vitro germination in culture media Peter´s 1.0 g L -1 and Peter´s 0.5 g L -1 without (0 g L -1 ) and with (3.0 g L -1 ) activated charcoal after an acclimatization period of three months in Biomix Floreira ® (S1) and Biomix Floreira ® + sand (S2) acclimatization substrates.Tabela 6. Matéria seca da parte aérea (MSPA) e matéria seca de raiz (MSR) de plântulas de Hamatocactus setipinus obtidas a partir de germinação in vitro nos meios de cultivo Peter´s 1.0 g L -1 e Peter´s 0,5 g L -1 sem (0 g L -1 ) e com (3.0 g L -1 ) carvão ativado após três meses de aclimatização nos substratos Biomix Floreira ® (S1) e Biomix Floreira ® + areia (S2).

Table 4 .
Root length (RL) and root number (RN Upper-case letters compare culture media and lower-case letters compare substrates.Means followed by the same letters do not differ significantly at 5% probability by Tukey test.Advances in Ornamental Horticulture and Landscaping V. 21, N o .2,2015, p. xx -case letters compare culture media and lower-case letters compare substrates.Means followed by the same letters do not differ significantly at 5% probability by Tukey test. Upper Upper-case letters compare culture media and lower-case letters compare substrates.Means followed by the same letters do not differ significantly at 5% probability by Turkey Test