Seed Germination and Production of Erythrina mulungu and Erythrina velutina Plantlets

Species of the genus Erythrina are frequently used as ornamental plants and in projects to restore degraded areas. Also, extracts from its shoots and stems are popularly used as a herbal medicine with validated activity on the central nervous system. The objective of this study was to evaluate E. mulungu and E. velutina seed germination and seedling development potential aiming commercial scale production of those species. Seeds stored for one or twelve months at 10 ̊C ± 2 ̊C were sown in sand, soil or Plantmax® substrates and evaluated for germination and seedling development. Subsequently, seedlings sprouted in Plantmax® were transferred to polyethylene bags and kept in greenhouse, under direct sunlight for plant development (plant height, stem diameter and root length pivoting) evaluations. Four-month-old plantlets were transplanted to the field and after a period of one year the collar diameter and shoot height (of each plant were measured. Seed germination rates of both species grown in soil and in Plantmax® were significantly high (over 80%). Storing seeds for 1 month did not inhibit germination. However, seeds stored for twelve months had germination reduced by more than a quarter. The development of plants in greenhouse and in the field was satisfactory, indicating the viability of producing E. mulungu and E. velutina on a commercial scale, in order to meet the expanding market demand for herbal medicines.


Introduction
The plant genus Erythrina [Leguminosae-Papilionoideae (Fabaceae)] comprises approximately 115 species distributed all around the Neotropics, across South Africa, and all over the Himalayas and southern United States [1].
In Brazil, the most customarily found species are Erythrina velutina Willd.and Erythrina mulungu Mart.Ex Benth., which are well known for their multipurpose use.Those species are used as ornamental plants.Because of its deep pivoting root system and rapid growth, it is also used in the restoration of degraded areas and in riparian vegetation recovery.Erythrina is considered a pioneer species in ecological succession [2]- [5].The sedative action of the Erythrina tea that is popularly consumed in Brazil has been reported in ethnobotanical studies [6] [7].Moreover, numerous pharmacological studies carried out with animals validated the anxiolytic, anticonvulsant and antinociceptive effects of the hydroalcoholic extract of E. velutina and E. mulungu stem bark and flowers [8]- [17] However, even being a species relevant to the development of scientific researches, especially considering its diversity of use and validated therapeutic efficacy, only a few studies on Erythrina seed germination characteristics and production of seedlings have been carried out and those works are restricted to seed physical dormancy caused by its water-impermeable seed coat [18]- [21].
In view of the importance of studies aiming the large scale production of Erythrina species for further establishment of sustainable management and production of plantlets with commercial purposes, in this work the germination potential of E. mulungu and E. velutina seeds and plant development in greenhouse and in field conditions were evaluated in order to achieve this plant mass production and enable its use in flora recovery programs and also to provide crude material for the development of phytopharmaceutical formulations.
To evaluate seed viability, E. velutina and E. mulungu seeds (1200 un) were packed separately in kraft paper bags (200 seeds of each species per bag) and stored for 1 and 12 months in a dry cold chamber (30% relative humidity) and controlled temperature (10˚C ± 2˚C).
Before sowing, seeds were immersed for 20 h in benomyl solution (1% w/v) under continuous agitation (100 rpm), dipped for 1 hour in calcium hypochlorite solution (0.5% w/v) and washed with autoclaved distilled water.After disinfestation seeds were mechanically scarified (at the opposite end of the embryo) using electric scarifier (Dremel-3957CT-multpro 10,000 RPM).Following scarification, seeds were soaked for 24 h in autoclaved distilled water under agitation (100 rpm).
To evaluate seed germination 3 different experiments were carried out in polystyrene trays containing soil, sand, or Plantmax ® substrate.The trays were kept for 30 days in phytotronics germination chamber (MA 1403/UR Marconi ® ) under 28˚C ± 2˚C temperature and 60% relative humidity.For each experiment E. velutina and E. mulungu seeds stored for 1 and 12 months (4 treatments with 50 seeds per repetition) were sowed (at 1 cm profundity) in sand, soil or Plantmax ® .The experiment was conducted in a completely randomized design and germination speed and seedling emergence rates were evaluated daily during 1 month, using as a parameter of germination the hypocotyl length ± 2 cm.
Germination was evaluated calculating percentage of emerged seedlings and the speed of emergence index was determined according to the formula proposed by Maguire [22], IVE = G1/N1 G2/N2 + + ... + Gn / Nn where: G1, G2, Gn = number of seedlings germinated in the first, second, until the last count and N1, N2, Nn = number of days from the first, second until the last count.
Plants grown in Plantmax ® substrate for 30 days were transplanted to polyethylene bags (15 × 25 cm) containing soil and cattle manure (3:1) as substrate, kept in greenhouse under direct sunlight and irrigated once a day during 4 months.The collar diameter, shoot height and pivoting root system elongation of each plant were measured 2 and 4 months after the transplant of seedlings.Experiments were carried out in a randomized block design with 100 plantlets of each species divided into 4 groups of twenty five plants.After cultivation in polybags containing soil and cattle manure substrate for a period of 4 months the plantlets were transplanted to field in pits of 40 × 40 × 40 cm, fertilized with 2 kg•m 2 of cattle manure observing the spacing of 3 × 3 m in the medicinal plant cultivation area of the University of Ribeirão Preto, located in the city of Jardinópolis (SP).During the period one year the plantlets were watered twice a week and hand weeding was performed every two months.After one year of field cultivation the development of E. velutina and E. mulungu plants was evaluated measuring collar diameter and shoot height of each plant.

Statistical Analysis
The experimental design adopted was the randomized block design in a 3 × 2 factorial structure with 3 replications and 20 plants per plot.Obtained data were compared by analysis of variance (ANOVA) using the software SISVAR V.4.3 and the means were compared by the Scott-Knott test at a level of significance of 5%.

Results and Discussion
Mechanical scarification was an efficient method for breaking tegument dormancy of E. velutina and E. mulungu seeds, since it improved seed imbibition and germination between the fourth and fifteenth day.Obtained results corroborate those reported by Silva et al. [18] on the validation of mechanical scarification as an efficient method for breaking dormancy of Erythrina seeds.According to Rolston [23] and Carvalho & Nakagawa [24], the seed dormancy of leguminous plants is related to impermeability of seed coat to water due to the hard seed coat structure which restrict the entry of moisture into the seeds and so mechanical scarification facilitates germination.Lazarotto et al. [25] reported that seeds of Erythrina crista-galli presented dormancy and low percentage of germination on the tenth week after anthesis.
Examining E. mulungu and E. velutina seed germination it was evidenced that according to the classification of cotyledons characteristics proposed by Miquel [26] those species are phanero-epigeal-reserve type (PER) Evaluating germination of E. velutina seeds stored for different periods of time and sown in different substrates it was observed that germination rates of seeds stored for one month ranged between 85.8 and 91.6%, and there was no statistical difference between the treatments (Table 1).However, seeds stored for twelve months showed significant reduction on germination ratios regardless the substrate used.Even though, a reduction in the germination speed index (GSI) was observed for seeds stored for twelve months in relation to seeds stored for 1 month, in either one of the three types of substrate tested, the difference was not significant (Table 2).The GSI (4.39) obtained for seeds sown in sand was similar to the results (GSI 4 and GSI 5.1) reported by Cardoso et al. [27] and Matthew et al. [28], respectively, when investigating the influence of sowing position and depth on germination of E. velutina seeds sown in seed boxes containing sand substrate and kept in a greenhouse.
Bento et al. [29] investigating the physiological quality of E. velutina seeds observed homogeneity among germination tests, germination speed index and accelerated aging.Both, data obtained by those authors and data recorded in this present work may be used as reference when focusing on large-scale production of E. velutina plantlets for commercial purposes.Regarding E. mulungu, higher germination percentage (84.99%)was achieved with seeds sown in Plantmax ® substrate if compared with germination rates verified for seeds sown in soil and in sand substrates (54.16 and 63.33%), respectively.Independently of the type of substrate tested the GSIs did not statistically differ between them.As for the species E. velutina, storage for twelve months resulted in the reduction of seed germination for all tested substrates (Tables 1 and 2).
Medeiros & Zanon [30] reported that germination of Machaerium stipitatum (Leguminosae-Papilionoideae) seeds stored at 10˚C was reduced by more than 40%, comparable to what happened with E. velutina and E. mulungu seeds in this study.
Values recorded for collar diameter, shoot height and root elongation of plantlets grown in polybags in the greenhouse are shown in Table 3.During the evaluations it was possible to observe total development of the plants, which grew rapidly and presented statistical differences between species.Comparing E. mulungu and E. velutina plantlets after 2 and 4 months of cultivation it was observed that plant height of four-month-old plants was enhanced three times.However, no significant differences were observed for root elongation in plants cultured.
Considering that the slow growth of aerial parts of tree species has been constantly reported, obtained results can be extremely interesting, especially if deeming large-scale production when the main goal is to produce higher number of quality plants.RAMOS et al. [31] reported that Amburana cearensis, an important species for the herbal industry after 4 months of cultivation under intense sunlight presented showed enhanced shoot height (15).Moreover, among the variables to determine plant development during initial phase of growth, one important characteristic is shoot elongation.According to Carneiro [32] shoot height is particularly correlated to the development of plantlets transplanted to field.
Values obtained for collar diameter root elongation also demonstrated the effective development of the seedlings (Table 3).Plantlets cultured under direct sunlight, did not present stem etiolation and a reasonable growth of the main root was observed.For both species after 4-month cultivation, the average values recorded for those two variables almost doubled, compared to 2-month-old plantlets (Table 3).
According to Carneiro [32], species which present superior stem diameter development assure better-quality plantlets, more resistant to adaptation to field conditions.Collar diameter ratio is very important when evaluating the plant potential to survive and grow after transplanting to field.
E. velutina shoot height in the first four months of cultivation was superior to E. mulungu, but 12 months after transplanting to field the difference for that variable was not significant, though E. velutina collar diameter values were significantly higher compared to E. mulungu (Table 3).
After 2 months of culture, the occurrence of Rhizobium nodules on the radicular system of 98% of the plants of the two species was observed.Those nodules measured between 0.1 and 0.8 cm and were located in the hypocotyl region.The symbiosis between Rhizobium and species of the genus E. crista-galli, E. variegata (syn.E. indica) and E.costaricensis has been extensively investigated [33]- [35] but this is the first report of Rhizobium nodules on E. mulungu and E. velutina roots.
The symbiosis between Rhizobium and plant species is very interesting because it propitiates biological nitrogen fixation and that may have been one of the factors that enhanced E. velutina and E. mulungu growth.However additional specific studies are necessary to corroborate those findings.

Conclusion
There is no complexity in producing E. velutina and E. mulungu plantlets.Moreover, besides the interest on the chemical, pharmacological and clinical properties regarding those species, large-scale production are viable and simple to be implemented in order to meet the market demands for herbal medicines.

Table 1 .
Mean germination rates (%) of E. velutina and E. mulungu seeds stored for 1 or 12 months and sown in three different substrates.
Means followed by the same lowercase letters in the vertical and uppercase letters in the horizontal do not statistically differ from each other by Scott-Knott (p < 0.05).

Table 2 .
Average data of germination speed index of E. velutina and E. mulungu seeds stored for 1 or 12 months and sown in three different substrates.Means followed by the same lowercase letters in the vertical and uppercase letters in the horizontal do not statistically differ from each other by Scott-Knott (p < 0.05).

Table 3 .
Average data of development of 2, 4 and 12-month-old E. velutina and E. mulungu plants.Data not obtained.Means followed by the same letters in the vertical do not statistically differ from each other by Scott-Knott (p < 0.05).