Vol. 12, No.2, 2012.pmd 
Artículo científico                                                         Biotecnología Vegetal Vol. 12, No. 2: 77-83, abril-junio, 2012 


ISSN 2074-8647 (Versión electrónica)

ln vitro propagation of Hylocereus purpusii Britton & Rose, a mexican species in danger of extinction

Manuel de Feria1*, Daniel Rojas2, Maité Chávez1, Mireya Reyna2, Elisa Quiala1, Juan Solís2, Florentina Zurita2. *Authorforcorrespondence

instituto de Biotecnología de las Plantas, Universidad Central 'Marta Abreu' de Las Villas, Carretera a Camajuaní km 5.5, Santa Clara, CP 54 830 Cuba. e-mail: mdeferia@ibp.co.cu

2Centro Universitario de la Ciénega, Universidad de Guadalajara, Av. Universidad 1115, Ocotlán, CP 47 820, Jalisco, México e-mail: drojas@cuci.udg.mx

ABSTRACT

The purpose ofthis workwasto evalúate different conditions and culture parametersforthein vitro establishment and multiplicaron of Hylocereus purpusii. Seeds were used as plant material and a workflowwas developed as an alternative forthe propagation and recovery ofthis species in danger of extinction. ln the establishment phase, the best result was obtained in the treatment with 1% NaOCI for 15 minutes achieving a 90% of germinated seeds and a final 77.7% of in vitro established plants. ln the multiplicaron phase when combining 8.88 uM 6-BAP with different concentrations of IAA, a significant increment in the length of the shoots was observedwith respecttothe control (no addition of IAA). Acombination of8.88 uM 6-BAP with 3.42 uM IAA was selected as the best multiplication condition reaching a multiplicaron rate of 8.8. ln the acclimatization phase, after two months of culture in ex vitro conditions the survival was higher than 98% and after six months of growth, the plants were transplanted to field.

Key words: cactus; disinfection; growth regulators; seed germination

RESUMEN

El propósito de este trabajo fue evaluar diferentes condiciones y parámetros de cultivo para el establecimiento in vitro y multiplicación de Hylocereus purpusii. El protocolo de trabajo se desarrolló como una alternativa para la propagación y la recuperación de esta especie en peligro de extinción. Se utilizaron semillas como material vegetal inicial. En la fase de establecimiento, el mejor resultado se obtuvo en el tratamiento con hipoclorito de sodio al 1% durante 15 minutos. Se logró un 90% de semillas germinadas y finalmente un 77.7% de plantas establecidas in vitro. En la fase de multiplicación cuando se combinaron 8.88 uM de 6-BAP con diferentes concentraciones de AIA, se observó un incremento significativo en la longitud de los brotes con respecto al control (sin adición de AIA) La combinación de 8 88 uM de 6-BAP con 3 42 uM de AlAfue seleccionada como la mejor para la fase de multiplicación y para alcanzar un coeficiente de 88. En la fase de aclimatización tras dos meses de cultivo en condiciones ex vitro, la supervivencia fue superior al 98%. Después de seis meses de crecimiento, las plantas fueron transplantadas a campo.

Palabras clave: cactus, desinfección, reguladores del crecimiento

INTRODUCTION

Cacti are very common plants in Mexican landscapes. The country has the privilege to have the biggest quantity of species ofthis family around the world. Great quantities of cactus species are present in the daily diet of several human communities, either as fresh fruit or vegetable.

Among the broad range of cacti species, cultivation of members of the Hylocereus genus has recently become amenable in different countries. Hylocereus plants constitute an

alternative in regions where climatic factors are obstacles for fresh fruits production (Mohamed-Yasseen,2002). Duetotheirproper adaptation to wild conditions, their productive and economic potential (Legaría et al. 2005) offers the possibility of cultivation in semi-arid regions (Chávez, 2002). Moreover, Hylocereus fruits contain a high nutritional valué (Castillo et al. 2005).

Potentially, the propagation of Hylocereus plants can be achieved by two strategies: 1) from steam sections and 2) from seeds. The first

has as prerequisite having a large number of élite donor plants which is extremely difficult considering the small number of individuáis available in natural conditions. In the case of propagation from seeds, very few studies have been carried out about it. Propagation from seeds has, as main drawback, the large lapse of time that mediates from germination until harvest (3 to 4 years) (Mizrahi et al., 2004). Other limitation of the use of seeds is related to the environmental conditions of arid and semi-arid regions, where the water is a restrictive factor for germination (Osorio et al., 2001).

In the particular case of H. purpusii (Pitahaya purple), the main problems for its propagation, using traditional methods (see 1 and 2 above), are related with the low availability of plant material and its sexual autoincompatibility (Tel etai, 2004; Castillo et ai, 2005). In this sense, in vitro propagation can be an alternative method to multiply selected genotypes of H. purpusii.

In vitro propagation in the Hylocereus genus has not been well described. Authors such as Johnson and Emino (1979), mentioned the potential of tissue culture for the propagation of H. calcaratus. Mohamed-Yasseen (2002) described his results in H. undatus. Pelah et al. (2002) worked with Selinecereus megalanthus, while Cuellar et al. (2006) were able to establish H. undatus in vitro, always using seeds as starting material. However, there is no report of in vitro studies with H. purpusii so far.

The objective of this work was to evalúate different conditions and culture parameters for the in vitro propagation of H. purpusii from seeds. The in vitro culture of H. purpussi is a possible alternative for the propagation and recovery of this species in danger of extinction.

MATERIALS AND METHODS

In vitro establishment

Seeds were obtained from fresh fruits of wild Pitahaya purple plants at the semitropical zone of the southern part of Puebla, México. Seeds extraction was done by removing the flesh from the fruits carefully. These were further placed in distilled water with constant agitation. Washing was carried out five times, until the seeds were free of mucilaginous material and acquired a brilliant black color.

A basal culture médium of inorganic salts proposed by Murashige and Skoog (1962) was used for in vitro seeds germination and plant establishment. This médium was supplemented with 20 g M sucrose and 2 g M Phytagel. Finally, pH was adjusted to 5.8.

Fifteen milliliters of culture media were dispensed per glass tube and sterilized during 15 minutes in an autoclave at 1.2 kg crrr1 of pressureand 121°C.

One seed was placed per glass tube with a total of 30 seeds per treatment. Glass tubes were kept at 28 ± 2°C in a room with a photosynthetic photon flux density (PPFD) between 38 and 47.5 umol rrr2s-1.

For the statistical processing of the data, the SPSS package for Windows versión 16.0 was used. Significance levéis were determined by a simple varince analysis (ANOVA) and the differences among mean valúes were statistically assesed by a Duncan múltiple ranges test.For the variables expressed in percentages, differences among valúes were determined using a two samples proportion test (p<0 05) by the statistical packaqe STATISTIX veTsionl.O In all the experiments (see below), visual evaluations were carried out daily until 35 days to determine the germination time of the seeds under in vitro conditions. The number of germinated seeds and established plants was determined as well.

Effectofsodium hypochlorite concentration and immersion time on seeds germination

The effects of the immersion time on seeds germination in different sodium hypochlorite concentrations were evaluated. Firstly, an immersion time was fixed to 15 min and three sodium hypochlorite concentrations were assayed: 1,1.5 and 2%. In a second part of the experiment, the sodium hypochlorite concentration with the highest germination percentage was assayed at 5, 10 and 15 minutes. All the combinations were cultured in the basal médium mentioned before with a 100% MS salts.

Effect of MS salts germination

concentration on seeds

Afterwards the interest was focused on knowing whetherthe reduction of MS salt had an effect on seeds germination. Basal media containing a 25, 50, 75 and 100% of MS salts were evaluated taking into account the number of germinated seeds after 35 days of culture.

In vitro multiplication

Effect ofdifferent concentrations of6-BAP and the type of explant For the in vitro multiplicación the basal culture médium was composed of 100% MS salts, 30 g f1 sucrose, pH 5.8 and 2 g f1 Phytagel. The plant material used in the multiplication phase was apical shoots with two subcultures and an approximate length of 5 cm. In this phase, different concentrations of 6-BAP (0,2.22,4.44, 6.66, 8.88,11.1 and 13.32 uM) were evaluated. The multiplication rate was used as evaluation criteria in all the treatments.

From the results of the previous analysis, three concentration of 6-BAP with the best results splited into selected to determine the influence of the explant type on the multiplication rate. Shoots were halves obtaining two new explants called apex and base. Twenty explants of each type were used per treatment and after 40 days of culture the number of shoots per explant was counted.

Effect of the combination between different concentrations of 6-BAP and IAA

Finally, in this phase, the effect of different concentrations and combinations of 6-BAP (6.66; 8.88 and 11.1 uM) and indolacetic acid (IAA) (1.14; 2.28 and 3.42 uM) was also studied. After 40 days of culture, number of shoots per explant, shoot length, (cm) fresh and dry weight of the shoots pertreatment and the relationship dry weigh-fresh weigh were determined.

The plants obtained from the previous experiment were placed in a culture médium withoutgrowth regulators, with 100% MS salts, 30 g I1 sucrose, pH 5.8 and 2 g f1 Phytagel to induce roots formation. After 30 days of culture in these conditions, plants were transferred to ex vitro conditions.

RESULTS AND DISCUSSION

In vitro establishment

Effect ofsodium hypochlorite concentration and immersion time on seeds germination

Seeds germination took place between the 5th and 12thday of culture, butagreatpercentage (76.6%) germinated between the 8th and 9th days. The increase in NaOCI concentration affected germination, although between the treatments with 1.5 and 2% differences were nosignificant(Tablel).

The best results were obtained in the treatment with 1% NaOCI and an immersion time of 15 minutes where the 90% of the seeds germinated. However, when the concentration of NaOCI increased to 1.5 and 2%, some plants died (21.7 and 20.1%, respectively) as a consequence of seeds damage.

When combining different immersion times (5, 10, 15 minutes) in a solution of 1.0% NaOCI, there were no significative differences in the germination percentage and in vitro establishment of the plants.

However, the treatment with 5 minutes, presented 13.3% of microbial contamination, and henee, it was decided to use the treatment with 1.0% NaOCI for 10 minutes.

obtained the best germination percentage (81.4%) with 25% MS salts. These results confirm the influence of the genotype in the response to similar in vitro culture conditions.

Effect of MS salts germination concentration on seeds In vitro multiplication

Differences were not on seeds germination among the treatments with 25, 50,75 and 100% MS salts in the culture médium. In Figure 1, plants with 8, 16 and 25 days of culture are shown.

Different results have been described in other cactus species regarding in vitro seeds germination. Quiala et al. (2004) germinated seeds of Pilosocereus robinii and the best results (91.4%) were obtained with 50% MS salts. In the species Melocactus actinacanthus same authors

Effect of different concentrations of6-BAP and the type of explant

The results demostrated that 6-BAP had effect on in vitro multiplication of Hylocereusporpusii.

The best multiplication rate (6.7) and newshoots length (1.75 cm) were obtained (Table 2 and Fig. 2e) in the treatment with 11.1 uM of 6-BAP, the largest number of shoots (8.3) was reached in the treatment with 8.88 uM of 6-BAP butthey showed an average in length of 0.67 cm which limited their multiplication.

 

Figura 1. In vitro plant of Hylocereus purpusii germinated with 100% MS salts at different times after seed germination: a) 8 days, b) 16 day c) 25 days.

Table 2. Different responses during shoots development of Hylocereus purpusii in vitro plants after 40 days of culture using different 6-BAP concentrations.

When the cactus species Ariocarpus kotschoubeyanus was multiplied by Moebius et al. (2003), shoots development depended on the presence of 6-BAP in the culture médium.The largest number of shoots was obtained when the concentration of this growth regulator oscillated between 4.44 and 13.32 uM. Similar results were obtained in our study, with H. purpusii.

Many species cultivated in vitro (including cacti) form roots in the absence of growth regulators. This response was observed in the species Ariocarpus kotschoubeyanus by Moebius et al. (2003) and Burdyn et al. (2006) in Aloysia polystachya. However, in our study H. purpusii formed as an average up to 2.5 roots per plant when cultivated in auxins depletion and with a concetration of up to 4.44 uM 6-BAP (Fig. 2 a, b, c).

It is well known that the explant type influences the in vitro multiplicaron of many species (Dhar and Joshi, 2005). This study included two explants types called apex and base (see Materials and Methods). In all the evaluated treatments, the first shoots were observed after three weeks of culture starting from both aüex and base exülants In the treatment with 0 uM of 6-BAP the presence of roots in the exDlants faDex and base} coincided with the formation of new shoots. On the rnntrarv in the treatments with 6-BAP no roots were observed.

New shoots formation increased proportionally with the adition of 6-BAP. In the treatment with 11.1 uM 6-BAP the greatest number of shoots per explant was obtained on average. Similarly to previous experiments, shoots were poorly developed which limited their use in the in vitro propagation.

Similar responses were described by Kumar et al. (2005) when working with the species Holarrhena antidysenterica and by Liu et al. (2006) with the species Rhodiola fastigiata.

In the control treatment (0 uM 6-BAP), apex explants hardly emitted new shoots, while base explants activated some areoles located where the cut was done and formed shoots.

According tothe multiplication rate obtained and the length of the shoots emmited by both type of explants, the treatment with 8.88 uM of 6-BAP was selected as the best one.

Effect of the combination between different concentrations of 6-BAP and IAA

When combining 8.88 uM of 6-BAP with different concentrations of IAA, a significant increase in the length of the emmited shoots was observed respect to the control treatment (no addition of IAA) (Table 3). A combination of 8.88 uM of 6-BAP with 3.42 uM of IAA was selected as the best multiplication condition giving a multiplication rate of 8.8.

Means with differentlettersin the same column differs forp <0.05according to Duncan Liu etal. (2006) reported in R. fastigiata the best results in the multiplication rate when combining concentrations of cytokinins and auxins, maintaining a favorable proportion to the cytokinins.

After two months of culture under ex vitro conditions, survival was higher than 98% and after six months of culture (Fig. 3) plants were transplantedtothefield.

This results were the first reports worldwide on the in vitro propagation oí Hylocereus purpusii. The studies in the multiplication phase demonstrated that it is possible to propágate this species with good multiplication rates.

However, it is important to assist the in vitro response of this species with different concentrations of 6-BAP and combinations of this growth regulator with IAA.

Acknowledgements

We would like to thank Dion Daniels who collaborated in the English versión.

REFERENCES

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(Recibido 5-2-2012; Aceptado 13-3-2012)



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