An improved method for in vitro regeneration of common bean (Phaseolus vulgaris L.)
Método eficiente de regeneración in vitro de frijol común (Phaseolus vulgaris L.)
Anareli Quintero–Jiménez, Elsa Espinosa–Huerta, J. Alberto Acosta–Gallegos, H. Salvador Guzmán–Maldonado, M. Alejandra Mora–Avilés*
Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. Unidad de Biotecnología de Plantas, km 7.5, Carretera Celaya–San Miguel de Allende. Apartado Postal 112. 38110 Celaya, Guanajuato. *Author for correspondence: (mora.alejandra@inifap.gob.mx).
]]> Received: October, 2008.
Abstract
In vitro regeneration of common bean (Phaseolus vulgaris L.) is a requirement for genetic transformation which involves induction and development to the whole plant. Success in regeneration of common bean from tissue organ culture has been achieved to some extent in the last years. However, genotype effects in regeneration response as well as efficiency and reproducibility have been a limiting factor. Embryos, from four different varieties, were excised from sterilized mature seeds and cultured in Murashige and Skoog (MS) or Gamborg (GM) media containing 6–benzylaminopurine (BAP) (10 mg–1) and adenine (A) (0 or 20 mg–1). Efficient regeneration was achieved when inducing formation of differentiation of cells like bud clusters at the internodal segment of the embryo axes in the four varieties studied. One way analysis of variance and Tukey media comparison (p≤0.05) showed that regeneration efficiency varied considerably between the two basic media. GM media provided high bud cluster formation (97.8 to 100 %) and full plant regeneration (93 %), whereas MS medium showed lower bud cluster formation (15 to 73 %), and full plant regeneration (29 %). It is provided evidence of how the culture media and growth regulators influence the regeneration of common bean, when seeking for an efficient transformation protocol.
Key words: Adenine, bud clusters, embryonic axes, gamborg medium, organogenesis.
Resumen
La regeneración in vitro de frijol común (Phaseolus vulgaris L.) es un requisito para la transformación genética que involucra inducción y desarrollo de una planta completa. La regeneración del frijol común a partir del cultivo de tejidos se ha alcanzado con cierto grado de éxito en los últimos años. Sin embargo, los efectos del genotipo en la capacidad regenerativa, así como la eficiencia y la reproductibilidad han sido factores limitantes. Se extrajeron embriones, de cuatro variedades diferentes, de semillas maduras esterilizadas y cultivadas en medios de cultivo Murashige y Skoog (MS) o Gamborg (GM) con 6–bencilaminopurina (BAP) (10 mg L–1) y adenina (A) (0 o 20 mg L–1). La regeneración eficiente se obtuvo al inducir la formación de células diferenciadas como grupos de yemas en el segmento internodal de los ejes embrionarios en las cuatro variedades estudiadas. El análisis de varianza de una vía y la comparación de medias por la prueba de Tukey (p≤0.05) mostró que la eficiencia de regeneración varió considerablemente entre los dos medios de cultivo básicos. El cultivo GM produjo una alta formación de grupos de yemas (97.8 a 100 %) y regeneración de plantas completas (93 %), mientras que el cultivo MS mostró una menor formación de grupos de yemas (15 a 73 %) y regeneración de plantas completas (29 %). Esto proporciona evidencia sobre cómo los medios de cultivo y los reguladores del crecimiento influyen en la regeneración del frijol común, cuando se busca un protocolo de transformación eficiente.
Palabras clave: adenina, grupos de yemas, ejes embrionarios, medio Gamborg, organogénesis.
]]>INTRODUCTION
Common bean is a staple food and one of the main sources of protein in the diet of population from Latin America and East Africa (Cruz de Carvalho et al, 2000). Common bean breeding programs intend to incorporate characteristics such as drought tolerance, resistance to pests and diseases, as well as increased nutritional quality (CIAT, 2008). Currently, several genetic engineering techniques are used to improve these characteristics however; common bean regeneration using published methods was not easily reproducible (Dillen et al, 2000; Cruz de Carvalho et al, 2000; Veltcheva et al., 2002).
Heterologous or homoeologous gene transfer is becoming a common approach in genetic improvement programs. This process, together with traditional plant breeding methods, could accelerate the development of new cultivars with specific characteristics such as drought resistance, acid soil adaptation, upright architecture, long shelf life and nutritional quality (Aragao et al, 2002).
The basis of the regeneration of common bean with high efficiency in a broad spectrum genetic material will allow including genetic transformation as part of the common bean breeding programs, now in progress by our group. Improvement of regeneration system protocol is focused on increasing the array of races of common bean in which the protocol can be applied. These genotypes are part of our common bean national breeding program due to their agronomical and quality advantages.
The objective of the present study was to develop an improved and reproducible method for organogenic bud induction from embryonic axis of four common bean (Phaseolus vulgaris L.) cultivars.
MATERIALS AND METHODS
Plant material
Mature seeds four common bean cultivars G13637 Apetito (G1) (CIAT), ICA Palmar G4523 (IP) (CIAT), Pinto Saltillo (PS) (Reg. No. FRI–035–161181) (Castillo et al, 1997) and Flor de Mayo Anita (FMA) (1494–FRI–032–220302/C) (Castellanos et al, 2003) were sterilized with gas chlorine (commercial chlorine (Cloralex®) and 12N HC1 (v/v 5:0.16) in a desiccator and they were used as explants donors.
]]> Culture mediaInduction and multiplication media (IMM)
The media (IMM) consisted of GM (Gamborg et al., 1968) or MS (Murashige and Skoog, 1962) amended with myo–inositol (100 mg L–1), pyridoxine (1 mg L–1), thiamine (10 mg L–1), sucrose (2 %) Phytagel® (2.8 mg L–1), benzyl aminopurine–HCl (10 mg L–1) and adenine hemisulphate [0 mg L–1 (GM0 or MS0) or 20 mg L–1 (GMA or MSA)]; these last two as a grown regulators, pH 5.8 adjusted with 1N NaOH.
Embryos were cultivated for 5 d in IMM. Then, meristematic shoots and roots were removed and embryonic axes were cultivated in the same media. Explants were transferred to fresh IMM every two weeks until bud differentiation. Regeneration efficiency was measured as the number of embryos with bud cluster divided by the number of embryos times 100. Experimental treatments consisted of nine petri dishes containing 10 embryos each, per cultivar.
Elongation and footing medium (ERM)
The medium (ERM) consisted of the same components as the IMM, with no growth regulators. Bud clusters with differentiated shoots were transferred to ERM for complete development, elongation and rooting. Growth conditions were the same as the previous stage. In vitro regenerated plantlets were acclimatized and grown in greenhouse at 22–25 °C, photoperiod 16 h light and 8 h darkness with a light quality in photosynthetic active radiation 400–700 nm.
RESULTS AND DISCUSSION
The results of the present work improve the in vitro regeneration protocol published by our group (Delgado–Sánchez et al, 2006). Previous reports of regeneration of P. vulgaris needed improve the efficiency during differentiation of transformed lines. Some variables like selection of explants, growth conditions, addition of hormones and basal culture (MS or GM) and differentiation route modify the in vitro regeneration process (Zambre et al, 2001; De Clercq et al, 2002; Veltcheva etal, 2002).
Bud cluster formation
]]> Embryos were aseptically cultivated in IMM with no contamination and no damage derived from an efficient sterilization process. Both, GM and MS basal media induced two meristematic bud clusters (0.5 mm wide) per embryonic axis in all four common bean cultivars showing an intense green color (Figures 1C; 1I ). There were differences (p≤0.05) in bud cluster formation rates among the four cultivars in GMO and GMA (Table 1). IP and FMA formed organogenic bud clusters after 4d in culture after escisión (AE) (93–5 and 96.7 %), while cultivars PS and G1 formed bud clusters after 4 d in culture AE (94.4 and 98.9 %) at the internode area.Embryonic axes cultivated in MSO and MSA treatments showed more than 90 % of bud cluster formation after 10 d in culture AE (Figure 1I); however, oxidation was observed in the tissue (Figure 1J), reducing further shoot differentiation. FMA showed high bud cluster formation efficiency in this media (61–73 %); however, this was not the case for the other cultivars that had a reduced efficiency G1 (31.1 %), IP (28.8 to 46.7 %) and PS (15.6 to 17–8%) (Table 1). Differences in response were found in FMA and IP (p≤0.05) cultivated in MSO or MSA; however, no differences were observed for cultivars G1 and PS cultivated in MS media, suggesting no significant effect of adenine in regeneration induction (Table 1).
Shoot differentiation
Bud clusters in GMA or GMO media increased in size within 8 d AE (1.5 mm wide × 1 mm height) (Figure 1D). Shoot differentiation started at 24 d AE showing a range of 21–26 shoots per bud cluster (Figure 1E) and forming differentiated leaves of 6 mm wide (Figure 1F, Table 1). However, the shoot differentiation started at 40 d AE in MSA or MSO media, forming 2 to 8 shoots per bud cluster. More than 50 % of the embryonic axes and bud clusters were completely oxidized in the MS media decreasing shoot development in all genotypes (Figure 1L). The differences in basal media were evident in embryonic axis response. MS medium used in most of the studies has been less effective than GM medium regardless of concentration in growth regulators (Mohamed et al., 1991). Apparently, a differential in macroelements concentration of GM vs. MS is a key component for shoot differentiation.
Addition of adenine hemisulphate was reported for somatic embryogenesis induction and in organogenic bud cluster formation (Delgado–Sánchez et al., 2006). However, in owr study, there was no significant effect on bud cluster formation or shoot development when it was combined with BAP regardless of basal media.
Shoot differentiation in GM media as opposed to other reports where differentiation route based on organogénesis of P. acutifolius and P. vulgaris showed several drawbacks, such as no differentiation of initial structures (Zambre et al., 1998; Dillen et al., 2000). Analogous to our observations, Muñoz et al. (personal communication) reported absence of apical and root meristems, induced de novo shoot formation as clusters or organogenic calli in MS basal medium added with thidiazuron and indol acetic acid, followed by transfers to low BAP (1 mg L–1) concentrations for shoot maintaining and development.
Plant development
In our previous report, the efficiency of full plant regeneration and development (number of regenerated plants/number of induced shoots X 100) was 83 % for FJM (Delgado–Sánchez et al, 2006). Here, we report an efficiency of 83 to 100 % depending on the cultivar in GM basal media (Figure 1H). In both reports, FMA tested had an increase in regeneration efficiency. Organogenic response in both reports was consistent, providing evidence of repeatability for cultivars G1, IP and PS 25–50 % to 97.8 %.
Fully developed plants (Figure 1I) transferred to greenhouse, showed a similar phenotypic appearance as plants from seed germination. Flowering, pod filling and morphology of the regenerated plants showed typical genotype characteristics.
Cultivar response
]]> Regeneration efficiency for FMA and G1 lines was higher in all four treatments compared to IP and PS (Table 1). GM basal medium induced higher bud cluster formation (90 to 100 %), shoot differentiation and plant development efficiency (83–100 %) than MS basal medium (13 to 50 %) in all four cultivars (p≤0.05). No differences were observed when adenine was included in either of basal media (p=0.208) at any of the stages of differentiation and development of the regeneration process (Table 1).
CONCLUSIONS
The protocol described here showed an improved organogenic differentiation response using embryonic axis; besides this protocol may be adapted to a other cultivars in México. The procedure is reproducible resulting in the development of whole plants comparable to germinated plants. Finally, this regeneration protocol provides a reliable protocol during genetic transformation of common bean in breeding programs.
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