Multiple Shoot Formation from Shoot Apex Cultures of Phaseolus vulgaris L.::-)

I. S. MARTINS and M. R. SONDAHL

Dept. Genetics, Institute of Agronomy, P.O. Box 28, Campinas, SP, Brazil

Received July 9, 1983 . Accepted December 29, 1983

Summary

Shoot apexes of 33 bean cultivars (Phaseolus vulgaris) were cultivated in order to explore pos­sible genotypic differences pertaining to in vitro plant regeneration. This explant source was found to be very promising during preliminary tests. Shoot apex cultures of these 33 cultivars were established on B5 medium in the presence of BA (0.5 /lM). Callus, roots, and/or buds were found according to the genotype. Adventitious bud development varied according to the cul­ture conditions (growth room or shaded greenhouse) and genotypes. This methodology was further explored with the cultivar Palmital Precoce whereby 276 buds were recovered from a 2,4-D x BA diallelic design with an average of 15 buds/shoot apex. Full plants of «Palmital Pre­coce» were raised following tertiary culture in the presence of GA3 and NAA.

Key words: Phaseolus vulgaris, shoot apex cultures, shoot development, adventitious buds, bean plantlets.

Introduction

Examples of recovery of leguminous plants in vitro have been described from me­ristem (apical and axillary) development of Arachis hypogaea, Cicer arietinum, Pha­seolus vulgaris, Vigna unguiculata (Kartha et aI., 1981); Glycine max (Cheng et aI., 1980; Kartha et aI., 1981); Lotus comiculatus (Thomes, 1979); Pisum sativum (Kartha et aI., 1974); Viciafaba (Aubry et aI., 1975; Galzy and Hamoui, 1981). This paper pre­sents a protocol for plant recovery from shoot apex cultures of Phaseolus vulgaris.

Material and Methods

Seeds of 33 cultivars of Phaseolus vulgaris L (Table 1) from the bean germplasm bank of the Institute of Agronomy, Campinas, Brazil, were germinated aseptically (ethanol 70% - 10 min; NaOCI3.9 % - 20 min). After 7 days of dark incubation, shoot apexes (apical meristem includ­ing 3-4 leaf primordia, ca. 1-2mm long) were used to establish cultures on B5 medium (Gam­borg et aI., 1968), supplemented with 0.5/lM of BA. This primary culture was maintained in a growth room at 26±3 °C under a 12h photoperiod_ After 45 days, the tissues were transferred to a secondary medium without BA. Part of the culture flasks (10 replicates of each cultivar) were kept in the growth room and the remaining flasks (10 replicates) were transferred to a

*) This research was partially supported by CNPq and FAPESP.

Abbreviations: BA = 6-benzylaminopurine; KIN = 6-furfurylaminopurine; 2,4-D = 2,4-di­chlorophenoxyacetic acid; NAA = alpha-naphthaleneacetic acid; GA3 = gibberellic acid.

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206 1. S. MARTINS and M. R. SONDAHL

shaded bench in a greenhouse. After further 40 days of culture, 12 cultivars were subcultured on the following tertiary medium: MS medium (Murashige and Skoog, 1962) supplemented by 1 I'M of NAA and 2 I'M of GA3. All media had the pH adjusted to 5.5 before autoclaving.

Shoot apexes of P. vulgaris cv. Palmital Precoce were cultivated on a B5 solid medium using a diallelic design (16 treatments, 10 replicates) containing BA (0.5-1.0-2.0-5.0JLM) and 2,4-D (0-0.025-0.050-0.100 JLM). After 4 weeks of primary culture under controlled conditions, the tissues were subcultured to the same basic medium without growth regulators and transferred to a shaded bench in a greenhouse. Five to six weeks later, all the multiple buds formed were subcultured to the following tertiary medium: NfuN03 (10.3mM), KN03 (18.8mM), KH2P04 (0.63mM), CaCh·2H20 (1.5mM), MgS04'7H20 (0.75mM), Na:zEDTA (O.lmM), FeS04 . 7H20 (0.1 mM), 0.1 x micronutrients of MS, nicotinic acid (30 I'M), thiamine (30 JLM), pyridoxine (15 I'M), myo-inositol (550 I'M), cystein (200 I'M), sucrose (2 %), NAA (0.5 I'M), KIN (2.5 I'M), agar 0.6 %, and the pH adjusted to 5.5. The developing shoots were transferred to a mixture of sandlVermiculite (2: 3) containing the tertiary medium without sucrose.

Results and Discussion

There was a great deal of variation during primary culture of the 33 P. vulgaris cul­tivars. Callus formation, profused root growth and/or bud development were ob­served (Table 1). Following secondary culture, it was possible to discriminate the dif­ferential morphogenetic capacity of the bean germplasm studied. Out of 33 cultivars, only 12 materials produced multiple buds (Carioca, Roxote, Rosinha, Palmital Pre­coce, 60-Dias, Jules, Roxinho, Puebla-153, Bico de Ouro, Fartura, Mulatinho, Linea-17). Higher frequency of multiple buds was observed in the cultivars kept in the greenhouse in contrast to the ones in the growth room. The cultures of «Palmital Pre­coce», «60-Dias», and «Linea-17» presented a frequency of 15.6, 8.7, and 8.0 buds/ shoot apex in the greenhouse conditions, respectively. These three cultivars produced only 5.0, 5.2, and 2.6 buds/shoot apex when cultivated in the growth room. Using the methodology described, only «Palmital Precoce» developed plandets (2 em high) during tertiary culture. These results demonstrate once again the germplasm reaction to different media for bud differentiation and growth as discussed for Lotus spp. (Nii­zeki and Grant, 1971), alfalfa (Walker et aI., 1978), red clover (Phillips and Collins, 1979; Horvath Beach and Smith, 1979), and pea (Malmberg, 1979).

Table 1: Morphogenetic response of 33 bean cultivars after 45 days of shoot apex culture on the presence of 0.5 I'M of BA.

Bean cultivar

Roxinho, Puebla-153, Linea-17, Brasil Palmital Precoce, Rosinha, Jules, Carioca, Roxote, 60-Dias Linea-29, Coco BIanchi, Catu, Actopan, N-203, Vul. Cavalo Xaxin, Red Kloud, Mamoninha Sujo, Nep-2, Bayo Bico de Ouro, Fartura

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Remarks

Bud development and root differentiation Bud development, no roots

Single shoots, no roots

Little bud development, callus formation Bud development, callus formation and root differentiation

Shoot apex cultures of Phaseolus vulgaris 207

Fig. 1: Multiple bud development and plantlet formation in Phaseolus vulgaris cv. Palmital Pre­coce. (A) Callus formation, root differentiation, bud and plantlet development during se­condary culture (growth regulator free medium) according to the exposition of different treat­ments during primary culture (diallelic design). (B) Developing shoot after 4 weeks on tertiary culture; note the asynchroneous growth of these shoots derived from one single shoot apex cul­ture. (C) A bean plant with ca. 12 weeks old recovered from multiple bud formation, cultivated in sand/vermiculite substrate in a shaded protion of the greenhouse.

A diallelic design was set up with shoot apexes of «Palmital Precoce» to optimize a defined culture medium for plant recovery in P. vulgaris. The evaluation of this ex­periment during secondary culture (absence of growth regulators) clearly shows the effect of the primary medium on shoot apexes of beans (Fig. 1 A). The exposition to BA (0.5 JLM) and 2,4-D (0.025-0.050 JLM) during primary culture brought about the best development of apical meristems. Multiple adventitious buds were observed with the following interactions: BA (F-2-S JLM) combined with 2,4-D (0-0.025-0.050-0.100 JLM). One experiment gave a total of 276 adventitious buds fol­lowing the interactions just described. There were minor differences of bud induction among all growth regulator interactions studied whereby an average of 15 buds/ shoot apex were produced. Larger callus formation in the presence of BA (2-5 JLM) and 2,4-D (0.05-O.10JLM) were noticed. Multiple adventitious buds cultivated in tertiary medium produced shoots after 4 weeks (Fig. 1 B). Most of these shoots de­veloped normal leaves and roots at the 8th week of tertiary culture. These plantlets

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208 1. S. MARTINS and M. R. SONDAHL

were then transferred to sand Vermiculite vials containing the tertiary medium with­out sucrose. Several trifoliate leaves developed following an additional 4-5 weeks in culture (Fig. 1 C). These data add new information for further control of in vitro plant regeneration among bean tissues. This technique may be applied for virus eli­mination and germplasm exchange.

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