In vitro Culture of Shoot Apical Meristems of Various Phaseolus Species and Cultivars

In vitro Culture of Shoot Apical Meristems of Various PhaseD/us Species and Cultivars 1)

Plant Biotechnology Institute, National Research Council, Saskatoon, Saskatchewan, Canada SlN OW9 Received July 7, 1984 . Accepted December 28, 1984

Summary In vitro morphogenetic responses of shoot apical meristems of 3 species of Phaseolus were studied under various temperature regimes as well as hormone combinations. Meristems of Pha· seolus vulgaris exhibited the best response in terms of shoot regeneration (65 %) when cultured on media supplemented with 10.0 t-tM each of BA and IAA or IBA at a constant temperature of 26°C. The efficiency of bud and shoot regeneration was different with various cultivars of P. vulgaris, P. coccineus and P. lunatus. Except for 10.0 JLM BA, other concentrations of cytokinins or auxins alone failed to induce bud or shoot regeneration. Even at the optimal level of BA (10.0 JLM), differential responses were observed in plant regeneration efficiency (0 -100 %). Meristems of P. lunatus failed to produce shoots and plantlets under all conditions tested. An elevation of mannitol and sucrose concentrations in the media drastically reduced the growth of plantlets regenerated from meristems (up to 95 %). Sucrose alone, at 5 % level, appeared to be adequate to bring about growth reduction and thus facilitate short-term preservation of Pha· seolus meristems. Key words: Phaseolus vulgaris, Phaseolus coccineus, Phaseolus lunatus, in vitro storage, meristem culture.

Introduction The expression of organogenesis in vitro is a reflection of the intrinsic genetic constitution of the explants and therefore, meristem culture could be used to elucidate such differences at the species/cultivar level. Meristem culture has also been shown to be of application in mass propagation, elimination of viral pathogens including seedborne viral infections in legumes, germplasm preservation and consequently exchange of genetic material (Kartha, 1982). Germplasm maintenance in Phaseolus is generally accomplished through storage of seeds. However, seed-borne viruses are re-

1) NRCC No. 24316. 2) Visiting Scientist, Tissue Culture Laboratory, Instituto de Biologfa, Universidad Nacional Autonoma de Mexico, Mexico, 20 D.F. 3) To whom requests for reprints should be addressed. Abbreviations: BA, 6-Benzyladenine; NAA, 1-Naphthaleneacetic acid; IBA, lndole-3-butyric acid; IAA, Indole-3-acetic acid; KIN, Kinetin (K), ZEA, Zeatin (Z); 2iP, (2-Isopentenyl) adenine; GA 3, Gibberellic acid.

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ported to occur in Phaseolus germplasm collections. The ensuing danger to Phaseolus seed collections has been emphasized only recently (Hampton, 1982). Short and long-term preservation of virus-free germplasm is best accomplished by cryopreservation of meristems; but this technique remains to be developed for Phaseolus and, therefore, alternate strategies must be developed. Induction of reduced growth of meristems in vitro by application of mannitol and sucrose has been suggested as one strategy (Westcott, 1981 a, b; Kartha, 1982). The use of such compounds in short-term preservation of meristems of tropical species sensitive to chilling has been proposed (Kartha, 1982). The present study was undertaken to evaluate the morphogenetic responses of shoot apical meristems of Phaseolus spp. in order to ascertain the genotypic variation, if any, between species and within species, which might affect the culture requirements. The study also examined the possibility of utilizing such osmotic stress inducers as sucrose and mannitol in the short-term preservation of meristems from two cultivars of P. coccineus.

Materials and Methods Seeds of Phaseolus vulgaris L., P. coccineus L., and P. lunatus L. (cultivars are listed in Table 1) were rinsed in 70 % ethanol for 2 min and desinfected in 1.2 % sodium hypochlorite for 20 min on a gyratory shaker at 150rpm. Subsequently, the seeds were thoroughly washed four times with sterile distilled water and germinated at 25°C in darkness on moistened cotton contained in sterile 110 ml-glass jars. After one week, shoot apical meristems, 0.4 and 0.5 mm in length, containing a pair of leaf primordia were aseptically isolated and cultured in 110 ml-glass jars on 30 ml of nutrient medium (3 meristems/jar and 7 jars/treatment). The nutrient medium (MS) consisted of mineral salts according to Murashige and Skoog (1962), 3 % sucrose, vitamins as in B5 medium {Gamborg et al., 1968),0.8% agar and supplemented with 10.0J.tM concentrations of BA, KIN, ZEA, and 2iP, alone or in combination with NAA, IAA and IBA. The pH of the medium was adjusted to 5.8 with KOH or HCI prior to adding the agar. The medium was autoclaved at 138 Kpa for 20 min. Indoleacetic acid and GA3 were added to the medium filter-sterile. Only the subculture medium contained GA 3. The meristems were incubated at 3 temperature and photoperiod regimes (details given in Figs. 1-4 and Tables). The effect of osmotic stress inducers was tested with 2 cultivars of P. coccineus by culturing the meristems on medium supplemented with 10.0 J.tM BA in the presence of several concentrations of sucrose and/or mannitol. These meristems were stored for 3 months at 26°C constant at 16 h-photoperiods and subsequently recultured on osmotic stress-free medium. At least 20 meristems were cultured under each treatment. After being in culture for 5 weeks, the buds which had grown to 10 mm or more in length were considered as shoots. Rooting on the shoots was induced by reculturing the shoots on half strength MS medium containing 1.0 J.tM NAA (Kartha et aI., 1974) and the regenerated plants were transferred to pots and grown to maturity. Results on the morphogenetic responses (meristems forming buds, shoots and total plants regenerated) were subjected to variance analysis and Duncan's multiple range test in order to substantiate the effect of species-cultivar differences.

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Results and Discussion a) The effect of temperature and hormones

A marked effect of temperature on shoot differentiation from bean meristems was noticed, the lower the temperature, the lesser was the tendency for the meristems to form buds and shoots. In all species studied, a constant temperature of 26°C appeared to yield the best response in terms of regeneration of shoots and buds (Figs. 1- 4 C). The results are in agreement with those reported previously for meristems of bean, soybean and cowpea (Kartha et aI., 1981). When bean meristems were cultured at a temperature of 26°C dayllS °C night, the responses were lower. At a temperature regime of 20°C dayllS °C night, total inhibition of shoot development was noted (Figs. 1- 4 A) thus signalling a high sensitivity of Phaseolus meristems to low temperature. This observation was unexpected because the optimal temperature range for the growth of P. vulgaris plants has been reported to be 15.6 to 21.6°C (Duke et al., 1981). The 20/15 °C temperature regime did not inhibit the differentiation of buds, especially when IAA and IBA were used (Figs. 1- 4 A), suggesting low temperature to affect adversely shoot development primarily. Among the various cytokinin-auxin combinations (Figs. 1-4), combinations of cytokinins and IAA induced more efficient plant regeneration than those combinations involving cytokinins and IBA or NAA. The effect of a particular cytokinin depended upon the specific genotype studied. Indoleacetic acid in combination with BA induced plant regeneration in the cv. Scarlet runner, Dwarf green stringless and Blue lake, but not in Red kidney, whereas IAA and KIN induced plant regeneration both at 26°/15 °C and 26°C, but at different frequencies. When IBA was combined with cytokinins, the meristems cultured at 26°C differentiated into plants at varying frequencies. Moreover, IBA in combination with ZEA yielded the best response in cv. Scarlet runner while the former with BA was best suited for Dwarf green stringless. Inhibition in shoot and bud differentiation was observed in 3 cases involving combinations of NAA and cytokinins (Figs. 1, 3 and 4); but not with the cv. Dwarf green stringless where shoot and bud regeneration were noted (Fig. 2). The cv. Red kidney exhibited the lowest regeneration capacity under our conditions and in some cases (Fig. 4) no growth of the meristems was noticed. Phaseolus vulgaris (Dwarf stringless green) and P. coccineus (Scarlet runner) responded similarly although each showed distinct differences (Figs. 1 and 2). In contrast, P. lunatus, cv. Jaspeado did not grow in vitro at any of the tested hormone combinations and temperature regimes. These results indicated the influence of species and cultivar in eliciting the morphogenetic responses from bean meristems. Meristems from different cultivars of Phaseolus spp. (Table 1) except cv. Ayocote negro were cultured on nutrient medium devoid of growth hormones as well as in the presence of 10.0 fLM levels of auxins and cytokinins alone. Plant regeneration was obtained in those cases where BA was used. Kartha et al. (1981) obtained plant regeneration in P. vulgaris cv. Dwarf green stringless when the meristems were cultured on

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Fig. 3: P. vulgaris cv. Blue lake. Fig. 4: P. vulgaris cv. Red kidney. Figs. 1-4: Effect of interaction of several cytokinins and auxins, at concentrations of 10.0 pM each, at 3 different temperature regimes (A, 20°C day/iS °C night; B, 26 °C day/iS °C night; and C, 26 °C constant) on the morphogenetic responses of Phaseolus meristems. At least 20 meristems were cultured for each treatment and incubated at 2000 lx, 16 h-photoperiods.

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hormone-free medium at an elevated light intensity of 7500 Ix. In the experiments reported here where a reduced light intensity of 2000 Ix was employed, the meristems, instead of differentiating into plants, either turned green or produced only callus. A similar effect on the alteration of morphogenetic response by the light intensity employed has been documented also in the immature leaflet cultures of Pisum sativum (Rubluo et aI., 1982). Table 1: Morphogenetic Responses of Meristems of Phaseolus spp. on Media with 10 /LM BA and Shoot Development in Subculturing Media (1.0 /LM NAA + 0.1 ~ GA3)!). Species and Cultivars

Growth Responses:

% Buds Developing Shoots on Subculturing medium

% of Meristems

Forming Calli with:

P. coccineus Scarlet runner Ayocote negro

P. vulgaris

Dwarf green Stringless Blue lake Red kidney

Total Plant Regeneration 2)

(%)

Buds

Shoots

Z100e( + + + +?) 65 a(+ + +)

15e 50a

100 SO

100e

S5 b ( + ++ +) 90be( + + +)

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The two P. coccineus cultivars have different patterns of response in bud and shoot regeneration, but the buds readily elongate to form shoots upon reculture on a medium containing 1.0/LM NAA and O.l/LM GA3 (subculturing medium of Kartha et al., 1981). In P. vulgaris, the difference in bud and shoot differentiation was more striking. In cv. Dwarf green stringless, no shoot formation was obtained but instead the meristems produced multiple buds whereas in other cultivars, both shoot and bud differentiation occurred. All the buds of Dwarf green stringless developed into shoots in subculturing medium, but other cultivars failed to do so, instead they developed into either leafy shoots or tiny plantlets which were unsuitable to be grown to maturity. The requirements for successful meristem culture can vary between species and within species. Our results show that this general statement applies also to Phaseolus. In spite of the fact that, in most cases, bud and shoot formation was successfully induced, the degree of development and frequency of response (0-100 % shoot regeneration) vary from species to species and even from cultivar to cultivar, as evidenced

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by the statistical analyses (Table 1). This difference is attributable to the intrinsic genetic constitution at the species and cultivar level. Genotypic differences in the regeneration capacity of grain legumes (Malmberg, 1979; Rubluo et aI., 1982) and a differential genotypic response to auxins in Phaseolus hypocotyl tissues have been reported (Mok and Mok, 1977). Similarly, the influence of genotype in eliciting various morphogenetic responses were also noted for meristems of potato (Westcott et aI., 1977), several grass species (Dale, 1977) and Trifolium (Cheyne and Dale, 1980; Bhojwani, 1981). Benzyladenine has been considered to be an ideal cytokinin for meristem cultures of several species such as, tomato (Kartha et aI., 1977), strawberry (Kartha et al., 1980), soybean and bean (Kartha et aI., 1981) and Trifolium (Bhojwani, 1981). However, differential responses were noted at the species and cultivar level. A drastic example of failure in obtaining plant regeneration from meristem is provided by P. lu· natus cv. Jaspeado in which only callus growth could be induced. Considering P. lunatus to be very sensitive to excessive salt concentrations (Baudet et aI., 1981) our attempts to culture the meristems on various media with reduced salt concentrations were also unsuccessful in inducing shoot regeneration in this species. b) Effect of osmotic stress inducers

The effect of addition of sucrose and mannitol to the storage medium on the survival of meristems of 2 cultivars of P. coccineus is presented in Table 2. In general, the growth rates were strongly reduced in the storage media and differential responses were noticed in the reculture media. The absence of sucrose in the media (not shown in Table 2) suppressed all visible growth in meristems of both the cultivars regardless of the presence of mannitol. When cultured on media without the stress inducers (control; 3% sucrose, 0% mannitol), 50% of the meristems of cv. Ayocote negro developed shoots, 25±0.2 (S.E.) mm long, in about 5 weeks (Table 1); but after 3 months storage, about 50 % of the shoots with callus turned brown and eventually died while the rest grew to an average size of 103±8.5mm (Table 2). On the other hand, in cv. Scarlet runner, after 5 weeks of culture, although profuse bud formation was observed in only 15 % of the meristems, the buds developed into shoots attaining an average size of 22.4±0.2 mm (Table 1). However, after 3 months storage in this osmotic-free medium, some of the buds developed into shoots with well developed leaves and roots with an average shoot length of 95.3±6.9 mm (Table 2). When the meristems of both cultivars were cultured directly on the storage media containing the stress inducers, the morphogenetic potential was either retarded or suppressed. Consequently, wherever shoots were developed, they were consistently smaller than those in the control (Table 2) and a clear growth inhibition was apparent. Higher concentrations of mannitol (9 and 11 %) exhibited toxic effects regardless of sucrose levels employed and no plant regeneration was obtained upon reculturing the ]. Plant Physiol. Vol. 119. pp. 425-433 (1985)

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Table 2: Growth Responses of Phaseolus coccineus Meristems Cultured and Stored for Three Months in Presence of Sucrose and Mannitol and Recultured on Media with 10 p.M BAa). Concentration

(%)

Storage Media Callus + Buds

(%) Sucrose Mannitol 3 3

o (Control)

3 3 3

3 5 7 9 11

5 5 5 5 5 5

0 3 5 7 9 11

3

Reculture Media

Callus + Shoots

(%)

Length of Shoots (mm)±S.E.

Growth Reduction (%)

(%)

A

B

A

B

A

B

61 78 28 39 17

67 100 76 67

22 22 17 17

67 71 57 33

67 67 78 72 17

100 100

56 44 56 28

86 57

A

B

A

B

A

61 78 28 39 17

67 76 81 33

22 22

67 43 14

103.50±8.54 95.35±6.92 16.50± 1.32 13.94±0.61 10.50 ± 0.43

84

85 89

72 67 78 72

62 52

28 22 44

33 28

5.50±0.22 20.06±4.29 5.25±0.62 18.11±2.09 5.44±0.33

95 95 95

79 81

B

Callus + Buds

Plant Regeneration

(%)

a) Storage conditions: 26°C constant; 2000 Ix; 16 h-photoperiods. A = cv. Ayocote negro; B = cv. Scarlet runner.

meristems on stress-free medium. Furthermore, the presence of mannitol appeared to be not essential in order to reduce the growth rates and plant regeneration of Phaseo/us meristems under the conditions tested. On the other hand, cv. Scarlett runner was more susceptible when exposed to 5 % sucrose combined with mannitol (Table2). Low water potential has been indicated as a factor that provokes a lag period in plant cell populations resulting in growth arrest (Handa et al., 1982). Sucrose and mannitol have been suggested as inducers of low osmotic potential (Eriksson et aI., 1978) and their use in short to medium-term storage of valuable germplasm emphasized (Kartha, 1982). The results presented in Table 2 indicated that the growth inhibition occurred as a result of the presence of sucrose more than by mannitol. Westcott (1981 a, b) also reported that the survival of potato shoot tip cultures increased by elevating the sucrose levels in storage media and that higher concentrations of mannitol were toxic to single nodal cultures. Similarly, Schilde-Rentschler et al. (1982), working with potato shoot apices on media with added sucrose and mannitol, observed that the growth responses improved with increased sucrose levels. These results agreed with ours except that the presence of mannitol apparently was not necessary in the storage media of P. coccineus. In contrast, in sweet potato meristems, a combination of 5 % each of sucrose and mannitol resulted in a drastic growth inhibition within 3 months (Kartha, 1982). These results are in good agreement with those reported here. Fur-

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thermore, since sweet potato is a species sensitive to chilling as is Phaseolus, the potential use of osmotic stress inducers might aid in the short to medium-term preservation of species sensitive to chilling. Acknowledgements The authors wish to thank Mr. Victor Chavez, Botanical Gardens, Institute of Biology, National University of Mexico for the supply of seeds of P. coccineus cv. Ayocote negro, and Dr. Mroginski for critical review of the manuscript. References BAUDET, J. c., J. A. DUKE, M. GOLDEN, C. F. REED, R. MARECHAL, K. O. RACHlE, J. SMARTT, and J. K. P. WEDER: Phaseolus lunatus L. In: DUKE, J. A. (ed.): Handbook of legumes of world economic importance, 191-195. Plenum Press, New York, 1981. BHO]WANI, S. S.: A tissue culture method for propagation and low temperature storage of Trifo· lium repens genotypes. Physiol. Plant. 52, 187-190 (1981). CHEYNE, V. A. and P. J. DALE: Shoot tip culture in forage legumes. Plant Sci. Lett. 19,303-309 (1980). DALE, P. J.: Meristem tip culture in Lolium, Festuca, Phleum and Dactylis. Plant Sci. Lett. 9, 333-338 (1977). DUKE, J. A., P. H. GRAHAM, C. F. REED, J. SMARTT, and J. K. P. WEDER: Phaseolus vulgaris. In: DUKE, J. A. (ed.): Handbook of legumes of world economic importance, 195-200. Plenum Press, New York, 1981. ERIKSSON, T., K. GLIMELIUS, and A. WALLIN: Protoplast isolation, cultivation and development. In: THORPE, T. A. (eds.): Frontiers of Plant Tissue Culture, 131-139. Int. Association for Plant Tissue Culture, U. of Calgary, Canada, 1978. GAMBORG, O. L., R. A. MILLER, and K. OJIMA: Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell. Res. 50, 151-158 (1968). HANDA, A. K., R. A. BREssAM, S. HANDA, and P. M. HASEGAWA: Characteristics of cultured tomato cells after prolonged exposure to medium containing Polyethylene-Glycol. Plant Physiol. 69, 514-521 (1982). HAMPTON, R.: Importance of seedborne virus in crop germplasm. Plant Dis. 66, 977-978 (1982). KARTHA, K. K., O. L. GAMBORG, and F. CONSTABEL: Regeneration of pea (Pisum sativum L.) plants from shoot apical meristems. Z. Pflanzenphysiol. 72, 172-176 (1974). KARTHA, K. K., S. CHAMPOUX, O. L. GAMBORG, and K. PAHL: In vitro propagation of tomato by shoot apical meristem culture. J. Amer. Soc. Hort. Sci. 102,346-349 (1977). KARTHA, K. K., N. L. LEUNG, and K. PAHL: Cryopreservation of strawberry meristems and mass propagation of plantlets. J. Amer. Soc. Hort. Sci. 105, 481-484 (1980). KARTHA, K. K., K. PAHL, N. L. LEUNG, and L. MROGINSKI: Plant regeneration from meristems of grain legumes: soybean, cowpea, peanut, chickpea and bean. Can. J. Bot. 59, 1671-1679 (1981). KARTHA, K. K.: Genepool conservation through tissue culture. In: RAo, A. N. (ed.): Tissue culture of economically important plants. Proc. Int. Symp., 213-218. National University, Singapore, 1982. MALMBERG, R. L.: Regeneration of whole plants from callus culture of diverse genetic lines of Pisum sativum L. Planta 146, 243-244 (1979). MOK, M. C. and D. W. S. MOK: Genotypic responses to auxins in tissue cultures of Phaseolus. Physiol. Plant. 40, 261- 264 (1977).

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MURASHIGE, T. and F. SKOOG: A revised medium for rapid growth and bioassay with tobacco tissue cultures. Phyiol. Plant. 15, 473-497 (1962). RUBLUO, A., L. MROGINSKI, and K. hlRTHA: Morphogenetic responses of pea leaflets cultured in vitro. In: FUJIWARA, A. (ed.): Plant Tissue Culture 1982, 151-152. The Japanese Association for Plant Tissue Culture, Tokyo, 1982. SCHILDE-RENTSCHLER, L., N. ESTRADA, R. ESPINOZA, and R. LIZARRAGA: In vitro storage and distribution of potato germplasm. In: FUJIWARA, A. (ed.): Plant Tissue Culture 1982,781-782. The Japanese Association for Plant Tissue Culture, Tokyo, 1982. WESTCOTT, R. J., G. G. HENSHAW, and W. M. ROCA: Tissue culture storage of potato germplasm: culture initiation and plant regeneration. Plant Sci. Lett. 9, 309-315 (1977). WESTCOTT, R. J.: Tissue culture storage of potato germplasm. 1 Minimal growth storage. Potato Res. 24, 331-342 (1981 a). Tissue culture storage of potato germplasm. 2 Use of growth retardants. Potato Res. 24, 343-352 (1981 b).

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