PlantCeU Reports
Plant Cell Reports (1992) 11:163-168
9 Springer-Verlag1992
Somatic embryogenesis and shoot regeneration from intact seedlings of Phaseolus acutifolius A., P. aureus (L.) Wilczek, P. coccineus L., and P. wrightii L. Kamal A. Malik and Praveen K. Saxena Department of Horticultural Science, University of Guelph, Guelph, Ont. N1G 2Wl, Canada Received January 6, 1992/Revised version received February 14, 1992 - Communicated by E Constabel
A rapid, one-step procedure has been developed for inducing direct organogenesis and somatic embryogenesis in cultures of Phaseolus coccineus L., P. acutifolius A., P. aureus L. [Vigna radiata L. Wilczek] and P. wrightii L. Development of somatic embryos and shoot buds occurred within 6-8 weeks of culture from intact seedlings raised on MS (Murashige and Skoog 1962) medium supplemented with N6benzylaminopurine (BAP). Shoot buds or embryoids originated from subepidermal tissue of the regions adjacent to the shoot apex, hypocotyl and cotyledonary axils. While P. acutifolius and P. aureus were regenerated via shoot formation and P. wrightii by somatic embryogenesis, both embryogenesis and shoot regeneration were observed in P. coccineus. Relatively higher levels of BAP, 50-80/~M, were found to be optimal for inducing regeneration while lower concentrations were ineffective. About 40-70 shoots and 70-250 somatic embryos were produced per responding seedling. Regenerated shoots and somatic embryos developed into whole plants on a basal medium or the one supplemented with 1/zM naphthaleneacetic acid. Abstract.
Key words: N6-benzylaminopurine; Grain legumes; Organogenesis; Phaseolus; Somatic embryogenesis Introduction
An efficient and reproducible procedure for the regeneration of plants from cell and tissue cultures is a prerequisite for the application of gene transfer methods in crop improvement. However, in spite of a considerable progress in developing tissue culture Correspondence to: P.K. Saxena
protocols in a variety of plant species, grain legumes in general, and the species of Phaseolus in particular, have remained recalcitrant to regeneration in vitro (Mroginski and Kartha 1985). Crocomo et al. (1!)76) recovered two plantlets from callus cultures of Phaseolus vulgaris using a medium supplemented with phytohormones and aqueous bean seed extract of an undefined composition. Since then, plant development has been achieved from cultured apical and axillary meristems of P. vulgaris (Kartha et al. 1981; Martins and Sondahl 1984a) and via de novo organogenesis in Phaseolus lunatus (Sreedhar and Mehta 1984), P. coccineus (Allavena and Rossetti 1986) and P. vulgaris (McClean and Grafton 1989; Malik and Saxena 1991; Franklin et al. 1991). Early stages of somatic embryo differentiation were also observed in cell cultures of P. vulgaris (Allavena 1984; Martins and Sondahl 1984b; Saunders et al. 1987) but whole plant regeneration from embryo-like structures was accomplished in P. acutifolius (Kumar et al. 1988) and P. coccineus (Angelini and Allavena 1989). However, the process of regeneration in these studies was slow and produced generally low frequencies of shoot regeneration or somatic embryogenesis. In addition, the protocols :require the use of immature embryonic tissue (Angelini and Allavena 1989) or extensive manipulation of cells (Kumar et al. 1988), for establishing morphogenic cultures. In this communication, we describe a simple procedure for inducing high-frequency organogenesis and somatic embryogenesis from mature seeds of :several Phaseolus species. The process of induction and expression of morphogenesis is accomplished in a single step of
164 direct seed germination in a BAP-supplemented medium. Culture conditions reported here may be generally applicable to other difficult to regenerate plant species. Materials and methods Seed Culture and Plant Regeneration Seeds of Phaseolus coccineus L. were obtained from (A.E. MeKenzie Co., Brandon, Man.) and those of P. acutifolius, P. aureus, and P. wrightii were kindly provided by Dr. T. E. Michaels (Department of Crop Science, University of Guelph, Ont.). Prior to surface sterilization, seeds were immersed in concentrated sulfuric acid for 60 see for softening the waxy seed coat and washed twice with sterile water. Seeds were immersed in ethanol for 1-3 min and then in a 1% solution of sodium hypochlorite for 20 min; during this period they were continuously stirred with a magnetic bar. Thereafter, seeds were washed five times with sterile water, dried on a sterile paper towel, and cultured in Magenta culture vessels (Magenta Corp., Chicago, Ii1., USA; 5 seeds per 300-ml box) containing 45 ml of the culture medium. The nutrient medium contained macro- and micro-nutrients of the Murashige and Skoog (1962) (MS) medium, vitamins according to Gamborg et al. (1968), 3 % sucrose, 0.25 % Gelrite ( Scott Laboratories, Carson, Cal., USA), and Nr-benzylaminopurine (BAP) (0-80 #M). The pH of the media was adjusted to 5.7 with KOH or HC1 prior to autoclaving at 0.122 MPa for 20 rain. All cultures were incubated at 25~ in darkness for the first two weeks and later transferred to light (40-60 #mol.m-2.s~; 16-h photoperiod) provided by cool-white fluorescent lamps (Phillips Canada, Scarborough, Ont.). Regenerated shoots and embryo-derived shoots were excised from the mother seedling after 4-8 weeks of seed germination and subeultured on MS medium containing 1 #M naphthaleneaeetie acid for inducing root formation. Somatic embryos were directly subcultured on MS basal medium for further development. Regenerated shoots developed a prolific root system within four weeks of culture. Rooted plantlets were washed thoroughly with distilled water, transferred to autoclaved peat pellets (2.5 era; Ball superior, Mississauga, Ont.), and after two more weeks of growth in a growth chamber at 24~ under 16-h photoperiod (25-40 /~mol.m-2.sl), were transferred to soil in the greenhouse. For each concentration of BAP, about 50 seeds were raised and those showing uniform germination (usually about 30-40) were considered for the final score. The data presented here are averages obtained from 30 seedlings per treatment and are expressed as percentages of seedlings showing organogenesis and-or somatic embryogenesis and the numbers of shoots or somatic embryos regenerated per seedling. All experiments were repeated three times.
Histology For histological examination, seedling explants bearing regenerated shoot buds of Phaseolus coccineus were excised from the cultures raised on a medium containing 80/xM BAP. Paraffin embedding of tissue samples was performed as described by Johansen (1940). Longitudinal sections of 10 /~ thickness were cut using a Spencer 820 mierotome (American Optical Corporation, Buffalo, NY, USA) and resulting paraffin
ribbons were passed through a series of deparaffinizing solutions followed by staining with Aleian Green and Safranin solution (Johansen, 1940). The sections were examined under light microscope (Zeiss photomieroseope III, Carl Zeiss, Germany).
Results
Germination of seeds of all four species of Phaseolus was not affected by the presence of BAP in the culture medium and occurred within a week. The frequency of seed germination for different species varied between 80 and 100% (Table 1 and 2). Table 1. The efficacy of NLbenzylaminopurine in inducing direct differentiation of shoots in cultures of intact seedlings of Phaseolus acutifolius, 1". aureus, and 1". cocdneus. Species
Conc. Germination
Regeneration"
No.
Shoot (pM)
%
%
per seedling~
30 50 80
85 80 70
20 70 60
5 41 38
30 50 80
90 85 70
-" 78 70
40 52
30 50 80
90 85 85
15 25 85
11 30 73
e. acutifolius
P. aureu$
P. coccineus
~ percentages of seedlings showing regeneration of shoots b= total number of shoots produced from tissues of the
cotyledon axll and shoot apex r
Figs. 1-9. Direct differentiation of shoots and somatic embryos from intact seedling of Phaseolus species. 1. A control seedling. 2. Differentiation of shoots in P. aculifolius from tissues in the regions of shoot apex (arrow) and axils (arrow). 3. Cotyledonary node region of the same shoot-producing seedling (Fig. 2). 4. A embryogenie seedling of P. coceineus. Note the emergence of somatic embryos in the hypoeotyl region (arrow). Cotyledons are denoted by Ct. Fig. 5-7. Various developmental stages of somatic embryos observed in cultured seedlings of P. cor and P. wrightii. Note the presence of heart-shaped (arrows) and eotyledonary somatic embryos. 8. Germination of somatic embryos of P. wrightii to develop into shoots while still attached to the mother seedling. 9. Regenerated plants of P. wrightii. Bars in 1 = 5mm, 2 = 10ram, 3 = 0.5ram, 4 = 2mm, 5 = 1.2ram, 6 = 0.5ram, 7 = 0.5ram, 8 = 2ram, 9 = 15ram.
165
166
Figs. 10-12. Histology of a shoot-producing seedlings of Phaseolus coccineus. 10. and 11. Mierograph of a longitudinal section of the nodal region showing differentiation of shoot buds. Note the initiation (Pig. 10; arrow) and further development (Fig. 11; arrow) of shoot buds in the subepidermal regions and the absence of any connection with the original vascular tissue. 12. A mierograph of longitudinal section from the base of eotyledonary node. Note the presence of well developed shoot buds with leaf primordia (arrow). Bar in 10 = 0.3mm, 11 = 85 #m, 12 = 85/~m
In the initial eight to ten days, no drastic differences were noted in the growth of seedlings grown in media supplemented with or without BAP. All seedlings developed single shoots with cotyledons and a pair of primary leaves. However, root systems showed marked differences: plants grown in medium devoid of BAP had well developed primary and secondary roots which in the presence of BAP showed poor growth. Pronounced differences in growth of seedlings in the presence and the absence of BAP were observed after 4-6 weeks of culture. Control plants were twice the size of those grown in BAP-enriched media and the apical meristem of these plants grew extensively developing many leaves (Fig. 1). The cotyledonary axillary buds also developed into shoots. By contrast, primary shoot apices of seedlings raised on media containing BAP ceased grow, turned yellow, and underwent necrosis. In cultures of P. acutifolius, P. coccineus and P. aureus, several shoot buds differentiated from the tissue surrounding the suppressed apices. Similarly, axillary shoots did not grow further, but the areas adjacent to the cotyledonary node became meristematic and produced numerous shoot buds (Figs. 2 and 3).
The frequency of shoot-producing seedlings and the average number of shoots were highest on media containing 50 or 80/~M BAP (Table 1), while lower concentrations were ineffective (data not shown). About 40-70 shoots were recovered from the seedlings of P. acutifoIius, P. aureus and P. coccineus under optimal culture conditions. The pattern of the development of regenerated shoots was similar in all three species. The seedlings of P. coccineus produced somatic embryos in the hypocotyl region (Fig. 4); somatic embryogenesis occurred 4-6 weeks after the differentiation of adventitious shoots. The seedlings of P. wrightii, on the other hand, showed differentiation of somatic embryos from peripheral tissue of the apical region. In both species, representative developmental stages of somatic embryogenesis ranging from the globular, heartTable 2. Induction o f somatic embryogenesis in cultures of intact seedlings of Phaseolus coccineus and P. wrightii by N 6benzylaminopurine. Species
Cone. Germination Regeneration" No. Somatic embryos per
O,M)
(%)
(%)
seedling~
30 50 80
95 85 80
-r 10 80
70 250
30 50 80
95 100 80
38 90
80 130
P. coccineus
P. wrightii
= Percentageof seedlingsshowing somatic embryogenesis b = Average number of somatic embryos per seedling
167 shaped to cotyledonary stature were present (Figs. (5-7). Somatic embryos matured on the mother seedling and germinated to develop into well formed shoots (Fig. 8). Alternatively, they could be excised, germinated and grown into whole plants (Fig. 9). The differentiation of somatic embryos, like that of shoots, was also induced by higher concentrations of BAP, 50 and 80 #M (Table 2). Lower concentrations (1-40 /zM) evoked no response and at concentrations beyond the optimum (90-120/zM), the shoots or somatic embryos were highly vitrified and showed stunted growth (data not shown). About 50% of the regenerated shoots and somatic embryos were able to develop prolific roots. The plantlets survived transplant to soil and formed apparently normal plants (Fig. 9). Histology of the shoot-producing seedlings revealed that the regenerated shoots had their origin in the subepidermal tissue (Fig. 10 and 11; arrow). Shoot regeneration was found to be direct i.e., without any callus formation and different stages of shoot organization were clearly visible in the longitudinal sections through the shoot-producing region of the cotyledonary node (Figs. 10-12). No direct vascular connections between the mother tissue and the regenerated shoot buds were found to: exist indicating de novo origin of developing shoots. Discussion
Success in regenerating Phaseolus species has been achieved only recently. In the first report of direct differentiation of shoots from bean seedlings, McClean and Grafton (1989) used cotyledonary node explants. In this method, originally developed for soybean (Wright et al. 1986; Hinchee et al. 1988), seeds were germinated in the presence of 5 /~M BAP and the cotyledonary nodes excised after 12-14 days. Axillary buds were removed by scraping the axis and surrounding region prior to culture on a medium of the same BAP complement (5#M), for shoot regeneration. It was proposed that the excision (wounding) may be responsible for meristem initiation, and the presence of BAP, for further development of shoots since the removal of developing buds was found to be associated with continued bud proliferation. Franklin et al. (1991) also used cotyledonary node explants but these included axillary buds and a portion of the cotyledons. Young cotyledonary node explants were shown to produce optimum shoot regeneration in
pea also (Jackson and Hobbs 1990). However, about 4-10 shoots were recovered in these studies. In Phaseolus coccineus, both organogenesis and somatic embryogenesis were reported in cultures of immature cotyledons; a preculture treatment of the explants with abscisic acid was found to be essential for shoot formation and the degree of regeneration was expressed as percentage of explants showing regeneration centres consisting of at least one shoot bud (Allavena and Rossetti 1986; Angelini and Allavena 1989). Earlier, we reported the promotive role of direct culture of seeds in the presence of BAP in subsequent shoot regeneration from leaf explants of Phaseolus vulgaris (Malik and Saxena 1991). In these experiments, a relatively lower concentration of BAP (5 /~M) was used in the medium for seed germination and explant culture. The results of the present study clearly demonstrate that the application of higher concentrations (50 and 80 /xM) of BAP induces a high frequency of organogenesis and somatic embryogenesis in all tested species of Phaseolus (Table 1 and 2). Histological examination revealed that shoots developed de novo from subepidermal tissues of the cotyledonary node. The pattern of shoot origin and development was very similar to that previously described for bean (McClean and Grafton 1989) and pea (Jackson and Hobbs 1990). Thus, it seems that higher levels of BAP during seed culture and the maintenance of morphological integrity of the seedlings may be responsible for high efficiency of regeneration in our experiments. In previous studies, the seeds were germinated either in the absence or in the presence of a low concentration of BAP (5 /zM); concentrations higher than 5 tzM were applied only during the culture of excised explants and were found to have action maxima between 5-15 /zM. The relationships among high cytokinin concentration, structural intactness of the seedlings and observed de novo differentiation of shoots and somatic embryos are being investigated in our laboratory. An interesting aspect of this study is the simultaneous occurrence of organogenesis and somatic embryogenesis in P. coccineus. Angelini and Allavena (1989) also reported the development of shoot buds and somatic embryos in response to 2-naphthoxyacetic acid and 2-isopentenyladenine. In our experiments, both shoots and somatic embryos developed in the presence of BAP alone. According to the theory of hormonal regulation of
168 morphogenesis, all types of growth and differentiation responses are mediated by relative amounts of growth regulators, mainly phytohormones (Skoog and Miller 1957). It is likely that the auxin:cytokinin complement required for regeneration of shoots or somatic embryos becomes available to the competent cells of the seedling as a result of exogenous application of BAP and endogenous auxins. Biosynthesis and transport of auxin(s) have been shown to influence the growth of axillary buds in cultured stem segments of Phaseolus vulgaris (Tamas et al. 1989). Besides the typical auxin:cytokinin combinations, another growth regulator, thidiazuron- a substituted phenylurea known to elicit cytokinin-like activity- also induced regeneration of shoots and somatic embryos from bean and geranium seedlings, respectively, suggesting the involvement of endogenous phytohormones (Malik and Saxena 1992; Saxena et al. unpublished). Although we have been able to apply seed culture method for regenerating a number of crop species, the mechanism of the induction and expression of morphogenesis from intact seedlings remains unresolved. In conclusion, the present study describes a simple procedure for inducing organogenesis, somatic embryogenesis, and plant regeneration in three Phaseolus crops, P. coccineus, P. acutifolius, and P. aureus and a wild bean, P. wrightii. Since the differentiation occurs on an intact seedling, the number of manipulations required to induce regeneration is reduced to one in comparison to several in procedures involving culture of immature embryo, leaf or seedling explants and callus (Mathews 1987; Kumar et al. 1988; Angelini and Allavena 1989). Moreover, the use of mature seeds (present investigation) avoids labour-intensive optimization of factors associated with explant culture and, thus, may significantly reduce the cost of maintaining the source plants in the green house. In addition, direct morphogenic differentiation offers many advantages as an experimental system, for example, in the study of biochemical and molecular events in organ determination and development, and the mechanism of action of growth hormones involved. The rapidity and high frequency of direct morphogenesis routinely obtainable in seed cultures are expected to facilitate stable, Agrobacterium-mediated or direct, genetic transformation of Phaseolus species in which success has so far been rather limited (Leon et al.
1991; McClean et al. 1991).
Acknowledgements. This research was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada. We thank Dr. R. Gill for helpful discussions and S. Saxena for technical assistance. References Allavena A (1984) In: Handbook of Plant Cell Culture, WR Sharp, DA Evans, PV Ammirato, Y Yamada (eds) Macmillan, New York. 137-168 Allavena A, Rossetti L (1989) Ann. Rep. Bean Improv. Coop. 29:132-133 Angelini RR, Allavena A (1989) Plant Ceil Tissue Organ Culture. 19:167-174 Crocomo OJ, Sharp WR, Peters JC (1976) Z. Pflanzenphysiol. 78:458460 Franklin CI, Treveu N, Gronzales RA, Dixon RA (1991) Plant Cell Tissue Organ Culture 24:199-206 Gamborg OL, Miller RA, Ojima K (1968) J. Exp. Res. 50: 151-158 Jackson JA, Hobbs SLA (1990) In Vitro Cell Dev. Biol. 26: 835-838 Johansen DA (1940) Plant mieroteehnique. McGraw-Hill, New York Hinehee MAW, Connor-Ward DV, Newell CA, MeDormell RE, Sato SJ, Gasser CS, Fisehhoff DA, Re DB, Fraley RT, Horsch RB (1988) Bio/Teehnol. 6:3915-3922 Kartha KK, Pahl K, Leung NL, Mroginski LA (1981) Can. J. Bot. 59:1671-1679 Kumar AS, Gramborg OL, Nabors MW (1988) Plant Cell Rep. 7:322-325 Leon P, Planckaert F, Walbot V (1991) Plant Physiol. 95: 968972 Malik KA, Saxena PK (1991) Planta 184:148-150 Malik KA, Saxena PK (1992) Planta (in press) Martins IS, Sondahl MR (1984a) Turrialba 34:157-161 Martins IS, Sondahl MR (1984b) J. Plant Physiol. 117:97-103 Mathews H (1987) Plant Cell Tissue Organ Culture 11:233-240 McClean P, Chee P, Held B, Semental J, Drong RF,SlightomJ(1991) Plant Ceil Tissue Organ Culture 60:131-138 MeClean P, Grafton KF (1989) Plant Sei. 60:117-122 Mroginski LA, Kartha KK (1985) Plant Breed. Rev. 2:215-264 Murashige T, Skoog F (1962) Physiol. Plant. 15:473-497 Saunders JW, Hosfield GL, Levi A (1987) Plant Cell Rep. 6: 46-49 Skoog F, Miller, CO (1957) Sym. $oe. Exp. Biol. 11:118-131 Sreedhar D, Mehta AR (1984) Ind. J. Biol. 22:345-346 Tamas IA, Schlossberg-Jacobs JL, Lira L, Friedman LB, Barone CC (1989) Plant Growth Regulation 8:165-183 Wright MS, Carnes MG, Hinehee MA, Davis GC, Koehler SM, Williams MH, Colburn SM, Pierson PE (1986) In: Cell Culture and Somatic Cell Genetics of Plants, Vol. 3, Vasil IK, (ed) Academic Press, Orlando. 111-120
Plant Cell Reports (1992) 11:163-168
9 Springer-Verlag1992
Somatic embryogenesis and shoot regeneration from intact seedlings of Phaseolus acutifolius A., P. aureus (L.) Wilczek, P. coccineus L., and P. wrightii L. Kamal A. Malik and Praveen K. Saxena Department of Horticultural Science, University of Guelph, Guelph, Ont. N1G 2Wl, Canada Received January 6, 1992/Revised version received February 14, 1992 - Communicated by E Constabel
A rapid, one-step procedure has been developed for inducing direct organogenesis and somatic embryogenesis in cultures of Phaseolus coccineus L., P. acutifolius A., P. aureus L. [Vigna radiata L. Wilczek] and P. wrightii L. Development of somatic embryos and shoot buds occurred within 6-8 weeks of culture from intact seedlings raised on MS (Murashige and Skoog 1962) medium supplemented with N6benzylaminopurine (BAP). Shoot buds or embryoids originated from subepidermal tissue of the regions adjacent to the shoot apex, hypocotyl and cotyledonary axils. While P. acutifolius and P. aureus were regenerated via shoot formation and P. wrightii by somatic embryogenesis, both embryogenesis and shoot regeneration were observed in P. coccineus. Relatively higher levels of BAP, 50-80/~M, were found to be optimal for inducing regeneration while lower concentrations were ineffective. About 40-70 shoots and 70-250 somatic embryos were produced per responding seedling. Regenerated shoots and somatic embryos developed into whole plants on a basal medium or the one supplemented with 1/zM naphthaleneacetic acid. Abstract.
Key words: N6-benzylaminopurine; Grain legumes; Organogenesis; Phaseolus; Somatic embryogenesis Introduction
An efficient and reproducible procedure for the regeneration of plants from cell and tissue cultures is a prerequisite for the application of gene transfer methods in crop improvement. However, in spite of a considerable progress in developing tissue culture Correspondence to: P.K. Saxena
protocols in a variety of plant species, grain legumes in general, and the species of Phaseolus in particular, have remained recalcitrant to regeneration in vitro (Mroginski and Kartha 1985). Crocomo et al. (1!)76) recovered two plantlets from callus cultures of Phaseolus vulgaris using a medium supplemented with phytohormones and aqueous bean seed extract of an undefined composition. Since then, plant development has been achieved from cultured apical and axillary meristems of P. vulgaris (Kartha et al. 1981; Martins and Sondahl 1984a) and via de novo organogenesis in Phaseolus lunatus (Sreedhar and Mehta 1984), P. coccineus (Allavena and Rossetti 1986) and P. vulgaris (McClean and Grafton 1989; Malik and Saxena 1991; Franklin et al. 1991). Early stages of somatic embryo differentiation were also observed in cell cultures of P. vulgaris (Allavena 1984; Martins and Sondahl 1984b; Saunders et al. 1987) but whole plant regeneration from embryo-like structures was accomplished in P. acutifolius (Kumar et al. 1988) and P. coccineus (Angelini and Allavena 1989). However, the process of regeneration in these studies was slow and produced generally low frequencies of shoot regeneration or somatic embryogenesis. In addition, the protocols :require the use of immature embryonic tissue (Angelini and Allavena 1989) or extensive manipulation of cells (Kumar et al. 1988), for establishing morphogenic cultures. In this communication, we describe a simple procedure for inducing high-frequency organogenesis and somatic embryogenesis from mature seeds of :several Phaseolus species. The process of induction and expression of morphogenesis is accomplished in a single step of
164 direct seed germination in a BAP-supplemented medium. Culture conditions reported here may be generally applicable to other difficult to regenerate plant species. Materials and methods Seed Culture and Plant Regeneration Seeds of Phaseolus coccineus L. were obtained from (A.E. MeKenzie Co., Brandon, Man.) and those of P. acutifolius, P. aureus, and P. wrightii were kindly provided by Dr. T. E. Michaels (Department of Crop Science, University of Guelph, Ont.). Prior to surface sterilization, seeds were immersed in concentrated sulfuric acid for 60 see for softening the waxy seed coat and washed twice with sterile water. Seeds were immersed in ethanol for 1-3 min and then in a 1% solution of sodium hypochlorite for 20 min; during this period they were continuously stirred with a magnetic bar. Thereafter, seeds were washed five times with sterile water, dried on a sterile paper towel, and cultured in Magenta culture vessels (Magenta Corp., Chicago, Ii1., USA; 5 seeds per 300-ml box) containing 45 ml of the culture medium. The nutrient medium contained macro- and micro-nutrients of the Murashige and Skoog (1962) (MS) medium, vitamins according to Gamborg et al. (1968), 3 % sucrose, 0.25 % Gelrite ( Scott Laboratories, Carson, Cal., USA), and Nr-benzylaminopurine (BAP) (0-80 #M). The pH of the media was adjusted to 5.7 with KOH or HC1 prior to autoclaving at 0.122 MPa for 20 rain. All cultures were incubated at 25~ in darkness for the first two weeks and later transferred to light (40-60 #mol.m-2.s~; 16-h photoperiod) provided by cool-white fluorescent lamps (Phillips Canada, Scarborough, Ont.). Regenerated shoots and embryo-derived shoots were excised from the mother seedling after 4-8 weeks of seed germination and subeultured on MS medium containing 1 #M naphthaleneaeetie acid for inducing root formation. Somatic embryos were directly subcultured on MS basal medium for further development. Regenerated shoots developed a prolific root system within four weeks of culture. Rooted plantlets were washed thoroughly with distilled water, transferred to autoclaved peat pellets (2.5 era; Ball superior, Mississauga, Ont.), and after two more weeks of growth in a growth chamber at 24~ under 16-h photoperiod (25-40 /~mol.m-2.sl), were transferred to soil in the greenhouse. For each concentration of BAP, about 50 seeds were raised and those showing uniform germination (usually about 30-40) were considered for the final score. The data presented here are averages obtained from 30 seedlings per treatment and are expressed as percentages of seedlings showing organogenesis and-or somatic embryogenesis and the numbers of shoots or somatic embryos regenerated per seedling. All experiments were repeated three times.
Histology For histological examination, seedling explants bearing regenerated shoot buds of Phaseolus coccineus were excised from the cultures raised on a medium containing 80/xM BAP. Paraffin embedding of tissue samples was performed as described by Johansen (1940). Longitudinal sections of 10 /~ thickness were cut using a Spencer 820 mierotome (American Optical Corporation, Buffalo, NY, USA) and resulting paraffin
ribbons were passed through a series of deparaffinizing solutions followed by staining with Aleian Green and Safranin solution (Johansen, 1940). The sections were examined under light microscope (Zeiss photomieroseope III, Carl Zeiss, Germany).
Results
Germination of seeds of all four species of Phaseolus was not affected by the presence of BAP in the culture medium and occurred within a week. The frequency of seed germination for different species varied between 80 and 100% (Table 1 and 2). Table 1. The efficacy of NLbenzylaminopurine in inducing direct differentiation of shoots in cultures of intact seedlings of Phaseolus acutifolius, 1". aureus, and 1". cocdneus. Species
Conc. Germination
Regeneration"
No.
Shoot (pM)
%
%
per seedling~
30 50 80
85 80 70
20 70 60
5 41 38
30 50 80
90 85 70
-" 78 70
40 52
30 50 80
90 85 85
15 25 85
11 30 73
e. acutifolius
P. aureu$
P. coccineus
~ percentages of seedlings showing regeneration of shoots b= total number of shoots produced from tissues of the
cotyledon axll and shoot apex r
Figs. 1-9. Direct differentiation of shoots and somatic embryos from intact seedling of Phaseolus species. 1. A control seedling. 2. Differentiation of shoots in P. aculifolius from tissues in the regions of shoot apex (arrow) and axils (arrow). 3. Cotyledonary node region of the same shoot-producing seedling (Fig. 2). 4. A embryogenie seedling of P. coceineus. Note the emergence of somatic embryos in the hypoeotyl region (arrow). Cotyledons are denoted by Ct. Fig. 5-7. Various developmental stages of somatic embryos observed in cultured seedlings of P. cor and P. wrightii. Note the presence of heart-shaped (arrows) and eotyledonary somatic embryos. 8. Germination of somatic embryos of P. wrightii to develop into shoots while still attached to the mother seedling. 9. Regenerated plants of P. wrightii. Bars in 1 = 5mm, 2 = 10ram, 3 = 0.5ram, 4 = 2mm, 5 = 1.2ram, 6 = 0.5ram, 7 = 0.5ram, 8 = 2ram, 9 = 15ram.
165
166
Figs. 10-12. Histology of a shoot-producing seedlings of Phaseolus coccineus. 10. and 11. Mierograph of a longitudinal section of the nodal region showing differentiation of shoot buds. Note the initiation (Pig. 10; arrow) and further development (Fig. 11; arrow) of shoot buds in the subepidermal regions and the absence of any connection with the original vascular tissue. 12. A mierograph of longitudinal section from the base of eotyledonary node. Note the presence of well developed shoot buds with leaf primordia (arrow). Bar in 10 = 0.3mm, 11 = 85 #m, 12 = 85/~m
In the initial eight to ten days, no drastic differences were noted in the growth of seedlings grown in media supplemented with or without BAP. All seedlings developed single shoots with cotyledons and a pair of primary leaves. However, root systems showed marked differences: plants grown in medium devoid of BAP had well developed primary and secondary roots which in the presence of BAP showed poor growth. Pronounced differences in growth of seedlings in the presence and the absence of BAP were observed after 4-6 weeks of culture. Control plants were twice the size of those grown in BAP-enriched media and the apical meristem of these plants grew extensively developing many leaves (Fig. 1). The cotyledonary axillary buds also developed into shoots. By contrast, primary shoot apices of seedlings raised on media containing BAP ceased grow, turned yellow, and underwent necrosis. In cultures of P. acutifolius, P. coccineus and P. aureus, several shoot buds differentiated from the tissue surrounding the suppressed apices. Similarly, axillary shoots did not grow further, but the areas adjacent to the cotyledonary node became meristematic and produced numerous shoot buds (Figs. 2 and 3).
The frequency of shoot-producing seedlings and the average number of shoots were highest on media containing 50 or 80/~M BAP (Table 1), while lower concentrations were ineffective (data not shown). About 40-70 shoots were recovered from the seedlings of P. acutifoIius, P. aureus and P. coccineus under optimal culture conditions. The pattern of the development of regenerated shoots was similar in all three species. The seedlings of P. coccineus produced somatic embryos in the hypocotyl region (Fig. 4); somatic embryogenesis occurred 4-6 weeks after the differentiation of adventitious shoots. The seedlings of P. wrightii, on the other hand, showed differentiation of somatic embryos from peripheral tissue of the apical region. In both species, representative developmental stages of somatic embryogenesis ranging from the globular, heartTable 2. Induction o f somatic embryogenesis in cultures of intact seedlings of Phaseolus coccineus and P. wrightii by N 6benzylaminopurine. Species
Cone. Germination Regeneration" No. Somatic embryos per
O,M)
(%)
(%)
seedling~
30 50 80
95 85 80
-r 10 80
70 250
30 50 80
95 100 80
38 90
80 130
P. coccineus
P. wrightii
= Percentageof seedlingsshowing somatic embryogenesis b = Average number of somatic embryos per seedling
167 shaped to cotyledonary stature were present (Figs. (5-7). Somatic embryos matured on the mother seedling and germinated to develop into well formed shoots (Fig. 8). Alternatively, they could be excised, germinated and grown into whole plants (Fig. 9). The differentiation of somatic embryos, like that of shoots, was also induced by higher concentrations of BAP, 50 and 80 #M (Table 2). Lower concentrations (1-40 /zM) evoked no response and at concentrations beyond the optimum (90-120/zM), the shoots or somatic embryos were highly vitrified and showed stunted growth (data not shown). About 50% of the regenerated shoots and somatic embryos were able to develop prolific roots. The plantlets survived transplant to soil and formed apparently normal plants (Fig. 9). Histology of the shoot-producing seedlings revealed that the regenerated shoots had their origin in the subepidermal tissue (Fig. 10 and 11; arrow). Shoot regeneration was found to be direct i.e., without any callus formation and different stages of shoot organization were clearly visible in the longitudinal sections through the shoot-producing region of the cotyledonary node (Figs. 10-12). No direct vascular connections between the mother tissue and the regenerated shoot buds were found to: exist indicating de novo origin of developing shoots. Discussion
Success in regenerating Phaseolus species has been achieved only recently. In the first report of direct differentiation of shoots from bean seedlings, McClean and Grafton (1989) used cotyledonary node explants. In this method, originally developed for soybean (Wright et al. 1986; Hinchee et al. 1988), seeds were germinated in the presence of 5 /~M BAP and the cotyledonary nodes excised after 12-14 days. Axillary buds were removed by scraping the axis and surrounding region prior to culture on a medium of the same BAP complement (5#M), for shoot regeneration. It was proposed that the excision (wounding) may be responsible for meristem initiation, and the presence of BAP, for further development of shoots since the removal of developing buds was found to be associated with continued bud proliferation. Franklin et al. (1991) also used cotyledonary node explants but these included axillary buds and a portion of the cotyledons. Young cotyledonary node explants were shown to produce optimum shoot regeneration in
pea also (Jackson and Hobbs 1990). However, about 4-10 shoots were recovered in these studies. In Phaseolus coccineus, both organogenesis and somatic embryogenesis were reported in cultures of immature cotyledons; a preculture treatment of the explants with abscisic acid was found to be essential for shoot formation and the degree of regeneration was expressed as percentage of explants showing regeneration centres consisting of at least one shoot bud (Allavena and Rossetti 1986; Angelini and Allavena 1989). Earlier, we reported the promotive role of direct culture of seeds in the presence of BAP in subsequent shoot regeneration from leaf explants of Phaseolus vulgaris (Malik and Saxena 1991). In these experiments, a relatively lower concentration of BAP (5 /~M) was used in the medium for seed germination and explant culture. The results of the present study clearly demonstrate that the application of higher concentrations (50 and 80 /xM) of BAP induces a high frequency of organogenesis and somatic embryogenesis in all tested species of Phaseolus (Table 1 and 2). Histological examination revealed that shoots developed de novo from subepidermal tissues of the cotyledonary node. The pattern of shoot origin and development was very similar to that previously described for bean (McClean and Grafton 1989) and pea (Jackson and Hobbs 1990). Thus, it seems that higher levels of BAP during seed culture and the maintenance of morphological integrity of the seedlings may be responsible for high efficiency of regeneration in our experiments. In previous studies, the seeds were germinated either in the absence or in the presence of a low concentration of BAP (5 /zM); concentrations higher than 5 tzM were applied only during the culture of excised explants and were found to have action maxima between 5-15 /zM. The relationships among high cytokinin concentration, structural intactness of the seedlings and observed de novo differentiation of shoots and somatic embryos are being investigated in our laboratory. An interesting aspect of this study is the simultaneous occurrence of organogenesis and somatic embryogenesis in P. coccineus. Angelini and Allavena (1989) also reported the development of shoot buds and somatic embryos in response to 2-naphthoxyacetic acid and 2-isopentenyladenine. In our experiments, both shoots and somatic embryos developed in the presence of BAP alone. According to the theory of hormonal regulation of
168 morphogenesis, all types of growth and differentiation responses are mediated by relative amounts of growth regulators, mainly phytohormones (Skoog and Miller 1957). It is likely that the auxin:cytokinin complement required for regeneration of shoots or somatic embryos becomes available to the competent cells of the seedling as a result of exogenous application of BAP and endogenous auxins. Biosynthesis and transport of auxin(s) have been shown to influence the growth of axillary buds in cultured stem segments of Phaseolus vulgaris (Tamas et al. 1989). Besides the typical auxin:cytokinin combinations, another growth regulator, thidiazuron- a substituted phenylurea known to elicit cytokinin-like activity- also induced regeneration of shoots and somatic embryos from bean and geranium seedlings, respectively, suggesting the involvement of endogenous phytohormones (Malik and Saxena 1992; Saxena et al. unpublished). Although we have been able to apply seed culture method for regenerating a number of crop species, the mechanism of the induction and expression of morphogenesis from intact seedlings remains unresolved. In conclusion, the present study describes a simple procedure for inducing organogenesis, somatic embryogenesis, and plant regeneration in three Phaseolus crops, P. coccineus, P. acutifolius, and P. aureus and a wild bean, P. wrightii. Since the differentiation occurs on an intact seedling, the number of manipulations required to induce regeneration is reduced to one in comparison to several in procedures involving culture of immature embryo, leaf or seedling explants and callus (Mathews 1987; Kumar et al. 1988; Angelini and Allavena 1989). Moreover, the use of mature seeds (present investigation) avoids labour-intensive optimization of factors associated with explant culture and, thus, may significantly reduce the cost of maintaining the source plants in the green house. In addition, direct morphogenic differentiation offers many advantages as an experimental system, for example, in the study of biochemical and molecular events in organ determination and development, and the mechanism of action of growth hormones involved. The rapidity and high frequency of direct morphogenesis routinely obtainable in seed cultures are expected to facilitate stable, Agrobacterium-mediated or direct, genetic transformation of Phaseolus species in which success has so far been rather limited (Leon et al.
1991; McClean et al. 1991).
Acknowledgements. This research was supported by operating grants from the Natural Sciences and Engineering Research Council of Canada. We thank Dr. R. Gill for helpful discussions and S. Saxena for technical assistance. References Allavena A (1984) In: Handbook of Plant Cell Culture, WR Sharp, DA Evans, PV Ammirato, Y Yamada (eds) Macmillan, New York. 137-168 Allavena A, Rossetti L (1989) Ann. Rep. Bean Improv. Coop. 29:132-133 Angelini RR, Allavena A (1989) Plant Ceil Tissue Organ Culture. 19:167-174 Crocomo OJ, Sharp WR, Peters JC (1976) Z. Pflanzenphysiol. 78:458460 Franklin CI, Treveu N, Gronzales RA, Dixon RA (1991) Plant Cell Tissue Organ Culture 24:199-206 Gamborg OL, Miller RA, Ojima K (1968) J. Exp. Res. 50: 151-158 Jackson JA, Hobbs SLA (1990) In Vitro Cell Dev. Biol. 26: 835-838 Johansen DA (1940) Plant mieroteehnique. McGraw-Hill, New York Hinehee MAW, Connor-Ward DV, Newell CA, MeDormell RE, Sato SJ, Gasser CS, Fisehhoff DA, Re DB, Fraley RT, Horsch RB (1988) Bio/Teehnol. 6:3915-3922 Kartha KK, Pahl K, Leung NL, Mroginski LA (1981) Can. J. Bot. 59:1671-1679 Kumar AS, Gramborg OL, Nabors MW (1988) Plant Cell Rep. 7:322-325 Leon P, Planckaert F, Walbot V (1991) Plant Physiol. 95: 968972 Malik KA, Saxena PK (1991) Planta 184:148-150 Malik KA, Saxena PK (1992) Planta (in press) Martins IS, Sondahl MR (1984a) Turrialba 34:157-161 Martins IS, Sondahl MR (1984b) J. Plant Physiol. 117:97-103 Mathews H (1987) Plant Cell Tissue Organ Culture 11:233-240 McClean P, Chee P, Held B, Semental J, Drong RF,SlightomJ(1991) Plant Ceil Tissue Organ Culture 60:131-138 MeClean P, Grafton KF (1989) Plant Sei. 60:117-122 Mroginski LA, Kartha KK (1985) Plant Breed. Rev. 2:215-264 Murashige T, Skoog F (1962) Physiol. Plant. 15:473-497 Saunders JW, Hosfield GL, Levi A (1987) Plant Cell Rep. 6: 46-49 Skoog F, Miller, CO (1957) Sym. $oe. Exp. Biol. 11:118-131 Sreedhar D, Mehta AR (1984) Ind. J. Biol. 22:345-346 Tamas IA, Schlossberg-Jacobs JL, Lira L, Friedman LB, Barone CC (1989) Plant Growth Regulation 8:165-183 Wright MS, Carnes MG, Hinehee MA, Davis GC, Koehler SM, Williams MH, Colburn SM, Pierson PE (1986) In: Cell Culture and Somatic Cell Genetics of Plants, Vol. 3, Vasil IK, (ed) Academic Press, Orlando. 111-120
