Plant Cell Reports (1981) 1 : 64-66

PlantCell Reports

© Springer-Verlag 1981

Regeneration of Pea (Pisum sativum L. cv. Century) Plants by in vitro Culture of Immature Leaflets* L. A. Mroginski** and K. K. Kartha Prairie Regional Laboratory, National Research Council, Saskatoon, Saskatchewan, Canada S7N OW9 Received September 16, 1981 ; October 21, 1981

ABSTRACT In v i t r o regeneration of plants from immature l e a f l e t s of 3 day-old pea (Pisum sativum L. cv. Century) seedlings was stucIi-e-d--u-n~defined n u t r i t i o n a l , hormonal and environmental conditions. Immature l e a f l e t s isolated from the second and t h i r d apical leaves of a s e p t i c a l l y germinated seeds were cultured on MS medium containing vitamins as in B5 medium, 3% sucrose, 0.8% agar and supplemented with O.l, l , and IO ~M concentrations of naphthaleneacetic acid (NAA) and l and lO vH levels of benzyladenine (BA) in various combinations. Shoot regeneration from the primary callus occurred w i t h i n 45 to 90 days of c u l t u r e in most of the hormone combinations. Although the number of c a l l i producing shoots was maximal at lO ~M levels of NAA and BA, m u l t i p l e shoot regeneration was predominant at a combination of O.l ~M NAA and I0 ~M BA. Indoleacetic acid (IAA) and k i n e t i n (K), both at lO uH, also induced shoot regeneration. No shoots were regenerated when lO day-old l e a f l e t s were used as explants. Root production generally occurred on non-shoot regenerating calli. Roots were induced to d i f f e r e n t i a t e by t r a n s f e r r i n g the regenerated shoots onto h a l f - s t r e n gth B5 medium supplemented with 1 ~H NAA. INTRODUCTION One of the basic requirements for employing the technique of t i s s u e , c e l l and protoplast c u l t u r e in plant breeding is to e i t h e r make use of or devise an in v i t r o system which would permit whole plant regeneration. With pea, successful plant regenerat i o n from in v i t r o cultured meristems (Kartha et a l . , 1974) made i t possible to extend the technique towards virus e l i m i n a t i o n (Kartha and Gamborg, 1979) and germplasm preservation by cryogenic methods (Kartha et a l . , 1979). However, the f a i l u r e in achieving plant regeneration in pea through e i t h e r organogenesis or embryogenesis when explants other than meristems were used (McComb, 1977; Jacobsen et a l . , 1980) res t r i c t e d the a p p l i c a t i o n of in v i t r o techniques aimed at induction of genetic v a r i a b i l i t y and selection f o r crop improvement. Attempts have been made in the past to resolve

t h i s problem. Two reports described procedures for inducing plant regeneration in pea tissue culture. Although plants have been successfully regenerated by c u l t u r i n g macerated shoot apices (Gamborg et a l . , 1974) and epicotyl segments of germinated seeds (Halmberg, 1979), i t was not certain whether or not the shoots were induced de novo or arose from pree x i s t i n g meristems or meristematic l o c i of the explants. Shoot formation de novo may have been realized with a v a r i e t y of legume tissue cultures including pea (Thomas and Wernicke, 1978; Kameya and Hidholm, 1981). The present i n v e s t i g a t i o n was prompted by the observation of Haskins and Kartha (1980) that the primordial leaves of cryopreserved pea meristem-explants were p r e f e r e n t i a l l y a l i v e and that the constituent c e l l s of these leaves had the propensity f o r d i v i s i o n and eventual d i f f e r e n t i a t i o n into shoots in v i t r o . This paper describes the c u l t u r a l conditions employed in regenerating pea plants in v i t r o by c u l t u r i n g immature l e a f l e t s under defined environmental conditions. MATERIALS AND METHODS Seeds of pea (Pisum sativum L. cv. CenturY) were surface s t e r i l i z e d in 70% ethanol for 2 min., f o l lowed by immersion in 1.2% sodium hypochlorite on a gyratory shaker (150 rpm) for 20 min., and subsequently rinsed several times with s t e r i l e double dist i l l e d water. The seeds were germinated in s t e r i l e lO0 ml glass jars on humidified cotton (6-7 seeds/ j a r ) in the dark at 26°C. Seedlings, 3 days old, were again disinfected by immersion in 0.6% sodium hypochlorite for lO min. on a gyratory shaker (150 rpm) and were thoroughly washed 3 times in double d i s t i l l e d water. Yellowish-white immature l e a f l e t s from the second and t h i r d apical leaves were aseptic a l l y dissected under a stereomicroscope. Approximately two-thirds of t h e i r d i s t a l portion were placed in lO0 ml glass jars (2-4 e x p l a n t s / j a r ) on 33 ml of n u t r i e n t medium s o l i d i f i e d with 0.8% agar. The jars were covered with screw caps, sealed with Parafilm MR and incubated at photoperiods of 16 h (40 l~.m-2 supplied by fluorescent and incandescent lamps), 20°C and 70% r e l a t i v e humidity. In one experiment l e a f l e t s

*NRCC # 19712 ** V i s i t i n g S c i e n t i s t , supported by a research f e l l o w s h i p from "Consejo Nacional de Investigaciones Cient{ficas y T6cnicas (Rep. Argentina)". Permanent address: Facultad de Ciencias Agrarias, IBONE, Casilla de Correos 209, Corrientes (3400), Argentina.

0721-7714/81/0001/0064/$ 01.00

65 of lO day-old seedlings (seeds germinated in the dark and subsequently transferred to l i g h t ) were employed as source of explants. The n u t r i e n t medium consisted of major and minor salts a f t e r Hurashige and Skoog (1962), 3% sucrose, 0.8% Difco agar and vitamins as in B5 medium (Gamborg et a l . , 1968). Naphthaleneacetic acid (NAA) or 3indoleacetic acid (IAA) and 6-benzyladenine (BA) or k i n e t i n (K) were added in various combinations and concentrations. The pH of the medium was adjusted to 5.8 with NaOH or HCl p r i o r to adding the agar. The jars were covered with screw caps and autoclaved at 1.46 kg/cm2 f o r 20 min. Wherever IAA was employed, : i t was added to the medium f i l t e r - s t e r i l e . Each treatment consisted of 4 r e p l i c a t e s and each experiment was repeated at least 3 times. RESULTS AND DISCUSSION

Figs. I-4. Regeneration of pea plants by in v i t r o culture of immature l e a f l e t s . Bar represents l cm. Fig. I. Bud regeneration in lO ~M each of IAA and K. Fig. 2. M u l t i p l e shoot regeneration in O.l ~H NAA + lO ~M BA a f t e r 80 days of culture. Fi 9. 3. Root formation from callus in lO ~M NAA + l vM BA a f t e r 80 days of culture. Fig. 4. Root formation on i n d i v i dually excised and subcultured shoots on h a l f - s t r e n gth B5 medium supplemented with l mM NAA.

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The e a r l i e s t v i s i b l e response, w i t h i n 3-5 days of culture, was a gradual greening in over 60-90% of the explants. This process was a t t r i b u t a b l e to the concentration of NAA and BA; the higher the concentrat i o n , the stronger the greening, and viceversa. Further growth of the green explants i n t o callus was slow, a f t e r l month of culture the r e l a t i v e size of callus to the o r i g i n a l explants was in order of 3 or 5. Within 45 days structures resembling bud primordia were detected in some c a l l i , e s p e c i a l l y those on media supplemented with lO ~H each of NAA and BA or IAA and K (Fig. l ) . Shoot production occurred w i t h i n 45-90 days of c u l t u r e , they were mostly regenerated in clusters (Fig. 2). In some c a l l i , roots were formed, but in most cases rooting occurred on c a l l i which did not produce shoots (Fig. 3). Figure 5 summarizes the morphogenetic responses of explants to 3 concentrations of NAA and 2 concentrations of BA a f t e r 90 days of culture. I t appeared that both NAA and BA must be supplied ill adequate levels in order to obtain the highest production of c a l l i capable of regenerating shoots. Under the present experimental conditions the percentage of c a l l i with shoots was greatest when NAA and BA were employed at lO vH levels. Root formation only occurred in 2 out of 6 combinations of NAA and BA and, in both cases, higher levels of NAA (lO ~H)were used. As regards shoot formation, NAA and BA at O.l ~H and lO ~M l e v e l s , r e s p e c t i v e l y , were more e f f e c t i v e than other hormone levels (Fig. 6). Independent of NAA concentrations used, a concentration of BA at lO ~H was more e f f e c t i v e for m u l t i p l e shoot regenera-

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Fig. 5. Morphogenetic response of pea l e a f l e t explants to combinations of NAA and BA a f t e r 90 days of culture.

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Fig. 6. Effect of various combinations of NAA and BA on the number of shoots per callus, a f t e r 90 days of culture. Only shoots of more than l cm in length were scored.

66 Table l .

Effect of the age of the pea seedlings used for explants on shoot regeneration from primary callus (cultured with lO ~N each NAA and BA)

Age of seedlings (days)

leaflet length (mm)

number of l e a f l e t explants cultured

l e a f l e t explants forming c a l l i + shoots (%)

3

0.7-2.0

32

37.5

lO

lO-15

24

0.0

tion than at the l ~M l e v e l . Table l summarizes the results obtained a f t e r 75 days of cultu{e when explants of 3 and lO day-old l e a f l e t s were incubated with media containing lO ~M each of NAA and BA. Shoot regeneration occurred from the 3 day-old explants only. Since root production on regenerated shoots did not occur, i t became necessary to subculture the shoots on h a l f - s t r e n g t h B5 medium supplemented with l ~rl NAA according to Kartha et al. (1974). On t h i s medium rooting of regenerated shoots was r e a d i l y achieved (Fig. 4) and, a f t e r 30 days, the p l a n t l e t s were transplanted to s o i l . In conclusion t h i s work demonstrates, for the f i r s t time, a procedure f o r the successful regeneration of pea plants by in v i t r o c u l t u r e of explants from immature l e a f l e t s u~der defined conditions. This process is r e a d i l y achieved in two sequential stages: i ) shoot regeneration from callus and i i ) induction of roots on regenerated shoots. The e n t i r e process of producing transplantable pea p l a n t l e t s takes about 4-4½ months. The age of the l e a f l e t s appeared to be the c r i t i c a l factor. I t is d i f f i c u l t to f i n d an explanation for t h i s phenomenon except to postulate possible differences in the physiological status among both kinds of explants employed. The r e s u l t s reported in t h i s paper concerning the potential of the primordial leaves to regenerate in v i t r o into p l a n t l e t s are in agreement with the observation of Haskins and Kartha (1980). Moreover, s i m i l a r results were obtained with peanut (Arachis hypogaea), i . e . , p l a n t l e t s were regenerated in v i t r o by culture of immature l e a f l e t s (Mroginski et a l . , 1981). However, immature l e a f l e t s of other grain legumes such as chickpea (Cicer arietinum), cowpea ( ~ u n g u i c u l a t a ) bean (Phaseolus v u l q a r i s ) , and soybean (Glycine max) did not e x h i b i t the propensity for shoot regeneration in culture (our unpublished r e s u l t s ) . Therefore, the morphogenetic capacity of the immature leaves may be r e s t r i c t e d to certain grain legumes and not be a p p l i cable in general.

ACKNOWLEDGEMENTS The authors wish to express t h e i r appreciation to A. Lutzko and E. Knapp f o r the i l l u s t r a t i o n s . REFERENCES Gamborg OL, M i l l e r RA, Ojima K (1968) Exp. Cell Res. 50: 151-158 Gamborg OL, Constabel F, Shyluk JP (1974) Physiol. Plant. 30: 125-128 Haskins RH, Kartha KK (1980) Can. J. Bot. 58: 833840 Jacobsen HJ, Ingensiep HW, Herlt M, Kaul NLH (1980) In: Sala F, Parisi B, Cella R, C i f e r r i 0 (eds.) Plant Cell Cultures: Results and Perspectives, Elsevier/North Holland Biomedical Press. pp 319324 Kameya T, Widholm J (Ig81) Plant Sci. Lett. 21: 289294 Kartha KK, Gamborg OL, Constabel F (1974} Z. Pflanzenphysiol. 72: 172-176 Kartha KK, Gamborg OL (1979) In: Maraite H, Meyer JA (eds.) Diseases of t r o p i c a l food crops, Univers i t e Catholique de Louvain, Belgium, pp 267-283 Kartha KK, Leung NL, Gamborg OL (1979) Plant Sci. Lett. 15: 7-15 rlalmberg RL (1979) Planta 146: 243-244 McComb AJ (1977) In: S u t c l i f f e JF, Pate JS (eds.) The physiology of the garden pea, Academic Press, London, pp 235-263 Mroginski LA, Kartha KK Shyluk JP (1981) Can. J. Bot. 59: 826-830 Murashige T, Skoog F (1962) Physiol. Plant. 15: 473497 Thomas E, l~ernicke I~ (1978) In: Thorpe TA (ed.) Frontiers of Plant Tissue Culture 1978. Univers i t y of Calgary, Canada, pp 403-410