Plant Cell Reports (1992) 11:44 47
9 Springer-Verlag 1992
High frequency adventitious shoot regeneration from immature cotyledons of pea (Pisum sativum L.) Sebahattin Ozcan, Mehdi Barghchi, Simon Firek, and John Draper Department of Botany, University of Leicester, Leicester LEI 7RH, U K Received September 26, 1991/Revised version received December 2, 1991 - Communicated by I. Potrykus
ABSTRACT A procedure has been developed which allows high frequency adventitious shoot regeneration from immature cotyledons of pea. Prolific shoot regeneration occurred following an initial callus growth on a Murashige and Skoog (MS) medium containing 0.5 mg/l 6-benzyleminopurine (BAP) and 4 mg/l ~-naphthaleneacetic acid (NAA). Cotyledon explants proximal to the embryonic axis had the highest regeneration potential,however, the presence of an embryonic axis inhibited adventitious shoot regeneration. Addition of silver nitrate (AgNOs) to the medium did not promote the number of regenerated shoots but resulted in shoots with well developed tendrils and large stipules which had a reduced rooting capacity. Regenerated shoots rooted readily (80-90~) in half strength MS medium containing 1 mg/l indole-butyric acid (IBA) and further established well in compost.
INTRODUCTION C o n v e n t i o n a l s e e d legume b r e e d i n g programmes can be improved and complemented w i t h i n v i t r o g e n e t i c manipulation methods if an efficient plant regeneration is available. Adventitious shoot regeneration and somatic embryogenesis has been reported for pea in recent years (Gemborg et al. 1974, Mroginski and Kartha 1981, Hussey and Gunn 1984, Rubluo et al. 1984, Kysely et al. 1987, Natali and, Cavallani 1987, Puonti-Kaerlas and Erikson 1988, Lehminger-Martens and Jacobsen 1989, Kysely and Jacobsen 1990, De Kathen and Jacobsen 1990, Puonti-Kaerlas et al. 1990, Nauerby et al. 1991) from a range of explants (immature leaflets, shoot apices, epicotyls, protoplasts, nodal thin cell layer segments, immature embryos and cotyledons) cultured in vitro. However, in these reports regeneration was rather slow and had a low frequency. Although transgenic fertile pea plants have been reported previously (Puonti-Kaerlas et a2. 1990), the regeneration system they describe required many steps on media with different hormone combinations over several months. Therefore, inefficient regeneration systems seem to be the greatest barrier to the efficient production of transgenic pea plants. The present research aims are to provide tissue culture techniques for the potential genetic manipulation of pea through i~robacteriuu~mediated transformation or direct gene transfer techniques and for somaclonal variation studies in the future. This paper reports the morphogenic potential of cotyledon explants at various developmental stages.
Offprint requests to: J. Draper
MATERIALS AND METHODS Plant material: Pea cultivars "Orb" and "Consort", supplied by Sharpes International Limited, UK were raised in a greenhouse under a 18/6 hour light/dark (Osram high pressure sodium lamps, 400W) and a 22/16 "C day/night temperature regime. Seed pods were harvested at various stages of development and sterilized in 20~ commercial bleach ("Domestos") for 20 min in 500 ml glass jars and then washed 3 times in sterilized tap water. Seeds were removed from the sterile pods in a laminar air flow hood. After removal of the seed coat, the embryonic axis was gently prised off the split seed and axillary meristems at the area of attachment of the cotyledons to the embryo axis were removed under a dissecting microscope. The whole, or parts of the cotyledons, ranging in size from 1 to 9 mm and also the embryonic axes (Fig. i) were cultured on an appropriate medium for adventitious shoot regeneration. The cultures were grown at 24 "C under cool white fluorescent light (4000 lux) with a 16 h photoperiod. Shoots were excised and rooted in agar-solidified medium (xl/2 MS) supplemented with indole-butyric acid (IBA). For regeneration experiments each treatment had three replicates consisting of 100XI0 mm Petri dishes each containing 5-10 cotyledon explants. For rooting experiments each treatment had three replicates consisting of 125 ml glass jars containing 3-5 explants. All experiments were repeated at least once and the results were pooled. ~dia: MS mineral salts and vitamins (Murashige and Skoog 1962) containing 0.7% agar and 3~ sucrose were used as a basal medium. This basal medium was supplemented with 0-4 mg/1 6-benzylaminopurine (BAP), and 0-8 mg/l ~-naphthaleneacetic acid or 0-8 mg/l IBA. Silver nitrate (AgNO3) (5 and i0 mg/l) was also added to some media to study its effect on morphogenesis in pea cotyledons. The medium was adjusted to pH 5.6 with IN NaOH or IN HCI before
a u t o c l a v i n g a t 120 "C, 1.4 kg/cm z f o r 20 min. RESULTS Shoot regeneration potential o f different pea organ ewplauts. Various organ explants (at different
developmental stages) were investigated in order to establish efficient adventitious shoot regeneration. Hoot, epicotyl segments, immature leaflets and shoot tips from in vitro grown seedlings, and mature and immature embryos and cotyledons from green. house-grown plants were examined. The effect of varying the concentration of cytokinins and auxins in the growth media was also investigated.
Figure I. Different immature embryo parts cultured for adventitious shoot regeneration. A. Embryonic axis B. One-third proximal part of cotyledon with embryonic axis. C. One-third proximal part of cotyledon. D. Two-third distal part of cotyledon. E. Whole cotyledon. Figure 2. Adventitious shoot initials preceded by as early stage of callus growth at the proximal end of cotyledon. Bar=Imm.
Figure 3. Developing prolific adventitious shoots. Bar=1 mm. Figure 4. Elongated adventitious shoots with very well developed tendrils and larger stipules on the cotyledons cultured on the regeneration medium containing 5 mg/l AgNO 3. Bar=5 mm. Figure 5. Excised shoots growing on shoot elongation medium with 5 mg/l AgNO s (left) and without AgNO s. Bar=l cm.
Adventitious shoots were not observed on root, epicotyl and shoot tip explants from in vitro grown seedlings but very good callus growth occurred on most explasts. Organogenesis and embryogenesis from shoot apices, using different cultivars and media to those used in the present study, have already been reported (Gamborg et al 1974, Kysely et a L 1987, Kysely and Jacobsen 1990). However, our preliminary experiments showed that shoot growth from the existing meristems on shoot tip explants suppressed any adventitious shoot development. A small number of adventitious shoots were regenerated on 25~ of leaflet explants. Rapid and prolific adventitious shoot development occurred on the im~Bature cotyledon and therefore it was decided to concentrate on this explant. The orientation of the cotyledon explants to the medium surface did appear to influence shoot development; with the distal end placed onto the agar, the regeneration frequency increased, but it was found to be essential to ensure that the explants were always well in contact with medium. Incubation in the dark and changes in sugar concentration did not appear to influence shoot development. Effect Of deve]ol~-J~ta] stage and t.~]~ o f cotyJedoa explant o n shoot regeneration. Whole cotyledon explants smaller than 3 mm across bleached and did not grow, whilst fully mature cotyledons produced only a little callus and no shoots in vitro. Shoot
organogenesis was best achieved in cotyledon explants which had grown to their full size (approximately 15 days after pollination) but were still green and very soft in texture. Any remaining axillary meristems developed into large shoots within 4-5 days in culture and were easily recognised and discarded. Within 5 days callus appeared at the wound site (where the embryonic axis had been dissected off) and shoot initials were present within 10-15 days (Fig. 2). Shoot regeneration was always preceded by an early stage of callus growth which appeared Table I. Adventitious shoot regeneration from different part of immature cotyledons and embryonic axis of pea (P. sativum cvs Orb. and Consort) after 5-6 weeks in culture on MS media supplemented with 0.5 mg/l BAP and 4 mg/l NAA. Explant (See Fig. i)
Explant Explant Explant Explant Explant
A B C D E
% cotyledons responding•
Mean no. of shoots /cotyledon*•
0 , 15• 81• 13• 75•
0 12• 75• 0 77•
0.0 2.9• 15.6• 2.7• 9.4•
*Each value represents the mean • replications each with 5-10 explants. ~From cotyledons which regenerated shoots
0.0 2.7• 11.7• 0.0 8.0 SE
46 predominantly at the wound site (Fig. 2). This compact morphogenic callus was yellowish to green in colour. Shoot formation was highest in the region of the cotyledon proximal to the embryonic axis, indicating a polarity in the regeneration potential of cotyledon explants (Fig. I, Table I). The presence of the embryonic axis (Fig. i, explant B) inhibited adventitious shoot regeneration. Two-third distal parts of cotyledons (Fig. I, explant D) produced less shoots than one-thlrd proximal parts of cotyledon (Fig. i, explant C) where prolific shoot regeneration occurred (Fig. 3). Effect o f Bs~z~, Nat4 end IBA c o n c e n t r a t i o n s on s h o o t development from immature pea cotyledons. Adventitious shoots developed in a range of media supplemented with BAP and NAA or IBA (Table 2). NAA was superior to IBA; high concentrations of IBA also appeared to increase the frequency of stunted shoots. The highest shoot regeneration capacity was achieved in a medium supplemented with 0.5 mg/l BAP and 4 mg/l NAA in cultivar "Orb". BAP alone also stimulated shoot development. In some cases, especially with high BAP and low NAA or IBA, a few shoots became dominant and then inhibited the elongation of others. In some explants, especially in the media with low BAP and high NAA or IBA concentrations, a very large number of shoot buds were produced but shoot elongation was delayed. With respect to cultivars, Orb seemed to have a better regeneration capacity than Consort (Table 2). Shoot elongation. Shoot elongation was best achieved when the regenerating part of the explant was cut into smaller pieces containing 3-4 shoots and subcultured to the basal medium supplemented with 1 mg/l BAP and 0.25 mg/l NAA for further growth. The developing shoots had sturdy growth end small leaves or stipules. Effect of AKNOs on shoot regeneration. In recent years, there has been increasing evidence that growth and differentiation of plant cells and tissues in vitro can be affected considerably by the action of ethylene. It has been suggested that AgNO s stimulates adventitious shoot regeneration by inhibiting ethylene action. It was found that addition of AgNO s had no effect on shoot numbers but increased the number of elongated shoots on explents Table 2. Adventitious shoot regeneration from immature cotyledon explants of pea (P. sativum cvs. Or5 and Consort) after 5-6 weeks in culture on MS media supplemented with growth regulators. Growth regulator (mg/l)
% cotyledon responding•
0 2 4 0.25 1 4 4 0.5 i 0.5 1 0.25 1 4 4 0.5 I 0.5 1
0 0 0 0.25 0.25 0.25 1 4 4 8 8 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0.25 0.25 0.25 1 4 4 8 8
50• 88• 90• 79• 82• 85• 83• 77• 78• 93• 56• 82• 72• 78• 81• 74• 54• 67• 60•
Consort 58• 86• 85• 45• 87• 66• 65• 68• 66• 69• 51• 62• 68• 80• 83• 72• 58• 62• 57•
Mean no. of shoots /cotyledon*'Z• Orb 2.2• 10.0• 9.1z2.1 10.6• 8.7• 12.1• 10.3• 17.5• 15.8• 9.0• 9.7• 5.3• 7.4• 8.2• 8.4• 4.6• 4.1• 5.4• 5.3•
Consort 3.9• 6.0• 5.9• 4.3• 6.5• 8.5• 11.2• 8.1• 9.1• 8.0• 6.1• 5.2• 4.9• 5.6• 7.2• 4.4• 5.4• 4.7• 4.7•
*Each value represents the mean • SE of 6 replications each with 5-10 explant. ~0ne-third proximal part of the cotyledon (Fig. I, explant C) was used as cultured explant. ZFrom cotyledons which regenerated shoots.
containing adventitious shoot primordia in "Orb" end "Consort" cotyledons (Fig. 4). However, shoots in the presence of AgNO s developed larger stipules end tendrils in vitro (Fig. 4 end 5). Root development in regenerated shoots. Regenerated shoot tips were excised (10-20 mm length) and rooted readily within 7-10 days in half strength basal medium supplemented with 0.25-1 mg/l IBA (Table 3). Rooted shoots established well in potting mix. Many of the shoots regenerated on the medium containing AgNO s did not produce any roots and root numbers per shoot were lower (Table 3). Table 3. In vitro rooting of pea (P. satlvum cv. Orb) after 2 weeks in half strength MS medla supplemented with IBA. IBA (mg/l) 0.01 0.25 I 1.01 2.5* 1.02
% rooting 65• 82• 90• 74• 25•
Root no. /shoot 2.4• 3.4• 3.3• 3.3• 2.0•
Root length(mm)/ shoot 76• 96• 40• 26• 19•
*Each value represents the mean • SE of 6 replications each with 3-5 explants. IShoots regenerated on MS medium with I mg/l BAP and 0.25 mg/l NAA Zshoots regenerated on MS medium with i mg/l BAP , 0.25 mg/l NAA and 5 mg/l AgNO 3. DISCUSSION Adventitious shoot regeneration from cotyledon explants has been reported in pea (Natali and Cavallini 1987) and in several other species (Angelini and Allavena 1989, Mante et 81. 1989) indicating the potential of cotyledons as a suitable material for plant regeneration. In the present study shoot regeneration best occurred from callus produced on the one-third part of the cotyledon explant which was proximal to the embryonic axis (see Fig. I, explant C). Callus produced on the two-third distal part cotyledon explants regenerated only a few shoots (Fig. I, explant D). This indicated that the origin of explant is vitally important in establishing an efficient regeneration method in pea. It also suggests that a polar phenomenon affecting morphogenesis exists in the pea cotyledon explants. Similar observations were made in apple and soybean cotyledons (Kouider e t a ] . 1984, Manta et al. 1989). The reason for the polarity of regeneration potential in these explants is not clear. Maximum shoot regeneration was achieved using fully grown cotyledons prior to full maturity and not from younger or mature cotyledons as reported for bean (Angelini end Allavena 1989). This indicates that the developmental stage of the explant has a crucial effect on its response to regeneration treatments. The influence of the developmental stage of zygotic embryo explants on somatic embryogenesis in other seed legumes (Lippman and Lippman 1984, Lazzeri et a]. 1985, Ranch eta]. 1985, Barwale eta]. 1986) and pea has been reported previously (Kysely and Jacobsen 1990). Adventitious shoot regeneration was inhibited by the presence of the embryonic axis similar to reports i n soybean (Mante et a]. 1989), although some callus and adventitious shoots developed on the site where the cotyledons were attached to the embryonic axis (Explant B and Table I). The reason for this inhibition is not clear, but factors relating to dominancy of the axis or the presence of vascular connections of the cotyledons to the axis could be partly responsible. The embryo axis (Fig. I, explant A and Table I) produced mainly callus at the basal end of the embryo. Some green meristematic spots were visible but they failed to develop into proper shoot primordia. In contrast, Natali and Cavallini
47 (1987), using a similar medium and a material from field grown plants found that embryo axes gave a better response than cotyledons for shoot regeneration in pea. In this report a maximum 37~ of the embryo axis produced shoots but the number of the shoots regenerated per explant was not stated. They also reported that morphogenetic potential of pea immature embryo and cotyledon explants is often dependent on the genotype used, and the two cultivars used in the present study were not included in this previous work. The origin of the material, different cultivars and the excision method they used might have resulted in a poor regeneration response in cotyledons. It was somewhat suprising that one-third proximal part of cotyledons were very responsive for shoot regeneration on a range of medium, including media with a high auxin to cytokinin in ratio (Table 2). A very similar result was found in another legume, mung bean (Mathews 1987). In this study the highest regeneration response (80~) was observed at the proximal end of the cotyledon on a MS medium with a high auxin (5 ~M BAP) to cytokinin (20 ~M IBA) ratio. The reason why shoot regeneration is so high on media with a high auxin to cytokinin ratio could be related the fact that there is no direct contact between the medium and the wound surface where regeneration occurs.
The regeneration of shoots from callus tissue has been improved in some species by addition of AgNO z to the medium in order to inhibit ethylene action (Purnhauser et 81. 1987, C h i e t al. 1990, Roustan et al. 1990). It has been suggested that in non-morphogenic callus cultures, auxin - induced ethylene production may be responsible for the suppression of shoot regeneration. However AgNO 3 did not improve the number of adventitious shoots in pea but tended to produce shoots with large, well developed stipules and tendrils, which are morphological characteristics of a more mature plant. The poor rooting response of shoots grown in the presence of AgNOz was also been noted in Brassica juncea cv. India Mustard ( C h i e t al. 1990). In many species it is common for mature shoots to have a poorer rooting potential as compared to juvenile material. The possible effect of AgNO s on the physiological or morphological characteristics of pea has not been reported before. The procedure we have developed for the adventitious regeneration of shoots from immature cotyledons of pea is simple, rapid and reproducible. Large numbers of shoots (over 30/explant) can be produced from an initial callus growth on the cotyledon within 3-4 weeks. These features make this an ideal regeneration system to be adopted in transformation and somaclonal variant selection studies.
The authors gratefully acknowledge receipt of experimental and financial assistance from Sharpes International Limited, UK. S. Ozcan was supported by a grant from the University of Ankara, Turkey. REFERENCES A n g e l i n i P~, A l l a v e n a A (1989) P l a n t C e l l , T i s s u e and Organ C u l t u r e 19:167-174 Barwale UB, Kerns HR, Widholm JM (1986) Planta 167:473-481 Chi G-L, Barfield DG, Sim GE, Pau EC (1990) Plant Cell Reports 9:195-198 De Kathen A, Jacobsen H-J (1990) Plant Cell Reports 9:276-279 Gamborg OL, C o n s t a b e l F, Shyluk JP (1974) P h y s i o l . Plant. 30:125-128 Hussey G, Gunn HV (1984) Plant Science Letters 37:143-148 Kysely W, Myers JR, Lazzeri PA, Collins GB, Jacobsen H-J (1987) Plant Cell Reports 6:305-308 Kysely W, J a c o b s e n H-J (1990) P l a n t C e l l , T i s s u e and Organ C u l t u r e 20:7-14 Koulder M, Korban SS, S k i r v i n I~M, ChuMC (1984) Ame~ Soc.Hort. Scl 109(3):381-385 L a z z e r i PA, H i l d e b r a n d DF, C o l l i n s GB (1985) P l a n t Mol. B i o l . Rep. 3 ( 4 ) : 1 6 0 - 1 6 7 Lehminger-Martens R, Jacobsen H-J (1989) P l a n t C e l l Reports 8:379-382 Lippman B, Lippman G (1984) Plant Cell Reports 3:215-218 Mante S, Scorza E, Cordts J (1989) In V i t r o C e l l u l a r & Developmental Biology 2 5 ( 4 ) : 3 8 5 - 3 8 8 Mathews H (1987) Plant Cell, Tissue and Organ Culture 11:233-240 Mroginski LA, Kartha KK (1981) Plant Cell Reports 1:64-66 Murashige T, Skoog F (1962) Physiol. Plant. 15:473-497 Natali L, Cavallini A (1987) Plant Breeding 99:172-176 Nauerby B, Madsen M, Christiansen J, Wyndaele R (1991) P l a n t C e l l Reports 9:676-679 P u o n t i - K a e r l a s J, E r i k s s o n T (1988) P l a n t C e l l Reports 7:242-245 P u o n t i - K a e r l a s J , E r l k s s o n T, Engstrom P (1990) TheoDAppl. Genet. 80:246-252 P u r n h a u s e r L, Medgyesy P, Czako M, Dix JP, Marton L (1987) P l a n t C e l l Reports 6 : 1 - 4 Ranch JP, Oglesby L, Z e i l i n s k i AC (1985) In V i t r o C e l l u l a r & Developmental Biology 2 1 ( 1 1 ) : 6 5 3 - 6 5 8 Roustau JP, Latehe A, F a l l o t J (1990) P l a n t Science 67:89-95 Rubluo A, Kartha KK, Mroginski LA, Dyck J (1984) J. P l a n t P h y s i o l . 117:119-130