PlantCell Reports
Plant Cell Reports (1993) 13:107-110
9 Springer-Verlag 1993
In vitro morphogenetic responses and plant regeneration from pepper (Capsicum annuum L. cv. Early California Wonder) seedling explants Aly I. A . Ebida 1,2 and Ching-yeh Hu 1 1 Biology Department, William Paterson College, Wayne, NJ 07470, USA 2 Present address: Plant Production Department, Faculty of Agriculture, Alexandria University, Egypt
Received 27 April 1993/Revised version received 25 August 1993 - Communicated by G.C. Phillips
Abstract. Explants from 13-d old pepper (Capsicum annuum, L. cv. Early California Wonder) seedlings were cultured in Murashige and Skoog (MS) medium supplemented with different levels of 1naphthalene acetic acid (NAA) and 6-benzylamino purine (BAP). Multiple shoot-budS proliferated from the cut surfaces of cotyledon, shoot-tip and hypocotyl explants in one month. The best NAA to BAP combinations (mg/l : mg/l) for multiple shoot-bud regeneration of the above three explant types were 0.1 : 5.0, 0.0 : 5.0, and 0.1 : 10.0, respectively. Root explants did not express any new morphogenetic response in all hormonal combinations tested. Regenerated shoot-buds were excised from the explant and cultured in 1/2X or 1X MS medium supplemented with different levels of Indole-3-acetic acid (IAA) or NAA. When cultured in full strength MS medium with 0.5 mg/1 IAA or 0.4 mg/I NAA, 70% of the buds rooted in one month. Plantlets were established successfully ex vitro under greenhouse mist and grown to maturity.
Introduction Pepper is one of the worlds' most important vegetable crops. The pathological problem that poses a great threat to cultivated peppers is viral infection. Pepper, therefore, should be a prime target to receive viral coat protein genes via gene transfer technology. Despite world-wide efforts (Liu et al., 1990; Wang et al., 1991) no transgenic pepper plant has yet been reported. This is likely due to the imperfection of in vitro pepper regeneration procedures. Pepper in vitro studies have been reviewed by Morrison et al. (1986), and Fari (1986). Its in vitro regeneration has been reported by numerous laboratories via protoplast cultures (Saxena et al., 1981; Diaz ct al., 1988), anther cultures (Vagera and Havranek, 1985; Munyon et al., 1989) and seedling explant organogenesis. The most effective reported method in pepper regeneration is through organogenesis of shoot-buds from seedling cotyledon and hypocotyl. Whole plant regeneration has been achieved in numerous cultivars with this procedure (Fari and Czako, 1981; Sripichitt et al., 1987; Subhash and Prolaram, 1987; Agrawal et al., 1988; Subhash and Christopher, 1988; Agrawal et al, 1989; Jacobs and Stephens, 1990; Arroyo and Revilla, 1991; Wang et al., 1991). Unfortunately, there are intervarietal differences in pepper explants responding to the various rates and/or combinations of plant growth regulators (Jacobs and Stephcns, 1990; Fortunato and Tudisco, 1991). Such data forced workers to conclude that cultivar-and-tissue-speeific media may be necessary in optimizing the pepper regeneration procedure (Hilliard and Stephens, 1989; Ochoa-Alejo and IretaCorrespondence to: C.-Y. Hu
Moreno, 1990). We are interested in introducing TMV coat protein into sweet pepper cv. Early California Wonder. Low frequencies (5-11%) of leaf-like structures have been obtained from the cxplants of this cuitivar by Lin et aL (1990). They were unable to induce rooting and plantlet regeneration from these Structures. To our knowledge there is no satisfactory regeneration protocol developed for this cultivar. We are reporting here the results of our initial work on this cultivar - the development of a reliable regeneration protocol.
Materials and methods Seeds of bell sweet pepper (Capsicum annuum L. cv. Early California Wonder) were obtained from Agway Inc. (Syracuse, N.Y.). The in vitro culture procedure was divided into three stages in this study, namely: germination, regeneration and rooting. A stage of ex vitro planting-out followed. A complete randomized factorial design was used for the regeneration and rooting stages. Culture tubes, 16 X 2 cm, containing 15 ml of sterile media were used. Culture media were adjusted to pH 5.7 and autoclaved in the tubes for 15 rain at 1.05 kg/cm 2. Cultures were incubated at 28~ With the exception of the germination stage, they were under 16 h photoperiod using approximately 70 ~uE m2s -] cool white fluorescent light. This study was carried out twice. Germination: Seeds were surface disinfested in 3% (v/v) CIorox containing a small amount of surfactant, Tween 80, for 15 min and rinsed with three changes of sterile distilled water. One sterile seed was placed in each culture tube containing one-half strength MS (Mnrashige and Skoog, 1962) medium with 2 % (w/v) sucrose and 0.7% (w/v) agar (U. S. Biochemical Corp., Cleveland, Ohio). Cultures were initially incubated in darkness for 10 d and then transferred to a 16 h photoperiod for an additional 3 d. Regeneration: Each germinated sterile seedling was sectioned into four different explants as shown in Fig. 1A. One explant was introduced into each culture tube. MS medium, with 3% sucrose and 0.4% agar, was used as the basal medium. Various concentrations of BAP (1.25, 2.5, 5.0, and 10.0 mg/I) and NAA (0.0, 0.1, 0.5, and 2.5 rag/l) were supplemented to this basal medium in combinations. Ten replicates of each explant type were used in each hormonal combination treatment. The morphogenetic responses were evaluated after one month incubation. Rooting: Shoot-buds at various developmental stages were excised from explants at the end of one month for rooting
108 experiments. Approximately 50% of the shOot-buds came from cotyledon explants, and 25% each came from shoot-tip and hypocotyl explants. They were pooled and randomly assigned to different treatments with one bud in each culture tube. Full and one-half strength MS medium with 3 % sucrose and 0.7% a g a r w e r e tested. NAA (0.0, 0.1, 0.2 or 0.4 mg/l) or IAA (0.0, 0.25, 0.5, or 1.0 mg/l) was supplemented to the medium to induce rooting. Twenty replicates were used in each medium/hormonal combination treatment. Rooting percentage was recorded after one month incubation. Planting-out: The first 15 large, rooted buds (R 0 plantlets) from various rooting treatments were transplanted ex vitro, after washing off the agar, into 12.7 cm square plastic pots filled with TERRALITE Vegetable Plug Mix (E.C. Geiger Inc., Harlegsville, PA). The pots were initially placed under intermittent mist underneath a greenhouse bench for 2 weeks. They were then placed on top of the bench and watered/fertilized manually as needed.
Table 1: Morphogenetic responses of the Early California Wonder pepper seedling explants cultured in MS medium supplemented with photohormones. Data scored at the end of an 1-month incubation. Each datum consists of 20 repications, the combination of two identical experiments. HORMONES (mg/l)
NAA
BAP
0.0
1. Cotyledon explants. The best bud regeneration treatment obtained in this study was by incubation of cotyledon explants in the basal medium supplemented with 5.0 mg/I BAP and 0.1 mg/l NAA. A large n u m b e r ofadventitiousshoot-buds per explant along with moderate amount of callus were produced in this treatment. The protuberance of bud initials appeared at the cut surfaces after 9 d incubation. Leafy shoot-buds became apparent by 16 d (Fig. 1B). A smaller n u m b e r of shoot-buds per explant were produced when auxin level was increased to 0.5 mg/I NAA. Only caili were induced when the highest auxin level (2.5 mg/l NAA) was used at all cytokinin levels tested. 2. Shoot-tip explants. The highest shoot-bud n u m b e r appeared at an exogenous cytokinin level of 5.0 mg/I BAP with no auxin added. Basipetal movement apparently brought the endogenous auxin from apical b u d to the cut end of the hypocotyl where most of the adventitious shoot-buds developed. Regeneration frequently took place at the cut ends of both hypocotyl and cotyledon (Fig. 1C). Fewer shoot-buds regenerated per explant when 0.1 mg/l NAA was added. No callus was observed on the treatments that expressed shoot-bud organogenesis. Only callus and roots, but no shoot-bud, were produced when the exogenous auxin was increased to 0.5 mg/l NAA. No growth was detected when high exogenous auxin (2.5 mg/I NAA) was added. 3. Hypocotyl explants. Hypocotyl tissue was not a likely site for hormone bio-synthesis. Both exogenous auxin and cytokinin were required for its shoot-bud organogenesis. Adventitious shoot-buds initiated only when 0.1 mg/l of NAA was supplemented to medium containing BAP. A high exogenous cytokinin level of 10.0 mg/l BAP was needed to induce the optimum shoot-bud regeneration. Shootbud organogenesis has been elicited at the upper, or both upper and lower cut ends of the explant (Fig. 1D). Rhizogenesis took place when the exogenous auxin level reached 0.5 mg/I of NAA. Like the cotyledon explants, callus was produced for all treatments except the one supplemented with the lowest cytokinin level and no auxin. 4. Root explants. No bud nor callus was produced by root explants. Small b r a n c h roots grew out at all cytokinin levels tested when the auxin levels were lower than 2.5 mg/I. They all turned brown and died in less than one month.
Shoot-Tip
TYPE Hypocotyl
Morph Resp*
Adv Buds**
Morph Adv Resp Buds
Morph Resp
1.25 2.50 5.00 10.00
B*C ÷+* B*÷C + B**C *
0/10 I0/I0 I0/I0 10/10
R÷ B*÷R * B .... B "÷
0/10 I0/I0 10/10 10/10
C÷ C~ C~
0.i
1.25 2.50 5.00 i0.00
0/i0 B*C ÷** I0/I0 B++++C++ I 0 / i 0 B+*+C+~ 1 0 / 1 0
R+ B *÷ B +++ B +++
0/i0 i0/i0 I0/i0 10/10
C*+R *~ B*C ÷* B*C ~+ B++C+
0.5
1.25 2.50 5.00 I0.00
C ÷++ B+C ÷+* B÷*C ÷÷ B+C +++
C+÷+R÷ C÷+~R ÷ C+++R ÷ C+~R +
2.5
1.25 2.50 5.00 10.00
C .... C ++ C ** C +~
Results and discussion A. Morphogenesis of Different Types of Explants: Morphogenetic responses of various explant types are summarized in Table 1. The key responses of each explant type are pointed out below:
EXPLANT Cotyledon
C ÷**
0/I0 i0/I0 i0/i0 I0/i0 0/10 0/10 0/i0 0/10
Adv Buds
Root Morph Adv Resp Buds
0/10 0/I0 0/I0 0/10
R+ R* R+ R+
0/10 0/I0 0/I0 0/10
0/i0 i0/I0 i0/I0 i0/I0
R+ R+ R* R+
0/I0 0/i0 0/i0 0/i0
R+ R÷ R÷ R+
0/i0 0/I0 0/I0 0/i0
0/I0 0/I0 0/I0 0/i0
C++B * C++R ÷ C++R * C +"
0/I0 0/I0 0/I0 0/I0
0/10 0/10 0/10 0/10
C ++ C*" C*" C *÷
0/10 0/10 0110 0/10
0/10 0/10 0110 0/10
Nature of morphogenetic responses: - = no new structure produced; B = adveutitiou~ shoot-buds; C = calli; R = short roots. Level of response: ÷ = low; ÷+ = moderate; ~ = high; ++++= intense. ~* # of explants regenerating adventitious shoot-buds / Total # of explants. *
O u r observationson morphogenetic responses by different types of explants were generally in agreement with those reported in the literature (Gunay and Rao, 1978; Fari and Czako, 1981; Phillips and Hubstenberger, 1985; Agrawal et al., 1989; Ochoa-Alejo and Garcia-Bautista, 1990). B. Adventitious Shoot-Bud Induction: Based on the survey of a large n u m b e r of species, Hu and Wang (1983) concluded that BAP and NAA were the most effective cytokinins and auxins, respectively, for inducing shoot-bud orgauogenesis. Consequently, we used these two hormones to supplement the regeneration medium. Most published works on pepper shoot-bud induction, however, used IAA instead NAA as their exogenous auxin. This may partially account for the low (511%) level of regeneration obtained by Liu et al. (1990) on the same eultivar compared to 100% regeneration obtained in numerous treatments in this study. The reported effective hormonal levels for pepper shoot-bud induction were between 1 to 7 mg/I for BAP and around 1 mg/l for IAA. The optimum BAP levels we identified in this study were within this range. A slightly lower optimum auxin level (0.1 mg/l NAA) was determined in our study. This might be due to cultivar differences as well as to the stronger auxin we used. Phillips and Hubstcnberger (1985) were the only group that employed drastic hormonal levels: 50 mg/I BAP and 0.05 mg/! IAA. In addition to the continuous light and high incubation temperature (28.5°C), such a high eytokinin and low auxin levels might be the main factor that accounted for the unique, long-term multiple-bud proliferation capacity they obtained. Although callus growth was common at the explant cut surfaces, it appeared that the shoot-buds were regenerated directly from the explant tissues without the intervention of callus growth. Most authors reported the same neo-formation events, with histological evidence provided by Agrawal et al. (1989). Workers, in general (e.g. Phillips and Hubstenberger, 1985; Agrawal et a11989), are also in agreement with our results that the unorganized calli from
109
Fig. 1. In vitro r e g e n e r a t i o n a n d e x vitro R 0 plantlet d e v e l o p m e n t of E a r l y C a l i f o r n i a W o n d e r pepper. A. F o u r types o f explants sectioned f r o m a 13-d old seedling: 1 - cotyledon, 2 - shoot tip, 3 hypocotyl a n d 4 - root explants. B-D. Multiple adventitious shootb u d s proliferated f r o m t h e e x p l a n t cut s u r f a c e s after~approximately
one m o n t h i n c u b a t i o n in t h e r e g e n e r a t i o n m e d i u m , c = cotyledon, h = hypocotyl. B. A r e g e n e r a t i n g cotyledon e x p l a n t in MS m e d i u m with 5 mg/! B A P a n d 0.1 rag/! NAA. Cotyledon length = 11 ram. C. A shoot-tip e x p l a n t (incubated in the s a m e m e d i u m as in B) r e g e n e r a t i n g at the c u t s u r f a c e s of both cotyledons a n d hypocotyL
110 Hypocotyl length = 5 mm. D. A hypocotyl explant, in MS medium with 10 mg/l BAP and 0.1 mg/l NAA, showing regeneration around the periphery of the 6 mm wide cut surface. E. An in vitro rooted plantlet incubated for one month in the regeneration medium, followed by one month in the rooting medium of full-strength MS supplemented with 0.4 mg/l NAA, ready to be transplanted ex vitro. Each division (on the upper left side of the culture tube wall) = 1 mm. F. An ex vitro established R0 plantlet (in a greenhouse for 3 weeks). G. M a t u r e plants reared in 22.86 cm pots in a greenhouse for 4.5 months. I. Normal-looking fruits produced by the mature plants.
pepper seedling explants were not capable of de novo shoot-bud regeneration. Shoot-buds on the same explant were at different developmental stages. They continued to grow in the same medium for 2 to 3 months. New bud initials continued to evolve after a few subcultures (data not shown). But the growth rate and the potential for initiating of new buds seemed to diminish quickly after about 3 subcultures. C. Rooting and ex vitro plantlet development: Data on rhizogenesis from adventitious shoot-buds are shown in Table 2. A 70% rooting resulted when 0.5 mg/I IAA or 0.4 mg/I NAA was supplemented to the full strength MS medium. Based on LSD analysis, such rooting percentages were significantly higher (P = 0.05) than those from auxin-free media.
Table 2. Effect of auxin and MS medium strength on the rooting of regenerated shoot-buds of the Early California Wonder pepper. N = 20 / treatment. Rooting % were recorded after one month incubation. Treatment Auxin MS Cone. Mean 1
2
3
4
IAA*
1.0 X 0.5 X Mean
30 35 32.5
60 60 60.0
70 45 57.5
40 40 40.0
50.0 45.0 47.5
NAA ~
1.0 X 0.5 X Mean
40 30 35.0
40 40 40.0
50 40 45.0
70 60 65.0
50.0 42.5 46.3
For IAA experiment: Auxin concentrations (mg/I) of Treatment 1 through 4 were 0.00, 0.25, 0.5 and 1.0, respectively. LSD at 0.05 level for medium strengths = 11.81, for auxin concentrations = 23.78, and for medium strengths x auxin concentrations = 33.63. ~* For NAA experiment: Auxin concentrations (mg/I) of Treatment 1 through 4 were 0.0, 0.1, 0.2, and 0.4, respectively. LSD at 0.05 level for medium strengths = 11.94, for auxin concentrations = 23.88, and for medium strengths x auxin concentrations = 33.77.
In order to confine the size of the rooting experiments, we did not include the "bud origin" in our factorial design. Instead, we pooled buds from the three explant sources. This, unfortunately, resulted in the loss of valuable information as to which explant type provided buds that rooted most readily, which explant type did each R 0 plant originate from and which explant type was the most desirable for plantlet production. Liu et al. (1990) failed to obtain plantlets from regenerated shoot-buds of Early California Wonder and five more pepper cultivars because of their inability to produce normal elongated shoots for rooting. Instead, we rooted the isolated rosette buds directly. Once in the greenhouse, these rooted buds elongated and
grew into normal plants. Arroyo and Revilla (1991) also rooted the rosette buds to obtain plantlets. The first 15 large, rooted buds (Fig. 1E) from various rooting treatments were transplanted ex vitro and reared in pots in greenhouse (Fig. 1F). Thirteen of them survived and grew into fruit-bearing mature plants (Fig. 1G). All our 13 regenerated plants appeared normal. They started to produce flowers after growing in the greenhouse for about 13 weeks and continuously produced normal sized and shaped fruits (Fig. 1H). The crowns of them were killed during the winter due to a greenhouse heating failure. Nine of them recovered after pruning back and continued to produce fruits up to now - 10 month after the first flowering. O u r resultant R 0 plants and their fruits appear normal. No somaclonal variation was detected by Arroyo and Revilla (1991) in their regenerated shoot-bud derived pepper plants. Such normal appearances might be due to the fact that the shoot-buds were initiated adventitiouslywithout the intervention of callus growth (for supporting data, see Agrawal et al., 1989). This may be an advantageous feature for our transgenic project on the Early California W o n d e r pepper currently underway.
Acknowledgements This work was supported in part by U.S. Peace Fellowship to Egypt and the Assigned Release Time program of Wm. Paterson College. Thanks is due to Prof. Robert Callaham for his critical reading of this manuscript.
References Agrawal S, C h a n d r a N, Kothari SL (1988) C n r r Sci 57:1347-1349 Agrawal S, C h a n d r a N, Kothari SL (1989) Plant Cell Tiss Org Cult 16:57-55 Arroyo R, Revilla MA (1991) Plant Cell Rep 10:414-416 Diaz I, Moreno R, Power JB (1988) Progr. Plant Cell Rep 7:210-212 Fari M (1986) In: Baj~j u (ed) Biotechnology in Agriculture and Forestry, vol 2, Chapter 4. Springer-Verlag, Berlin Heidelberg, pp 345-362 Fari M, Czako M (1981) Sci Hort (Amst) 15:207-214 Fortunato IM, Tudisco M (1991) Capsicum Newslett 10:59-60 G u n a y AL, Rao PS (1978)Plant Sci Lett 11:365-372 Hilliard JL, Stephens CT (1989) In Vitro 25(3 Pt.2):28A (Abstr) Hu CY, Wang P J (1983) In: Evans DA, Sharp WR, Ammirato PV, Yamada Y (eds) Handbook on Plant Cell Culture, vol I, MacMillan, NY, pp 177-227 Jacobs JL, Stephens CT (1990)HortScience 25:120 Liu W, P a r r o t t WA, Hildebrand DF, Collins GB, Williams EG (1990) Plant Cell Rep 9:360-364 Morrison RA, Koning RE, Evans DA (1986) In: Evans DA, Sharp WR, Ammirato PV (eds) Handbook on Plant Cell Culture, vol IV, MacMillan, NY, pp 552-573 Munyon IP, Hubstenberger JF, Phillips GC (1989) In Vitro Cel Dev Biol 25:293-296 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Ochoa-Alejo N, Garcia-Bautista MAR (1990) Turrialba 40:311-318 Oehoa-Alejo N, Ireta-Moreno L (1990) Sei Hurt 42:21-28 Phillips GC, Hubstenberger J F (1985) Plant Cell Tiss Org Cult 4:261-270 Saxena PK, Gill R, Rashid A, Maheshwari SC (1981) Protoplasma 108:357-360 Sripiehitt P, Nawata E, Shigenaga S (1987) J a p J Breed 37:133-142 Subhash K, Christopher T (1988) C u r r Sei 57:99-100 Subhash K, Prolaram B (1987) C u r r Sci 56:1289-1291 Vagera J, H a v r a n e k P (1985) Biol Plant 27:10-21 Wang YW, Yang MZ, Pan NS, Chen ZL (1991) Acta Bot Sinica 33:780-786
Plant Cell Reports (1993) 13:107-110
9 Springer-Verlag 1993
In vitro morphogenetic responses and plant regeneration from pepper (Capsicum annuum L. cv. Early California Wonder) seedling explants Aly I. A . Ebida 1,2 and Ching-yeh Hu 1 1 Biology Department, William Paterson College, Wayne, NJ 07470, USA 2 Present address: Plant Production Department, Faculty of Agriculture, Alexandria University, Egypt
Received 27 April 1993/Revised version received 25 August 1993 - Communicated by G.C. Phillips
Abstract. Explants from 13-d old pepper (Capsicum annuum, L. cv. Early California Wonder) seedlings were cultured in Murashige and Skoog (MS) medium supplemented with different levels of 1naphthalene acetic acid (NAA) and 6-benzylamino purine (BAP). Multiple shoot-budS proliferated from the cut surfaces of cotyledon, shoot-tip and hypocotyl explants in one month. The best NAA to BAP combinations (mg/l : mg/l) for multiple shoot-bud regeneration of the above three explant types were 0.1 : 5.0, 0.0 : 5.0, and 0.1 : 10.0, respectively. Root explants did not express any new morphogenetic response in all hormonal combinations tested. Regenerated shoot-buds were excised from the explant and cultured in 1/2X or 1X MS medium supplemented with different levels of Indole-3-acetic acid (IAA) or NAA. When cultured in full strength MS medium with 0.5 mg/1 IAA or 0.4 mg/I NAA, 70% of the buds rooted in one month. Plantlets were established successfully ex vitro under greenhouse mist and grown to maturity.
Introduction Pepper is one of the worlds' most important vegetable crops. The pathological problem that poses a great threat to cultivated peppers is viral infection. Pepper, therefore, should be a prime target to receive viral coat protein genes via gene transfer technology. Despite world-wide efforts (Liu et al., 1990; Wang et al., 1991) no transgenic pepper plant has yet been reported. This is likely due to the imperfection of in vitro pepper regeneration procedures. Pepper in vitro studies have been reviewed by Morrison et al. (1986), and Fari (1986). Its in vitro regeneration has been reported by numerous laboratories via protoplast cultures (Saxena et al., 1981; Diaz ct al., 1988), anther cultures (Vagera and Havranek, 1985; Munyon et al., 1989) and seedling explant organogenesis. The most effective reported method in pepper regeneration is through organogenesis of shoot-buds from seedling cotyledon and hypocotyl. Whole plant regeneration has been achieved in numerous cultivars with this procedure (Fari and Czako, 1981; Sripichitt et al., 1987; Subhash and Prolaram, 1987; Agrawal et al., 1988; Subhash and Christopher, 1988; Agrawal et al, 1989; Jacobs and Stephens, 1990; Arroyo and Revilla, 1991; Wang et al., 1991). Unfortunately, there are intervarietal differences in pepper explants responding to the various rates and/or combinations of plant growth regulators (Jacobs and Stephcns, 1990; Fortunato and Tudisco, 1991). Such data forced workers to conclude that cultivar-and-tissue-speeific media may be necessary in optimizing the pepper regeneration procedure (Hilliard and Stephens, 1989; Ochoa-Alejo and IretaCorrespondence to: C.-Y. Hu
Moreno, 1990). We are interested in introducing TMV coat protein into sweet pepper cv. Early California Wonder. Low frequencies (5-11%) of leaf-like structures have been obtained from the cxplants of this cuitivar by Lin et aL (1990). They were unable to induce rooting and plantlet regeneration from these Structures. To our knowledge there is no satisfactory regeneration protocol developed for this cultivar. We are reporting here the results of our initial work on this cultivar - the development of a reliable regeneration protocol.
Materials and methods Seeds of bell sweet pepper (Capsicum annuum L. cv. Early California Wonder) were obtained from Agway Inc. (Syracuse, N.Y.). The in vitro culture procedure was divided into three stages in this study, namely: germination, regeneration and rooting. A stage of ex vitro planting-out followed. A complete randomized factorial design was used for the regeneration and rooting stages. Culture tubes, 16 X 2 cm, containing 15 ml of sterile media were used. Culture media were adjusted to pH 5.7 and autoclaved in the tubes for 15 rain at 1.05 kg/cm 2. Cultures were incubated at 28~ With the exception of the germination stage, they were under 16 h photoperiod using approximately 70 ~uE m2s -] cool white fluorescent light. This study was carried out twice. Germination: Seeds were surface disinfested in 3% (v/v) CIorox containing a small amount of surfactant, Tween 80, for 15 min and rinsed with three changes of sterile distilled water. One sterile seed was placed in each culture tube containing one-half strength MS (Mnrashige and Skoog, 1962) medium with 2 % (w/v) sucrose and 0.7% (w/v) agar (U. S. Biochemical Corp., Cleveland, Ohio). Cultures were initially incubated in darkness for 10 d and then transferred to a 16 h photoperiod for an additional 3 d. Regeneration: Each germinated sterile seedling was sectioned into four different explants as shown in Fig. 1A. One explant was introduced into each culture tube. MS medium, with 3% sucrose and 0.4% agar, was used as the basal medium. Various concentrations of BAP (1.25, 2.5, 5.0, and 10.0 mg/I) and NAA (0.0, 0.1, 0.5, and 2.5 rag/l) were supplemented to this basal medium in combinations. Ten replicates of each explant type were used in each hormonal combination treatment. The morphogenetic responses were evaluated after one month incubation. Rooting: Shoot-buds at various developmental stages were excised from explants at the end of one month for rooting
108 experiments. Approximately 50% of the shOot-buds came from cotyledon explants, and 25% each came from shoot-tip and hypocotyl explants. They were pooled and randomly assigned to different treatments with one bud in each culture tube. Full and one-half strength MS medium with 3 % sucrose and 0.7% a g a r w e r e tested. NAA (0.0, 0.1, 0.2 or 0.4 mg/l) or IAA (0.0, 0.25, 0.5, or 1.0 mg/l) was supplemented to the medium to induce rooting. Twenty replicates were used in each medium/hormonal combination treatment. Rooting percentage was recorded after one month incubation. Planting-out: The first 15 large, rooted buds (R 0 plantlets) from various rooting treatments were transplanted ex vitro, after washing off the agar, into 12.7 cm square plastic pots filled with TERRALITE Vegetable Plug Mix (E.C. Geiger Inc., Harlegsville, PA). The pots were initially placed under intermittent mist underneath a greenhouse bench for 2 weeks. They were then placed on top of the bench and watered/fertilized manually as needed.
Table 1: Morphogenetic responses of the Early California Wonder pepper seedling explants cultured in MS medium supplemented with photohormones. Data scored at the end of an 1-month incubation. Each datum consists of 20 repications, the combination of two identical experiments. HORMONES (mg/l)
NAA
BAP
0.0
1. Cotyledon explants. The best bud regeneration treatment obtained in this study was by incubation of cotyledon explants in the basal medium supplemented with 5.0 mg/I BAP and 0.1 mg/l NAA. A large n u m b e r ofadventitiousshoot-buds per explant along with moderate amount of callus were produced in this treatment. The protuberance of bud initials appeared at the cut surfaces after 9 d incubation. Leafy shoot-buds became apparent by 16 d (Fig. 1B). A smaller n u m b e r of shoot-buds per explant were produced when auxin level was increased to 0.5 mg/I NAA. Only caili were induced when the highest auxin level (2.5 mg/l NAA) was used at all cytokinin levels tested. 2. Shoot-tip explants. The highest shoot-bud n u m b e r appeared at an exogenous cytokinin level of 5.0 mg/I BAP with no auxin added. Basipetal movement apparently brought the endogenous auxin from apical b u d to the cut end of the hypocotyl where most of the adventitious shoot-buds developed. Regeneration frequently took place at the cut ends of both hypocotyl and cotyledon (Fig. 1C). Fewer shoot-buds regenerated per explant when 0.1 mg/l NAA was added. No callus was observed on the treatments that expressed shoot-bud organogenesis. Only callus and roots, but no shoot-bud, were produced when the exogenous auxin was increased to 0.5 mg/l NAA. No growth was detected when high exogenous auxin (2.5 mg/I NAA) was added. 3. Hypocotyl explants. Hypocotyl tissue was not a likely site for hormone bio-synthesis. Both exogenous auxin and cytokinin were required for its shoot-bud organogenesis. Adventitious shoot-buds initiated only when 0.1 mg/l of NAA was supplemented to medium containing BAP. A high exogenous cytokinin level of 10.0 mg/l BAP was needed to induce the optimum shoot-bud regeneration. Shootbud organogenesis has been elicited at the upper, or both upper and lower cut ends of the explant (Fig. 1D). Rhizogenesis took place when the exogenous auxin level reached 0.5 mg/I of NAA. Like the cotyledon explants, callus was produced for all treatments except the one supplemented with the lowest cytokinin level and no auxin. 4. Root explants. No bud nor callus was produced by root explants. Small b r a n c h roots grew out at all cytokinin levels tested when the auxin levels were lower than 2.5 mg/I. They all turned brown and died in less than one month.
Shoot-Tip
TYPE Hypocotyl
Morph Resp*
Adv Buds**
Morph Adv Resp Buds
Morph Resp
1.25 2.50 5.00 10.00
B*C ÷+* B*÷C + B**C *
0/10 I0/I0 I0/I0 10/10
R÷ B*÷R * B .... B "÷
0/10 I0/I0 10/10 10/10
C÷ C~ C~
0.i
1.25 2.50 5.00 i0.00
0/i0 B*C ÷** I0/I0 B++++C++ I 0 / i 0 B+*+C+~ 1 0 / 1 0
R+ B *÷ B +++ B +++
0/i0 i0/i0 I0/i0 10/10
C*+R *~ B*C ÷* B*C ~+ B++C+
0.5
1.25 2.50 5.00 I0.00
C ÷++ B+C ÷+* B÷*C ÷÷ B+C +++
C+÷+R÷ C÷+~R ÷ C+++R ÷ C+~R +
2.5
1.25 2.50 5.00 10.00
C .... C ++ C ** C +~
Results and discussion A. Morphogenesis of Different Types of Explants: Morphogenetic responses of various explant types are summarized in Table 1. The key responses of each explant type are pointed out below:
EXPLANT Cotyledon
C ÷**
0/I0 i0/I0 i0/i0 I0/i0 0/10 0/10 0/i0 0/10
Adv Buds
Root Morph Adv Resp Buds
0/10 0/I0 0/I0 0/10
R+ R* R+ R+
0/10 0/I0 0/I0 0/10
0/i0 i0/I0 i0/I0 i0/I0
R+ R+ R* R+
0/I0 0/i0 0/i0 0/i0
R+ R÷ R÷ R+
0/i0 0/I0 0/I0 0/i0
0/I0 0/I0 0/I0 0/i0
C++B * C++R ÷ C++R * C +"
0/I0 0/I0 0/I0 0/I0
0/10 0/10 0/10 0/10
C ++ C*" C*" C *÷
0/10 0/10 0110 0/10
0/10 0/10 0110 0/10
Nature of morphogenetic responses: - = no new structure produced; B = adveutitiou~ shoot-buds; C = calli; R = short roots. Level of response: ÷ = low; ÷+ = moderate; ~ = high; ++++= intense. ~* # of explants regenerating adventitious shoot-buds / Total # of explants. *
O u r observationson morphogenetic responses by different types of explants were generally in agreement with those reported in the literature (Gunay and Rao, 1978; Fari and Czako, 1981; Phillips and Hubstenberger, 1985; Agrawal et al., 1989; Ochoa-Alejo and Garcia-Bautista, 1990). B. Adventitious Shoot-Bud Induction: Based on the survey of a large n u m b e r of species, Hu and Wang (1983) concluded that BAP and NAA were the most effective cytokinins and auxins, respectively, for inducing shoot-bud orgauogenesis. Consequently, we used these two hormones to supplement the regeneration medium. Most published works on pepper shoot-bud induction, however, used IAA instead NAA as their exogenous auxin. This may partially account for the low (511%) level of regeneration obtained by Liu et al. (1990) on the same eultivar compared to 100% regeneration obtained in numerous treatments in this study. The reported effective hormonal levels for pepper shoot-bud induction were between 1 to 7 mg/I for BAP and around 1 mg/l for IAA. The optimum BAP levels we identified in this study were within this range. A slightly lower optimum auxin level (0.1 mg/l NAA) was determined in our study. This might be due to cultivar differences as well as to the stronger auxin we used. Phillips and Hubstcnberger (1985) were the only group that employed drastic hormonal levels: 50 mg/I BAP and 0.05 mg/! IAA. In addition to the continuous light and high incubation temperature (28.5°C), such a high eytokinin and low auxin levels might be the main factor that accounted for the unique, long-term multiple-bud proliferation capacity they obtained. Although callus growth was common at the explant cut surfaces, it appeared that the shoot-buds were regenerated directly from the explant tissues without the intervention of callus growth. Most authors reported the same neo-formation events, with histological evidence provided by Agrawal et al. (1989). Workers, in general (e.g. Phillips and Hubstenberger, 1985; Agrawal et a11989), are also in agreement with our results that the unorganized calli from
109
Fig. 1. In vitro r e g e n e r a t i o n a n d e x vitro R 0 plantlet d e v e l o p m e n t of E a r l y C a l i f o r n i a W o n d e r pepper. A. F o u r types o f explants sectioned f r o m a 13-d old seedling: 1 - cotyledon, 2 - shoot tip, 3 hypocotyl a n d 4 - root explants. B-D. Multiple adventitious shootb u d s proliferated f r o m t h e e x p l a n t cut s u r f a c e s after~approximately
one m o n t h i n c u b a t i o n in t h e r e g e n e r a t i o n m e d i u m , c = cotyledon, h = hypocotyl. B. A r e g e n e r a t i n g cotyledon e x p l a n t in MS m e d i u m with 5 mg/! B A P a n d 0.1 rag/! NAA. Cotyledon length = 11 ram. C. A shoot-tip e x p l a n t (incubated in the s a m e m e d i u m as in B) r e g e n e r a t i n g at the c u t s u r f a c e s of both cotyledons a n d hypocotyL
110 Hypocotyl length = 5 mm. D. A hypocotyl explant, in MS medium with 10 mg/l BAP and 0.1 mg/l NAA, showing regeneration around the periphery of the 6 mm wide cut surface. E. An in vitro rooted plantlet incubated for one month in the regeneration medium, followed by one month in the rooting medium of full-strength MS supplemented with 0.4 mg/l NAA, ready to be transplanted ex vitro. Each division (on the upper left side of the culture tube wall) = 1 mm. F. An ex vitro established R0 plantlet (in a greenhouse for 3 weeks). G. M a t u r e plants reared in 22.86 cm pots in a greenhouse for 4.5 months. I. Normal-looking fruits produced by the mature plants.
pepper seedling explants were not capable of de novo shoot-bud regeneration. Shoot-buds on the same explant were at different developmental stages. They continued to grow in the same medium for 2 to 3 months. New bud initials continued to evolve after a few subcultures (data not shown). But the growth rate and the potential for initiating of new buds seemed to diminish quickly after about 3 subcultures. C. Rooting and ex vitro plantlet development: Data on rhizogenesis from adventitious shoot-buds are shown in Table 2. A 70% rooting resulted when 0.5 mg/I IAA or 0.4 mg/I NAA was supplemented to the full strength MS medium. Based on LSD analysis, such rooting percentages were significantly higher (P = 0.05) than those from auxin-free media.
Table 2. Effect of auxin and MS medium strength on the rooting of regenerated shoot-buds of the Early California Wonder pepper. N = 20 / treatment. Rooting % were recorded after one month incubation. Treatment Auxin MS Cone. Mean 1
2
3
4
IAA*
1.0 X 0.5 X Mean
30 35 32.5
60 60 60.0
70 45 57.5
40 40 40.0
50.0 45.0 47.5
NAA ~
1.0 X 0.5 X Mean
40 30 35.0
40 40 40.0
50 40 45.0
70 60 65.0
50.0 42.5 46.3
For IAA experiment: Auxin concentrations (mg/I) of Treatment 1 through 4 were 0.00, 0.25, 0.5 and 1.0, respectively. LSD at 0.05 level for medium strengths = 11.81, for auxin concentrations = 23.78, and for medium strengths x auxin concentrations = 33.63. ~* For NAA experiment: Auxin concentrations (mg/I) of Treatment 1 through 4 were 0.0, 0.1, 0.2, and 0.4, respectively. LSD at 0.05 level for medium strengths = 11.94, for auxin concentrations = 23.88, and for medium strengths x auxin concentrations = 33.77.
In order to confine the size of the rooting experiments, we did not include the "bud origin" in our factorial design. Instead, we pooled buds from the three explant sources. This, unfortunately, resulted in the loss of valuable information as to which explant type provided buds that rooted most readily, which explant type did each R 0 plant originate from and which explant type was the most desirable for plantlet production. Liu et al. (1990) failed to obtain plantlets from regenerated shoot-buds of Early California Wonder and five more pepper cultivars because of their inability to produce normal elongated shoots for rooting. Instead, we rooted the isolated rosette buds directly. Once in the greenhouse, these rooted buds elongated and
grew into normal plants. Arroyo and Revilla (1991) also rooted the rosette buds to obtain plantlets. The first 15 large, rooted buds (Fig. 1E) from various rooting treatments were transplanted ex vitro and reared in pots in greenhouse (Fig. 1F). Thirteen of them survived and grew into fruit-bearing mature plants (Fig. 1G). All our 13 regenerated plants appeared normal. They started to produce flowers after growing in the greenhouse for about 13 weeks and continuously produced normal sized and shaped fruits (Fig. 1H). The crowns of them were killed during the winter due to a greenhouse heating failure. Nine of them recovered after pruning back and continued to produce fruits up to now - 10 month after the first flowering. O u r resultant R 0 plants and their fruits appear normal. No somaclonal variation was detected by Arroyo and Revilla (1991) in their regenerated shoot-bud derived pepper plants. Such normal appearances might be due to the fact that the shoot-buds were initiated adventitiouslywithout the intervention of callus growth (for supporting data, see Agrawal et al., 1989). This may be an advantageous feature for our transgenic project on the Early California W o n d e r pepper currently underway.
Acknowledgements This work was supported in part by U.S. Peace Fellowship to Egypt and the Assigned Release Time program of Wm. Paterson College. Thanks is due to Prof. Robert Callaham for his critical reading of this manuscript.
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