Plant Cell Reports
Plant Cell Reports (1996) 16:226-231
© Springer-VerlagI996
An improved and reliable chili pepper (Capsicum annuum L.) plant regeneration method Rafael Ramirez-Malag6n and Neftali Ochoa-Alejo Departamento de [ngenieria Gen~tica de Plantas, Unidad de Biotecnologia e Ingenieria Genetica de Plantas, Centro de Investigaei6n y de Estudios Avanzados de1 IPN, Apartado Postal 629; 36500-Irapuato, Gto., M6xico Received 9 October 1995/Revised version received 13 June 1996 - Communicated by G. C. Phillips
Abstract, In this work we report a new method for in vitro chili pepper (Capsicum annuum L.) plant regeneration based on shoot formation from wounded hypocotyls. Chili pepper seeds were surface sterilized and germinated on agar (0.8%) at 25 _+ 2oc in the dark. Five factors that may influence shoot regeneration were studied: age of seedlings, hypocotyl wounding site, time elapsed between wounding the hypocotyls and decapitation of seedlings, culture media and cultivars. In order to study the influence of the first three factors on shoot regeneration, the apical, middle or basal hypocotyl regions of seedlings of cv. Mulato Bajio at different stages of development (9, 15, 16, 21 and 28 d old) were wounded with a syringe needle, and the seedlings were cultured on MS semisolid medium without growth regulators at 25 +_2oc under a 16/8 h light/dark photoperiod (daylight fluorescent lamps; 35 #mol m -2 s-1) until decapitation. The seedlings were decapitated (3 mm below the cotyledons) at different times after wounding (0, 2, 4, 10, 12 and 14 d), and each explant was evaluated for bud and shoot formation (> 5 mm in length) at the wounded site after 30 d of incubation. In general, seedlings at the stage of curved hypocotyl (9 d old) wounded in the apical region of hypocotyl were the best explants for shoot regeneration when inoculated on culture medium without growth regulators. Decapitation after wounding also influenced the shoot regeneration efficiency, with 10-14 d being the best period. Up to 90% shoot regeneration in cv. Mulato Bajio was obtained under these conditions. Statistically significant differences were observed for shoot formation among 21 cultivars tested. Regeneration of whole plants was achieved by rooting the shoots with indole-3-butyric acid pulses of 60 mg L-1 for 3 h and then subculturing on MS medium without growth regulators. Abbreviations: BA - N6-Benzyladenine; IAA - indole3-acetic acid; IBA indole-3-butyric acid; MS Murashige and Skoog (1962) medium; TDZ Correspondence to: N. Ochoa-Alejo
thidiazuron. Key words: Capsicum annuum, organogenesis, plant regeneration.
chili
pepper,
Introduction
The establishment of an efficient in vitro plant regeneration system is a fundamental prerequisite for plant genetic engineering. Chili pepper is an important horticultural crop that belongs to the Solanaceae family. In general, it has long been recognized that cells, tissues and organs from members of this family (tobacco, potato, tomato, petunia) undergo morphogenesis and in vitro plant regeneration easily. Organogenesis and embryogenesis in chili pepper tissues have been reported by several authors (Gunay and Rao 1978; Fari and Czak6 1981; Philips and Hubstenberger 1985; Agrawal et al. 1989; Ochoa-Alejo and Garcia-Bautista 1990; Ochoa-Alejo and Ireta-Moreno 1990; Ebida and Hu 1993; Ezura et al. 1993; Harini and Sita 1993). However, unlike other solanaceous species, chili pepper has been a recalcitrant species with regard to its capacity for in vitro plant regeneration (Liu et al. 1990). Previously, we reported a novel chili pepper plant regeneration system based on shoot formation using rooted hypocotyls cultured upside down on shoot-inducing medium, and the subsequent rooting of shoots (Valera-Montero and Ochoa-Alejo 1992). This method recently was found to be appropriate for in vitro plant regeneration using several bell pepper genotypes (Sz~lsz et al. 1995). In this communication we report an improved organogenic method for in vitro chili pepper plant regeneration using injured and decapitated seedlings from different cultivars cultured on medium without growth regulators. Materials and methods Plant materials and explants. Seeds of chiti pepper (Capsicum annuum L.) were surface sterilized by
227 soaking them in a commercial bleach solution (20% v/v + 0.1% Tween 20) for 20 min. The seeds were rinsed 3 times with sterile distilled water and inoculated onto a sterile gel prepared with 0.8% agar, without supplemental organic or mineral nutrients, and adjusted to pH 5.8. Seeds were incubated in the dark at 25 _+2oC for 9-16 d, depending on the stage needed for each treatment. The developmental stages tested were as follows (Fig. la): Stage 1, seedlings with curved hypocotyls (9 d old); Stage 2, etiolated seedlings (!5 d old); Stage 3, green seedlings (9 d in the dark and 7 d in the light with a 16 h lightJ8 h dark photoperiod; daylight fluorescent lamps, 35 p.mol m-2 s-l); Stage 4, green seedlings with the first pair of well developed leaves (9 d in the dark/12 d in the light); Stage 5, green seedlings with two pairs of well developed leaves (9 d in the dark/19 d in the light). Explant treatment. A total of 480 seedlings (cv. Mulato Bajio) for each developmental stage were used and the seedlings were divided into 3 groups each of 160. Hypocotyls from seedlings of each group were wounded by puncturing them with a sterile syringe needle (gauge: 0.72 mm; length: 32 mm) as shown in Figure lb. The selected sites for wounding were: apical region of the hypocotyls (3 to 5 mm below the cotyledons); middle region of the hypocotyls; or basal region of the hypocotyls. Explant culture. Groups of 160 seedlings were subdivided into 8 subgroups each of 20 seedlings, and these were distributed in 4 bottles (135 ml with 20 ml of culture medium) containing 5 seedlings each. The culture medium used in these experiments was MS semisolid medium (Murashige and Skoog 1962) without growth regulators, pH 5.8 and gelled with Phytagel (3 g L-I; Sigma Chemical Co). The seedlings of each group were decapitated by cutting the shoot 3 mm below the cotyledons at different times (0, 2, 4, 6, 8, 10, 12 and 14 d after the date of wounding) and were subcultured on fresh medium lacking growth regulators. Experimental design. A factorial experiment (3 wounding sites x 5 developmental stages x 8 decapitation dates = 120 treatments) was established with 4 replicates (each bottle as a replicate) and 5 explants per replicate. The mean number of buds and/or shoots per hypocotyl, percentage of hypocotyls that produced buds and/or shoots, and the percentage of explants that formed shoots > 5 mm in length were recorded 30 d after decapitation. Analysis of variance for the factorial experiment, and mean separation for each variable and factor within each variable, were carried out using the Tukey test at 5% level. Influence of culture medium and cultivar on shoot regeneration. Using the data obtained from the factorial experiment, bud and shoot formation in 21 chili pepper cultivars using 4 different culture media were investigated. Seedlings (9 d old) were wounded at the apical region of the hypocotyls and then decapitated 10 d after wounding. Decapitated seedlings were grown on MS medium with the following compositions: (1) without growth regulators (Ezura et aL 1993), (2) 1 pg L-1 thidiazuron (TDZ), (3) 1 mg L-1 benzyladenine (BA), and (4) 0.3 mg L-1 indoleacetic acid (IAA) plus 5 mg L-1 BA (Valera-Montero and Ochoa-Alejo 1992). In order to compare the shoot efficiency formation of this method with that of Ezura et al. (1993), a total of 50 explants from each of 12 chili pepper cultivars were prepared as reported by these authors and were inoculated on MS medium lacking growth regulators. Evaluations of response were carried out after 30 d of incubation under the same conditions described for the factorial experiment. Data were statistically analyzed by analysis of variance and the Tukey test at the level of 5%. In all experiments in which data on shoot regeneration was recorded, only shoots >_5 mm in length were recorded, as these shoots were the only ones capable of plant formation after rooting. Rooting of shoots. Regenerated shoots that reached > 5 mm in length were dissected from the hypocotyls and rooted on MS medium using an IBA pulse of 60 mg L-1 for 3 h. They were then subcultured on media without growth regulators. Rooted shoots were transferred to pots (1 L) containing peat moss, and developed into mature plants under greenhouse conditions. All experiments were performed at least twice.
Results and discussion
Influence of seedling age, wounding decapitation time on shoot formation
site
and
Injured hypocotyls were found to produce a small callus at the wounding site, and buds later formed that developed into shoots (Fig. lc, d). Analysis of variance for the factorial experiment showed statistical significance at the level of 0.01 for the percentage of hypocotyls regenerating buds and shoots. Bud and shoot regeneration was influenced, in decreasing order, by the hypocotyl wounding site, the developmental stage of seedlings, and the time elapsed between wounding and decapitation of seedlings (Table 1), as evidenced by analysis of variance data that showed sums of squares of 24.5, 22.3 and 3.6, respectively. In general, seedlings injured at the apical region of the hypocotyl exhibited the highest bud and shoot induction response, whereas those wounded at the basal zone showed the worst response. These results partially agree with those reported by Fari and Czako (1981) who observed that explants from the apical region of chili pepper hypocotyls produced buds, while the explants from the middle and the basal zones of hypocotyls produced only callus and roots when cultured on a bud inducing medium. Table 1. Effect of the wounding site, developmental stage of seedlings, and time of decapitation aEer wounding on shoot induction in chili pepper explants. % Explants with shoots Wounding site Apical 23.4a Middle 11.4b Basal 1.6c
Developmental stage* 1) 27.3 a 2) 21.0 b 3) 8.3 c 4) 4.4 c 5) 0.7 d
Decapitation time (Days after wounding) 14 12 10 8 6 4 2 0
41.3 47.5 40.7 39.6 35.8 23.7 30.4 20.1
ab a ab ab abc bc abc c
* Developmental stages have been indicated in Materials and methods and in Fig. la. Values are means of 800, 480 and 300 explants per each factor for wounding site, developmental stage and decapitation time experiments, respectively. Means with the same letter within columns are not significantly difeferent according to Tukey's studentized range test (alpha = 0.05).
Another important factor that was shown to influence bud and shoot formation was the developmental stage of the seedlings used as explants (Table 1). In this case, a trend was noted towards decreasing bud and shoot formation as the age of seedlings increased. Seedlings at the curved hypocotyl stage (9 d old; stage 1) were the explants that exhibited both the highest percentages of bud and shoot formation and the highest number of buds and shoots per explant. This system differs from that reported by Ezura et al. (1993), which used explants consisting of the proximal part of the hypocotyl and the radicle from mature seeds of chili pepper. In this system the best
228
Fig. 1. In vitro chili pepper plant regeneration from wounded and decapitated seedlings, a) Seedling explants at different developmental stages (from left to right: stages 1 to 5), b) seedlings showing the wounding site (from left to right: apical, middle and basal), c) decapitated seedling exhibiting shoot formation after 2 weeks of culture, d) explants bearing elongated shoots after 4 weeks of culture, e) regenerated plant, and f) regenerated plant transferred to soil.
229
shoot formation was observed when the seeds were first precultured for 5 h to 5 d on filter paper wetted with sterilized water, followed by cutting and culturing onto MS medium without growth regulators. Preculture periods longer than 5 d resulted in no shoot formation (Ezura et aL 1993). The third factor studied was the effect on shoot formation of decapitation of seedlings after wounding the hypocotyls (Table 1). It was noted that seedlings decapitated immediately after wounding were the explants that showed the lowest percentages of bud and shoot formation. The regeneration capacity generally increased as the time elapsed between wounding and decapitation increased. The maximum mean values for bud and shoot formation were observed when decapitation occurred 10-14 d after wounding, but there was no statistical difference between 2-14 d treatment. These results suggest that the presence of cotyledons has a direct effect on adventitious shoot formation. According to Ezura et al. (1993), the presence of radicle cells in the mature embryo axis explant was critical for organogenesis of adventitious buds. However, we obtained evidence suggesting that roots do not influence bud formation in hypocotyl tissues of chili pepper but rather have a stimulatory effect on bud elongation, an important step in shoot production and development (Valera-Montero and Ochoa-Alejo 1992). It is possible that both roots and cotyledons could supply some factors (growth regulators?) to the cells at the wounding site, which might exert a positive effect on in vitro shoot formation when seedlings or embryo axes of chili pepper are used as explants. Taking into account the mean values of bud and shoot formation in seedlings of cv. Mulato Bajio at the curved hypocotyl stage, which were wounded at the apical zone of the hypocotyl and decapitated 10 d after wounding, it was possible to get up to 90% of explants producing buds or shoots. Influence of culture medium and cultivars on shoot formation
The effects of culture medium on in vitro bud and shoot production in seedling explants from 21 chili pepper cultivars are summarized in Tables 2 and 3. The culture medium that induced the highest percentages and number of buds and shoots per explant was MS medium lacking growth regulators. Percentages of explants bearing buds and shoots on this culture medium ranged from 8 to 72, depending on the cultivar used, followed by medium 2 (2-60%), medium 3 (4-46%) and medium 4 (0-26%). The range of percentages of explants with shoots > 5 mm were 6-56%, 0-30%, 0-28% and 0-12% for media 1, 2, 3 and 4, respectively. We have previously shown that medium 4 was adequate to induce bud formation in chili pepper rooted hypocotyl explants cultured in an inverted position, and the buds were stimulated to elongate in media without growth regulators (Valera-
Montero and Ochoa-Aiejo 1992). However, in the system used in the present work, medium 4 was the least effective. Table 2. Influence of culture media on bud and shoot induction in 21 chili pepper cultivars. Culture medium
% Explants with buds and shoots
1 2 3 4
39.8 27.7 20.9 8.1
a b c d
% Explants with shoots*
26.8 a 12.6 b 7.9 c 2.2d
# Buds and shoots per explant 2.8 2.2 1.9 1.3
a b b b
Culture media composition: Medium 1 (MS without growth regulators), Medium 2 (MS + 1 #g L 1 TDZ), Medium 3 (MS + 1 mg L t BA), Medium 4 (MS + 0.3 mg L 4 IAA + 5 mg L 4 BA). Values are means of responses from 50 explants x 21 cultivars x culture medium. Column values with the same letter are not significantly different according to Turkey's studentized range test (alpha =
0.05).
* = Shoots > 5 mm in length.
Table 3. Influence of chili pepper cultivar on the induction of buds and/or shoots in wounded and decapitated seedings. Cultivar
% Explants with buds and shoots*
AnaheimTMR 23 Ancho Antocianina Ancho Compacto Ancho CR Ancho Dr Mora Ancho Esmeralda Ancho San Luis Caloro PS Cayenne College 64 L JalapeSo M Mulato Bajio Mulato 9222 Mulato 9236 Mulato9248 PasUla Morelia Piquin Serrano Milagro Serrano SQ 74 Serrano S 37 Wonder300 TMR
44 a 30 bcd 36 ab 25 cdef 23 cdef 7f 11 ef 16 ef 54 a 10 ef 43 ab 55 a 37 ab 32 bc 16 ef 18 ef 5f 7f 37 ab 5f 21 ef
Culture medium 1"* % Explants with: Buds and Shoots Shoots* 58 ab 46 abcd 46 abcd 38 defg 34 efgh 24 ghi 12 hi 42 bcdef 70 a 42 bcdef 56 abc 72 a 42 bcdef 44 abcde 34 efgh 32 fghi 18 hi 14 hi 44 cdef 8i 58 ab
46 ab 32 bcd 38 abc 26 def 24 cdef 8f 12 ef 18 ef 56 a 12 ef 48 ab 56 a 38 abc 34 abcd 18 ef 20 cdef 6f 8f 38 abc 6f 24 cdef
*= Mean values of responses from 50 explants x 4 culture media; composition of culture media as described in Materials and methods. **= Medium 1: MS without growth regulators. Fifty explants were used per treatment. ***= Shoots > 5 mm in length. Column values with the same letter are not significantly different according to Tukey's studentized range test (alpha = 0.05).
Data on bud and/or shoot formation in all tested chili pepper cultivars are shown in Table 3. Taking into account the mean percentages of buds and shoots produced by explants from 21 chili pepper cultivars grown on 4 culture media, it is possible to separate the cultivars as highly responsive (Mulato Bajio, Cayenne and Anaheim TMR 23 as a single group that overlaps with JalapeSo M, Mulato 9222,
230
Serrano SQ 74 and Ancho Compacto), intermediate (Mulato 9236, Mulato Antocianina, Ancho CR and Ancho Dr Mora) and low responsive (Wonder 300 TMR, Pasilla Morelia, Mulato 9248, Caloro PS, Ancho San Luis, College 64 L, Ancho Esmeralda, Serrano Milagro, Serrano S 37 and the wild type Piqufn). The number of buds and shoots per explant in chili pepper cultivars ranged from 0 to 3.4, but no significant differences were found for genotype x medium interactions, except in cvs. Serrano SQ 74 and Serrano S 37 (data not shown). Responses of chili pepper cultivars in medium 1 was also included in Table 3, since the highest bud and/or shoot formation was observed in this medium. The most responsive cultivars in terms of percentages of explants bearing buds and shoots were Mulato Bajio and Cayenne followed, as an overlapping group and in descending order, by Anaheim TMR 23, Wonder 300 TMR, Jalapefio M, Ancho Antocianina, Ancho Compacto and Mulato 9236. Some cultivars showed intermediate bud and shoot production (Caloro PS, College 64 L, Mulato 9222, Serrano SQ 74, Ancho CR, Ancho Dr Mora, Mulato 9248 and Pasilla Morelia), while others exhibited low regeneration capacity (Ancho Esmeralda, Piquin, Serrano Milagro, Ancho San Luis and Serrano S 37). Regarding shoot formation, in general the percentages of explants bearing shoots > 5 mm were lower than those that produced buds and shoots (range: 29 to 100 % with a mean of 67+20). It can be seen from Table 3 that Mulato Bajio and Cayenne cultivars exhibited the best response. Jalape5o M, Anaheim TMR 23, Ancho Compacto, Mulato 9222, Serrano SQ 74 and Mulato 9236 also showed good responses. Cultivars that displayed intermediate shoot formation capacity were Ancho Dr Mora, Pasilla Morelia, Wonder 300 TMR and Ancho CR, and those with low shoot capacity production were Caloro PS, Mulato 9248, Ancho San Luis, College 64 L, Ancho Esmeralda, Serrano Milagro, Piqu[n and Serrano S 37. Differences in bud and shoot forming capacity in tissues from cultivars of chili pepper cultured in vitro have been documented elsewhere (Ochoa-Alejo and Ireta-Moreno 1990). Shoot formation depends primarily on the cultivar used as the source of explants, and the culture medium is important for the expression of this capacity; cultivar Anaheim TMR 23 has consistently shown a high shoot forming capacity independent of the type of explant and culture medium employed, whereas Caloro cultivars have been found to be deficient in shoot formation in all tested media. In order to compare the bud and shoot induction efficiency of the method established in this study with that reported by Ezura et al. (1993), experiments with explants from 12 chili pepper cultivars were carried out (Table 4). No statistical differences were observed in the number of buds and shoots per explant produced by the two methods except for the cultivar Mulato 9248, which produced more buds and shoots with our method. The percentages of explants bearing buds and shoots
were generally higher in our method (7 out of 12 cultivars exhibited higher values). However, the most important advantage of our method is that a higher percentage of conversion of buds into well developed shoots (> 5 mm in length) was obtained (all 12 cultivars exhibited higher values). In our experience, shoots that do not reach this size after 4 weeks of incubation normally do not grow further and do not develop into plants (data not shown). Table 4. Comparative efficiency of bud and shoot induction in tissues from different chili pepper cultivars using the method described by Ezura et al. (1993) and method established in this work. Method used Cultivar
Ezura et al. (1993) %EBS %ES #BS
AnchoAntocianina Ancho CR Ancho San Luis Caloro PS Cayenne Jalapefio M Mulato 9236 Mulato 9248 Serrano S 37 Serrano Milagro Serrano SQ 74 Wonder300 TMR
46 28 10 14 12 30 6 0 16 6 60 22
16 22 0 6 2 4 2 0 0 2 24 12
1.6 1~8 1.5 2.0 2.6 1.6 1.5 0 3.0 2.8 3.1 1.7
This work %EBS 46 NS 38NS 12 NS 42** 70"* 56"* 44"* 34 ** 8 NS 14 NS 40 ** 58"*
%ES 32"* 26** 12"* 18"* 56"* 48"* 34"* 18 ** 6"* 8"* 38 ** 24"*
#BS 3.2 NS 2.7NS 3.0 NS 2.2NS 2,7 NS 2.5 NS 2.4 NS 2.3 ** 3.3 NS 2.6 NS 3.4 NS 2.3 NS
% EBS = Percentage of explants with buds and shoots; % ES = Percentage of explants with shoots >_ 5 mm; # BS = Number of buds + shoots per explant. Values are the means of responses from 50 explants per treatment. The culture medium was the semisolid MS medium without growth regulators for both methods. NS = Non significant; ** = Significant Z value at the 0.01 level, when comparing the method developed in this work to that of Ezura et al. (1993).
Rooting of shoots
Auxin pulses for rooting induction have been reported by several workers for different species (Arrillaga et al. 1991; Goldfarb et aL 1991; Mohammed and Vidaver 1988; Pythoud et al. 1986). In this study an IBA pulse of 60 mg L-1 in MS semisolid medium for 3 h was given to the explants, and they were subsequently cultured on MS medium without growth regulators, leading to rooting values of up to 80%. The rooted plants were transferred to pots with peat moss and grown in a greenhouse to maturity (Fig. 1 e,
f). In conclusion, we have demonstrated that the formation of buds and elongated shoots occurred at high efficiency by wounding the apical zone of hypocotyl tissues of chili pepper seedlings at the curved stage and decapitating these seedlings t0 to 14 d after culture on MS medium without growth regulators. The method described in this work is also more efficient and takes less time to regenerate whole chili pepper plants than that reported previously by our group (Valera-Montero and Ochoa-Alejo 1992). We have already tested this system for Agrobacterium - mediated transformation and some
231
transgenic plants have been regenerated that exhibited GUS expression and npt II gene integration as revealed by PCR (data not shown). Acknowledgements. We would like to thank Dr. Javier G. Salinas for the supply of seeds of C. a n n u u m . We are grateful to Dr. George J. Vandemark for critical review of the manuscript and to CONACYT (Mexico) for the fellowship to R. Ram[rez-Malag6n. This work was supported by CONACYT, project 3017N9306. References Agrawal S, Chandra N, Kothari SL (1989) Plant Cell Tissue Org Cult 16:47-55 Arrillaga I, Marzo T, Segura J (1991) Plant Cell Tissue Org Cult 27:341-348 Ebida AIA, Hu C (1993) Plant Cell Rep 13:107-110 Ezura H, Nishimiya S, Kasumi M (1993) Plant Cell Rep 12:676-680 Fari M, Czak6 M (1981) Sci Hort 15:207-213 Goldfarb B, Howe GT, Bailey LM, Strauss SH, Zaerr JB (1991) Plant Cell Rep 10:156-160 Gunay AL, Rao PS (1978) Plant Sci Lett 11:365-372 Harini I, Sita GL (1993) Plant Sci 89:107-112 Liu W, Parrott WA, Hildebrand DF, Collins GB, Williams EG (1990) Plant Cell Rep 9:360-364 Mohammed GH, Vidaver WE (1988) Plant Cell Tissue Org Cult 14:137-160 Murashige T, Skoog F (1962) Physiol Plant 15: 472497 Ochoa-Alejo N, Garcia-Bautista MAR (1990) Turrialba 40:311-318 Ochoa-Alejo N, Ireta-Moreno L (1990) Sci Hort 42: 21-28 Phillips GC, Hubstenberger JF (1985) Plant Cell Tissue Org Cult 4:261-269 Pythoud F, Buchala A J, Schmid A (1986) Physiol Plant 68:93-99 Szasz A, Nervo G, Fari M (1995) Plant Cell Rep 14: 666-669 Valera-Montero LL, Ochoa-Aiejo N (1992) Plant Sci 84:215-219
Plant Cell Reports (1996) 16:226-231
© Springer-VerlagI996
An improved and reliable chili pepper (Capsicum annuum L.) plant regeneration method Rafael Ramirez-Malag6n and Neftali Ochoa-Alejo Departamento de [ngenieria Gen~tica de Plantas, Unidad de Biotecnologia e Ingenieria Genetica de Plantas, Centro de Investigaei6n y de Estudios Avanzados de1 IPN, Apartado Postal 629; 36500-Irapuato, Gto., M6xico Received 9 October 1995/Revised version received 13 June 1996 - Communicated by G. C. Phillips
Abstract, In this work we report a new method for in vitro chili pepper (Capsicum annuum L.) plant regeneration based on shoot formation from wounded hypocotyls. Chili pepper seeds were surface sterilized and germinated on agar (0.8%) at 25 _+ 2oc in the dark. Five factors that may influence shoot regeneration were studied: age of seedlings, hypocotyl wounding site, time elapsed between wounding the hypocotyls and decapitation of seedlings, culture media and cultivars. In order to study the influence of the first three factors on shoot regeneration, the apical, middle or basal hypocotyl regions of seedlings of cv. Mulato Bajio at different stages of development (9, 15, 16, 21 and 28 d old) were wounded with a syringe needle, and the seedlings were cultured on MS semisolid medium without growth regulators at 25 +_2oc under a 16/8 h light/dark photoperiod (daylight fluorescent lamps; 35 #mol m -2 s-1) until decapitation. The seedlings were decapitated (3 mm below the cotyledons) at different times after wounding (0, 2, 4, 10, 12 and 14 d), and each explant was evaluated for bud and shoot formation (> 5 mm in length) at the wounded site after 30 d of incubation. In general, seedlings at the stage of curved hypocotyl (9 d old) wounded in the apical region of hypocotyl were the best explants for shoot regeneration when inoculated on culture medium without growth regulators. Decapitation after wounding also influenced the shoot regeneration efficiency, with 10-14 d being the best period. Up to 90% shoot regeneration in cv. Mulato Bajio was obtained under these conditions. Statistically significant differences were observed for shoot formation among 21 cultivars tested. Regeneration of whole plants was achieved by rooting the shoots with indole-3-butyric acid pulses of 60 mg L-1 for 3 h and then subculturing on MS medium without growth regulators. Abbreviations: BA - N6-Benzyladenine; IAA - indole3-acetic acid; IBA indole-3-butyric acid; MS Murashige and Skoog (1962) medium; TDZ Correspondence to: N. Ochoa-Alejo
thidiazuron. Key words: Capsicum annuum, organogenesis, plant regeneration.
chili
pepper,
Introduction
The establishment of an efficient in vitro plant regeneration system is a fundamental prerequisite for plant genetic engineering. Chili pepper is an important horticultural crop that belongs to the Solanaceae family. In general, it has long been recognized that cells, tissues and organs from members of this family (tobacco, potato, tomato, petunia) undergo morphogenesis and in vitro plant regeneration easily. Organogenesis and embryogenesis in chili pepper tissues have been reported by several authors (Gunay and Rao 1978; Fari and Czak6 1981; Philips and Hubstenberger 1985; Agrawal et al. 1989; Ochoa-Alejo and Garcia-Bautista 1990; Ochoa-Alejo and Ireta-Moreno 1990; Ebida and Hu 1993; Ezura et al. 1993; Harini and Sita 1993). However, unlike other solanaceous species, chili pepper has been a recalcitrant species with regard to its capacity for in vitro plant regeneration (Liu et al. 1990). Previously, we reported a novel chili pepper plant regeneration system based on shoot formation using rooted hypocotyls cultured upside down on shoot-inducing medium, and the subsequent rooting of shoots (Valera-Montero and Ochoa-Alejo 1992). This method recently was found to be appropriate for in vitro plant regeneration using several bell pepper genotypes (Sz~lsz et al. 1995). In this communication we report an improved organogenic method for in vitro chili pepper plant regeneration using injured and decapitated seedlings from different cultivars cultured on medium without growth regulators. Materials and methods Plant materials and explants. Seeds of chiti pepper (Capsicum annuum L.) were surface sterilized by
227 soaking them in a commercial bleach solution (20% v/v + 0.1% Tween 20) for 20 min. The seeds were rinsed 3 times with sterile distilled water and inoculated onto a sterile gel prepared with 0.8% agar, without supplemental organic or mineral nutrients, and adjusted to pH 5.8. Seeds were incubated in the dark at 25 _+2oC for 9-16 d, depending on the stage needed for each treatment. The developmental stages tested were as follows (Fig. la): Stage 1, seedlings with curved hypocotyls (9 d old); Stage 2, etiolated seedlings (!5 d old); Stage 3, green seedlings (9 d in the dark and 7 d in the light with a 16 h lightJ8 h dark photoperiod; daylight fluorescent lamps, 35 p.mol m-2 s-l); Stage 4, green seedlings with the first pair of well developed leaves (9 d in the dark/12 d in the light); Stage 5, green seedlings with two pairs of well developed leaves (9 d in the dark/19 d in the light). Explant treatment. A total of 480 seedlings (cv. Mulato Bajio) for each developmental stage were used and the seedlings were divided into 3 groups each of 160. Hypocotyls from seedlings of each group were wounded by puncturing them with a sterile syringe needle (gauge: 0.72 mm; length: 32 mm) as shown in Figure lb. The selected sites for wounding were: apical region of the hypocotyls (3 to 5 mm below the cotyledons); middle region of the hypocotyls; or basal region of the hypocotyls. Explant culture. Groups of 160 seedlings were subdivided into 8 subgroups each of 20 seedlings, and these were distributed in 4 bottles (135 ml with 20 ml of culture medium) containing 5 seedlings each. The culture medium used in these experiments was MS semisolid medium (Murashige and Skoog 1962) without growth regulators, pH 5.8 and gelled with Phytagel (3 g L-I; Sigma Chemical Co). The seedlings of each group were decapitated by cutting the shoot 3 mm below the cotyledons at different times (0, 2, 4, 6, 8, 10, 12 and 14 d after the date of wounding) and were subcultured on fresh medium lacking growth regulators. Experimental design. A factorial experiment (3 wounding sites x 5 developmental stages x 8 decapitation dates = 120 treatments) was established with 4 replicates (each bottle as a replicate) and 5 explants per replicate. The mean number of buds and/or shoots per hypocotyl, percentage of hypocotyls that produced buds and/or shoots, and the percentage of explants that formed shoots > 5 mm in length were recorded 30 d after decapitation. Analysis of variance for the factorial experiment, and mean separation for each variable and factor within each variable, were carried out using the Tukey test at 5% level. Influence of culture medium and cultivar on shoot regeneration. Using the data obtained from the factorial experiment, bud and shoot formation in 21 chili pepper cultivars using 4 different culture media were investigated. Seedlings (9 d old) were wounded at the apical region of the hypocotyls and then decapitated 10 d after wounding. Decapitated seedlings were grown on MS medium with the following compositions: (1) without growth regulators (Ezura et aL 1993), (2) 1 pg L-1 thidiazuron (TDZ), (3) 1 mg L-1 benzyladenine (BA), and (4) 0.3 mg L-1 indoleacetic acid (IAA) plus 5 mg L-1 BA (Valera-Montero and Ochoa-Alejo 1992). In order to compare the shoot efficiency formation of this method with that of Ezura et al. (1993), a total of 50 explants from each of 12 chili pepper cultivars were prepared as reported by these authors and were inoculated on MS medium lacking growth regulators. Evaluations of response were carried out after 30 d of incubation under the same conditions described for the factorial experiment. Data were statistically analyzed by analysis of variance and the Tukey test at the level of 5%. In all experiments in which data on shoot regeneration was recorded, only shoots >_5 mm in length were recorded, as these shoots were the only ones capable of plant formation after rooting. Rooting of shoots. Regenerated shoots that reached > 5 mm in length were dissected from the hypocotyls and rooted on MS medium using an IBA pulse of 60 mg L-1 for 3 h. They were then subcultured on media without growth regulators. Rooted shoots were transferred to pots (1 L) containing peat moss, and developed into mature plants under greenhouse conditions. All experiments were performed at least twice.
Results and discussion
Influence of seedling age, wounding decapitation time on shoot formation
site
and
Injured hypocotyls were found to produce a small callus at the wounding site, and buds later formed that developed into shoots (Fig. lc, d). Analysis of variance for the factorial experiment showed statistical significance at the level of 0.01 for the percentage of hypocotyls regenerating buds and shoots. Bud and shoot regeneration was influenced, in decreasing order, by the hypocotyl wounding site, the developmental stage of seedlings, and the time elapsed between wounding and decapitation of seedlings (Table 1), as evidenced by analysis of variance data that showed sums of squares of 24.5, 22.3 and 3.6, respectively. In general, seedlings injured at the apical region of the hypocotyl exhibited the highest bud and shoot induction response, whereas those wounded at the basal zone showed the worst response. These results partially agree with those reported by Fari and Czako (1981) who observed that explants from the apical region of chili pepper hypocotyls produced buds, while the explants from the middle and the basal zones of hypocotyls produced only callus and roots when cultured on a bud inducing medium. Table 1. Effect of the wounding site, developmental stage of seedlings, and time of decapitation aEer wounding on shoot induction in chili pepper explants. % Explants with shoots Wounding site Apical 23.4a Middle 11.4b Basal 1.6c
Developmental stage* 1) 27.3 a 2) 21.0 b 3) 8.3 c 4) 4.4 c 5) 0.7 d
Decapitation time (Days after wounding) 14 12 10 8 6 4 2 0
41.3 47.5 40.7 39.6 35.8 23.7 30.4 20.1
ab a ab ab abc bc abc c
* Developmental stages have been indicated in Materials and methods and in Fig. la. Values are means of 800, 480 and 300 explants per each factor for wounding site, developmental stage and decapitation time experiments, respectively. Means with the same letter within columns are not significantly difeferent according to Tukey's studentized range test (alpha = 0.05).
Another important factor that was shown to influence bud and shoot formation was the developmental stage of the seedlings used as explants (Table 1). In this case, a trend was noted towards decreasing bud and shoot formation as the age of seedlings increased. Seedlings at the curved hypocotyl stage (9 d old; stage 1) were the explants that exhibited both the highest percentages of bud and shoot formation and the highest number of buds and shoots per explant. This system differs from that reported by Ezura et al. (1993), which used explants consisting of the proximal part of the hypocotyl and the radicle from mature seeds of chili pepper. In this system the best
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Fig. 1. In vitro chili pepper plant regeneration from wounded and decapitated seedlings, a) Seedling explants at different developmental stages (from left to right: stages 1 to 5), b) seedlings showing the wounding site (from left to right: apical, middle and basal), c) decapitated seedling exhibiting shoot formation after 2 weeks of culture, d) explants bearing elongated shoots after 4 weeks of culture, e) regenerated plant, and f) regenerated plant transferred to soil.
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shoot formation was observed when the seeds were first precultured for 5 h to 5 d on filter paper wetted with sterilized water, followed by cutting and culturing onto MS medium without growth regulators. Preculture periods longer than 5 d resulted in no shoot formation (Ezura et aL 1993). The third factor studied was the effect on shoot formation of decapitation of seedlings after wounding the hypocotyls (Table 1). It was noted that seedlings decapitated immediately after wounding were the explants that showed the lowest percentages of bud and shoot formation. The regeneration capacity generally increased as the time elapsed between wounding and decapitation increased. The maximum mean values for bud and shoot formation were observed when decapitation occurred 10-14 d after wounding, but there was no statistical difference between 2-14 d treatment. These results suggest that the presence of cotyledons has a direct effect on adventitious shoot formation. According to Ezura et al. (1993), the presence of radicle cells in the mature embryo axis explant was critical for organogenesis of adventitious buds. However, we obtained evidence suggesting that roots do not influence bud formation in hypocotyl tissues of chili pepper but rather have a stimulatory effect on bud elongation, an important step in shoot production and development (Valera-Montero and Ochoa-Alejo 1992). It is possible that both roots and cotyledons could supply some factors (growth regulators?) to the cells at the wounding site, which might exert a positive effect on in vitro shoot formation when seedlings or embryo axes of chili pepper are used as explants. Taking into account the mean values of bud and shoot formation in seedlings of cv. Mulato Bajio at the curved hypocotyl stage, which were wounded at the apical zone of the hypocotyl and decapitated 10 d after wounding, it was possible to get up to 90% of explants producing buds or shoots. Influence of culture medium and cultivars on shoot formation
The effects of culture medium on in vitro bud and shoot production in seedling explants from 21 chili pepper cultivars are summarized in Tables 2 and 3. The culture medium that induced the highest percentages and number of buds and shoots per explant was MS medium lacking growth regulators. Percentages of explants bearing buds and shoots on this culture medium ranged from 8 to 72, depending on the cultivar used, followed by medium 2 (2-60%), medium 3 (4-46%) and medium 4 (0-26%). The range of percentages of explants with shoots > 5 mm were 6-56%, 0-30%, 0-28% and 0-12% for media 1, 2, 3 and 4, respectively. We have previously shown that medium 4 was adequate to induce bud formation in chili pepper rooted hypocotyl explants cultured in an inverted position, and the buds were stimulated to elongate in media without growth regulators (Valera-
Montero and Ochoa-Aiejo 1992). However, in the system used in the present work, medium 4 was the least effective. Table 2. Influence of culture media on bud and shoot induction in 21 chili pepper cultivars. Culture medium
% Explants with buds and shoots
1 2 3 4
39.8 27.7 20.9 8.1
a b c d
% Explants with shoots*
26.8 a 12.6 b 7.9 c 2.2d
# Buds and shoots per explant 2.8 2.2 1.9 1.3
a b b b
Culture media composition: Medium 1 (MS without growth regulators), Medium 2 (MS + 1 #g L 1 TDZ), Medium 3 (MS + 1 mg L t BA), Medium 4 (MS + 0.3 mg L 4 IAA + 5 mg L 4 BA). Values are means of responses from 50 explants x 21 cultivars x culture medium. Column values with the same letter are not significantly different according to Turkey's studentized range test (alpha =
0.05).
* = Shoots > 5 mm in length.
Table 3. Influence of chili pepper cultivar on the induction of buds and/or shoots in wounded and decapitated seedings. Cultivar
% Explants with buds and shoots*
AnaheimTMR 23 Ancho Antocianina Ancho Compacto Ancho CR Ancho Dr Mora Ancho Esmeralda Ancho San Luis Caloro PS Cayenne College 64 L JalapeSo M Mulato Bajio Mulato 9222 Mulato 9236 Mulato9248 PasUla Morelia Piquin Serrano Milagro Serrano SQ 74 Serrano S 37 Wonder300 TMR
44 a 30 bcd 36 ab 25 cdef 23 cdef 7f 11 ef 16 ef 54 a 10 ef 43 ab 55 a 37 ab 32 bc 16 ef 18 ef 5f 7f 37 ab 5f 21 ef
Culture medium 1"* % Explants with: Buds and Shoots Shoots* 58 ab 46 abcd 46 abcd 38 defg 34 efgh 24 ghi 12 hi 42 bcdef 70 a 42 bcdef 56 abc 72 a 42 bcdef 44 abcde 34 efgh 32 fghi 18 hi 14 hi 44 cdef 8i 58 ab
46 ab 32 bcd 38 abc 26 def 24 cdef 8f 12 ef 18 ef 56 a 12 ef 48 ab 56 a 38 abc 34 abcd 18 ef 20 cdef 6f 8f 38 abc 6f 24 cdef
*= Mean values of responses from 50 explants x 4 culture media; composition of culture media as described in Materials and methods. **= Medium 1: MS without growth regulators. Fifty explants were used per treatment. ***= Shoots > 5 mm in length. Column values with the same letter are not significantly different according to Tukey's studentized range test (alpha = 0.05).
Data on bud and/or shoot formation in all tested chili pepper cultivars are shown in Table 3. Taking into account the mean percentages of buds and shoots produced by explants from 21 chili pepper cultivars grown on 4 culture media, it is possible to separate the cultivars as highly responsive (Mulato Bajio, Cayenne and Anaheim TMR 23 as a single group that overlaps with JalapeSo M, Mulato 9222,
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Serrano SQ 74 and Ancho Compacto), intermediate (Mulato 9236, Mulato Antocianina, Ancho CR and Ancho Dr Mora) and low responsive (Wonder 300 TMR, Pasilla Morelia, Mulato 9248, Caloro PS, Ancho San Luis, College 64 L, Ancho Esmeralda, Serrano Milagro, Serrano S 37 and the wild type Piqufn). The number of buds and shoots per explant in chili pepper cultivars ranged from 0 to 3.4, but no significant differences were found for genotype x medium interactions, except in cvs. Serrano SQ 74 and Serrano S 37 (data not shown). Responses of chili pepper cultivars in medium 1 was also included in Table 3, since the highest bud and/or shoot formation was observed in this medium. The most responsive cultivars in terms of percentages of explants bearing buds and shoots were Mulato Bajio and Cayenne followed, as an overlapping group and in descending order, by Anaheim TMR 23, Wonder 300 TMR, Jalapefio M, Ancho Antocianina, Ancho Compacto and Mulato 9236. Some cultivars showed intermediate bud and shoot production (Caloro PS, College 64 L, Mulato 9222, Serrano SQ 74, Ancho CR, Ancho Dr Mora, Mulato 9248 and Pasilla Morelia), while others exhibited low regeneration capacity (Ancho Esmeralda, Piquin, Serrano Milagro, Ancho San Luis and Serrano S 37). Regarding shoot formation, in general the percentages of explants bearing shoots > 5 mm were lower than those that produced buds and shoots (range: 29 to 100 % with a mean of 67+20). It can be seen from Table 3 that Mulato Bajio and Cayenne cultivars exhibited the best response. Jalape5o M, Anaheim TMR 23, Ancho Compacto, Mulato 9222, Serrano SQ 74 and Mulato 9236 also showed good responses. Cultivars that displayed intermediate shoot formation capacity were Ancho Dr Mora, Pasilla Morelia, Wonder 300 TMR and Ancho CR, and those with low shoot capacity production were Caloro PS, Mulato 9248, Ancho San Luis, College 64 L, Ancho Esmeralda, Serrano Milagro, Piqu[n and Serrano S 37. Differences in bud and shoot forming capacity in tissues from cultivars of chili pepper cultured in vitro have been documented elsewhere (Ochoa-Alejo and Ireta-Moreno 1990). Shoot formation depends primarily on the cultivar used as the source of explants, and the culture medium is important for the expression of this capacity; cultivar Anaheim TMR 23 has consistently shown a high shoot forming capacity independent of the type of explant and culture medium employed, whereas Caloro cultivars have been found to be deficient in shoot formation in all tested media. In order to compare the bud and shoot induction efficiency of the method established in this study with that reported by Ezura et al. (1993), experiments with explants from 12 chili pepper cultivars were carried out (Table 4). No statistical differences were observed in the number of buds and shoots per explant produced by the two methods except for the cultivar Mulato 9248, which produced more buds and shoots with our method. The percentages of explants bearing buds and shoots
were generally higher in our method (7 out of 12 cultivars exhibited higher values). However, the most important advantage of our method is that a higher percentage of conversion of buds into well developed shoots (> 5 mm in length) was obtained (all 12 cultivars exhibited higher values). In our experience, shoots that do not reach this size after 4 weeks of incubation normally do not grow further and do not develop into plants (data not shown). Table 4. Comparative efficiency of bud and shoot induction in tissues from different chili pepper cultivars using the method described by Ezura et al. (1993) and method established in this work. Method used Cultivar
Ezura et al. (1993) %EBS %ES #BS
AnchoAntocianina Ancho CR Ancho San Luis Caloro PS Cayenne Jalapefio M Mulato 9236 Mulato 9248 Serrano S 37 Serrano Milagro Serrano SQ 74 Wonder300 TMR
46 28 10 14 12 30 6 0 16 6 60 22
16 22 0 6 2 4 2 0 0 2 24 12
1.6 1~8 1.5 2.0 2.6 1.6 1.5 0 3.0 2.8 3.1 1.7
This work %EBS 46 NS 38NS 12 NS 42** 70"* 56"* 44"* 34 ** 8 NS 14 NS 40 ** 58"*
%ES 32"* 26** 12"* 18"* 56"* 48"* 34"* 18 ** 6"* 8"* 38 ** 24"*
#BS 3.2 NS 2.7NS 3.0 NS 2.2NS 2,7 NS 2.5 NS 2.4 NS 2.3 ** 3.3 NS 2.6 NS 3.4 NS 2.3 NS
% EBS = Percentage of explants with buds and shoots; % ES = Percentage of explants with shoots >_ 5 mm; # BS = Number of buds + shoots per explant. Values are the means of responses from 50 explants per treatment. The culture medium was the semisolid MS medium without growth regulators for both methods. NS = Non significant; ** = Significant Z value at the 0.01 level, when comparing the method developed in this work to that of Ezura et al. (1993).
Rooting of shoots
Auxin pulses for rooting induction have been reported by several workers for different species (Arrillaga et al. 1991; Goldfarb et aL 1991; Mohammed and Vidaver 1988; Pythoud et al. 1986). In this study an IBA pulse of 60 mg L-1 in MS semisolid medium for 3 h was given to the explants, and they were subsequently cultured on MS medium without growth regulators, leading to rooting values of up to 80%. The rooted plants were transferred to pots with peat moss and grown in a greenhouse to maturity (Fig. 1 e,
f). In conclusion, we have demonstrated that the formation of buds and elongated shoots occurred at high efficiency by wounding the apical zone of hypocotyl tissues of chili pepper seedlings at the curved stage and decapitating these seedlings t0 to 14 d after culture on MS medium without growth regulators. The method described in this work is also more efficient and takes less time to regenerate whole chili pepper plants than that reported previously by our group (Valera-Montero and Ochoa-Alejo 1992). We have already tested this system for Agrobacterium - mediated transformation and some
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transgenic plants have been regenerated that exhibited GUS expression and npt II gene integration as revealed by PCR (data not shown). Acknowledgements. We would like to thank Dr. Javier G. Salinas for the supply of seeds of C. a n n u u m . We are grateful to Dr. George J. Vandemark for critical review of the manuscript and to CONACYT (Mexico) for the fellowship to R. Ram[rez-Malag6n. This work was supported by CONACYT, project 3017N9306. References Agrawal S, Chandra N, Kothari SL (1989) Plant Cell Tissue Org Cult 16:47-55 Arrillaga I, Marzo T, Segura J (1991) Plant Cell Tissue Org Cult 27:341-348 Ebida AIA, Hu C (1993) Plant Cell Rep 13:107-110 Ezura H, Nishimiya S, Kasumi M (1993) Plant Cell Rep 12:676-680 Fari M, Czak6 M (1981) Sci Hort 15:207-213 Goldfarb B, Howe GT, Bailey LM, Strauss SH, Zaerr JB (1991) Plant Cell Rep 10:156-160 Gunay AL, Rao PS (1978) Plant Sci Lett 11:365-372 Harini I, Sita GL (1993) Plant Sci 89:107-112 Liu W, Parrott WA, Hildebrand DF, Collins GB, Williams EG (1990) Plant Cell Rep 9:360-364 Mohammed GH, Vidaver WE (1988) Plant Cell Tissue Org Cult 14:137-160 Murashige T, Skoog F (1962) Physiol Plant 15: 472497 Ochoa-Alejo N, Garcia-Bautista MAR (1990) Turrialba 40:311-318 Ochoa-Alejo N, Ireta-Moreno L (1990) Sci Hort 42: 21-28 Phillips GC, Hubstenberger JF (1985) Plant Cell Tissue Org Cult 4:261-269 Pythoud F, Buchala A J, Schmid A (1986) Physiol Plant 68:93-99 Szasz A, Nervo G, Fari M (1995) Plant Cell Rep 14: 666-669 Valera-Montero LL, Ochoa-Aiejo N (1992) Plant Sci 84:215-219
