Plant Cell Reports (1996) 15:536-540
Induction of direct somatic embryogenesis and plant regeneration in pepper (Capsicum annuum L.) Maria L. Binzel 1, N. Sankhla 1, 2, Sangeeta Joshi 2, and Daksha Sankhla 2 Texas Agricultural Experiment Station, Texas A&M University Research and Extension Center, 1380 A&M Circle, E1 Paso, Texas 79927-5020, USA 2 j. N. Vyas University, Jodhpur 342001, India Received 17 March 1995/Revised version received 19 September 1995 - Communicated by G. C. Phillips
Summary. Pepper (cv. New Mexico - 6 and Rajur Hirapur) plants were regenerated fu immature zygotic embryos via direct somatic embryogenesis. Somatic embryos were formed directly, without any intervening callus, on the zygotic enibryo apex, embryo axis and cotyledons on Murashige and Skoog's (MS) mextinln containing 2,4-D (418 /~M), thidiazuron (10/zM) and a high concentration of sucrose (6-10%). The best response was observed on MS medium containhag 2,4-D (9 I~M); coconut water (10%) and high sucrose (8%). The entire process of induction and maturation of the embryos was completexl on the same medhnn. Histological exanffnation indicateA that secondary embryogenesls also occurred directly from the primary somatic embryos. Differentiation of elnbryos was nonsynchronous, and some embryos were swollen mad distorted with fasciation. More than 70% of the mature normal somatic embryos germinated readily on MS medium containing GA 3 or TDZ; alone and in combination, and following transfer to pots developed into normal plants. Abbreviations: CM, Coconut lnilk; 2,4-D, 2,4diclflorophonoxyacetic acid; GA3, gibberellic acid; MS, Mnraslffge and Skoog (1962) mexlium; NAA, napthaleneacetic acid; TDZ, thidiazalron
Introduction Plant transformation and gene cloning are beconffng unportant tools in plant ilnprovement via genetic engineering. However, developlnent of an efficient and reprcxtucible tissue culture regeneration protocol is the first step in utilizing the power and potential of this new teclmology. Although excellent progress has been lnade in obtaining transge~ffc plants from many species of the Solanaceae, pepper has laggexl behind most likely due to tmavailability of an efficient regeneration protocol (Lin et al. 1990; Ebida and Hu 1993). Correspondence to: M. L. Binzel
In pepper, based on organogenesis of diverse explants, several tissue culture protocols have been described (Phillips and Hubstenberger 1985; Agrawal et al. 1989; Ochoa-Alejo and Ireta-Moreno 1990; Arroyo and Revilla 1991; Valera-Montero and Ochoa-Alejo 1992; Ebida and Hu 1993; F~,ri and Andr~isfalvy 1994). However, the intervarietal differences in regeneration from various explants are highly pronounced. Therefore, cultivar and tissue specific media have been devised to optimize regeneration for specific cultivars (FAri and Andr~isfalvy 1994). Additionally, in several cultivars, aclffeving the elongation of in vitro fonnexl shoot buds in itself poses a col~siderable challenge becanse the ill-def'med buds or shootlike stmctnres either resist elongation or produce rosettes of distorted leaves which generally do not produce normal shoots (Arroyo and Revilla 1991; Valera-Montero and Ochoa-Alejo 1992). Numerous plant Sl~zies have been reported to be capable of forming somatic embryos from diverse explants (Williams and Maheswaran 1986; Kiyosue et al. 1993). Somatic embryogenesis offers distinct advantages over regeneration via organogenesis (Willianls and Maheswaran 1986). For peppers, tiffs could be a method of choice because of the difficulties encountered in elongation of in vitro organogelffC shoot buds. Surprisingly, except for a recent report on pepper cv. California Wonder (Harini and Lakslmff Sita 1993), little infornmtion is available on somatic embryogenesis ha peppers. Further, since somatic embryos develophlg directly from organs without any intervening callus tend to be genetically more uniform than those generated via callus, direct somatic embryogenesis may be preferred in the unprovement of crops via bioteclmology (Maheswaran and Williams 1984). Tiffs report describes the initial results of our work on the development of a regeneration protocol via direct somatic elnl)ryogenesis from hnmatnre embryos of two cnltivars of peppers.
537 Materials and Methods Two cultivars of pepper (Capsicum ammum L) were used as experimental material, viz., New Mexico - 6 (NM-6), a non-pungent long green chile; and Rajpur Hirapur (RH), a pungent type chile. NM6 is extensively grown in New Mexico and the southwestern region of the USA, while cv. RH is highly popular in northwestern semi-arid regions of India. Plants were grown in the greenhouse separately to prevent cross pollination. Fully elongated green, red-green and red fruits were collected from these plants. The fi'uits were surface sterilized with commercial clorox (5.25 % active ingredient) for 10 min and then washed thoroughly with sterile water. Further sterilization was accomplished by dipping the fruits in 90% ethanol for 1 min followed by repeated washing with autoclaved water. Fruits were then opened aseptically in a laminar flow bood and the developing seeds were separated fi'om the placenta using a sterile scalpel. The immature seeds were ~wface sterilized by immersing in 20% commercial clorox for 1-2 rain followed by thorough rinsing in sterile Water. Excised embryos of various sizes (4-10 ram) were used as primary explants. The procedure used to isolate intact immature embryos fi'om the developing seeds was as follows. "I'he sterile seeds were stored in autoclaved water under the laminar flow hood following removal from the fi'uil. The seed coat was peeled starting from the radicle end towards the cotyledonary end keeping the seed vertical. Then, a slight pressure was exerted on the cotyledonary side of the seed to force the embryo out of the seed coat and endosperm. Any adhering piece of seed coat or endosperm was then carefully removed using a pair of fine forceps. With some practice and patience it was possible to isolate a sufficient number of intact embryos for the experiment. The basal nutrient medium (BM) contained Murashige and Skoog (1962) salts and organic components. The embryo induction medium consisted of BM supplemented with 2-10% sucrose, 1-10 uM thidiazuron (TDZ), 4.5-18 ~M 2,4-D or 5.4-21.5 ~M NAA and 0.25% Phytagel. Based on earlier results (Harini and Lakshmi Sita 1993) an experiment was also conducted in which the excised embryos were cultured on BM supplemented with 8% sucrose, 10% coconut water, 9 uM 2,4-D and 10 ~M TDZ singly and in combination. The pH of the medium was adjusted to 5.8 prior to autoclaving. The cultures were maintained at Z5__2~ under a white fluorescent light bank (40-50 x~tnol m -~ s-~) with a 16 h photoperiod. The embryos, in groups of 6 each, were cultured iu 6 cm polystyrene petri dishes containing 15 ml of the desired media. For each treatment, 24 embryos were cultured and each experime,a was repeated at least three times. The cultures were observed regularly, and the data recorded every week. Depending on the induction media used. clumps of somatic embryos at various stages of development were clearly visible within 15-21 d. The frequency of cultures showing embryo induction and the number of embryos per explant were recorded after 30d. The embryos matured on the induction medium itself. After 24 d of culture the cluster of embryos were transferred to baby food jars containing germination media. The germination media consisted of BM, 2% sucrose, 10 ~M AgNO3, and 2.8 ~M GA 3 or 0.05 ~M TDZ alone or in combination. After the embryos germinated, and developed to a height of 4-5 cm, the plantlets were transferred to Magenta G7 boxes containing sterile soil:vermlculite (3:1) and moistened with 20 ml of liquid BM. After 8-10 d, the cap of the boxes was replaced wlth a piece of polyethylene film with 4-5 holes. After an additional acclimation of 7-10 d, the plantlets were transferred to pots and placed in the greenhouse where they developed into normal plants. For histological examination, the samples were fixed in formalin/acetic acid/alcohol (FAA) and stored in 70% ethanol. These samples were processed for paraffin embedding as described by
Jd~ansen (1940). Sections of 10 ~m thickness were cut using a Spencer microtome. The ribbons containing the sections were passed through a series of deparaffinizing and dehydrating solutions and stained in toluidine blue. The sections were examined under a light microscope (Olympus Model BHS) equipped with an automatic photomicrographic system (PM- I 0ADS). Data were analyzed using SAS (SAS Institute 1990). Mean separation was determined by using Duncan's multiple range test. Results and Discussion In cv. RH, cotyledonary stage e m b r y o s o f 7-8 m m w e r e the only ones that r e s p o n d e d well. ha cv. N M - 6 , the e m b r y o s (6-7 n m 0 excised from fruits that w e r e t u r n i n g red gave the best reslxmse. In contrast, in p e p p e r cv. C a l i f o r n i a W o n d e r i n m m t u r e e m b r y o s r a n g i n g f r o m 3-10 m m in length r e s p o n d e d to f o r m somatic e m b r y o s (Harini and Lakslmfi Sita 1993). Specificity ha explant d e v e l o p m e n t a l stage with r e s p e c t to somatic e m b r y o g e n e s i s has b e e n reported previously (Ln and Vasil 1982; Hazra et al. 1989). F u r t h e r , we observed flint in cv. N M - 6 it was not n e c e s s a r y to excise the e l n b r y o s completely f r o m the seed. T h e e m b r y o s that w e r e cultured t o g e t h e r with t h e e n d o s p e r m and the i n t e g u m e n t , with only the outer seeA coat r e m o v e d , also reslxmded eqtmlly well. In y o u n g or u n r e s p o n s i v e e m b r y o s the i n t e g m n e n t soon b e c a m e black, and ulthnately necrosis set in (unpublisheA data). As reported for peanut ( H a z r a et al. 1989), the expansion o f c o t y l e d o n s and g r o w t h o f the e m b r y o axis o f pepper r e m a i n e d highly s u p p r e s s e d ha the p r e s e n c e o f auxins. No somatic
embryosw e r e
o b s e r v e d in the p r e s e n c e o f N A A or
2,4-D w h e n used in c o m b i n a t i o n with sucrose, but without T D Z or o t h e r s n p p l e m e n t s . S h n i l a r l y , somatic e m b r y o s w e r e not f o r m e d in the p r e s e n c e o f T D Z (1-10 ~ M ) alone. In the p r e s e n c e o f T D Z , the cotyledons e x p a n d e d e n o r m o u s l y , b o t h hypocotyl and cotyledons turned green, elongation g r o w t h o f the hypocotyl r e m a i n e d highly arrested, mad after s o m e swelling several e x t r e m e l y small shoot-buds were initiated f r o m the h y p o c o t y l and cotylexlon o f t h e excised e m b r y o s (data not shown). Unusual elongation of only one cotyledon, w h i c h was in contact with t h e mexlium, was c o m m o n l y o b s e r v e d in the p r e s e n c e of T D Z . T D Z induced e x p a n s i o n o f cotyledons, and shoot fonnation was almost completely s u p p r e s s e d in the p r e s e n c e of 2,4-D ha the medhtln. At a low c o n c e n t r a t i o n o f sucrose, some loose, fluffy calhts w~s formed f r o m the root, m o r e so in c w R H . In general, o n c e callus was initiated, the induction o f somatic e m b r y o s did not occur. T h e c o n c e n t r a t i o n o f s u c r o s e in the m e d i n m was also found to h a v e a p r o f o u n d influence o n somatic embryogenesis. In the p r e s e n t study, in cv. N M - 6 the best r e s p o n s e was o b s e r v e d with 8 % sucrose, wlfile in cv. R H 1 0 % s u c r o s e was found m o r e effective (Table 1). The c o n c e n t r a t i o n o f s u c r o s e in the m e d i n m has b e e n s h o w n to
538 Table 1. Effect of sucrose mad 2,4-D on pepper somatic embryogenesis after 30 d of culture." Sucrose(N)
2, 4-D O~M) 4
9 % Explants Nnmber of % Explants Number of forming embryos/ fomfing embryos/ somatic explant somatic explant emb_ry_os____ 0nean ) eml~:y_os (Inean)_~ NM-6 2 0 Cb 0 C 4 2t'* 1-2(1) 5e 1-2(1) 6 4e 1-2(1) 12c 1-3(2) 8 10cd 1-2(2) 30a 3-7(5) 10 8d 1-2(2) 25a 3-5 (3) RH 2 0 C 0 C 4 0 C 0 C 6 8e 1-2(1) 18d 1-3(2) 8 20d 1-3(2) 40b 4-7(4) __ 10 3~lc~__ 1-~(2) 55a__ 4-9(6) "The basal medium contained MS salts and vitamins + TDZ (10~zM) UMostly callus was formed *Means with different letters ate significant at the 5% level by Duncan's nmltiple range test be quite hnportant for the induction of somatic embryogenesis ha several other plants (Lazzeri et al. 1988; Eapen and George 1993; Elnons et al. 1993; Patti et al. 1994). Emons et al. (1993) demonstratexl the hnportance of manipnlating the balance of sucrose, mannitol, L-proline, ABA and GA 3 ha hnproving the frequency and uniformity of somatic embryo matttration ha maize. According to these authors sucrose appears to have a dual role. In addition to its osmotic effect, it is also necessary as a carbon source for both calhts maintenance and embryo maturation, while mmmitol acts as an agent that maintains the embryogenic competence. ha the presence of TDZ and 2,4-D, beginning at 15-20 d in culture, small globular structures were conspictmnsly visible on the excised embryos (Fig. la, b). Generally, these structures first appearexl at or near the shoot apex, but soon such protuberances also appeareA Oll the short, stttbby hypocotyl as well as o n cotylexlons (Fig. la). Dnring the ensuing 7-10 d these protrusions developexl into somatic embryos (Fig. lc, d). The embryos were tbrmexl directly without any intervening callus, although some callus was formed from the root region of the excisexl embryos and the wounded portion of the hypocotyl. As in peanut (Hazra et al. 1988), the development of somatic embryos was asynchronous. As a result, various stages of embryo development could be observext in the same cluster of embryos originating from an explant. Some embryos were swollen and distortexl, and fhsciation of embryos resulting in multiple embryos was quite frequent (Fig. le). The fasciation of eml, ryos lnay have been due to an extremely close proximity of embryo initiation sites on the explants. In addition, frequently the primary somatic embryos developing on the cnltttrexl innnatnre zygotic embryos also showexl signs
18 % Explant forming sonmtic ~_anllk'yos 0 3ef Bed 20b 18b 0 0 15d 35bc 48a~
Number of embryos/ explant (mean) C 1-2(1) 1-2(1) 2-4(2) 1-3 (2) C C 1-2(1) 3-5(3) 3-7(4)
of secondary embryogenesis. It was necessary to transfer the somatic embryos to the gemfination meAia within 21-28 d. Otherwise, dexlifferentiation set in, resulting in callns fommtion. Table 2. Effect of tlfidiazatron and coconut water on pepper somafimemb~yogenesis. cv. Mexlia" % Explants Nnmber of forming embryos/ somatic explant e|nl3ryo.q (menn) __ NM-6 BM + CM 60a* 4-9(6) BM + TDZ 30b 3-7(5) BM + CM + TDZ 351, 2-5(2) RH
BM + CM BM + TDZ BM + CM + TDZ
85a 40b 45b
5-12(8) 4-7(4) 3-6(3)
aBM = MS + 8% sucrose + 9~M 2, 4-D, CM = 10%, TDZ = 10~M "means with different letters are significant at 5% level by Duncan's nmltlple range test Histology of somatic embryo producing regions of nnmature zygotic embryos colffirmexl that the induction and development processes were embryogenic and not organogenic ha nature (Fig. 2a-f). Development of somatic embryos appearexl to progress through typical globular, heart-shapexl, torpezlo-shaped mad cotylexlonary stages of embryo development. The globular (Fig. 2a, b) and torpedoshapexl stages (Fig. 2d, e) were seen more frequently than the heart-shapezl stage (Fig. 2c). The morphology of the somatic embryos was apparently silnilar to the zygotic elnbryos. An examination of the sections further indicatexl
539 Figure 1. Somatic embryogenesis from immature zygotic embryos of pepper: ai A cluster of embryos forming on the cotyledons on MS + 2,4-D (9 #M) + CM (10%) + sucrose (8%) after 21 d in culture (bar = 1 mm, this also applies to figs. lb -le); b) embryos originating from the shootapex on MS + 2,4-D (9/.aM) + TDZ (10/.aM) + sucrose (8%) after 15 d; e) embryos arising from the embryonic axis on MS + 2,4-D (9/.aM) + CM (10%) + sucrose(10%) after 15 d (ev. RH); d) an almost mature embryoon MS + 2,4-D (9 #M) + CM (10%) + TDZ (10 glvl) + sucrose (8%) after 30 d (cv. NM-6); e) two fasciated embryos showinggemainationon MS + sucrose (2%) + AgNO3 (10/.aM) + GA3 (2.8 /zM) after 10 d (cv. NM-6); f) germinating somatic embryos cv. NM-6 with roots on MS + TDZ (0.05 gM) after 15 d (bar = 1 cm); g) an 8 week old plant of cv. NM-6 derived from somatic embryo growing in 6 cm pot containing soil: vermiculite 0:l). (Harini and Lakshnfi Sita 1993). In several plants, the process of sonratic embryo hfitiation reqnires the presence of 2,4-D alone (Ammirato 1983; Baker taxi Wetzstein 1994), wlfile in others presence of both auxin and cytokinin is required (Kiss et al. 1992; Zhou et al. 1994). In pepper, somatic embryos were produced either in tile presence of 2,4-D mxl TDZ, or 2,4-D and cocomlt water, but not with 2,4-D alone. This suggests that in peppers both 2,4-D and cytokinins are necessary for somatic embryogenesis.
tlmt repetitive embryogenesis (Fig. 20, exhibitexl by somatic embryos, may be initiated as early as during the globular stage of embryo development (Fig. 2a). Interestingly, in some developing somatic embryos, originating in the presence of TDZ, development of shoot buds via orgemogenesis was also distinctly observexl (Fig. 20. ha cotnparison to TDZ and 2,4-D, the mexlia containing coconut water and 2,4-D induced a greater percentage emhryogenesis and mean number of embryos (Tahle 2). Even the rate of induction of somatic embryos was hastened on tiffs medinm. Clumps of white mature somatic emhryos were visible within 15-21 d. Addition of TDZ to coconut water mad 2,4-D contailfing mexlia greatly reduced the incidence of somatic embryogenesis (Table 2). The role of type and concentration of auxin nsezl ill tile ilxluction of somatic embryos is well documented (Ammirato 1983; Eapen mid George 1993; Baker and Wetzstein 1994). ha the current hwe~stigation, the presence of 2,4-D was found necessary for induction of somatic embryogenesis, while N A A proved completely ineffective. These resnlts are sinfilar to those repoi'ted for pepper cv. California Wonder (Harini and Lakshini Sita 1993) and peanut (Hazra et al. 1989). In the pepper cultivars usexl in this study, as in cv. California Wonder, coconnt water containing media was fonnd to be more effective in inducing somatic embryogenesis. However, the yield of somatic embryos in onr cultivars wa~s much lower than for cv. Califbrnia Wonder
ThMiazuron (N-phenyl-N'-l,2,3-thiadiazol-5-yl-nrea) is a snbstituted phenyl urea derivative which exhibits a considerably higher degree of activity than purine-based cytokinhas (Mok et al. 1987; Huetteman and Preece 1993). In several plmat systems, when used alone or in combination with an auxin, it induces shoot organogenesis and/or somatic embryogenesis (Malik and Saxena 1992, Sankhla et al. 1994). In some plants, TDZ can effectively replace the reqtfirement of auxin and (or) cytokinin in induction of somatic embryogenesis (Gill and Saxena 1992; 1993). However, in pepper, TDZ alone was not sufficient to bring about somatic embryogenesis, although it frequently cruised the fornmtion of shoot buds on both hypocotyl and cotylexlons. Somatic embryos were formed only if 2,4-D was also present in the mexlium. In many plants, initiation and maturation of somatic emhryos occurs in two or more steps, mad involves SlZe.cific media designs (Kysley et al. 1987; Nagarajan et al. 1986; Eapen and George 1993; Emons et al. 1993). On the other hand, the results from our studies as well as from an earlier study (Harini aixl LaEslmfi Sita 1993) indicate that in peppers the entire process of embryo initiation and development is a one step process. More than 70% of the mature embryos, separated from the explant, germinated readily on the medium containing GA 3 or TDZ alone or in combination (Fig. If). Witlffn 2-3 d, the embryos turned green, and in another 5-7 d converted into plantlets which grew into normal plants (Fig. lg). Even a group of unseparated embryos, when transferred to gemfination medium, started to gennhaate while still on the original explant. Becanse the
~iiiiiiiii. . . .
Figure 2. Histological examination of the in vitro development of somatic embryos in pepper: a) a section of cotyledon showing origin of a globular embryo in ev. RH, note the initiation of two additional embryos at the base, and meristematie activity at the top (bar = 125 #m, this also applies to fig. 2b); b) a magnified view of well developed globular embryo on an older somatic embryo in ev. NM-6; e) a heart shaped embryo Of cv. RH, note the formation of an additional embryo (bar = 250 #m, this also applies to fig. 2d); d) a torpedo-shaped embryo of cv. NM-6; e) a late torpedo-shaped embryo ofev. RH (bar = 500 /zm, this also applies to fig. 20;.0 a cluster of somatic embryos showing repetitive embryogeny in ev. NM-6, note the larger embryo on the left showing a developing shoot bud.
iiiiiiiiiiiii!iiiiiiiiii ~;; i ~'iiiiJ~igiiiiiiiii
d e v e l o p m e n t , s o m e exhibitexl p r e c o c i o u s g e n n i n a t i e n . T h e e l o n g a t i n g root with a tuft o f root hairs was clearly evident on fllese e m b r y o s . T h e plantlets d e v e l o p i n g in the p r e s e n c e o f T D Z in t h e g e r m i n a t i o n ntexliunt w e r e fotmd to b e m o r e r o b u s t and h e a l t h y , with expandexl cotylexlons and thick roots. E v e n the m o r p h o l o g i c a l l y a b n o r m a l e m b r y o s g r e w e x t e n s i v e l y a n d attainexl u n u s u a l l y large d i m e n s i o n s , a l t h o u g h n o m m l plemts w e r e not producexl. Recent results indicate float modifications in culture mexlia comlx)sifions couplexl to matnration e n h a n c h ] g treatlnents c a n significantly h n p r o v e the quality, f r e q n e n c y and tmifomfity o f s o m a t i c e m b r y o s ( E m o n s et al. 1993; L e c o u t e u x et al. 1993; Sherry mad M c K e r s i e 1993). Maniptflation o f b a l a n c e o f A B A , G A , L-proline, mm-mitol oaad s u c r o s e has provexl e s p e c i a l l y effective in sotne species (Etnons et al. 1993). Further exl;eriments aimext at fine-ttming the p e p p e r sotnatic embryogenesis System, m~d encottraging t n o r e cells within an e x p l a n t to follow the path o f e m b r y o g e n e s i s , are c u r r e n t l y b e i n g ptwsuexl. Acknowledgements. This work was supported by a grant provided by TAES (Research Enhancement Program) to MLB, We gralel'ully acknowledge the help of Dr. L.F. Mayberry, University of Texas, El Paso with histological studies. We also thank Kendra Rumbaugb for her excellent technical assistance.
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