Plant Cell Reports
Plant Cell Reports (1996) 16:204-209
© Springer-Verlag1996
High frequency adventitious shoot regeneration from excised leaves of Paulownia spp. cultured in vitro C. Dimps Rao, Chong-Jin Goh, and Prakash P. Kumar Tissue Culture Laboratory, Department of Botany, National University of Singapore, Lower Kent Ridge Road, Singapore 119 260 Received 8 March 1996/Revised version received 12 June 1996 - Communicated by A. Kornamine
Abstract
and fast-growing timber trees (Zhu et al., 1986). which is indigenous to China, has been naturalized in Japan, Australia, Brazil, Europe and the United States of America. The growth habitat extends from the temperate to near-tropical zones in China. P a u l o w n i a yields a multiple-use wood, its leaves and flowers are used in medicine, as fertilizer and fodder (Zhu et al., 1986). Hence, it has been hailed as the 'Miracle Tree'. Recently, there has been increased interest in this genus because of its possible use in reforestation of nutrient-poor soils (Marcotrigiano and Jagannathan, 1988). Paulownia,
High frequency, direct regeneration of shoots was induced in leaf cultures of Paulownia tomentosa, P. fortunei x P. tomentosa and P. kawakamii. The optimum culture medium for the leaf explants derived from shoot cultures was Murashige-Skoog (MS) medium supplemented with 10 gM indole-3-acetic acid and 50 gM benzyladenine. Up to 40 shoots were obtained over a 4 month culture period from each leaf explant. Rooting occurred spontaneously in the shoots that were about 1 cm tall when subcultured on phytohormone-free MS medium. The plantlets could be transplanted successfully. Some of the transplanted P. tomentosa plantlets flowered in the greenhouse one year after transplanting. The protocol is suitable not only for rapid multiplication of the various species of Paulownia, but also for analytical studies associated with adventitious shoot regeneration.
Introduction Successful application of tissue culture technology for plant propagation and plant improvement involves the regeneration of plants from cultured cells or tissues at high frequencies. Plant tissue culture depends on the control of morphogenesis which is influenced by several factors, namely, species variability, kinds of tissue, nutritional components of the medium, growth regulators and culture environment (Murashige and Skoog, 1962; Kumar et al., 1987; Thorpe et al., 1991). Two of the basic strategies used for micropropagation of woody species are direct regeneration and indirect regeneration via an intermediate callus phase (Thorpe et al., 1991), Indirect regeneration often results in somaclonal variations, making this strategy less desirable for large-scale clonal multiplication (Marcotrigiano and Jagannathan, 1988; Thorpe et al., 1991). Therefore, direct regeneration, which involves morphogenesis without an intermediate callus phase, is a more reliable method for clonal propagation. is an economically important genus in the family Scrophulariaceae, with 9 species of very adaptable
Paulownia
Correspondence to: P. P. Kumar
Although Paulownia can be propagated from seeds or root and stem cuttings (Zhu et al., 1986) these methods are not suitable for generating large numbers of planting material of selected elite trees. Therefore, the major objective of this study was to establish an efficient protocol for micropropagation of Paulownia species which will also be suitable for analytical studies on morphogenesis.
Materials a n d m e t h o d s materials: Seeds of P a u l o w n i a tomentosa, the hybrid P. fortunei x P. tomentosa and P. kawakamii were obtained from the Chinese Academy of Forestry, Beijing, China. The seeds were manually dewinged and surface sterilized using 5% AgNO3 (Marcotrigiano and Stimart, 1983). They were then soaked in sterile water at 40°C for 10 min and at about 25°C for 24 h (Zhu et al., 1986). Subsequently, the seeds were surface dried for 1 h in the laminar flow cabinet and placed in petri dishes (90 mm diameter) containing 25 ml of agar-solidified, full strength Murashige and Skoog (1962) basal medium (MS)2 for germination. After 2 to 3 weeks in the light (30 gmol m sq) the seedlings were about 1 cm in height and were transferred to GA7 (Sigma, USA) containers with MS basal medium. Plant
Fully expanded leaves with about 1.5 cm of petiole were excised near the axil leaving at least 0.5 cm petiolar stub on the stem. The leaves were cut in half, and the petiolar halves were placed with their cut ends partially embedded and their abaxial surfaces in contact with the medium.
205 Culture media and incubation conditions: The basal medium used for all the experiments was MS medium -1 (Murashige and Skoog, 1962) containing 30 g 1 sucrose and -1 + 2 g 1 Gelrite (Sigma Co.). The pH was adjusted to 5.8_0.1 prior to adding Gelrite, and the media were autoclaved (1.1kg -2 cm for 20 rain). Various phytohormones added to the culture media include tx-naphthaleneacetic acid (NAA), 2,4dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), benzyladenine (BA) and kinetin (KIN) at 0, 5, 10, 20 or 50 gM. IAA was filter sterilized and added to the cooled (-60°C), autoclaved media as required. Four to five explants were cultured per petri dish (90 mm diameter) with 25 ml of MS medium supplemented with factorial combinations of an auxin and a cytokinin. Cultures were incubated at 25_+2°C and 16 h photoperiod under cool white fluorescent lights with_2 alph°t°synthetic photon flux density of 30 to 40 mmol m s at the level of the cultures. Tissues were subcultured onto fresh media once every two weeks. Explants incubated on MS basal medium as control cultures were non-shoot-forming (NSF). Assessment of Growth: The number of explants with shoot-, root-, or callus formation at the petiolar as well as the laminar end was recorded on days 14 and 21 of culture. The amount of callus formed on the various explants was quantified based on the diameter of the callus mass (-, no callus formed; +, 0.5 cm). The number of explants exhibiting adventitious shoot formation was recorded under two categories: Stage I and Stage II. In Stage I, the adventitious buds were visible as dark green protuberances; in Stage II, the leaves were clearly visible. All experiments were of the completely randomized factorial design. There were 10 explants per treatment, and each experiment was repeated at least twice. The data presented are pooled means of 3 independent experiments. Rooting and Establishment of Shoots: Excised shoots were transferred to GA7 containers with 75 ml of full strength MS basal medium to induce rooting. Plantlets measuring about 6 cm in height and with 2 to 3 cm long roots were washed thoroughly with tap water. They were dipped in Benlate (0.6 g 11) suspension and transplanted to pots filled with a commercial potting mixture (containing peat, perlite, vermiculite; Smith's Soil Industries, Auckland, N.Z.) and sand (1:1 ratio). The pots were covered with clear plastic bags perforated with tiny holes for 2 to 3 weeks. They were kept in the shade and watered daily.
Results E s t a b l i s h m e n t of S h o o t Cultures: With all the three taxa, seed germination occurred about 1 to 2 weeks after sowing on MS basal medium. The seedlings were allowed to grow to 3 to 4 cm in height in GA 7 containers and the top cuttings were transferred to fresh MS basal medium for further growth. The excised shoots rooted in 2 to 3 weeks and the shoots elongated. The plantlets were thus multiplied for further experiments. These plantlets were maintained in culture for more than 3 years with constant subculture.
Adventitious shoot formation: In preliminary experiments aimed at identifying the best explant, shoottips, nodes, internodes, leaves and roots were taken from 2- to 3 - m o n t h - o l d aseptically g r o w n plantlets o f Paulownia spp. and cultured on MS medium supplemented with various phytohormones. The shoot-tip explants elongated into shoots with no adventitious shoot formation but axillary shoots sprouted from the nodes of these elongated shoots that were in culture for over 3 weeks. Axillary shoots were also initiated in the nodal explants. The internodes and roots became swollen but no other response was seen even after 21 days in culture. In leaf explants, the petiolar ends became swollen with dark green protuberances after 7-10 days in culture. These protuberances developed into shoots after 14 to 21 days of culture initiation. Therefore, petiolar halves of leaves were used in all further experiments. P. tomentosa: Leaf explants of P. tomentosa cultured on media supplemented with various combinations of 2,4D or N A A with B A or KIN developed soft, pale-yellow callus along the petiole and the veins o f the lamina in almost all the treatments (data not shown). Also, explants cultured on MS m e d i u m s u p p l e m e n t e d with high concentrations of N A A induced root formation. Thus, a different auxin, I A A , was used together with BA.
I A A on its own induced the formation of roots and some callus, while BA alone promoted callus production in all the explants (Table 1, Fig. 1). All the combinations of IAA and BA tested induced adventitious shoots to varying degrees, indicating that these p h y t o h o r m o n e s were necessary for shoot induction in leaf explants o f P. t o m e n t o s a (Table 1). Despite a high percentage o f Table 1: Morphogenetic responses of leaf explants of P. tomentosa cultured on MS medium with factorial combinations of IAA and BA. Calculations were based on the total number of explants cultured. D14: Scoring on the 14th day of culture. D21: Subculturing was carried out on the 14th day of culture and scoring was after a further 7 day incubation. IAA gM
BA pM
% F_xplanlswith Shoot buds Roots D 14 D21 (Staae I )
Callus
Degree of callus formed
(StaaeII
0
0
0.0
0.0
0.0
0.0
5 10 50
0 0 0
0.0 0.0 0.0
0.0 0.0 0.0
85.0 83.0 100.0
57.1 83.3 66.7
+ + +
0 0 0
5 10 50
8.4 8.4 12.9
0.0 8.4 8.4
0.0 0.0 0.0
100.0 100.0 100.0
++ +++ ++
5 5 5
5 10 50
47.2 71.4 61.3
58.4 50.0 73.8
0.0 0.0 0.0
100.0 100.0 100.0
+++ +++ +++
10 10 10
5 10 50
51.4 47.2 54.2
44.3 58.3 76.4
0.0 0.0 0.0
100.0 100.0 100.0
+++ +++ +++
50 50 50
5 10 50
14.3 54.2 54.9
21.4 25.0 92.9
0.0 0.0 0.0
100.0 100.0 100.0
+++ +++ +++
206 regeneration, shoots remained stunted and leaf expansion did not occur in 50 gM IAA with 50 p-M BA. The best treatment for shoot induction in this species was 10 gM IAA with 50 gM BA. However, shoot formation at the petiolar end was accompanied by profuse callus formation. f o r t u n e i x P. tomentosa: Roots were induced on the leaf explants by NAA alone, and by 10 p-M or 50 p-M of NAA together with BA (Table 2). Callus was induced in the presence of 10 ~tM or 50 gM NAA and all the concentrations of BA tested. Adventitious shoots were induced in 8.4% to 62.5% of the explants depending on the concentrations of NAA and BA in the medium (Table 2). Callus was also present at the petiolar ends where adventitious shoots were formed. P.
The best response was obtained with medium supplemented with 10 gM IAA and 50 gM BA where all the explants regenerated shoots. Also, callus formation in this treatment was minimal. The sequence of development of adventitious shoot buds in this species was similar to that in the other species studied except that the events were slower by about one week. Table 3: Morphogenetic responses of leaf explants of P, fortunei x P. tornentosa cultured on MS medium with factorial combinations of IAA and BA. Calculations were based on the total number of explants cultured. Data from 3 independent experiments were pooled. (D14 & D21 as in Table 1).
With IAA and BA treatments, there was no callus production during shoot formation (Fig. 2). Only 11.1% of the explants produced roots in IAA-supplemented cultures (Table 3). Adventitious shoot formation was highest in combinations (p.M) of IAA:BA at 5:50 and 10:50 (Table 3). The developing shoot buds were visible as dark green protuberances by day 10 (Stage I), leaves by day 14 and Stage II shoots between days 21 to 28. Table 2: Morphogenetic responses of leaf explants of P. fortunei x P. tomentosa cultured on MS medium with factorial combinations of NAA and BA, Calculations were based on the total number of explants cultured. (D14 and D21 as in Table 1). NAA BA gM gM
% Explantswith Shoot buds Roots D14 D21 (Stage I) (b-'lageII )
Callus
Degree of callus formed
0
0
0.0
0.0
8.3
0.0
5 10 50
0 0 0
0.0 0.0 0.0
0.0 0.0 0.0
0.0 85.0 100.0
0.0 0.0 0.0
0 0 0
5 10 50
0.0 8.4 12.9
0.0 8.4 8.4
0.0 0.0 0.0
0.0 0.0 0.0
5 5 5
5 10 50
25.0 37.5 37.5
62.5 50.0 62.5
37.5 0.0 0.0
12.5 0.0 25.0
+++
10 10 10
5 10 50
50.0 25.0 25.0
0.0 37.5 62,5
50.0 25.0 0.0
50.0 37.5 12.5
++ +++ +++
50 50 50
5 10 50
0.0 0.0 25.0
0.0 0.0 62.5
100.0 100.0 0.0
87.5 87.5 87.5
+++ +++ +++
IAA gM
BA pM
0
0
0,0
0.0
8.3
5 10 50
0 0 0
0,0 0.0 0.0
0.0 0.0 0.0
0.0 11.1 11.1
0 0 0
5 10 50
0.0 8.4 12.9
0.0 8.4 8.4
0.0 0.0 0.0
5 5 5
5 10 50
37.5 62,5 62.5
25.0 25.0 62.5
0.0 0.0 0.0
10 10 10
5 10 50
25.0 50.0 37.5
25.0 50.0 75.0
0.0 0.0 0.0
50 50 50
5 10 50
25.0 25.0 50.0
25.0 25.0 12.5
0.0 0.0 0.0
Table 4:
% Explantswith Shoot buds Roots D14 D21
Morphogenetic responses of leaf explants of P.
kawakamii cultured on MS medium with factorial combinations of
IAA and BA. Calculations were based on the total number of explants cultured. (D14 and D21 as in Table 1). IAA gM
BA gM
+++
P. kawakamii: Leaves of P. kawakamii cultured on NAA and BA showed profuse callus formation with little shoot formation. Profuse root production was observed on medium supplemented with 50 p-M IAA alone (Table 4). Shoots were induced in 12.5% of the explants cultured with 10 and 50 p.M BA. Combinations of IAA and BA resulted in a higher frequency of shoot differentiation. Some of the combinations (e.g., 10 p-M IAA and 10 p-M BA) induced shoot formation in up to 62.5% of explants, with simultaneous callus formation (Table 4, Fig. 3).
% F_.xp~ntswith Shoot buds Roots D14 D21 (Stage I ) (StageII)
Callus
0
0
0.0
0.0
0.0
0.0
5 10 50
0 0 0
0.0 0.0 0.0
0.0 0.0 0.0
25.0 62.5 100.0
0.0 0.0 0.0
0 0 0
5 10 50
0,0 0,0 0,0
0.0 12.5 12.5
0.0 0.0 0.0
0.0 0.0 0.0
5 5 5
5 10 50
37,5 25,0 50.0
25.0 37.5 37.5
0.0 0.0 0.0
0.0 12.5 25.0
10 10 10
5 10 50
25.0 87.5 87.5
12,5 62.5 100.0
0,0 0,0 0,0
25.0 50.0 0.0
50 50 50
5 10 50
37.5 62.5 50.0
12.5 25.O 37.5
0,0 0,0 0.0
25.0 0.0 0.0
Degree of callus formed
+ + ++ ++
207
Fig. 1: Formation of callus and multiple shoots at the petiolar end of leaf explants of P. tomentosa cultured on factorial combinations of IAA and BA. (Scale bar= 1 cm for Figs. 1-3). Fig. 2: Leaf explants of P. f o r t u n e i x P. tomentosa producing callus and multiple shoots on factorial combinations of IAA and BA. Fig. 3: Leaf explants of P. kawakamii producing callus and multiple shoots on factorial combinations of IAA and BA.
Fig. 4: Multiple shoot formation at the petiolar ends of leaf explants of P. kawakamii cultured on the optimal shoot-inducing medium, 10 ~tM IAA and 50 ~M BA. (a) Day 0 explant, (b) and (c) Day 21 explants with Stage II shoots. Some shoot buds were induced on the laminar cut ends also. Scale divisions are in mm. Fig. 5: Plantlet of P. t o m e n t o s a 6 months after transplanting. Scale bar=- 5 cm.
208
Shoot development and continued regeneration in leaf explants: In the three P a u l o w n i a species
Discussion
examined, removal of the dominant shoots led to elongation of the remaining shoots at a given regeneration site. The excised shoots developed further on phytohormone-free MS medium. Transferring the original explants to fresh, optimal shoot-inducing medium allowed further development of the remaining buds into shoots. Over a period of about three months, between 15 to 40 shoots could be obtained from each explant by periodically subculturing the original explant.
Phytohormones were the important factor affecting the rate of regeneration of de novo shoot buds from excised leaves of Paulownia species (Tables 1-4). Shoot differentiation from leaf explants of Paulownia species appears to be a function of cytokinin because auxin alone did not initiate shoot development. On the media with cytokinin alone, adventitious shoots were induced, but at a lower frequency compared to that on media supplemented with both auxin and cytokinin.
Adventitious shoots were also produced from the laminar end in almost all the treatments that induced shoot formation from the petiolar ends in P. kawakamii (Fig. 4). In P. tomentosa such regeneration occurred only in some treatments, and it was rarely observed in the hybrid. These shoots arose from the cut ends of the larger veins, but were not repetitively regenerative as the petiolar end regeneration sites.
The combined use of auxin and cytokinin improved the efficiency of regeneration, although this depended on the combinations and concentrations employed. The combination of IAA and BA was the most effective in inducing shoot regeneration without any callus induction (P. kawakamii, P. fortunei xP. tomentosa) or with minimal amount of callus (P. tomentosa). The optimal auxin to cytokinin ratios were 1:5 (but at 10 and 50 gM, respectively) and 1:10 (5 and 50 gM). Our earlier study with P. fortunei also indicated that the same ratios of auxin to cytokinin were optimal for shoot induction, but the best auxin was NAA (Rao et al., 1993). A similar requirement for both auxin and cytokinin was reported for the induction of shoots from hypocotyls of P. tomentosa (Marcotrigiano and Stimart, 1983). Regenerated shoots were stunted and there was no leaf expansion in the presence of 50 gM concentrations of auxins and cytokinins. 2,4-D induced callus formation along the length of the petiole and the main veins in all the concentrations and combinations (with BA or KIN) used. This clearly indicates that the types of auxin used can influence morphogenesis in different ways in different species.
Rooting
and
establishment
of
plantlets:
Rooting of excised shoots occurred spontaneously 2 to 3 weeks after subculture in about 90% of the shoots that were at least 6 mm tall. GA 7 containers with 75 ml of full strength MS basal medium were ideal for this stage of culture. Plantlets measuring at least 3 cm in height and with 2 to 3 cm long roots were successfully transplanted to pots. The plants were kept in the pots for 6 to t2 months (Fig. 5) before planting in the field.
Flowering in potted plants:
Some of the tissue culture-derived P. tomentosa plants that were established in the pot flowered spontaneously about one year after transplantation (Fig. 6). The flowers were complete and of normal size.
Our observation that the removal of dominant shoots developing on the primary explants results in an increase in the rate of elongation of more shoots is also consistent with observations from leaf cultures of other species, e.g., Mesembryanthemum sp. (Meiners et al., 1991). In the Paulownia leaf cultures, even when there was some degree of callus formation, the shoot buds arose directly from the explants and not via callus. There are relatively few reports of direct shoot bud regeneration from leaves of woody species compared to that in herbaceous species. These include mangosteen (Goh et al., 1994) and Populus sp. (Lee-Stadelmann et al., 1989). Many of the regeneration protocols for woody species include an intermediate callus phase as in Prunus (Mehra and Mehra, 1974), Betula pendula (Simola, 1985) and Punica granatum (Omura et al., 1987). We have succeeded in developing a direct shoot regeneration system for 3 species of Paulownia, namely, P. fortunei (Rao et al., 1993), P. tomentosa, P. k a w a k a m i i and a hybrid between P. fortunei and P. tomentosa. Fig. 6: Spontaneous flowering on a plantlet of P. tomentosa one year after transplanting.
The protocol described here for regeneration of de novo shoot buds from excised leaves is suitable for large-scale propagation of selected elite trees. Initial explants will
209 not be a limiting factor in the method described here because leaves are available in abundant numbers from shoot cultures that can be easily maintained as compared to hypocotyls and cotyledons (Marcotrigiano and Stimart, 1983; Jagannathan and Marcotrigiano, 1987) or shoot tips (Burger etal., 1985) used as explants in some of the previous studies with Paulownia spp. Moreover, although somatic embryo-like sturctures were reported in callus cultures of P. tomentosa, conversion of these to plantlets was very poor (Radojevic, 1979). Our protocol described here is more effective and convenient. Further, it appears to be suitable for most Paulownia species. In woody perennial species, the phenomenon of early flowering in tissue culture-derived plants such as that reported here can be exploited for conventional breeding programs. However, more work needs to be done in this area before this can be achieved routinely. A high frequency regeneration system such as the one described here is useful in evaluating the influence of different factors on de novo organogenesis. It will also facilitate the application of transformation techniques to P a u l o w n i a spp. Work on A g r o b a c t e r i u m - m e d i a t e d transformation is under way in our laboratory. Moreover, the petiolar ends of excised leaves of Paulownia spp. cultured under well-defined in vitro conditions seem ideal for physiological and molecular analyses of organogenesis.
References Burger DW, Liu L, Wu L (1985) HortSci., 20: 760-761. Goh C-J, Lakshmanan P, Loh C-S (1994) Plant Sci., 101: 173-180. Jagannathan L, Marcotrigiano M (1987) Plant Cell Tissue Organ Cult., 7: 227-236. Kumar PP, Reid DM, Thorpe TA (1987) Physiol. Plant., 69: 244-252. Lee-Stadelmann OY, Lee SW, Hackett WP, Read PE (1989) Plant Sci., 61: 263-272. Marcotrigiano M, Jagannathan L (1988) HortSci., 23: 226227. Marcotrigiano M, Stimart DP (1983) Plant Sci. Lett., 31: 303-310. Mehra A, Mehra PN (1974) Bot. Gaz., 135: 61-73. Meiners MS, Thomas JC, Bohnert HJ, Cushman JC (1991) Plant Cell Rep., 9: 563-566. Murashige T, Skoog F (1962) Physiol. Plant., 15: 473-493. Omura M, Matsuta N, Moriguchi T, Kozaki I (1987) HortSci., 22: 133-134. Radojevic L (1979) Z. Pflanzenphysiol, 91: 57-62. Rao CD, Gob C-J, Kumar PP (1993) In Vitro Cell. Dev. Biol., 29P: 72-76. Simola LK (1985) Sci. Horticult., 26: 77-85. Thorpe TA, Harry IS, Kumar PP (1991) Application of micropropagation to forestry. In: Micropropagation, technology and application. Debergh PC and Zimmerman RH (eds) Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 311-336. Zhu ZH, Chao CJ, Lu XY, Xiong YG (1986) Paulownia in China: Cultivation and utilization. Asian Network Biol. Sci. and Intl. Dev. Res. Ctr., Singapore.
Plant Cell Reports (1996) 16:204-209
© Springer-Verlag1996
High frequency adventitious shoot regeneration from excised leaves of Paulownia spp. cultured in vitro C. Dimps Rao, Chong-Jin Goh, and Prakash P. Kumar Tissue Culture Laboratory, Department of Botany, National University of Singapore, Lower Kent Ridge Road, Singapore 119 260 Received 8 March 1996/Revised version received 12 June 1996 - Communicated by A. Kornamine
Abstract
and fast-growing timber trees (Zhu et al., 1986). which is indigenous to China, has been naturalized in Japan, Australia, Brazil, Europe and the United States of America. The growth habitat extends from the temperate to near-tropical zones in China. P a u l o w n i a yields a multiple-use wood, its leaves and flowers are used in medicine, as fertilizer and fodder (Zhu et al., 1986). Hence, it has been hailed as the 'Miracle Tree'. Recently, there has been increased interest in this genus because of its possible use in reforestation of nutrient-poor soils (Marcotrigiano and Jagannathan, 1988). Paulownia,
High frequency, direct regeneration of shoots was induced in leaf cultures of Paulownia tomentosa, P. fortunei x P. tomentosa and P. kawakamii. The optimum culture medium for the leaf explants derived from shoot cultures was Murashige-Skoog (MS) medium supplemented with 10 gM indole-3-acetic acid and 50 gM benzyladenine. Up to 40 shoots were obtained over a 4 month culture period from each leaf explant. Rooting occurred spontaneously in the shoots that were about 1 cm tall when subcultured on phytohormone-free MS medium. The plantlets could be transplanted successfully. Some of the transplanted P. tomentosa plantlets flowered in the greenhouse one year after transplanting. The protocol is suitable not only for rapid multiplication of the various species of Paulownia, but also for analytical studies associated with adventitious shoot regeneration.
Introduction Successful application of tissue culture technology for plant propagation and plant improvement involves the regeneration of plants from cultured cells or tissues at high frequencies. Plant tissue culture depends on the control of morphogenesis which is influenced by several factors, namely, species variability, kinds of tissue, nutritional components of the medium, growth regulators and culture environment (Murashige and Skoog, 1962; Kumar et al., 1987; Thorpe et al., 1991). Two of the basic strategies used for micropropagation of woody species are direct regeneration and indirect regeneration via an intermediate callus phase (Thorpe et al., 1991), Indirect regeneration often results in somaclonal variations, making this strategy less desirable for large-scale clonal multiplication (Marcotrigiano and Jagannathan, 1988; Thorpe et al., 1991). Therefore, direct regeneration, which involves morphogenesis without an intermediate callus phase, is a more reliable method for clonal propagation. is an economically important genus in the family Scrophulariaceae, with 9 species of very adaptable
Paulownia
Correspondence to: P. P. Kumar
Although Paulownia can be propagated from seeds or root and stem cuttings (Zhu et al., 1986) these methods are not suitable for generating large numbers of planting material of selected elite trees. Therefore, the major objective of this study was to establish an efficient protocol for micropropagation of Paulownia species which will also be suitable for analytical studies on morphogenesis.
Materials a n d m e t h o d s materials: Seeds of P a u l o w n i a tomentosa, the hybrid P. fortunei x P. tomentosa and P. kawakamii were obtained from the Chinese Academy of Forestry, Beijing, China. The seeds were manually dewinged and surface sterilized using 5% AgNO3 (Marcotrigiano and Stimart, 1983). They were then soaked in sterile water at 40°C for 10 min and at about 25°C for 24 h (Zhu et al., 1986). Subsequently, the seeds were surface dried for 1 h in the laminar flow cabinet and placed in petri dishes (90 mm diameter) containing 25 ml of agar-solidified, full strength Murashige and Skoog (1962) basal medium (MS)2 for germination. After 2 to 3 weeks in the light (30 gmol m sq) the seedlings were about 1 cm in height and were transferred to GA7 (Sigma, USA) containers with MS basal medium. Plant
Fully expanded leaves with about 1.5 cm of petiole were excised near the axil leaving at least 0.5 cm petiolar stub on the stem. The leaves were cut in half, and the petiolar halves were placed with their cut ends partially embedded and their abaxial surfaces in contact with the medium.
205 Culture media and incubation conditions: The basal medium used for all the experiments was MS medium -1 (Murashige and Skoog, 1962) containing 30 g 1 sucrose and -1 + 2 g 1 Gelrite (Sigma Co.). The pH was adjusted to 5.8_0.1 prior to adding Gelrite, and the media were autoclaved (1.1kg -2 cm for 20 rain). Various phytohormones added to the culture media include tx-naphthaleneacetic acid (NAA), 2,4dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), benzyladenine (BA) and kinetin (KIN) at 0, 5, 10, 20 or 50 gM. IAA was filter sterilized and added to the cooled (-60°C), autoclaved media as required. Four to five explants were cultured per petri dish (90 mm diameter) with 25 ml of MS medium supplemented with factorial combinations of an auxin and a cytokinin. Cultures were incubated at 25_+2°C and 16 h photoperiod under cool white fluorescent lights with_2 alph°t°synthetic photon flux density of 30 to 40 mmol m s at the level of the cultures. Tissues were subcultured onto fresh media once every two weeks. Explants incubated on MS basal medium as control cultures were non-shoot-forming (NSF). Assessment of Growth: The number of explants with shoot-, root-, or callus formation at the petiolar as well as the laminar end was recorded on days 14 and 21 of culture. The amount of callus formed on the various explants was quantified based on the diameter of the callus mass (-, no callus formed; +, 0.5 cm). The number of explants exhibiting adventitious shoot formation was recorded under two categories: Stage I and Stage II. In Stage I, the adventitious buds were visible as dark green protuberances; in Stage II, the leaves were clearly visible. All experiments were of the completely randomized factorial design. There were 10 explants per treatment, and each experiment was repeated at least twice. The data presented are pooled means of 3 independent experiments. Rooting and Establishment of Shoots: Excised shoots were transferred to GA7 containers with 75 ml of full strength MS basal medium to induce rooting. Plantlets measuring about 6 cm in height and with 2 to 3 cm long roots were washed thoroughly with tap water. They were dipped in Benlate (0.6 g 11) suspension and transplanted to pots filled with a commercial potting mixture (containing peat, perlite, vermiculite; Smith's Soil Industries, Auckland, N.Z.) and sand (1:1 ratio). The pots were covered with clear plastic bags perforated with tiny holes for 2 to 3 weeks. They were kept in the shade and watered daily.
Results E s t a b l i s h m e n t of S h o o t Cultures: With all the three taxa, seed germination occurred about 1 to 2 weeks after sowing on MS basal medium. The seedlings were allowed to grow to 3 to 4 cm in height in GA 7 containers and the top cuttings were transferred to fresh MS basal medium for further growth. The excised shoots rooted in 2 to 3 weeks and the shoots elongated. The plantlets were thus multiplied for further experiments. These plantlets were maintained in culture for more than 3 years with constant subculture.
Adventitious shoot formation: In preliminary experiments aimed at identifying the best explant, shoottips, nodes, internodes, leaves and roots were taken from 2- to 3 - m o n t h - o l d aseptically g r o w n plantlets o f Paulownia spp. and cultured on MS medium supplemented with various phytohormones. The shoot-tip explants elongated into shoots with no adventitious shoot formation but axillary shoots sprouted from the nodes of these elongated shoots that were in culture for over 3 weeks. Axillary shoots were also initiated in the nodal explants. The internodes and roots became swollen but no other response was seen even after 21 days in culture. In leaf explants, the petiolar ends became swollen with dark green protuberances after 7-10 days in culture. These protuberances developed into shoots after 14 to 21 days of culture initiation. Therefore, petiolar halves of leaves were used in all further experiments. P. tomentosa: Leaf explants of P. tomentosa cultured on media supplemented with various combinations of 2,4D or N A A with B A or KIN developed soft, pale-yellow callus along the petiole and the veins o f the lamina in almost all the treatments (data not shown). Also, explants cultured on MS m e d i u m s u p p l e m e n t e d with high concentrations of N A A induced root formation. Thus, a different auxin, I A A , was used together with BA.
I A A on its own induced the formation of roots and some callus, while BA alone promoted callus production in all the explants (Table 1, Fig. 1). All the combinations of IAA and BA tested induced adventitious shoots to varying degrees, indicating that these p h y t o h o r m o n e s were necessary for shoot induction in leaf explants o f P. t o m e n t o s a (Table 1). Despite a high percentage o f Table 1: Morphogenetic responses of leaf explants of P. tomentosa cultured on MS medium with factorial combinations of IAA and BA. Calculations were based on the total number of explants cultured. D14: Scoring on the 14th day of culture. D21: Subculturing was carried out on the 14th day of culture and scoring was after a further 7 day incubation. IAA gM
BA pM
% F_xplanlswith Shoot buds Roots D 14 D21 (Staae I )
Callus
Degree of callus formed
(StaaeII
0
0
0.0
0.0
0.0
0.0
5 10 50
0 0 0
0.0 0.0 0.0
0.0 0.0 0.0
85.0 83.0 100.0
57.1 83.3 66.7
+ + +
0 0 0
5 10 50
8.4 8.4 12.9
0.0 8.4 8.4
0.0 0.0 0.0
100.0 100.0 100.0
++ +++ ++
5 5 5
5 10 50
47.2 71.4 61.3
58.4 50.0 73.8
0.0 0.0 0.0
100.0 100.0 100.0
+++ +++ +++
10 10 10
5 10 50
51.4 47.2 54.2
44.3 58.3 76.4
0.0 0.0 0.0
100.0 100.0 100.0
+++ +++ +++
50 50 50
5 10 50
14.3 54.2 54.9
21.4 25.0 92.9
0.0 0.0 0.0
100.0 100.0 100.0
+++ +++ +++
206 regeneration, shoots remained stunted and leaf expansion did not occur in 50 gM IAA with 50 p-M BA. The best treatment for shoot induction in this species was 10 gM IAA with 50 gM BA. However, shoot formation at the petiolar end was accompanied by profuse callus formation. f o r t u n e i x P. tomentosa: Roots were induced on the leaf explants by NAA alone, and by 10 p-M or 50 p-M of NAA together with BA (Table 2). Callus was induced in the presence of 10 ~tM or 50 gM NAA and all the concentrations of BA tested. Adventitious shoots were induced in 8.4% to 62.5% of the explants depending on the concentrations of NAA and BA in the medium (Table 2). Callus was also present at the petiolar ends where adventitious shoots were formed. P.
The best response was obtained with medium supplemented with 10 gM IAA and 50 gM BA where all the explants regenerated shoots. Also, callus formation in this treatment was minimal. The sequence of development of adventitious shoot buds in this species was similar to that in the other species studied except that the events were slower by about one week. Table 3: Morphogenetic responses of leaf explants of P, fortunei x P. tornentosa cultured on MS medium with factorial combinations of IAA and BA. Calculations were based on the total number of explants cultured. Data from 3 independent experiments were pooled. (D14 & D21 as in Table 1).
With IAA and BA treatments, there was no callus production during shoot formation (Fig. 2). Only 11.1% of the explants produced roots in IAA-supplemented cultures (Table 3). Adventitious shoot formation was highest in combinations (p.M) of IAA:BA at 5:50 and 10:50 (Table 3). The developing shoot buds were visible as dark green protuberances by day 10 (Stage I), leaves by day 14 and Stage II shoots between days 21 to 28. Table 2: Morphogenetic responses of leaf explants of P. fortunei x P. tomentosa cultured on MS medium with factorial combinations of NAA and BA, Calculations were based on the total number of explants cultured. (D14 and D21 as in Table 1). NAA BA gM gM
% Explantswith Shoot buds Roots D14 D21 (Stage I) (b-'lageII )
Callus
Degree of callus formed
0
0
0.0
0.0
8.3
0.0
5 10 50
0 0 0
0.0 0.0 0.0
0.0 0.0 0.0
0.0 85.0 100.0
0.0 0.0 0.0
0 0 0
5 10 50
0.0 8.4 12.9
0.0 8.4 8.4
0.0 0.0 0.0
0.0 0.0 0.0
5 5 5
5 10 50
25.0 37.5 37.5
62.5 50.0 62.5
37.5 0.0 0.0
12.5 0.0 25.0
+++
10 10 10
5 10 50
50.0 25.0 25.0
0.0 37.5 62,5
50.0 25.0 0.0
50.0 37.5 12.5
++ +++ +++
50 50 50
5 10 50
0.0 0.0 25.0
0.0 0.0 62.5
100.0 100.0 0.0
87.5 87.5 87.5
+++ +++ +++
IAA gM
BA pM
0
0
0,0
0.0
8.3
5 10 50
0 0 0
0,0 0.0 0.0
0.0 0.0 0.0
0.0 11.1 11.1
0 0 0
5 10 50
0.0 8.4 12.9
0.0 8.4 8.4
0.0 0.0 0.0
5 5 5
5 10 50
37.5 62,5 62.5
25.0 25.0 62.5
0.0 0.0 0.0
10 10 10
5 10 50
25.0 50.0 37.5
25.0 50.0 75.0
0.0 0.0 0.0
50 50 50
5 10 50
25.0 25.0 50.0
25.0 25.0 12.5
0.0 0.0 0.0
Table 4:
% Explantswith Shoot buds Roots D14 D21
Morphogenetic responses of leaf explants of P.
kawakamii cultured on MS medium with factorial combinations of
IAA and BA. Calculations were based on the total number of explants cultured. (D14 and D21 as in Table 1). IAA gM
BA gM
+++
P. kawakamii: Leaves of P. kawakamii cultured on NAA and BA showed profuse callus formation with little shoot formation. Profuse root production was observed on medium supplemented with 50 p-M IAA alone (Table 4). Shoots were induced in 12.5% of the explants cultured with 10 and 50 p.M BA. Combinations of IAA and BA resulted in a higher frequency of shoot differentiation. Some of the combinations (e.g., 10 p-M IAA and 10 p-M BA) induced shoot formation in up to 62.5% of explants, with simultaneous callus formation (Table 4, Fig. 3).
% F_.xp~ntswith Shoot buds Roots D14 D21 (Stage I ) (StageII)
Callus
0
0
0.0
0.0
0.0
0.0
5 10 50
0 0 0
0.0 0.0 0.0
0.0 0.0 0.0
25.0 62.5 100.0
0.0 0.0 0.0
0 0 0
5 10 50
0,0 0,0 0,0
0.0 12.5 12.5
0.0 0.0 0.0
0.0 0.0 0.0
5 5 5
5 10 50
37,5 25,0 50.0
25.0 37.5 37.5
0.0 0.0 0.0
0.0 12.5 25.0
10 10 10
5 10 50
25.0 87.5 87.5
12,5 62.5 100.0
0,0 0,0 0,0
25.0 50.0 0.0
50 50 50
5 10 50
37.5 62.5 50.0
12.5 25.O 37.5
0,0 0,0 0.0
25.0 0.0 0.0
Degree of callus formed
+ + ++ ++
207
Fig. 1: Formation of callus and multiple shoots at the petiolar end of leaf explants of P. tomentosa cultured on factorial combinations of IAA and BA. (Scale bar= 1 cm for Figs. 1-3). Fig. 2: Leaf explants of P. f o r t u n e i x P. tomentosa producing callus and multiple shoots on factorial combinations of IAA and BA. Fig. 3: Leaf explants of P. kawakamii producing callus and multiple shoots on factorial combinations of IAA and BA.
Fig. 4: Multiple shoot formation at the petiolar ends of leaf explants of P. kawakamii cultured on the optimal shoot-inducing medium, 10 ~tM IAA and 50 ~M BA. (a) Day 0 explant, (b) and (c) Day 21 explants with Stage II shoots. Some shoot buds were induced on the laminar cut ends also. Scale divisions are in mm. Fig. 5: Plantlet of P. t o m e n t o s a 6 months after transplanting. Scale bar=- 5 cm.
208
Shoot development and continued regeneration in leaf explants: In the three P a u l o w n i a species
Discussion
examined, removal of the dominant shoots led to elongation of the remaining shoots at a given regeneration site. The excised shoots developed further on phytohormone-free MS medium. Transferring the original explants to fresh, optimal shoot-inducing medium allowed further development of the remaining buds into shoots. Over a period of about three months, between 15 to 40 shoots could be obtained from each explant by periodically subculturing the original explant.
Phytohormones were the important factor affecting the rate of regeneration of de novo shoot buds from excised leaves of Paulownia species (Tables 1-4). Shoot differentiation from leaf explants of Paulownia species appears to be a function of cytokinin because auxin alone did not initiate shoot development. On the media with cytokinin alone, adventitious shoots were induced, but at a lower frequency compared to that on media supplemented with both auxin and cytokinin.
Adventitious shoots were also produced from the laminar end in almost all the treatments that induced shoot formation from the petiolar ends in P. kawakamii (Fig. 4). In P. tomentosa such regeneration occurred only in some treatments, and it was rarely observed in the hybrid. These shoots arose from the cut ends of the larger veins, but were not repetitively regenerative as the petiolar end regeneration sites.
The combined use of auxin and cytokinin improved the efficiency of regeneration, although this depended on the combinations and concentrations employed. The combination of IAA and BA was the most effective in inducing shoot regeneration without any callus induction (P. kawakamii, P. fortunei xP. tomentosa) or with minimal amount of callus (P. tomentosa). The optimal auxin to cytokinin ratios were 1:5 (but at 10 and 50 gM, respectively) and 1:10 (5 and 50 gM). Our earlier study with P. fortunei also indicated that the same ratios of auxin to cytokinin were optimal for shoot induction, but the best auxin was NAA (Rao et al., 1993). A similar requirement for both auxin and cytokinin was reported for the induction of shoots from hypocotyls of P. tomentosa (Marcotrigiano and Stimart, 1983). Regenerated shoots were stunted and there was no leaf expansion in the presence of 50 gM concentrations of auxins and cytokinins. 2,4-D induced callus formation along the length of the petiole and the main veins in all the concentrations and combinations (with BA or KIN) used. This clearly indicates that the types of auxin used can influence morphogenesis in different ways in different species.
Rooting
and
establishment
of
plantlets:
Rooting of excised shoots occurred spontaneously 2 to 3 weeks after subculture in about 90% of the shoots that were at least 6 mm tall. GA 7 containers with 75 ml of full strength MS basal medium were ideal for this stage of culture. Plantlets measuring at least 3 cm in height and with 2 to 3 cm long roots were successfully transplanted to pots. The plants were kept in the pots for 6 to t2 months (Fig. 5) before planting in the field.
Flowering in potted plants:
Some of the tissue culture-derived P. tomentosa plants that were established in the pot flowered spontaneously about one year after transplantation (Fig. 6). The flowers were complete and of normal size.
Our observation that the removal of dominant shoots developing on the primary explants results in an increase in the rate of elongation of more shoots is also consistent with observations from leaf cultures of other species, e.g., Mesembryanthemum sp. (Meiners et al., 1991). In the Paulownia leaf cultures, even when there was some degree of callus formation, the shoot buds arose directly from the explants and not via callus. There are relatively few reports of direct shoot bud regeneration from leaves of woody species compared to that in herbaceous species. These include mangosteen (Goh et al., 1994) and Populus sp. (Lee-Stadelmann et al., 1989). Many of the regeneration protocols for woody species include an intermediate callus phase as in Prunus (Mehra and Mehra, 1974), Betula pendula (Simola, 1985) and Punica granatum (Omura et al., 1987). We have succeeded in developing a direct shoot regeneration system for 3 species of Paulownia, namely, P. fortunei (Rao et al., 1993), P. tomentosa, P. k a w a k a m i i and a hybrid between P. fortunei and P. tomentosa. Fig. 6: Spontaneous flowering on a plantlet of P. tomentosa one year after transplanting.
The protocol described here for regeneration of de novo shoot buds from excised leaves is suitable for large-scale propagation of selected elite trees. Initial explants will
209 not be a limiting factor in the method described here because leaves are available in abundant numbers from shoot cultures that can be easily maintained as compared to hypocotyls and cotyledons (Marcotrigiano and Stimart, 1983; Jagannathan and Marcotrigiano, 1987) or shoot tips (Burger etal., 1985) used as explants in some of the previous studies with Paulownia spp. Moreover, although somatic embryo-like sturctures were reported in callus cultures of P. tomentosa, conversion of these to plantlets was very poor (Radojevic, 1979). Our protocol described here is more effective and convenient. Further, it appears to be suitable for most Paulownia species. In woody perennial species, the phenomenon of early flowering in tissue culture-derived plants such as that reported here can be exploited for conventional breeding programs. However, more work needs to be done in this area before this can be achieved routinely. A high frequency regeneration system such as the one described here is useful in evaluating the influence of different factors on de novo organogenesis. It will also facilitate the application of transformation techniques to P a u l o w n i a spp. Work on A g r o b a c t e r i u m - m e d i a t e d transformation is under way in our laboratory. Moreover, the petiolar ends of excised leaves of Paulownia spp. cultured under well-defined in vitro conditions seem ideal for physiological and molecular analyses of organogenesis.
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