Journal of Forestry Research, 12(4): 209-214 (2001)

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Callus induction from leaves of different paulownia species and its plantlet regeneration
FAN Guo-qiang, ZHAI Xiao-qiao, ZHAI Cui-juan, BI Hui-tao (Institute of Paulownia, Henan Agricultural University, Zhengzhou 450002, P R China)

Abstract:

The experiment was carried out on five different species of Paulownia for callus induction from leaves. MS medium

was adopted as basic medium, and from different combinations of NAA and BA the suitable media were determined for callus induction, bud differentiation, and root differentiation of five different species. MS+0.5NAA+4BA, MS+0.3NAA+2BA, MS+0.5NAA+4BA, MS+0.3NAA+6BA, and MS+0.3NAA+8BA were suitable media of callus inductions of leaves, respectively, for Paulownia tomentosa, Paulownia australis, Paulownia fortunei, Paulownia elongata and P. tmentosa x P. fortunei, and MS+0.3NAA+12BA, MS+0.3NAA+12BA, MS+0.5NAA+12BA, MS+0.5NAA+12BA, and MS+0.7NAA+12BA were suitable media for bud differentiation from leaf callus respectively for above five species. The rooting media was determined as 2MS+0.1NAA, 1/2MS+0.1NAA, 1/2MS, 1/2MS+0.3NAA, and 1/2MS+0.5NAA. These results provide reference data for breeding new fine varieties with different kinds of Paulownia protoplasts fusions. Keywords: Paulownia; Callus induction; Plantlet regeneration; Medium; Hormone CLC number: S792.43 Document code: A Article ID: 1007-662X(2001)04-0209-06

Materials and methods Introduction1

With the rapid development of science and technology, scientists have gotten many species of regenerated plantlets from their calli or protoplasts of different tree species with biotechnology (Huang 1990; Jiang 1988; Tang et al. 1998; Wang 1988; Wang et al. 1991, 1993, 1995; Wei et al. 1991, 1992; Zhuge et al. 2000; Becwer et al. 1990; Gupta & Durzan 1987; Hakman & Frowke 1987; Laine & David 1990; Schenk & Hildebrant 1972; Von Arnold &Woodward 1988). Plantlet regenerations from calli were an indispensable step in the regenerations of forest tree protoplasts or transgenic protoplasts. Paulownia is an important timber tree in China, which is used widely as architeculture material and furniture because of their short rotation and rapid growth. Although regenerated plantlet from P. tomentosa protoplast was studied, only little plantlets from induced calli was obtained (Wei et al. 1991); besides, there existed some differences in callus inductions of different species of Paulownia plant protoplasts and their plantlet regenerations. Here the callus inductions of different species of Paulownia plant leaves and their regenerated plantlets were studied, which lays a foundation for protoplast fusions among different Paulownia plant leaves and their plantlet regeneration. Materials The materials are seeds of Paulownia tomentosa (Thunb.) Steud, P. australis Gong Tong, P. fortunei (Seem.) Hemsl, P. elongata S.Y. Hu, and P. tomentosa x P. fortunei, which were picked and collected in Zhengzhou City, Henan Province in September 1998. Methods Surface sterilization of those seeds and seedling incubation Seeds were rinsed with 70% alcohol for 30 s and with 0.1% HgCl2 for 5 min orderly, and then washed 5 times with enough sterile distilled water, finally put on the PC culture medium in incubator. Eighty days later, Paulownia seedlings with 6-8 leaves were obtained, and their leaves were used as the materials for inducing callus. Medium for inducing callus from Paulownia leaves 1/2 MS, MS, B5, N6 and PC culture media modified adding some amount of NAA, 6-BA (briefly as BA), 25 gL-1 sugar and 8 gL-1 agar powder were used. First of all, the leaves of different species of Paulownia seedlings were cut into roughly pieces of 0.5cm x 1.0 cm (explants) and put in triangular bottles with media (10 bottles for each species, with 3 pieces of explants each bottle) for callus induction in darkness at temperature of 252 . When the calli grew to a certain size, they were transferred into an incubator with light intensity of 1 500 lx, light period of 16 h/d and temperature of 25 2 . Twenty days later, we took a close observation upon the calli, calculated their induction rate, and determined the suitable culture media for leaves of different Paulownia species.

Foundation Item: This paper was supported by National Nature Science Foundation of China (No. 39870631) and Nature Science Foundation of Henan Province (No. 994011100). Biography: FAN Guo-qiang (1964-), male, Ph. Doctor, Professor in Forestry Department of Henan Agriculture University. Zhengzhou 450002, P. R. China. E-mail: [email protected]. Received date: 2001-09-24 Responsible editor: Song Funan

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FAN Guo-qiang et al. suitable rooting media of those induced buds.

Medium for bud differentiation 3 Those induced calli were cut into about 1.0-cm pieces and put on the MS media adding 0.1~1.1 mgL-1 NAA (briefly as 0.1~1.1 NAA) and 2~12 mgL-1 BA for bud differentiation in light condition. One piece was put in each bottle, and 20 bottles were taken for the leaves of each species of Paulownia. Twenty days later the induction rates were calculated. Medium for rooting The induced buds with certain height were cut at their stem bases and transferred on the rooting media, and then incubated in the light condition for root induction. Twenty days later we observed the root generation and chose the
Table 1.

Results and analysis

Callus induction of leaves for different species of Paulownia The callus inductions of leaves of 5 different Paulownia species on different media were culture for 20 d (See Table 1). The induction rates of 5 species were highest on MS culture medium, and there were no calli induced on N6 culture medium. That is to say, the callus induction from the leaves of different species of Paulownia had significant difference if the culture media were different, and MS culture medium could be taken as the basic one for inducing calli from leaves of Paulownia.

Callus induction from the leaves of different Paulownia species on various media
Media 1/2MS MS Number of mean explants 28.2 26.4 27.6 27.6 28.2 26.4 26.4 25.8 28.2 25.8 26.4 27.6 26.4 26.4 26.4 26.4 27.6 25.2 26.4 25.2 25.8 26.4 28.2 26.4 26.4 Mean Number of induced callus 5.0 12.8 7.0 0 5.0 5.4 10.6 9.0 0 2.4 6.2 12.6 11.0 0 4.2 4.6 9.8 6.0 0 4.4 7.0 12.8 4.4 0 9.6 Induction ratio 17.7 46.8 25.1 0 17.4 20.5 40.2 34.9 0 9.3 23.5 45.7 41.7 0 16.0 17.4 35.5 23.8 0 17.5 27.1 48.5 15.6 0 36.4

Paulownia

P. tomentosa

B5 N6 PC 1/2MS MS

P. australis

B5 N6 PC 1/2MS MS

P. fortunei

B5 N6 PC 1/2MS MS

P. elongata

B5 N6 PC 1/2MS MS B5 N6 PC

P.

tomentosa

P.

fortunei

Note: The values of the number of mean explants in above table were mean numbers of explants on the media with different hormone combinations (0.3NAA+1BA, 0.3NAA+3BA, 0.3NAA+5BA, 0.3NAA+7BA, 0.3NAA+9BA).

Callus induction of leaves on MS media The callus induction of the leaves of different Paulownia species on MS media with different concentrations of hormones indicated that the induction rate declined with rising of BA concentrations when the NAA concentration was 0.1 mgL-1 (Table 2). The change of induction rate rose first and then declined at NAA concentration of 0.3~0.7 mgL-1. When NAA concentration was 0.9~1.1 mgL-1 with combination of any BA concentrations, the induction rate became

zero. These results clarified the callus induction of leaves of the 5 Paulownia species had similarity in heredity, but individual differences still existed in callus induction rates on the same culture medium. Moreover, callus of leaves of different Paulownia species could be divided into two catalogues: one was colorless transparent with brittle texture, the other was light-yellowish strumae with tight texture, and the later was used to proliferate buds. The callus inductions of leaves of different Paulownia species on MS culture media were shown in Fig.1 A~E.

Journal of Forestry Research, 12(4): 209-214 (2001)

Table 2. Calli induced from leaves of different Paulownia species on MS media
Induction rates P. tomentosa 43.3 18.5 12.5 0 0 0 66.7 83.3 50.0 14.2 0 0 33.3 92.5 50.0 12.5 0 0 16.7 55.6 12.5 0 0 0 0 18.5 14.3 0 0 0 0 0 0 0 0 0 P. australis 48.1 26.7 12.5 0 0 0 81.5 66.7 50.0 23.8 0 0 43.3 74.0 60.0 55.6 16.7 0 0 47.6 83.3 29.6 12.5 0 0 0 20.8 14.3 0 0 0 0 0 0 0 0 P. fortunei 60.0 37.5 19.0 0 0 0 37.0 88.3 55.5 23.3 0 0 37.0 85.7 50.0 12.0 0 0 0 23.8 41.7 50.0 60.0 16.6 0 11.1 20.8 33.3 19.0 0 0 0 0 0 0 0 P. elongata 11.1 0 0 0 0 0 16.7 47.6 87.5 33.3 11.1 0 14.2 83.3 33.3 11.1 0 0 0 20.0 40.0 18.5 0 0 0 0 6.6 12.5 0 0 0 0 0 0 0 0 P. tomentosa x P. fortunei 29.6 20.0 12.5 0 0 0 37.0 50.8 63.3 86.6 71.4 48.1 0 41.7 55.5 66.7 33.3 0 0 12.5 26.7 28.6 0 0 0 0 20.8 0 0 0 0 0 0 0 0 0

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Concentrations of hormones /mgL-1 0.1NAA+ 2BA 0.1NAA+ 4BA 0.1NAA+ 6BA 0.1NAA+ 8BA 0.1NAA+10BA 0.1NAA+12BA 0.3NAA+ 2BA 0.3NAA+ 4BA 0.3NAA+ 6BA 0.3NAA+ 8BA 0.3NAA+10BA 0.3NAA+12BA 0.5NAA+ 2BA 0.5NAA+ 4BA 0.5NAA+ 6BA 0.5NAA+ 8BA 0.5NAA+10BA 0.5NAA+12BA 0.7NAA+ 2BA 0.7NAA+ 4BA 0.7NAA+ 6BA 0.7NAA+ 8BA 0.7NAA+10BA 0.7NAA+12BA 0.9NAA+ 2BA 0.9NAA+ 4BA 0.9NAA+ 6BA 0.9NAA+ 8BA 0.9NAA+10BA 0.9NAA+12BA 1.1NAA+ 2BA 1.1NAA+ 4BA 1.1NAA+ 6BA 1.1NAA+ 8BA 1.1NAA+10BA 1.1NAA+12BA

Bud differentiation of leaves callus From Table 3, it can be seen that the bud differentiation rates of leaves callus of P. tomentosa on MS medium changed with the hormone concentrations. The bud differentiation rate decrease with BA concentration rising at NAA concentration of 0.1 mgL-1, increased with BA concentration rising at NAA concentrations of 0.3~0.5 mgL-1 and 0.7 mgL-1, but becomes zero at NAA concentrations of 0.9~1.1 mgL-1. When BA was 8 mgL-1, the bud differentiation rates declined gradually as NAA concentrations increasing; when -1 -1 BA was 10 mgL and 12 mgL , it tended to rise first and then decline with NAA concentration increasing. The bud differentiation rate was highest (94.7%) on MS+0.3 NAA+12 BA culture medium, so this medium was consid-

ered as the best culture medium for bud differentiation of P. tomentosa. For P. australis, the bud differentiation rate of leaves callus decreased with BA concentrations rising (from 8 to 12 mgL-1) at NAA concentration of 0.1 mgL-1, but increased with BA concentration rising at NAA concentration -1 of 0.3 mgL . When NAA concentrations was 0.5~0.7 -1 mgL and BA was 8~12 mgL-1, the bud differentiation rate was lower. At NAA concentrations of 0.9~1.1 mgL-1, with three combinations of BA concentrations, the bud differentiation rate was always zero. By comparing these bud differentiation rates, it could be seen that MS +0.3 NAA+12 BA medium was suitable for leaves callus of P. australis. Leaf callus of P. fortunei have higher bud differentiation rate on MS+0.3~0.5NAA+10~12BA medium. The bud dif-

212 ferentiation rates were always zero at NAA concentrations from 0.9 to 1.1 mgL-1, with three different BA concentrations. The MS+0.5 NAA+12 BA medium was the best culture medium for bud differentiation of leaf callus of P. fortunei, with a bud differentiation rate of 95%. The bud differentiation rates of P. elongata were always zero at NAA concentrations of 0.1, 0.3, 1.1 mgL-1, respecTable 3.

FAN Guo-qiang et al. tively, with the combination of the three BA concentrations. The bud differentiation mainly presented on MS+0.5~0.9 NAA+12 BA media, with a differentiation rate of 90% on MS+0.5 NAA+12 BA medium, which could be chosen as the medium for bud differentiations from leaves callus of P. elongata.

Buds induced from leaves callus of different Paulownia species on MS media with various hormone concentrations
P. tomentosa NC B 5 3 2 3 15 18 0 8 14 0 2 5 0 0 0 0 0 0 IR 25.0 15.0 10.5 15.0 83.3 94.7 0 42.1 77.8 0 10.5 29.4 0 0 0 0 0 0 P. australis NC 19 20 18 20 20 19 18 20 20 20 19 20 20 20 20 20 19 18 B 3 2 0 10 15 19 0 2 5 0 0 2 0 0 0 0 0 0 IR 15.6 10.0 0 50.0 75.0 100.0 0 10.0 25.0 0 0 10.0 0 0 0 0 0 0 NC 20 20 19 20 18 20 19 20 18 20 20 19 20 18 20 17 20 20 P. fortunei Bds 5 2 0 0 10 19 0 12 18 0 5 10 0 0 0 0 0 0 IR 25.0 10.0 0 0 55.6 95.0 0 60.0 100.0 0 25.0 52.6 0 0 0 0 0 0 P. elongata NC 20 19 20 20 18 19 20 17 20 19 20 20 18 20 19 17 20 19 B 0 0 0 0 0 0 0 10 18 0 13 15 0 8 3 0 0 0 IR 0 0 0 0 0 0 0 58.8 90.0 0 65.0 75.0 0 40.0 15.6 0 0 0 P. tomentosa x P. fortunei NC 20 20 19 18 20 20 20 19 17 20 18 20 20 19 17 18 20 20 B 0 0 0 0 5 8 0 6 10 0 10 18 0 4 6 0 0 0 IR 0 0 0 0 25.0 40.0 0 31.6 58.8 0 55.5 90.0 0 21.1 35.3 0 0 0

Concentrations of hormones /m gL-1 0.1NAA+ 8BA 0.1NAA+10BA 0.INAA+12BA 0.3NAA+ 8BA 0.3NAA+10BA 0.3NAA+12BA 0.5NAA+ 8BA 0.5NAA+10BA 0.5NAA+12BA 0.7NAA+ 8BA 0.7NAA+10BA 0.7NAA+12BA 0.9NAA+ 8BA 0.9NAA+10BA 0.9NAA+12BA 1.1NAA+ 8BA 1.1NAA+10BA 1.INAA+12BA

20 20 19 20 18 19 20 19 18 20 19 17 19 20 19 20 20 18

Note: NC: Number of callus; B: buds; IC: induction ratio.

The leaves callus of P. tomentosa x P. fortunei produced bud differentiation on MS+0.3~0.9 NAA+10~12 BA media, with a differentiation rate of 90% on MS+0.7 NAA+12 BA medium. But the differentiation rate was zero when NAA concentrations were 0.1 mgL-1 and 1.1 mgL-1, combining with any BA concentration respectively. For this reason, MS+0.7 NAA+12 BA medium was chosen as the suitable medium for the bud differentiation from leaves callus of P. tomentosa x P. fortunei. Differentiated buds from leaves callus of 5 Paulownia species were shown in Fig.1 (F~J). Root differentiation of differentiated buds Rooting of differentiated buds on 1/2MS (modified) media with various concentrations of NAA was shown in Table 4 and Fig.1 (K~P). The rooting rates of the differentiated buds for P. australis, P. fortunei, P. elongata and P. tomentosa x P. fortunei were all up to 100% on the media with 0, 0.1, 0.3, and 0.5 mgL-1 NAA, but there existed difference in number and length of root for different species on different concentrations of NAA. Based on this, the 1/2 MS+0.1 NAA, 1/2 MS+0.1 NAA, 1/2MS, 1/2MS+0.5 NAA, and 1/2MS+0.3 NAA media were chosen as the rooting media respectively for P. tomentosa, P. australis. P. fortunei. P. elongata and P. tomentosa x P. fortunei, respectively.

Table 4. Rooting of different Paulownia plantlet on 1/2MS media with various NAA concentrations
Paulownia plants NAA concentrations 0.0 0.1 0.3 0.5 0.0 0.1 0.3 0.5 0.0 0.1 0.3 0.5 0.0 0.1 0.3 0.5 0.0 0.1 0.3 0.5 Rooting percentage 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Number of mean roots 3 5 4 2 4 8 5 3 8 6 4 2 5 3 4 8 3 5 8 4 Mean length of roots 2.5 3.0 2.1 1.8 2.5 3.0 2.0 1.5 3.5 2.4 2.0 1.8 2.5 2.4 1.7 2.7 1.8 2.6 3.1 2.5

P. tomentosa

P. australis

P. fortunei

P. elongata

P. tomentosa x P. fortunei

Journal of Forestry Research, 12(4): 209-214 (2001)

213 with results reported before (He & Dai 1999; Jiang 1988; Shi 1995). The differences of plant species and their metabolic activity, especially hormone contents, resulted in requiring different hormone and concentration on culture media for callus induction from different species of plant tissues or organs (Huang 1990; Wei et al. 1991, 1992; Emily 1991; Julie 1986; Sticklen 1986; Von Arnold & Woodward 1988). Although the five species in this study were the plants in same genus, and especially P. australis, P. elongata and P. tomentosa x P. fortunei were all the hybrid varieties of P. tomentosa and P. fortunei (Fan et al. 2001), due to a long-term natural evolution, there exists differences in physical properties, biochemical metabolic level as well as hormone contents among them, and maybe this is main reason for that the most suitable media are different for callus inductions of the five species.

Discussions
The suitable culture media of different tissues or organs of plant to induce callus differed with the plants of different genus, family and species. Calli from tissues or organs of rice and wheat were induced on N6 culture medium with proper concentration of hormones (Chen 1980). Calli from leaves of Platanus occidenalis were induced on MS culture media (Sun 1982, 1991). In this study, we found that the callus induction rates of the leaves of P. tomentosa, P. australis, P. fortunei, P. elongata, and P. tomentosa x P. fortunei were the highest at MS culture medium; lower at 1/2MS, B5 and PC; and zero at N6. Therefore, MS could be chosen as the basic culture medium to induce calli from the leaves of the five species of Paulownia plants, which was consistent

Fig. 1

Callus inductions of leaves of different species of Paulownia and their plant regenerations
A~E F~J K~O Callus of P. tomentosa, P. australis, P. fortunei, P. elongata, P. tomentosa P. fortunei Proliferated buds from callus of P. tomentosa, P. australis, P. fortunei, P. elongata, P. tomentosa P. fortunei Regenerated plants of P. tomentosa, P. australis, P. fortunei, P. elongata, P. tomentosa P. fortunei

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Shi Shizheng. 1995. Primary study on Paulownia tissue culture [J].

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