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Shoot number and shoot size as affected by growth regulators in in vitro cultures of Spathiphyllum floribundum L.
Scientia Horticulturae 89 (2001) 227±236
Shoot number and shoot size as affected by growth regulators in in vitro cultures of Spathiphyllum ¯oribundum L. Rafael Ramirez-Malagona, Anatoli Borodanenkoa, Jose Luis Barrera-Guerraa, Neftali Ochoa-Alejob,* a
Instituto de Ciencias AgrõÂcolas, Universidad de Guanajuato, Apartado Postal 311, 36500 Irapuato, Gto., Mexico b Departamento de IngenierõÂa GeneÂtica de Plantas, Unidad de BiotecnologõÂa e IngenierõÂa GeneÂtica de Plantas, Centro de InvestigacioÂn y de Estudios Avanzados del I.P.N., CINVESTAV-Unidad Irapuato, Apartado Postal 629, 36500 Irapuato, Gto., Mexico Accepted 13 October 2000
Abstract The average number and average fresh weight of Spathiphyllum ¯oribundum cv. Petite axillary shoots grown on MS medium with three concentrations of BA (1, 2 and 5 mg l 1) in the absence of auxin or in combination with 0.05 mg l 1 NAA, 0.5 and 1.0 mg l 1 IAA or 0.005, 0.05 and 0.1 mg l 1 2,4-D were determined. In general, higher shoot number and fresh weight values were achieved in all tested levels of BA combined with 0.05 mg l 1 NAA than in combination with IAA or 2,4-D. The best response in terms of average shoot number (5.8), average fresh weight (116 mg/ explant), leaf number/plant (14.7), lateral shoot number/plant (5.3), plant diameter (14.2 cm), and plant height (13.0 cm) was observed in shoots cultured on medium containing 0:05 mg l 1 NAA 2 mg l 1 BA. The shoots produced in this treatment grew vigorously when the plants were acclimatized under greenhouse conditions. When shoots were grown on MS medium with the above combinations and added of 1 mg l 1 Kin, the highest shoot number (11.6 shoots per explant) was observed in the presence of 1 mg l 1 IAA 2 mg l 1 BA 1 mg l 1 Kin in which an average fresh weight of 39 mg/shoot was achieved. The importance of using a particular shoot size (minimum fresh weight) for better adaptation of plants to soil is discussed. Micropropagated plants of 20 mm Abbreviations: BA, benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid; IAA, indoleacetic acid; Kin, kinetin; NAA, naphthaleneacetic acid; PBA, 6-benzylamino-9-(2-tetrahydro-pyranyl)9H-purine * Corresponding author. Tel.: 52-462-39600/39654; fax: 52-462-45849. E-mail address: [email protected] (N. Ochoa-Alejo). 0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 2 3 6 - 3
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R. Ramirez-Malagon et al. / Scientia Horticulturae 89 (2001) 227±236
height and more than 66 mg weight grew best following acclimatization in the greenhouse. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Auxins; Cytokinins; Micropropagation; Shoot formation; Spathiphyllum ¯oribundum
1. Introduction Spathiphyllum ¯oribundum is an ornamental plant that due to its long lasting showy, white ¯owers and ease of growing has gained popularity in the tropical foliage industry. Spathiphyllum propagation can be done by seeds or in vitro multiplication. Tissue culture is becoming more utilized due to greater uniformity and the higher quality of plants demanded by the growers (Hennen and Hotchkiss, 1995). In spite of Spathiphyllum popularity among the foliage ornamental plants, and despite the increasing commercial demand of Spathiphyllum plants, only a few protocols for tissue culture propagation are published. The ®rst report on Spathiphyllum micropropagation was that of Fonnesbech and Fonnesbech (1979), who cultured leaf and stem explants from cv. Clevelandii on MS medium (Murashige and Skoog, 1962) supplemented with 2 mg l 1 PBA, and regenerated up to 9.1 shoots per explant. Werbrouck et al. (1995) studied the metabolism of BA related to the acclimatization of S. ¯oribundum. More recently, Watad et al. (1997) reported the micropropagation of Spathiphyllum using MS medium with 2 mg l 1 Kin and gelled with agar or with liquid medium and ¯oating raft membranes. We report the results of in vitro propagation of Spathiphyllum using a combination of two cytokinins with three different concentrations and combinations of auxins. The importance of taking into account not just the shoot yield (shoot average number), but also the size of the regenerated shoots (the fresh weight average of the regenerated plants) for acclimatization is discussed. 2. Materials and methods Plant material. Young Spathiphyllum plants, cv. Petite were obtained from a commercial nursery. Establishment of in vitro cultures. The leaves and roots were trimmed from the plants leaving just the central stem of each plant. Stems were washed in running tap water for 2 h, immersed in 70% ethanol for 60 s, and treated for 20 min in sodium hypochlorite solution (1.2%) containing 0.1% Tween 20. Next the stems were treated with Benomyl solution (2 g l 1; previously sterilized in autoclave for 20 min), followed by treatment with 1.2% sodium hypochlorite for 10 min. Stems were rinsed ®ve times with sterile distilled water in an air ¯ow cabinet and were
R. Ramirez-Malagon et al. / Scientia Horticulturae 89 (2001) 227±236
229
cut into three explants each containing one or two buds. The explants were placed in glass bottles (180 ml) containing 20 ml of MS medium supplemented with 2 mg l 1 BA 0:5 mg l 1 IAA, gelled with 0.8% (w/v) agar, and adjusted to pH 5.8. The cultures were incubated under a 16/8 h light/dark photoperiod (daylight ¯uorescent lamps, 35 mmol m 2 s 1) and at 25 2 C for 30 days. A second and a third 30-day reculture on the same culture medium were also carried out, until enough shoots were available to establish the planned experiments. Culture treatments with growth regulators. Shoots were individually separated and cultured on MS medium without growth regulators for 30 days until they reached 30 mm in length and a fresh weight average of 180 30 mg. The shoots were cultured in 180 ml glass bottles containing 20 ml MS medium with growth regulator treatments. Two experiments, each involving 22 treatments, were established: in the ®rst experiment, the growth regulator used was BA (1, 2 and 5 mg l 1) combined with: (1) no auxins, (2) 0.05 mg l 1 NAA, (3) 0.5 and 1.0 mg l 1 IAA, and (4) 0.005, 0.05 and 0.1 mg l 1 2,4-D; in addition, a control without growth regulators was included. The second experiment was established using the same auxin/cytokinin treatments as in the ®rst experiment, but in this case Kin (1 mg l 1) was added to each BA concentrations. Each experiment was carried out twice under a completely randomized design with seven replicates per treatment. Each replicate was represented by a culture bottle with four individual shoots rendering a group of 28 shoots per treatment. Clumps of shoots produced by each explant were weighed and the number of shoots per explant as well as the average fresh weight and number of shoots per treatment were recorded after 45 days of culture. Transference of plants to soil. Twenty ®ve plants from each treatment described above were transferred to pots with sterilized soil, acclimatized under greenhouse conditions and grown for 4 months. Plants from nine treatments were selected (because of space problems) and data on leaf number, lateral shoot number, plant diameter and plant height were recorded. Analysis of variance, mean separations and a correlation analysis were carried out for each experiment using the MSTATC program. 3. Results and discussion When BA was used as the sole growth regulator in the culture medium without the interaction of any auxin, a limited proliferation of explants with a maximum average of 1.8 shoots per cultured explant was observed (treatments 1±3, Table 1). Treatments with auxins alone were not included since preliminary tests had shown that these growth regulators induced only callus (data not shown). When BA was used with auxin (IAA, NAA or 2,4-D), the number of shoots per explant increased in comparison with either the control or treatments with only BA. An
230
Auxins (mg l 1)
1 mg l
1
BA
2 mg l
1
BA
5 mg l
1
BA
Shoot number per explant
Fresh weight (mg)
Shoot number per explant
Fresh weight (mg)
Shoot number per explant
Fresh weight (mg)
No auxin NAA, 0.05 IAA, 0.5 IAA, 1.0 2,4-D, 0.005 2,4-D, 0.05 2,4-D, 0.1
(1) 1.2 e (4) 5 abcd (7) 3.8 d (10) 4.1 cd (13) 4.5 bcd (16) 3.8 d (19) 4.4 bcd
180 B 112 CD 46 FGH 70 EF 64 EFG 58 EFGH 49 FGH
(2) 1.8 e (5) 5.8 abc (8) 4.4 bcd (11) 5.0 abcd (14) 4.2 bcd (17) 5.3 abcd (20) 4.5 bcd
190 B 116 C 64 EFG 49 FGH 71 EF 51 EFGH 51 EFGH
(3) 1.3 e (6) 5.9 abc (9) 4.6 bcd (12) 5.4 abcd (15) 5.3 abcd (18) 6.7 a (21) 6.0 ab
176 B 81 DE 37 GH 41 FGH 34 GH 30 H 29 H
Average Control
A 3:8 A 82:7 A 4:4 (22) shoot number per explant 1:0 e, fresh weight 223 A
A 84:5
A 5:0
A 61
a
Numbers in parenthesis indicate treatment number. Values are the means of 56 explants after 45 days of culture. Treatments with the same letter are not statistically different (LSD, P 0:05).
R. Ramirez-Malagon et al. / Scientia Horticulturae 89 (2001) 227±236
Table 1 Shoot number and fresh weight averages of axillary shoots of S. ¯oribundum cv. Petite cultured on MS medium with three concentrations of BA in combination with different levels of NAA, IAA and 2,4-Da
R. Ramirez-Malagon et al. / Scientia Horticulturae 89 (2001) 227±236
231
increase in the number of shoots was recorded when the level of BA was increased from 1 to 5 mg l 1, reaching the maximum average yield (6.7 shoots per explant) with 0.05 mg l 1 2,4-D and 5 mg l 1 BA (treatment 18, Table 1). Shoot fresh weight values were higher when the supplied auxin was NAA (0.05 mg l 1) combined with different BA levels. When Kin was added to each treatment, differences in responses were found to occur and the highest value for shoot number average was achieved with the treatment containing 1.0 mg l 1 IAA and 2 mg l 1 BA 1:0 mg l 1 Kin (11.6 shoots per explant) (Table 2), but this treatment yielded shoots with a fresh weight average of 39 mg (Table 2), while treatments with 0.005 mg l 1 2,4-D combined with BA and Kin yielded higher fresh weight averages compared to those obtained when Kin was not present in the culture medium (Table 1). The highest shoot fresh weight values were achieved when the explants were cultured on a medium lacking growth regulators (control) or supplemented with BA alone since only the original shoot or just few additional shoots were present after 45 days of culture. These shoots grew more than those inoculated on the treatments that induced a higher number of shoots. The average fresh weight of shoots is a factor to be considered for better growth and development of plants adapted to soil after in vitro propagation. According to our experience, this parameter is a function of both the length and the diameter of plants and could be used as a measure of vigor to ensure a better growth of micropropagated plants when they are transferred to soil under greenhouse conditions. The treatment that induced the highest multiplication rates (treatment 11, Table 2: 1 mg l 1 IAA 2 mg l 1 BA 1 mg l 1 Kin) was not the most adequate for commercial propagation of S. ¯oribundum. This treatment produced an average yield of 11.6 shoots per explant and a fresh weight average of 39 mg per shoot, but the plants were smaller in height and foliar diameter than those from other treatments (Table 3) after 4 months of growth in the greenhouse. Plants from treatment 11, grew slowly during the ®rst stages in the greenhouse due to their lower height (10 mm). In general, plants with an initial fresh weight ranging from 39 to 66 mg after the in vitro culture, produced the smallest plants after 4 months of growth in the greenhouse (Table 3). The best treatment for producing vigorous plants was the treatment 5 (0:05 mg l 1 NAA 2 mg l 1 BA) in which an average of 5.8 shoots per explant with a mean fresh weight of 116 mg per shoot and a length of at least 20 mm were achieved. The shoots produced in this treatment grew vigorously when the plants were acclimatized under greenhouse conditions (Fig. 1). When Kin was added to the culture medium, different responses were observed according to the auxins used. The fresh weight average decreased when NAA was present and an increase was observed when Kin was added to treatments with 0.005 mg l 1 2,4-D. Culture medium containing 0:05 mg l 1 NAA 1 mg l 1 BA 1 mg l 1 Kin increased the shoot number (7.3 shoots per explant) (Table 2). An inverse relationship between the
232
Auxins (mg l 1)
1 mg l
1
BA 1 mg l
1
Kin
2 mg l
1
BA 1 mg l
1
Kin
5 mg l
1
BA 1 mg l
1
Kin
Shoot number per explant
Fresh weight (mg)
Shoot number per explant
Fresh weight (mg)
Shoot number per explant
Fresh weight (mg)
No auxin NAA, 0.05 IAA, 0.5 IAA, 1.0 2,4-D, 0.005 2,4-D, 0.05 2,4-D, 0.1
(1) 2.1 gh (4) 7.3 b (7) 3.6 defg (10) 4.4 cde (13) 2.4 fgh (16) 4.7 cd (19) 3.7 defg
186 A 69 CD 45 DE 50 DE 97 BC 24 E 55 DE
(2) 2.7 efgh (5) 4.2 cdef (8) 3.8 cdefg (11) 11.6 a (14) 3.5 defg (17) 5.0 cd (20) 5.7 bc
170 A 68 CD 42 DE 39 DE 107 B 48 DE 41 DE
(3) 1.9 gh (6) 5.0 cd (9) 3.8 cdefg (12) 4.2 cdef (15) 4.3 cdfg (18) 4.9 cd (21) 4.9 cd
177 A 69 CD 95 BC 66 CD 105 B 50 DE 68 CD
Average Control
A 4:0 A 75 A 5:2 (22) shoot number per explant 1:0 h, fresh weight 187 A
A 73:5
A 4:1
A 90
a
Numbers in parenthesis indicate treatment number. Values are the means of 56 explants after 45 days of culture. Treatments with the same letter are not statistically different (LSD, P 0:05).
R. Ramirez-Malagon et al. / Scientia Horticulturae 89 (2001) 227±236
Table 2 Shoot number and fresh weight averages of axillary shoots of S. ¯oribundum cv. Petite cultured on MS medium with three concentrations of BA 1 mg Kin in combination with different levels of NAA, IAA and 2,4-Da
Treatments 0:05 mg l 0:05 mg l 0:05 mg l 1 mg l 1 mg l 1 mg l
1
1
1
NAA 1 mg l NAA 2 mg l 1 NAA 5 mg l
BA BA 1 BA
1
1
1
1
1
1
IAA 1 mg l IAA 2 mg l 1 IAA 5 mg l
0:005 mg l 0:005 mg l 0:005 mg l a
1
BA 1 mg l BA 1 mg l 1 BA 1 mg l
Kin Kin 1 Kin
1
1
1
1
1
1
2;4-D 1 mg l 2;4-D 2 mg l 1 2;4-D 5 mg l
BA 1 mg l BA 1 mg l 1 BA 1 mg l
Kin Kin 1 Kin
AFW (mg)
Leaf number/plant
Lateral shoots/plant
Plant diameter (cm)
Plant height (cm)
112 116 81
15.7 a 14.7 a 15.0 a
5.4 a 5.3 a 5.0 a
14.3 a 14.2 a 12.8 ab
14.1 ab 13.0 abc 12.1 bc
50 39 66
14.3 a 13.4 a 13.9 a
4.8 a 4.7 a 4.8 a
12.3 ab 11.8 b 12.4 ab
11.7 c 11.9 c 11.4 c
97 107 105
15.4 a 14.3 a 14.2 a
5.7 a 5.2 a 5.9 a
13.4 ab 13.0 ab 13.8 ab
12.2 bc 12.1 bc 12.0 c
Mean separation was run by column. Each value is the average of 25 plants. Data with the same letter are not statistically different (LSD, P 0:05). AFW: average fresh weight after in vitro culture and before starting greenhouse growing.
R. Ramirez-Malagon et al. / Scientia Horticulturae 89 (2001) 227±236
Table 3 Leaf number, lateral shoot number, height and plant diameter of S. ¯oribundum plants 4 months after transfer to soil from in vitro culturea
233
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R. Ramirez-Malagon et al. / Scientia Horticulturae 89 (2001) 227±236
Fig. 1. Spathiphyllum plants cultured in vitro with 0:05 mg l 1 NAA 2 mg l 1 BA: (a) two clumps of Spathiphyllum shoots after 45 days of in vitro culture; (b) plants after 30 days on MS medium lacking growth regulators and ready to be transferred to soil; (c) in vitro regenerated Spathiphyllum plant growing in soil.
number of regenerated shoots per explant and the fresh weight of the regenerated shoots was observed. A correlation analysis of values recorded 4 months after the acclimatization con®rmed this assessment (correlation coef®cient r 0:654). The size of in vitro plants is re¯ected in differences in vigor, stated as width and height of the developing plants. Table 3 shows that shoots with higher fresh weight values and with moderate multiplication rates achieved a better
R. Ramirez-Malagon et al. / Scientia Horticulturae 89 (2001) 227±236
235
development than those having higher multiplication rates of shoots, but exhibiting lower fresh weight values. In the present work, we observed that explants from NAA treatments with BA as the sole cytokinin (Tables 1 and 3), exhibited the best responses for height and plant diameter after 4 months in the greenhouse. Production of 7±11 shoots per explant is in the range of multiplication of many other plants cultured in vitro. There are species that respond with a higher multiplication rate (Ahroni et al., 1997), but some others multiply at lower rates than Spathiphyllum. Our results are in agreement with those reports published on the in vitro multiplication of Spathiphyllum. Fonnesbech and Fonnesbech (1979) reported that in vitro propagation of Spathiphyllum species (Clevelandii), using 2 mg l 1 PBA, produced 9.1 shoots per explant. This result is similar to the maximum average obtained in treatment 11 (Table 2). Watad et al. (1997) culturing in vitro plants of cultivar Petite obtained an average of 10.1 shoots per explant on a culture medium supplemented with 2 mg l 1 Kin and gelled with agar, and up to 14.4 shoots per explant with ¯oating liquid medium raft membranes. Some of the differences in shoot multiplication could be explained on the basis of differences in the Spathiphyllum cultivars used and also due to differences in growth conditions. 4. Conclusion In summary, the in vitro propagation of Spathiphyllum was promoted with different combinations and different concentrations of auxins and cytokinins with yields higher than 10 shoots per explant. However, not always the treatment with the highest shoot multiplication rate is the ideal for the propagation of the species. Sometimes, as is the case of the present work, a positive correlation is observed between the initial weight average of shoots from in vitro cultures and the growth of plants (measured as plant diameter and plant height) in the greenhouse. We have shown that shoot size is an important factor for further development in the greenhouse of the regenerated plants, and it is suggested that a better decision for growing Spathiphyllum plants from in vitro cultures could be the employment of treatments where a balance of high shoot regeneration combined with an adequate size (fresh weight) of regenerated shoots is achieved. Acknowledgements We would like to thank Dr. John Snape for his valuable comments during the preparation of the manuscript. This work was supported by a grant from Universidad de Guanajuato, Mexico.
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References Ahroni, A., Zuker, A., Rozen, Y., Shejtman, H., Vainstein, A., 1997. An ef®cient method for adventitious shoot regeneration from stem-segment explants of Gypsophila. Plant Cell Tiss. Org. Cult. 49, 101±106. Fonnesbech, M., Fonnesbech, A., 1979. In vitro propagation of Spathiphyllum. Sci. Hort. 10, 21±25. Hennen, G.R., Hotchkiss, S.E., 1995. Spathiphyllum: success for every market. GrowerTalks 59 (9), 31±36. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15, 473±497. Watad, A.A., Raghotama, K.G., Kochba, M., Nissim, A., Gaba, V., 1997. Micropropagation of Spathiphyllum and Syngonium is facilitated by use of interfacial membrane rafts. HortScience 32, 307±308. Werbrouck, S.P.O., VanderJeut, B., Dewitte, W., Prinsen, E., VanOnckelen, H.A., Debergh, P.C., 1995. The metabolism of benzyladenine in Spathiphyllum ¯oribundum ``Schot Petite'' in relation to acclimatization problems. Plant Cell Rep. 14, 662±665.
Shoot number and shoot size as affected by growth regulators in in vitro cultures of Spathiphyllum ¯oribundum L. Rafael Ramirez-Malagona, Anatoli Borodanenkoa, Jose Luis Barrera-Guerraa, Neftali Ochoa-Alejob,* a
Instituto de Ciencias AgrõÂcolas, Universidad de Guanajuato, Apartado Postal 311, 36500 Irapuato, Gto., Mexico b Departamento de IngenierõÂa GeneÂtica de Plantas, Unidad de BiotecnologõÂa e IngenierõÂa GeneÂtica de Plantas, Centro de InvestigacioÂn y de Estudios Avanzados del I.P.N., CINVESTAV-Unidad Irapuato, Apartado Postal 629, 36500 Irapuato, Gto., Mexico Accepted 13 October 2000
Abstract The average number and average fresh weight of Spathiphyllum ¯oribundum cv. Petite axillary shoots grown on MS medium with three concentrations of BA (1, 2 and 5 mg l 1) in the absence of auxin or in combination with 0.05 mg l 1 NAA, 0.5 and 1.0 mg l 1 IAA or 0.005, 0.05 and 0.1 mg l 1 2,4-D were determined. In general, higher shoot number and fresh weight values were achieved in all tested levels of BA combined with 0.05 mg l 1 NAA than in combination with IAA or 2,4-D. The best response in terms of average shoot number (5.8), average fresh weight (116 mg/ explant), leaf number/plant (14.7), lateral shoot number/plant (5.3), plant diameter (14.2 cm), and plant height (13.0 cm) was observed in shoots cultured on medium containing 0:05 mg l 1 NAA 2 mg l 1 BA. The shoots produced in this treatment grew vigorously when the plants were acclimatized under greenhouse conditions. When shoots were grown on MS medium with the above combinations and added of 1 mg l 1 Kin, the highest shoot number (11.6 shoots per explant) was observed in the presence of 1 mg l 1 IAA 2 mg l 1 BA 1 mg l 1 Kin in which an average fresh weight of 39 mg/shoot was achieved. The importance of using a particular shoot size (minimum fresh weight) for better adaptation of plants to soil is discussed. Micropropagated plants of 20 mm Abbreviations: BA, benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid; IAA, indoleacetic acid; Kin, kinetin; NAA, naphthaleneacetic acid; PBA, 6-benzylamino-9-(2-tetrahydro-pyranyl)9H-purine * Corresponding author. Tel.: 52-462-39600/39654; fax: 52-462-45849. E-mail address: [email protected] (N. Ochoa-Alejo). 0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 2 3 6 - 3
228
R. Ramirez-Malagon et al. / Scientia Horticulturae 89 (2001) 227±236
height and more than 66 mg weight grew best following acclimatization in the greenhouse. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Auxins; Cytokinins; Micropropagation; Shoot formation; Spathiphyllum ¯oribundum
1. Introduction Spathiphyllum ¯oribundum is an ornamental plant that due to its long lasting showy, white ¯owers and ease of growing has gained popularity in the tropical foliage industry. Spathiphyllum propagation can be done by seeds or in vitro multiplication. Tissue culture is becoming more utilized due to greater uniformity and the higher quality of plants demanded by the growers (Hennen and Hotchkiss, 1995). In spite of Spathiphyllum popularity among the foliage ornamental plants, and despite the increasing commercial demand of Spathiphyllum plants, only a few protocols for tissue culture propagation are published. The ®rst report on Spathiphyllum micropropagation was that of Fonnesbech and Fonnesbech (1979), who cultured leaf and stem explants from cv. Clevelandii on MS medium (Murashige and Skoog, 1962) supplemented with 2 mg l 1 PBA, and regenerated up to 9.1 shoots per explant. Werbrouck et al. (1995) studied the metabolism of BA related to the acclimatization of S. ¯oribundum. More recently, Watad et al. (1997) reported the micropropagation of Spathiphyllum using MS medium with 2 mg l 1 Kin and gelled with agar or with liquid medium and ¯oating raft membranes. We report the results of in vitro propagation of Spathiphyllum using a combination of two cytokinins with three different concentrations and combinations of auxins. The importance of taking into account not just the shoot yield (shoot average number), but also the size of the regenerated shoots (the fresh weight average of the regenerated plants) for acclimatization is discussed. 2. Materials and methods Plant material. Young Spathiphyllum plants, cv. Petite were obtained from a commercial nursery. Establishment of in vitro cultures. The leaves and roots were trimmed from the plants leaving just the central stem of each plant. Stems were washed in running tap water for 2 h, immersed in 70% ethanol for 60 s, and treated for 20 min in sodium hypochlorite solution (1.2%) containing 0.1% Tween 20. Next the stems were treated with Benomyl solution (2 g l 1; previously sterilized in autoclave for 20 min), followed by treatment with 1.2% sodium hypochlorite for 10 min. Stems were rinsed ®ve times with sterile distilled water in an air ¯ow cabinet and were
R. Ramirez-Malagon et al. / Scientia Horticulturae 89 (2001) 227±236
229
cut into three explants each containing one or two buds. The explants were placed in glass bottles (180 ml) containing 20 ml of MS medium supplemented with 2 mg l 1 BA 0:5 mg l 1 IAA, gelled with 0.8% (w/v) agar, and adjusted to pH 5.8. The cultures were incubated under a 16/8 h light/dark photoperiod (daylight ¯uorescent lamps, 35 mmol m 2 s 1) and at 25 2 C for 30 days. A second and a third 30-day reculture on the same culture medium were also carried out, until enough shoots were available to establish the planned experiments. Culture treatments with growth regulators. Shoots were individually separated and cultured on MS medium without growth regulators for 30 days until they reached 30 mm in length and a fresh weight average of 180 30 mg. The shoots were cultured in 180 ml glass bottles containing 20 ml MS medium with growth regulator treatments. Two experiments, each involving 22 treatments, were established: in the ®rst experiment, the growth regulator used was BA (1, 2 and 5 mg l 1) combined with: (1) no auxins, (2) 0.05 mg l 1 NAA, (3) 0.5 and 1.0 mg l 1 IAA, and (4) 0.005, 0.05 and 0.1 mg l 1 2,4-D; in addition, a control without growth regulators was included. The second experiment was established using the same auxin/cytokinin treatments as in the ®rst experiment, but in this case Kin (1 mg l 1) was added to each BA concentrations. Each experiment was carried out twice under a completely randomized design with seven replicates per treatment. Each replicate was represented by a culture bottle with four individual shoots rendering a group of 28 shoots per treatment. Clumps of shoots produced by each explant were weighed and the number of shoots per explant as well as the average fresh weight and number of shoots per treatment were recorded after 45 days of culture. Transference of plants to soil. Twenty ®ve plants from each treatment described above were transferred to pots with sterilized soil, acclimatized under greenhouse conditions and grown for 4 months. Plants from nine treatments were selected (because of space problems) and data on leaf number, lateral shoot number, plant diameter and plant height were recorded. Analysis of variance, mean separations and a correlation analysis were carried out for each experiment using the MSTATC program. 3. Results and discussion When BA was used as the sole growth regulator in the culture medium without the interaction of any auxin, a limited proliferation of explants with a maximum average of 1.8 shoots per cultured explant was observed (treatments 1±3, Table 1). Treatments with auxins alone were not included since preliminary tests had shown that these growth regulators induced only callus (data not shown). When BA was used with auxin (IAA, NAA or 2,4-D), the number of shoots per explant increased in comparison with either the control or treatments with only BA. An
230
Auxins (mg l 1)
1 mg l
1
BA
2 mg l
1
BA
5 mg l
1
BA
Shoot number per explant
Fresh weight (mg)
Shoot number per explant
Fresh weight (mg)
Shoot number per explant
Fresh weight (mg)
No auxin NAA, 0.05 IAA, 0.5 IAA, 1.0 2,4-D, 0.005 2,4-D, 0.05 2,4-D, 0.1
(1) 1.2 e (4) 5 abcd (7) 3.8 d (10) 4.1 cd (13) 4.5 bcd (16) 3.8 d (19) 4.4 bcd
180 B 112 CD 46 FGH 70 EF 64 EFG 58 EFGH 49 FGH
(2) 1.8 e (5) 5.8 abc (8) 4.4 bcd (11) 5.0 abcd (14) 4.2 bcd (17) 5.3 abcd (20) 4.5 bcd
190 B 116 C 64 EFG 49 FGH 71 EF 51 EFGH 51 EFGH
(3) 1.3 e (6) 5.9 abc (9) 4.6 bcd (12) 5.4 abcd (15) 5.3 abcd (18) 6.7 a (21) 6.0 ab
176 B 81 DE 37 GH 41 FGH 34 GH 30 H 29 H
Average Control
A 3:8 A 82:7 A 4:4 (22) shoot number per explant 1:0 e, fresh weight 223 A
A 84:5
A 5:0
A 61
a
Numbers in parenthesis indicate treatment number. Values are the means of 56 explants after 45 days of culture. Treatments with the same letter are not statistically different (LSD, P 0:05).
R. Ramirez-Malagon et al. / Scientia Horticulturae 89 (2001) 227±236
Table 1 Shoot number and fresh weight averages of axillary shoots of S. ¯oribundum cv. Petite cultured on MS medium with three concentrations of BA in combination with different levels of NAA, IAA and 2,4-Da
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increase in the number of shoots was recorded when the level of BA was increased from 1 to 5 mg l 1, reaching the maximum average yield (6.7 shoots per explant) with 0.05 mg l 1 2,4-D and 5 mg l 1 BA (treatment 18, Table 1). Shoot fresh weight values were higher when the supplied auxin was NAA (0.05 mg l 1) combined with different BA levels. When Kin was added to each treatment, differences in responses were found to occur and the highest value for shoot number average was achieved with the treatment containing 1.0 mg l 1 IAA and 2 mg l 1 BA 1:0 mg l 1 Kin (11.6 shoots per explant) (Table 2), but this treatment yielded shoots with a fresh weight average of 39 mg (Table 2), while treatments with 0.005 mg l 1 2,4-D combined with BA and Kin yielded higher fresh weight averages compared to those obtained when Kin was not present in the culture medium (Table 1). The highest shoot fresh weight values were achieved when the explants were cultured on a medium lacking growth regulators (control) or supplemented with BA alone since only the original shoot or just few additional shoots were present after 45 days of culture. These shoots grew more than those inoculated on the treatments that induced a higher number of shoots. The average fresh weight of shoots is a factor to be considered for better growth and development of plants adapted to soil after in vitro propagation. According to our experience, this parameter is a function of both the length and the diameter of plants and could be used as a measure of vigor to ensure a better growth of micropropagated plants when they are transferred to soil under greenhouse conditions. The treatment that induced the highest multiplication rates (treatment 11, Table 2: 1 mg l 1 IAA 2 mg l 1 BA 1 mg l 1 Kin) was not the most adequate for commercial propagation of S. ¯oribundum. This treatment produced an average yield of 11.6 shoots per explant and a fresh weight average of 39 mg per shoot, but the plants were smaller in height and foliar diameter than those from other treatments (Table 3) after 4 months of growth in the greenhouse. Plants from treatment 11, grew slowly during the ®rst stages in the greenhouse due to their lower height (10 mm). In general, plants with an initial fresh weight ranging from 39 to 66 mg after the in vitro culture, produced the smallest plants after 4 months of growth in the greenhouse (Table 3). The best treatment for producing vigorous plants was the treatment 5 (0:05 mg l 1 NAA 2 mg l 1 BA) in which an average of 5.8 shoots per explant with a mean fresh weight of 116 mg per shoot and a length of at least 20 mm were achieved. The shoots produced in this treatment grew vigorously when the plants were acclimatized under greenhouse conditions (Fig. 1). When Kin was added to the culture medium, different responses were observed according to the auxins used. The fresh weight average decreased when NAA was present and an increase was observed when Kin was added to treatments with 0.005 mg l 1 2,4-D. Culture medium containing 0:05 mg l 1 NAA 1 mg l 1 BA 1 mg l 1 Kin increased the shoot number (7.3 shoots per explant) (Table 2). An inverse relationship between the
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Auxins (mg l 1)
1 mg l
1
BA 1 mg l
1
Kin
2 mg l
1
BA 1 mg l
1
Kin
5 mg l
1
BA 1 mg l
1
Kin
Shoot number per explant
Fresh weight (mg)
Shoot number per explant
Fresh weight (mg)
Shoot number per explant
Fresh weight (mg)
No auxin NAA, 0.05 IAA, 0.5 IAA, 1.0 2,4-D, 0.005 2,4-D, 0.05 2,4-D, 0.1
(1) 2.1 gh (4) 7.3 b (7) 3.6 defg (10) 4.4 cde (13) 2.4 fgh (16) 4.7 cd (19) 3.7 defg
186 A 69 CD 45 DE 50 DE 97 BC 24 E 55 DE
(2) 2.7 efgh (5) 4.2 cdef (8) 3.8 cdefg (11) 11.6 a (14) 3.5 defg (17) 5.0 cd (20) 5.7 bc
170 A 68 CD 42 DE 39 DE 107 B 48 DE 41 DE
(3) 1.9 gh (6) 5.0 cd (9) 3.8 cdefg (12) 4.2 cdef (15) 4.3 cdfg (18) 4.9 cd (21) 4.9 cd
177 A 69 CD 95 BC 66 CD 105 B 50 DE 68 CD
Average Control
A 4:0 A 75 A 5:2 (22) shoot number per explant 1:0 h, fresh weight 187 A
A 73:5
A 4:1
A 90
a
Numbers in parenthesis indicate treatment number. Values are the means of 56 explants after 45 days of culture. Treatments with the same letter are not statistically different (LSD, P 0:05).
R. Ramirez-Malagon et al. / Scientia Horticulturae 89 (2001) 227±236
Table 2 Shoot number and fresh weight averages of axillary shoots of S. ¯oribundum cv. Petite cultured on MS medium with three concentrations of BA 1 mg Kin in combination with different levels of NAA, IAA and 2,4-Da
Treatments 0:05 mg l 0:05 mg l 0:05 mg l 1 mg l 1 mg l 1 mg l
1
1
1
NAA 1 mg l NAA 2 mg l 1 NAA 5 mg l
BA BA 1 BA
1
1
1
1
1
1
IAA 1 mg l IAA 2 mg l 1 IAA 5 mg l
0:005 mg l 0:005 mg l 0:005 mg l a
1
BA 1 mg l BA 1 mg l 1 BA 1 mg l
Kin Kin 1 Kin
1
1
1
1
1
1
2;4-D 1 mg l 2;4-D 2 mg l 1 2;4-D 5 mg l
BA 1 mg l BA 1 mg l 1 BA 1 mg l
Kin Kin 1 Kin
AFW (mg)
Leaf number/plant
Lateral shoots/plant
Plant diameter (cm)
Plant height (cm)
112 116 81
15.7 a 14.7 a 15.0 a
5.4 a 5.3 a 5.0 a
14.3 a 14.2 a 12.8 ab
14.1 ab 13.0 abc 12.1 bc
50 39 66
14.3 a 13.4 a 13.9 a
4.8 a 4.7 a 4.8 a
12.3 ab 11.8 b 12.4 ab
11.7 c 11.9 c 11.4 c
97 107 105
15.4 a 14.3 a 14.2 a
5.7 a 5.2 a 5.9 a
13.4 ab 13.0 ab 13.8 ab
12.2 bc 12.1 bc 12.0 c
Mean separation was run by column. Each value is the average of 25 plants. Data with the same letter are not statistically different (LSD, P 0:05). AFW: average fresh weight after in vitro culture and before starting greenhouse growing.
R. Ramirez-Malagon et al. / Scientia Horticulturae 89 (2001) 227±236
Table 3 Leaf number, lateral shoot number, height and plant diameter of S. ¯oribundum plants 4 months after transfer to soil from in vitro culturea
233
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R. Ramirez-Malagon et al. / Scientia Horticulturae 89 (2001) 227±236
Fig. 1. Spathiphyllum plants cultured in vitro with 0:05 mg l 1 NAA 2 mg l 1 BA: (a) two clumps of Spathiphyllum shoots after 45 days of in vitro culture; (b) plants after 30 days on MS medium lacking growth regulators and ready to be transferred to soil; (c) in vitro regenerated Spathiphyllum plant growing in soil.
number of regenerated shoots per explant and the fresh weight of the regenerated shoots was observed. A correlation analysis of values recorded 4 months after the acclimatization con®rmed this assessment (correlation coef®cient r 0:654). The size of in vitro plants is re¯ected in differences in vigor, stated as width and height of the developing plants. Table 3 shows that shoots with higher fresh weight values and with moderate multiplication rates achieved a better
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development than those having higher multiplication rates of shoots, but exhibiting lower fresh weight values. In the present work, we observed that explants from NAA treatments with BA as the sole cytokinin (Tables 1 and 3), exhibited the best responses for height and plant diameter after 4 months in the greenhouse. Production of 7±11 shoots per explant is in the range of multiplication of many other plants cultured in vitro. There are species that respond with a higher multiplication rate (Ahroni et al., 1997), but some others multiply at lower rates than Spathiphyllum. Our results are in agreement with those reports published on the in vitro multiplication of Spathiphyllum. Fonnesbech and Fonnesbech (1979) reported that in vitro propagation of Spathiphyllum species (Clevelandii), using 2 mg l 1 PBA, produced 9.1 shoots per explant. This result is similar to the maximum average obtained in treatment 11 (Table 2). Watad et al. (1997) culturing in vitro plants of cultivar Petite obtained an average of 10.1 shoots per explant on a culture medium supplemented with 2 mg l 1 Kin and gelled with agar, and up to 14.4 shoots per explant with ¯oating liquid medium raft membranes. Some of the differences in shoot multiplication could be explained on the basis of differences in the Spathiphyllum cultivars used and also due to differences in growth conditions. 4. Conclusion In summary, the in vitro propagation of Spathiphyllum was promoted with different combinations and different concentrations of auxins and cytokinins with yields higher than 10 shoots per explant. However, not always the treatment with the highest shoot multiplication rate is the ideal for the propagation of the species. Sometimes, as is the case of the present work, a positive correlation is observed between the initial weight average of shoots from in vitro cultures and the growth of plants (measured as plant diameter and plant height) in the greenhouse. We have shown that shoot size is an important factor for further development in the greenhouse of the regenerated plants, and it is suggested that a better decision for growing Spathiphyllum plants from in vitro cultures could be the employment of treatments where a balance of high shoot regeneration combined with an adequate size (fresh weight) of regenerated shoots is achieved. Acknowledgements We would like to thank Dr. John Snape for his valuable comments during the preparation of the manuscript. This work was supported by a grant from Universidad de Guanajuato, Mexico.
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