Plant Cell, Tissue and Organ Culture 69: 141–146, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.
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Plant regeneration from leaf-derived callus in Citrus grandis (pummelo): Effects of auxins in callus induction medium Huang Tao, Peng Shaolin, Dong Gaofeng, Zhang Lanying & Li Gengguang∗
South China Institute of Botany, the Chinese Academy of Sciences, Guangzhou, 510650, China (∗ requests for offprints: E-mail:
[email protected]) Received 24 January 2001; accepted in revised form 6 October 2001
Key words: auxins, benzyladenine, callus induction, leaf explant, root formation
Abstract Calli were induced from leaf explants of seedling in Citrus grandis (L.) Osbeck (pummelo) on MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4- D), 1-naphthaleneacetic acid (NAA), 2,4,5trichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic, 4-chlorophenoxyacetic acid, 4-methoxy-3,6dichlorobenzoic acid or 4-amino-3,5,6-trichloropicolinic acid. 2,4-D was most effective. Only green, compact calli induced by 2,4-D at low concentrations (0.9 and 4.5 µM) were capable of shoot formation and regenerated more than 13 shoots per callus on MS medium containing at least 6.66 µM benzyladenine (BA). Calli induced by other auxins did not regenerate shoots on MS medium containing BA at all concentrations studied. A multiplication rate of 5–7 shoots was achieved from shoot tip culture on MS medium with 0.89 µM BA. Roots developed when regenerated shoots were cultured on MS medium with 9.84 µM indole-3-butyric acid and 5.37 µM NAA. No response was obtained on mature leaves cultured on MS medium supplemented with the above mentioned auxins at various concentrations. Abbreviations: BA – benzyladenine; 4-CPA – 4-chlorophenoxyacetic acid; 2,4-D – 2,4-dichlorophenoxyacetic acid; dicamba – 4-methoxy-3,6-dichlorobenzoic acid; MCPA – 2-methyl-4-chlorophenoxyacetic; NAA – 1naphthaleneacetic acid; picloram – 4-amino-3,5,6-trichloropicolinic acid; 2,4,5-T – 2,4,5-trichlorophenoxyacetic acid
Introduction Citrus is an important fruit crop worldwide. The development of efficient tissue culture protocols is necessary for conservation and genetic improvement of citrus. Citrus grandis Osbeck is the largest citrus fruit and is widely grown in south Asia. There are only a few reports about tissue culture of this species compared to other citrus species. Rangan et al. (1968) reported tissue culture of nucellus in C. grandis. Chaturvedi and Mitra (1975) have studied shoot organogenesis from callus derived from the stem. Shoot development from leaf explants is valuable for research and applications of biotechnological methods. However, development of whole plantlets
from leaf explants taken from citrus trees is limited (Yelenosky, 1987). In C. grandis, few studies have been carried out on shoot development from leafderived callus (Chaturvedi and Mitra, 1974; Goh et al., 1995). A more efficient regeneration protocol should be set up when gene transfer technologies are to be used to improve this species. In this research, we have been searching for an efficient procedure for plant regeneration from leaf explants in C. grandis, in order to provide a basis for genetic improvement. We have studied effects of different auxins on callus initiation from leaf explants with following shoot development from those calli on MS medium supplemented with BA at various concentrations.
142 Materials and methods Plant materials and chemicals Seeds derived from Citrus grandis (L.) Osbeck cv. Shatian-Yu were surface disinfected in 70% ethanol for 2 min and 0.1% (w/v) HgCl2 for 10 min, then washed 3 times with sterile distilled water. Seeds from which the coat had been removed were cultured on half strength MS medium in culture tubes at 27 ◦ C with 16-h photoperiod under fluorescent light at 20 µmol m−2 s−1 . After 3 weeks culture, the expanded first true leaves of seedlings were collected for callus induction. Completely expanded mature leaves were collected from newly sprouting shoots in 10-year-old trees. Mature leaves were sterilized in 70% ethanol for 2 min and 0.2% (w/v) HgCl2 for 10 min, then washed with sterile distilled water three times. The leaves from seedlings or mature trees were then cut into pieces (4– 5 mm wide) transversely across the leaf lamina. Leaf segments were placed on the medium with the adaxial surface in contact with the medium. The auxins used in this research were obtained from Sigma. Callus induction The callus induction medium was the Murashige and Skoog medium (1962) supplemented with 2,4-D, NAA, 2,4,5-T, 4-CPA, MCPA, dicamba or picloram. The pH of the medium was adjusted to 5.8 with 1 N KOH before autoclaving at 121 ◦ C for 20 min. The auxins were added at various concentrations (Table 1) before autoclaving. The control was MS medium without auxin. The effect of 2,4-D in combination with NAA on callus induction was also studied as shown in Table 2. Four leaves were cut into about 20 segments and planted in a triangular flask. There were in total 5 triangular flasks for every treatment. Explants were transferred to the same, fresh medium every 20 days. The cultures were incubated at 25±2 ◦ C with 16-h photoperiod under fluorescent light at 20 µmol m−2 s−1 . Shoot differentiation After 50 days culture, calli excised from explants at suitable size (about 5×5×5 mm) were transfered to MS medium supplemented with BA in the range of 0.89, 2.22, 4.44, 6.66, 8.88 and 13.32 µM. Calli were cultured under the same environmental conditions as during callus induction and transfered to the fresh medium every 20 days. After shoots had developed from
calli, shoot tips were excised and cultured on MS medium supplemented with BA at 0.89, 1.78 or 2.66 µM. All experiments were repeated at least three times, and SEs were calculated.
Results and discussion Callus could be initiated from seedling leaf explants treated with all auxins examined. However, no callus was initiated from either seedling leaf explants on MS basal medium without auxins or mature leaf explants treated with auxins at all concentrations tested. Callusing frequency of seedling leaf explants depended on type and concentration of auxin. Table 1 shows the callusing frequency and callus growth after 50 days. 2,4-D induced high callusing frequency at all concentrations studied. At low concentrations (0.9 and 4.5 µM), 2,4-D induced two types of callus, viz. yellow friable and green compact (Figure 1). With more than 4.5 µM 2,4-D, only yellow friable callus was formed. Only the green compact callus was able to regenerate adventitious shoots after being transfered to differentiation medium supplemented with BA (Figure 2). In citrus, 2,4-D has been mostly used for callus induction from albedo tissue of fruits (Murashige and Tucker, 1969; Einset, 1978; Amo-Marco and Picazo, 1994), leaf (Goh et al., 1995), immature seed (Ling and Iwamasa, 1997), hypocotyl (Jumin and Nito, 1995, 1996) and undeveloped ovules (Gmitter and Moore, 1986). In some instances, NAA was also used for callus induction (Duran-vila et al., 1989; Nito and Iwamasa, 1990). Auxins other than 2,4- D and NAA have not received extensive attention in studies on citrus. Our results show that 2,4-D was most effective on callus induction. Although other auxins induced high callusing frequency at some concentrations, these calli cannot regenerate shoots on differentiation medium containing BA. NAA induced green compact callus as well as roots at the same time. These rooty calli did not regenerate shoots on MS medium containing BA. This is in agreement with an earlier report on switchgrasss that highly rooty calli cannot regenerate shoots (Denchev and Conger, 1995). At low concentration (1.1 µM), NAA only resulted in root formation from the midrib. With increased NAA concentration, roots were also induced from cut edges of leaf explants and the rooting rate as well as root number per explant increased. The roots, however, grew slowly at increased
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Figures 1–5. Figure 1. Green compact callus induced by 2,4-D from leaf explant of seedling in Citrus grandis (L.) Osbeck; Bar = 0.5 cm. Figure 2. Adventitious buds regeneration from leaf-derived callus induced by 2,4-D on MS medium containing BA; Bar = 0.5 cm. Figure 3. Adventitious shoots regeneration from shoot tip culture on MS medium containing 0.89 µM BA; Bar = 0.5 cm. Figure 4. Roots induction from shoot on MS medium containing 9.84 µM IBA and 5.37 µM NAA; Bar = 1.0 cm. Figure 5. Young plantlet of Citrus grandis (L.) osbeck transferred to soil. Bar = 1.5 cm.
144 Table 1. Effects of different auxins at various concentrations on induction and growth of callus and adventitious root formation from leaf explants from seedlings of C. grandis (L.) Osbeck Treatment
Concentration (µM)
Explants with callus (%)
Callus fresh weight (mg)
0.0
7.2±1.1
0.9
84.5±2.3
35.2±6.1
0.0
4.5
93.5±1.8
43.4±3.2
0.0
9.1 22.6 32.2
96.5±2.9 95.9±5.2 92.7±3.5
55.8±3.7 47.3±6.4 52.6±4.8
0.0 0.0 0.0
Compact, green Friable, yellow Compact, green Friable, yellow Friable, yellow Friable, yellow Friable, yellow
1.1 5.4 10.7 26.8 42.9
0.0 41.7±1.3 66.7±1.6 70.4±3.6 54.8±2.2
7.6±1.3 12.2±2.6 13.9±3.8 26.7±4.1 9.8±2.4
0.0 0.0 0.0 0.0 0.0
Friable, yellow Friable, yellow Friable, yellow Friable, yellow
NAA
1.1 5.4 10.7 26.9 42.9
7.4±0.8 38.2±1.1 96.7±4.1 92.2±3.4 91.7±1.5
23.4±4.3 34.7±2.9 53.4±5.3 65.7±4.9 58.2±3.1
22.2±0.8 44.1±1.3 33.3±5.2 44.4±2.1 75.0±3.4
1.2±0.1 1.4±0.2 2.1±0.2 3.4±0.5 3.6±0.3
3.0±0.4 2.1±0.3 1.8±0.2 1.0±0.2 0.5±0.1
Compact, green Compact, green Compact, green Compact, green Compact green
2,4,5-T
0.8 3.9 7.8 14.5 31.2
39.1±1.4 61.5±2.8 34.4±1.1 42.9±2.3 34.4±1.9
22.6±1.4 17.3±1.8 16.5±2.2 19.8±3.3 12.4±3.6
21.7±3.2 26.9±1.4 9.4±0.6 0.0 0.0
0.8±0.2 1.0±0.1 1.2±0.2
1.9±0.3 1.6±0.2 0.7±0.2
Compact, light green Compact, light green Friable, yellow Friable, yellow Friable, yellow
MCPA
1.0 5.0 10.0 25.0 40.0
33.3±0.9 61.9±1.8 95.2±6.7 97.2±1.8 98.1±0.7
24.2±3.5 27.3±4.5 16.6±2.8 22.7±1.8 31.4±5.3
55.5±5.2 0.0 0.0 0.0 0.0
1.4±0.2
3.2±0.5
Friable, yellow Friable, yellow Friable, yellow Friable, yellow Friable, yellow
Dicamba
0.9 4.5 9.1 22.6 32.2
13.8±1.7 62.1±2.4 50.0±2.3 14.3±1.7 0.0
20.8±2.4 29.6±1.9 16.4±3.8 11.8±2.3 8.1±1.1
3.4±0.5 0.0 0.0 0.0 0.0
1.3±0.2
2.6±0.3
Friable, yellow Compact, yellow Friable, yellow Friable, yellow
Picloram
0.8 4.2 8.3 20.7 33.1
11.1±1.5 35.7±2.3 59.3±4.6 55.8±3.7 0.0
12.5±1.6 15.9±3.2 14.1±2.7 16.4±3.6 6.5±1.0
18.5±1.3 0.0 0.0 0.0 0.0
1.0±0.1
1.6±0.4
Friable, yellow Friable, yellow Friable, yellow Friable, yellow
Control 2,4-D
4-CPA
Adventitious root formation Rooting (%)
No. roots per explant
Callus appearance
Root length (cm)
a Values represent treatment means of three replicates ± SE, each treatment had 96 leaf explants. b Fresh weight (including original fresh weight of explant before culture) was calculated as the mean weight of 12 calli ± SE.
145 Table 2. Effect of 2,4-D in combination with NAA on callus formation and adventitious root formation from leaf explants 2,4-D µM
NAA µM
Callus formation (%)
Root formation (%)
0.0 2.26 4.52 9.04
5.37 5.37 5.37 5.37
72.7±1.3 95.3±2.4 92.8±3.6 55.6±2.1
54.6±3.3 44.4±4.1 0.0 0.0
0.0 2.26 4.52 9.04
10.74 10.74 10.74 10.74
61.8±2.3 96.4±2.8 92.1±4.2 53.4±4.6
66.7±2,9 45.5±5.3 0.0 0.0
Values represent treatment means of three replicates±SE.; Each treatment had about 96 leaf explants.
NAA concentration (Table 1). The effect of NAA in combination with 2,4-D on callus induction was studied by increasing 2,4-D concentration while NAA was kept constant, with the result that the rooting rate of leaf explants decreased (Table 2). It is obvious that 2,4-D inhibited root development induced by NAA. Other auxins including 2,4,5-T, MCPA, dicamba and picloram only induced rooting at low concentrations (Table 1). Gill et al. (1994, 1995) reported that NAA is better in inducing embryogenic callus, while 2,4-D only induces non-embryogenic and friable callus that cannot regenerate shoots in ‘kinnow’ mandarin and C. reticulata. Charturvedi and Mitra (1975) found that callus induced from stems of C. grandis could regenerate shoots on differentiation medium, while the morphogenetic pattern of callus tissue shifted from shootbud differentiation to embryogenesis during prolonged culture on medium containing 2,4-D and NAA. It was suggested that 2,4- D and NAA have different roles on callus induction for various citrus species as well as at different culture stages. Here we show that 2,4-D is better than NAA in inducing regenerative callus; while the root development from leaf explants induced by NAA was inhibited by 2,4-D. Goh et al. (1995) reported that the leaf explants of Citrus grandis Osbeck cultured on medium with 4.52 µM 2,4-D and 4.44 µM BA produce yellowish-green, compact callus. Fifty percent of calli regenerated and an average of 5 shoots per callus was obtained on medium containing 4.44 µM BA. A multiplication rate of 2.6 shoots was obtained by culturing shoot tips on MS medium with 2.2 µM BA. Our results showed that 2,4-D alone can induce green compact callus of
Table 3. Effects of BA at various concentrations on shoot differentiation from compact green callus induced by 2,4-D from leaf explants BA µM
Shoot regeneration (%)
Shoots/per callus
0.89 2.22 4.44 6.66 8.88 13.32
0.0 0.0 0.0 66.7±2.4 87.5±4.2 83.3±1.9
0 0 0 13.2±2.2 21.1±3.1 17.4±2.6
Values represent treatment means of four replicates ± SE; Each treatment had 24 calli.
which about 87.5% can differentiate. Per callus 21 shoots were obtained on medium containing 8.88 µM BA (Table 3). At least 6.66 µM BA was necessary for shoot regeneration. Excised shoot tips cultured on MS medium containing 0.89 µM BA multiplicated 5–7 adventitious shoots per tip (Figure 3). Higher BA concentrations (1.78 and 2.67 µM) significantly decreased the number of adventitious shoots per tip (data not shown). Regenerated shoots were excised and cultured on MS medium supplemented with 9.84 µM IBA and 5.37 µM NAA for rooting. After 3 weeks, 90% of the shoots developed 2–3 roots per plantlet (Figure 4). These plantlets were transferred to soil with 93.5% survival rate. All of them grew normally and showed no morphological abnormalities (Figure 5).
Acknowledgements This research was surported by Guangdong Science Foundation grant (NO. 980478), Guangdong One Hundred Creative Engineering grant (NO. 99B05902X) and Chinese National Science Foundation grant (NO. 39870539).
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