In vitro plantlet regeneration from cotyledons of the tree-legume Leucaenaleucocephala

Havila Saafi and Dulal Borthakur*Department of Molecular Biosciences and Bioengineering, University of Hawaii, 1955 East-West Road,Honolulu, Hawaii 96822, USA; *Author for correspondence (e-mail: dulal@hawaii.edu; phone: (808)956-6600; fax: (808) 956-3542)

Received 18 December 2001; accepted in revised form 14 May 2002

Key words: Callus, Leucaena leucocephala, Organogenesis, Tissue culture

Abstract

Two plant regeneration methods applicable to Leucaena leucocephala were developed. In the first method, in-volving organogenesis via callus formation, cotyledon, hypocotyl and root segments were initiated on MS me-dium containing different concentrations of N6-benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), andnaphthaleneacetic acid (NAA). Both compact (type I) and friable (type II) calli were obtained from the cotyledonand hypocotyl explants treated with different concentrations of the growth regulators. Shoots were generated onlyfrom the friable calli formed from the cotyledon explants. The calli formed from the hypocotyl explants did notgenerate shoots and the root explants died without forming callus. Cotyledon explants from 3–4 day old seed-lings showed maximum callus induction compared to those from older seedlings. In a second method involvingdirect organogenesis, excised cotyledons were cultured on 1/2 MS medium containing 10–35 mg l−1 N6-benzy-ladenine (BA) for 7–14 days. Transfer of the cotyledons to regeneration medium containing low BA resulted incallus formation and subsequent shoot regeneration from the base of the excised cotyledon explants, with up to100% frequency. Regenerated shoots rooted best on a basal medium containing no growth regulators.

Introduction

Leucaena leucocephala is a fast-growing leguminoustree that grows in a wide range of soil types in manytropical and subtropical countries. It is considered tobe an important tree for agroforestry because it is re-sistant to diseases, can be cultivated easily and itsleaves can be used as fodder for farm animals. Fac-tors that limit the use of leucaena include poor toler-ance to acid or waterlogged soils, poor adaptation tocool temperatures and frost, and susceptibility to thepsyllid insect (Shelton and Brewbaker 1994). Theleaves of L. leucocephala are rich in protein but theyalso contain a toxin, known as mimosine (Jones1979). The mimosine concentration in the leaf can beas high as 8–10% on a dry weight basis (Soedarjo andBorthakur 1996). Efforts to develop L. leucocephalacultivars containing reduced amount of mimosine bytraditional breeding and selection methods have been

not successful since there are no mimosine-free Leu-caena species in nature. To develop new improvedcultivars of Leucaena with additional desirable char-acteristics such as mimosine-free shoots, better nutri-tional quality, and higher yield, it is necessary to de-velop genetic transformation methods for this treelegume. Genetic transformation using Agrobacteriumtumefaciens or the biolistic method requires an effi-cient tissue culture regeneration protocol. Tissue cul-ture work of this tree has been initiated in a few lab-oratories with limited success. Nodal explants ofgrowing L. leucocephala tree were used to generateshoots and roots without callus formation in tissueculture media (Peaseley and Collins 1980). Similarly,Glovak and Greatbatch (1981) generated whole plantsof L. leucocephala by transplanting small shoots fromyoung seedlings on tissue culture medium. Goyal etal. (1995) obtained shoot regeneration in tissue cul-ture from lateral bud explants. The goal of these pre-

279Plant Growth Regulation 38: 279–285, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

vious tissue culture studies was to develop an alter-native method of propagation for this tree via axillaryshoot proliferation rather than using tissue culture asan intermediate step for genetic transformation.Therefore, callus formation was not indicated in theseprevious reports. To achieve successful regenerationof adventitious shoots through callus formation, it isnecessary to search for tissues within the plant thatstill retain some totipotency. Evidence is accumulat-ing that only some tissues in certain states are acces-sible to either transformation or regeneration (Nagl etal. 1997). L. leucocephala is a hardy tree legume and,therefore, it may not be easy to regenerate shootsfrom any part of a growing seedling. The goal of thisresearch is to develop an efficient plant regenerationmethod through formation of adventitious shoots intissue culture, which is a prerequisite for developinga genetic transformation protocol for L. leucocephala.

Materials and methods

Plant materials

Seeds of L. leucocephala (cultivar K636) were ob-tained from Hawaii Agriculture Research Center(Aiea, Hawaii) and stored at 4 °C. The seeds werescarified by immersion in concentrated H2SO4 for 6min, followed by 5 washes in sterilized water. Tocompletely eliminate any contaminating bacteria, theseeds were then treated with 3.7% (w/v) sodium hy-pochlorite containing 60 �l of Tween-20 per 100 mlof the solution and agitated on a gyratory shaker at100 rpm for 20 min. The seeds were rinsed 5 timesand soaked in sterile water to imbibe overnight. Theimbibed seeds were selected and placed on 25 ml ofsolidified agar medium containing MS salts in petridishes until the radicles emerged from the seed coats(3 to 5 days). Once radicles emerged, the germinat-ing seeds were transferred to pre-germination me-dium in Magenta boxes (Magenta corp.). The pre-germination medium contained 1/2 MS salt and 1%agar but no growth regulators. The seedlings weregrown for 1 to 7 days after which the explants (roots,hypocotyls and cotyledons) were cultured.

Culture establishment and maintenance

Experiment 1: Shoot initiation through callusformationExplants used in shoot regeneration via callus forma-tion were cotyledons, 1-cm-long hypocotyl segments,and 1-cm-long root segments excised from 1- to7-day-old seedlings grown on pre-germination me-dium. For excision treatment studies, only cotyledonsections that were cut either longitudinally or trans-versely were used, while for age effect studies, wholecotyledons and 1-cm sections of roots and hypocotylswere used. For rooting tests, regenerated shoots over2 cm were excised and transferred to root inductionmedium in Magenta boxes sealed with parafilm.

The medium contained full strength MS (Murash-ige and Skoog 1962) basal salts in all stages. All me-dia were supplemented with 100 mg l−1 myoinositol,0.1 mg l−1 thiamine-HCl, 0.5 mg l−1 nicotinic acid,0.5 mg l−1 pyridoxine-HCl, 2.0 mg l−1 glycine, 30g l−1 sucrose and 10 g l−1 agar (Agar-agar/Gum agar,Sigma). The media were adjusted to pH 5.7–5.8 andautoclaved for 15 min at 121 °C and 1 × 105 Pa (1.1kg cm−2). All plant growth regulators were added be-fore autoclaving.

The callus induction medium was supplementedwith BA, 2,4-D and NAA in nine different combina-tions for the cotyledon explants and ten combinationsfor the hypocotyl and root explants (Table 1). Theexplants were maintained in the dark at 22 ± 1 °C.For shoot induction, the media were supplementedwith different concentrations of BA (Table 2). Thevarious concentrations of growth hormones in the cal-lus and shoot media were based on concentrationsused for other woody plants in the literature. After 2months, the explants were transferred to shoot induc-tion medium supplemented with eight different con-centrations of BA (Table 2), some in combinationwith NAA. The rooting medium contained NAA (1,2, 3 and 4 mg l−1), some with no growth regulators.For both shoot and root induction, the cultures weremaintained at 26 °C and 16 h photoperiods of coolwhite fluorescent light at 40 �mol m−2 s−1.

Experiment 2: Shoot initiation without callusformationAfter pre-germination, seed coats were removed andthe cotyledons were split-open to expose the embry-onic axes. The embryonic axes were surgically ex-cised and both embryonic axes and cotyledons werecultured on pre-induction medium with the adaxial

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side (internal side in intact seed) in contact with themedium. The pre-induction medium contained 1/2MS basal salts with different concentrations of BA.The explants were induced for adventitious shoot for-mation on pre-induction medium containing BA at10, 15, 20, 25 or 35 mg l−1 for 7 to 14 days beforetransferring to shoot induction medium. The shoot in-duction medium contained 1/2 MS basal salts withlow concentrations of growth regulators (see Table 3).The growing shoots were rooted on rooting mediumcontaining NAA (1, 2, 3 or 4 mg l−1) or no growthregulators. All cultures were maintained at 26 °C and16 h photoperiods of cool white fluorescent light at40 �mol m−2 s−1 for both shoot and root induction.

Statistical analysis

A one-way analysis of variances (ANOVA) was usedto test if there were significant differences betweenmeans obtained with different treatments at the 5%level of significance (p = 0.05). A chi-square test of

homogeneity was performed to compare rooting per-centages among different treatments.

Table 1. Effects of growth regulators on callus formation (experi-ment 1).

BA (mg l−1) 2,4-D (mg l−1) NAA (mg l−1) Mean % of

explants form-

ing callusa

Cotyledon

0.5 1.0 0 99.4 ± 1.51

.05 0.5 0 99.4 ± 1.51

1.0 0.1 0.1 100 ± 00

1.0 0.05 0 98.9 ± 3.02

0.1 0.5 0.1 99.4 ± 1.51

0.1 0.1 0 96.1 ± 4.10

0 2.0 0.1 97.1 ± 4.18

0.5 0.1 0 98.63 ± 3.78

4.5 0 1.0 99.4 ± 1.51

Hypocotyl

0.05 0.5 0.1 97.9 ± 4.18

0.05 0.5 0 98.0 ± 2.77

0.1 0.5 0.1 99.0 ± 2.65

0.5 0.05 0 97.8 ± 3.15

0.05 0.1 0.1 98.0 ± 2.77

0.5 0.5 0.1 98.4 ± 2.82

0.01 1.0 0 97.7 ± 2.98

2.0 0 0.1 98.2 ± 2.21

0.5 0.1 0 98.3 ± 2.98

4.5 0 1.0 99.0 ± 1.73

aValues represent means of 7 replicates (12 explants/plate) ± stan-dard deviations. Analysis of variances indicates that the mean per-centages are not significantly different at the 5% significance level.

Table 2. Effects of plant hormone on shoot formation (experiment1).

Shoot induction

hormones

Shoot frequency

(%)a

Shoot height (cm)b

1 mg l−1 BA 12.1 ± 2.85 2.4 ± 0.89

1.5 mg l−1 BA 5.6 ± 1.13 2.6 ± 0.55

2.0 mg l−1 BA 6.0 ± 0.82 3.2 ± 1.48

2.5 mg l−1 BA 2.8 ± 0.69 2.4 ± 0.55

5 mg l−1 BA 0 0

10 mg l−1 BA +

0.01 mg l−1 NAA

1.7 ± 0.76 2.8 ± 0.84

10 mg l−1 BA 2.6 ± 0.53 2.4 ± 0.89

0.5 mg l−1 BA + 1

mg l−1 NAA

0 0

aValues represent means of 7 replicates (5 explants/plate) ± stan-dard deviations and values are from experiment 1. bValues repre-sent means of 7 replicates (4 explants/magenta box) ± standarddeviations and values were measured a month after transferring toshoot induction media. The standard errors of difference betweentwo means for shoot frequency (%) and shoot height are 3.76 and0.17, respectively.

Table 3. Effect of growth regulators on subsequent shoot regener-ation from cotyledon explants of L. leucocephala (experiment 2).

BA (mg l−1) NAA (mg l−1) 2,4-D (mg l−1) Mean % of

explants form-

ing shootsa

0.5 0 0 80 ± 28.28

1 0 0 80 ± 28.28

1 0 0 86.17 ± 15.71

2 0 0 90 ± 14.14

5 0 0 77.8 ± 5.05

7 0.1 0 100 ± 0

10 0.01 0 100 ± 0

0.5 0 0.1 90 ± 14.14

2.5 0.93 0 50 ± 17.68

aValues represent means of 10 replicates (5 explants/plate) ± stan-dard deviations and values are from experiment 2. The measure-ments were taken a month after transferring to shoot inductionmedia. Analysis of variances indicates that the mean percentagesare significantly different at the 5% significance level. The stan-dard error of difference between two mean percentages of shootforming explants is 5.57.

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Results and discussion

Experiment 1The objective of the first experiment was to identifythe most effective combination of auxins (2,4-D andNAA) and cytokinin (BA) for the induction of shootsvia callus formation. Hypocotyl, root, and cotyledonexplants were cultured in the dark on callus inductionmedia containing different combinations of 2,4-D,NAA and BA. In an initial experiment, leaves of 2–3week-old seedlings were also cultured for callus in-duction. The leaves of the young seedlings were toothin and they withered without forming callus. Within2 weeks creamy or whitish and solid calli were ob-served at the wounded sites of the cotyledon and hy-pocotyl explants (Figures 1a and 1d). No calli wereobserved on the root explants. Hypocotyl and cotyle-don segments from 3- to 4-day-old seedlings culturedon callus induction medium formed callus with 100%efficiency. There were no significant differences incallus growth among explants excised from the mid-dle or either end of cotyledons from germinatedseeds. The shorter (1 cm) hypocotyl segments pro-duced more callus growth as compared to the longexplants (2–3 cm). Some of the explants producedroot-like structures during the callus induction stage,which indicates the high concentration of auxin. Ac-tively growing calli were subcultured twice (Figures1b and 1e), at 30-day intervals in the MS medium.Although there was no significant difference betweenthe combinations of 2,4-D, NAA and BA in the pro-duction of callus, a combination of 2,4-D 0.5 mg l−1

+ BA 0.05 mg l−1 + NAA 0.1 mg l−1 produced themaximum number of calli on the cotyledon explants.One of the major problems associated with the mi-cropropagation of L. leucocephala has been thebrowning of the explants. Such browning of explantshas also been observed in the explants of Brassicaspp. such as cabbage (Burnett et al. 1994; Jin et al.2000). These plants contain large quantities of phe-nolic compounds, which are oxidized by polyphenoloxidaxes and peroxidases and then produce thebrowning appearance of the explants (Sharma andRamamurthy 2000). These oxidized products arehighly reactive and lead to cell death. Some experi-ments were conducted to overcome this problem byadding AgN03 as an ethylene inhibitor (Burnett et al.1994). The browning observed in our initial experi-ments was greatly reduced when high concentrationsof BA (10–35 mg l−1) were applied for a shorter du-ration (7–14 days) in the preinitiation step (see exper-

iment 2). When AgNO3 (5–20 mg l−1) was also addedto the medium along with high concentrations of BA(10–35 mg l−1), no further reduction in the extent ofbrowning was observed.

The subcultured calli in the callus induction me-dium were compact and creamy or whitish in color.After 2 months of subculturing in the callus induc-

Figure 1. (a to f) Calli formed from hypocotyl explants on callusinduction medium (CIM) after 30 days (a), 60 days (b) and 90 days(c). Similarly, calli formed from cotyledon explants on CIM after30 days (d), 60 days (e) and 90 days (e) are shown. Shoots couldbe regenerated from friable calli of cotyledon explants after 90 days(g and h) in experiment 1. Shoots regenerated from the base of theexcised cotyledon explants that were preinitiated on high BA inexperiment 2 are shown (i and j).

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tion medium, the calli were transferred to a shoot in-duction medium containing different concentrationsof BA in combination with NAA (Table 2). Most calliin the shoot induction medium developed friable out-growths (Figures 1c and 1f), although some contin-ued to grow compactly. These friable calli were char-acterized by soft and yellowish to reddish-brownappearance and resembled the type II calli reportedin maize (Armstrong and Green 1985). Shoots wereregenerated only from the friable calli (Figures 1g and1h). The response to shoot regeneration was observedwithin 30 days after transferring to the shoot induc-tion medium. Friable calli initiated from compact cal-lus were also reported from Augustine grass (Kuo andSmith 1993), leek (Buiteveld et al. 1994), sorghum(Gendy et al. 1996) and Alstroemeria species (Lin etal. 2000). The highest percentage of shoots (12.1%)was produced on 1 mg l−1 BA. No shoot formationoccurred on 0.5 mg l−1 BA + 1 mg l−1 NAA and 5mg l−1 BA.

Experiment 2On the pre-germination medium, most seeds swelledand radicals emerged. This was the criterion by whichuniform viable seeds were selected for explanting.Although the excised embryonic axes and the cotyle-dons were both transferred to pre-induction media,only the cotyledons formed callus. The embryonicaxes elongated into shoot-like structures and died af-ter a month. Transfer of cotyledons from initiationmedium (high BA) to regeneration medium (low BA)stimulated callus formation and subsequent shoot re-generation. Callus formed within 14 days after trans-fer, at rates faster than in experiment 1. These calliresembled the type I callus described for maize (Arm-strong and Green 1985). Shoot regeneration com-menced within 2 weeks after transfer to the regener-ation medium and continued for up to 4 weeks. Sincethe embryonic axes were excised, shoots emergedonly from the proximal end (the embryonic axes at-tachment area) of the cotyledons (Figures 1i and 1j).To test whether shoots could regenerate from otherparts of the cotylelon, the distal parts of the cotyle-dons were also wounded. Callus formed in these partsbut shoots did not regenerate from these sites. Thusfrom these experiments it appears that shoots emergefrom the base of the cotyledons and not from the dis-tal parts. Close examination showed that these shootsregenerated from the base of the cotyledons and notfrom the calli. However, it is likely that the callusformed at the base of the cotyledon may have a role

in the shoot regeneration by providing a protectivelayer to the emerging shoots. Since these shootsformed directly from the cotyledons, the possibility ofsomaclonal variation arising from different calli isgreatly reduced. The highest percentage of shoot for-mation (100%) was found on medium containing 10mg l−1 of BA and multiple shoots appeared on 5mg l−1 BA (Table 3) in an average of 3–4 shoots perexplant. The differences in the mean percentages ofshoot formation among different treatments were sig-nificant at the 5% significance level.

When the shoots were induced to form roots inMagenta boxes containing rooting medium, one outof two shoots formed roots in 65.7% boxes, while in11.4% boxes, roots developed from both shoots. Chi-square test of homogeneity showed that the rootingpercentages among different boxes were not ran-domly distributed. However, there were no significantdifferences among treatments. Regenerated shootsrooted most readily on basal medium containingNAA (Table 4) but these roots were short, black incolor and died after a month. On the other hand, inmedium containing no growth regulators, the rootsformed were longer and healthier, and they continuedto grow even after a month. These roots were lightbrown in color with black root caps. The concentra-tions of auxins and cytokinins used for the tissue cul-ture of L. leucocephala also fall in the range of con-centrations for the tissue culture of other tree andlegume species (Table 5).

In conclusion, two protocols for shoot regenerationof L. leucocephala from cotyledon explants have beendeveloped. The second protocol, which involves di-rect organogenesis from the base of the cotyledon,resulted in 100% shoot regeneration, compared to a

Table 4. Effects of plant hormones on root formation.

Plant hormones Root frequency (%)a Number of roots per

shootb

1 mg l−1 NAA 49.4 ± 11.6 3.0 ± 2.0

2 mg l−1 NAA 34.7 ± 4.15 2.0 ± 0.82

3 mg l−1 NAA 49.1 ± 8.88 1.9 ± 0.69

4 mg l−1 NAA 48.4 ± 13.15 3.1 ± 0.69

No plant hormone 34.1 ± 4.56 2.7 ± 0.49

aValues represent means of 7 replicates (2 shoots/magenta box) ±standard deviations. bvalues represents means of 7 replicates ±standard deviations and measurements were taken after 6 weeks oftransferring to the root induction media. Analysis of variances in-dicates that the mean percentages of root formation and the num-ber of roots per shoot in different treatments are not significantlydifferent at the 5% significance level.

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maximum of 12% shoot regeneration obtained usingthe first method that involved indirect organogenesisvia callus formation. In the first protocol, shoots wereregenerated from friable calli induced on relativelylower concentrations of plant growth regulators,while in the second method, cotyledons were pre-ini-tiated on high BA (10 to 35 mg l−1) before transfer toshoot induction medium containing low BA (1–2mg l−1). Shoot regeneration required 13 weeks usingthe first protocol and 4–6 weeks with the second pro-tocol. To our knowledge, this is the first report ofsuccessful tissue culture plant regeneration for thisspecies. The establishment of an efficient tissue cul-ture regeneration system of L. leucocephala will beuseful for development of future genetic transforma-tion systems.

Acknowledgements

The project was funded by a USDA McIntire Grant.This is paper number 4607 of the College of Agricul-ture and Human Resources, University of Hawaii,Honolulu.

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