Plant Cell Reports

Plant Cell Reports (1991) 9:586 589

9 Springer-Verlag1991

Nodule culture and regeneration of Eucalyptus grandis hybrids E s a m Warrag 1,2, M . S . Lesney 1, and D.J. Rockwood 1 1 Department of Forestry, School of Forest Resources and Conservation, University of Florida, Gainesville, Florida 32611, USA 2 Present address: Faculty of Agriculture, Box 32, University of Khartoum, Khartoum North, Sudan

Received July 2, 1990/Revised version received August 15, 1990 - Communicated by [. K. Vasil

ABSTRACT

Ca llus

of three superior Eucalyptus hybrids was induced from immature scences, floral parts, shoot tips, zygotic embryos, and hypocotyl explants on various auxin (2,4-D or NAA) and cytokinin (kinetin) supplemented media. Hypocotyl callus initiated on 4 mg/l NAA and i mg/l kinetin formed massive nodular structures, and shoots and roots after four weeks on hormone free-medium. Callus from all other explants turned brown and died upon transfer t o hormone free or reduced hormone media. The nodular structures originating from hypocotyl-ca i lus were maintained by subculture for over three years and retained the ability to form thousands of shoots. Shoots were successfully rooted (98% rooting) and plantlets developed were transferred to mist-greenhouse and then to greenhouse conditions with 95% survival. Plantlets were grown for six months in the greenhouse without sign of abnormal growth. Key Words: Nodules, plantlets, morphogenesis Abbrevia tions : NAA, naphtha lene ace tic acid ;

2,4-D, 2,4-dichlorophenoxyacetic acid; IAA, indoleacetic acid; MS, Murashige and Skoog Medium (1962); IBA, indolebutyric acid INTRODUCTION

Efficient regeneration of elite Eucalyptus ~ Maid Ex Hill hybrids, and woody perenniaIs in general, depends on successful long term maintenance of tissues by subculture or storage, and regeneration of a large number of plantlets when needed. For this purpose, morphogenic callus that could be maintained for long periods of time without loss of the ability to regenerate through o r g a n o g e n e s is or somatic embryogenesis, would be suitable (Vasil and Vasil 1980). Recently, nodular tissues that form directly on explants or from callus and cell suspension culture were proposed as an alternative system parallel to somatic embryogenesis (Aitken-Christie et al. 1988, MeCown et al. 1988). Nodules have been observed in a number of species, but are Offprint requests to." M.S. Lesney

more common in woody plant species (McCown et al. 1988). Successful nodule culture with high regeneration potential for Pinus radiata and ~ w a s described by Ai tkenC-'6-~ti'e et al. [1988) and McCown et al. (1988), respectively. Regeneration has been obtained in many species of Eucalyptus through organogenesis in callus cultures. A nodular and red morphogenic callus from juvenile explants has been described by many authors (Aneja and Atal 1969, Kitahara and Caldas 1975, Lakashmi Sita 1986~ Bennett and McComb 1982, Durnad-Cresswell et al. 1982, Quyang et al. 1982, and McComb and Bennett 1986). Somatic embryogenesis has also been reported in a few Eucalyptus species. Eucalyptus ("leichow No.l") embryogenic callus was described as nodular and red pigmented and could be maintained for only a few weeks after induction (Quyang et a I. 198 2). Alternatively, in E. citriodora somatic embryos developed d-~ectly from cultured cotyledons, and formed an embryogenic mass that was maintained for three years with a high frequent X of regeneration by somatic embryogen esi s (Murali dharan and Mascarenhas 1987, Muralidharan et al. 1989). Embryolike structures, not found to regenerate lants, have been reported for E. grandis Lakashmi Sita 1986). This report describes a nodular culture and regeneration method developed using typical somatic embryogenesis procedures on superior Eucalyptus grandi s Maiden Ex. Hill hybrids, selected for their growth charact eri s tics and frost resilience (Meskimen et al. 1987). These hybrids were previous ly propagated successfully by enhancement of axillary shoots (Warrag et al. 1990).

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MATERIALS AND METHODS

Plant materials were collected from three pheno typically superior 5-year-old E. grandis hybrids (clone 28179 2814, and 2759 ~rom ~tudy ORNL-37 at Palmdale, Florida; Meskimen et al. 1987). Explants examined were immature inflorescences, floral parts (stamen filaments and ovary tissues) , zygotic embryos, hypocotyls of young

587 seedlings (from open pollinated seeds), shoot tips from 2-month-old sprouts and from shoots subcultured several times in a shoot multiplication medium (Warrag et al. 1990). Explants were surface sterilized and cultured in media with different hormonal treatments to induce callus. The callus was transferred to media with reduced hormonal concentrations to induce morphogenesis. The callus induction medium and the medium with reduced hormonal concentrations will be referred as primary and secondary media, respectively. The Kruskal-Wallis nonparametric one-way analysis of variance by ranks, was used to test the differences between treatments (SAS 1985). Primary media Twenty sterile explants from each type of clone were assigned to each treatment, 5 explants per plate. Hormonal treatments tested in the primary media were a factorial of either 2,4-dichlorophenoxyacetic acid (2,4-D at 1,2,3,4,6,8, and i0 mg/l) by kine tin (0.0, and 1.0 mg/l) , or naphthaleneacetic acid (NAA at 1,2,4 and 6 mg~l) by kinetin (0.0, 0.5, and 1.0 mg/l). The basal nutrient medium (BN) consisted of Murashige and Skoog's (1962) salts, Gamborg e t al. (1968) (BS) vitamins, supplemented w i t h 45 g/l sucrose, 200 mg/l glutamine, 10% coconut milk and 6.5 g/l agar. Media pH was adjusted to 5.5 before autoclaving. Cultures were kept in the dark at 25o-28 ~ C. After 4 weeks in culture, callus mass and compactness and root formation were scored in an increasing scale of 1-6 and the callus transferred to secondary media. Secondary media Callus produced from each initial explant was divided into three parts and subcultured into each of three secondary media trea tmen ts. The treatments were either hormone-free medium or 1/4 or 1/8 of the hormonal concentrations in the primary media (2,4-D or NAA plus kinetin). The basal nutrient medium was similar to that of the primary media except sucrose was reduced to 30 g/!. Cultures were kept under two light treatments: continuous or 12 hrs of light, with the intensity of average photon flux density of photosynthetically active radiation (PAR) of 35 umole m-2s-l. Nodular tissues produced on hormone free secondary medium, from callus of hypocotyl explants initiated on 4 mg/l NAA and i mg/l kinetin, were subcultured on maintenance media. Full and 1/4 strength basal nutrient media supplemented with I0, 20, 30, and 45 g/l of sucrose were tested to identify the best combination for maintenance. Shoot formation in the three clones was tested in a combination of kinetin (0.0, 0.I, 0.2, and 0.5 rag/l) and NAA (0.0, 0.I, and 0.5 rag/l). Rooting of 120 shoots (40 per clone) was tested on 1/4 MS salts medium with 20 g/l of sucrose supplemented with indolebutyric acid (IBA) at 0.0, 0.2, 0.5, and 1.0 mg/l. Plantlets (30 per clone) were transferred to mist greenhouse for three weeks and then to normal greenhouse conditions where they were allowed to grow for six months. Induction of nodular tissues was repeated twice with 20 hypocotyl explants on primary media with 4 mg/l NAA and i mg/l kinetin and secondary media without any hormones. Three genetic

lines have been retained and subcultured as nodular tissues since 1987. RESULTS AND DISCUSSION

Primary media Type of explant and hormonal concentration of the 2,4-D in the primary media influenced the amount, color, and compactness of the callus produced. The amount and compactness of the callus produced at the base of the peduncle in immature inflorescences was significantly variable (Table i). It was observed in up to I0 mg/l 2,4-D with or without kinetin with significantly higher mass on 2 mg/l 2,4-D and no kinetin. Compactness of the callus decreased with increasing 2,4-D concentration. Stamen filaments produced friable callus~ compared to the immature inflorescences, on 1-2 mg/l 2, 4-I) and I mg/l kine tin. Higher 2, 4-D concentrations resulted in browning and death of the stamen filaments. Cultured ovaries produced dark exudates, presumably due to injuries from the extraction procedures, that resulted in death. Shoot tips, from coppice or subcultured shoots in multiplication medium, produced callus in medium supplemented with up to 6 mg/l 2,4-D (Table I). Similar to immature inflorescences, compactness of the callus increased with decreasing 2,4-D concentrations and with the presence of kinetin. Adventitious roots were observed in 10% of the explants after 3 weeks on media containing 1-2 mg/l 2,4-D with no kinetin (Table i). Higher concentrations of 2,4-D (6 mg/1) killed the shoot tips or produced a small brown mass of callus that died soon after formation. The zygotic embryos and hypocotyls from one-week-old seedlings produced more nodular callus with occasional red pigmentation and adventitious root .production in 1-4 mg/l 2,4-D with no kin e tin. Using NAA in the primary medium produced less variable callus than 2,4-D treatments. Immature inflorescence callus was nodular and creamy yellow with no significant di ffe rence be tween the treatments. Stamen filaments produced here again friable callus on 1-3 mg/l NAA and i mg/l kinetin. Shoot tips from coppice or subcultured shoots produced nodular callus and adventitious roots on the kinetin free media, while zygotic embryos produced large numbers of roots. Hypocotyls produced nodular and red pigmented callus on 2-4 mg/l NAA and i mg/l kinetin. The callus increased in size and root differentiation was observed by the end of the fourth week on 50% of the explants. Secondary Media Morphogenesis was observed from callus of hypocotyls while callus of immature inf lorescences, floral parts, zygotic embryos, and shoots tips turned brown and died after 4-6 weeks. The hypocotyl callus initiated on 4 mg/l NAA and I mg/l kinetin shewed an increase in red pigmented areas which differentiated into massive numbers of shoot primordia and roots on the hormonefree medium under continuous light conditions (Figure I). Also, white, smoothsurfaced nodular tissues developed around

588 the shoots and root differentiating areas (Figure 2). These nodular tissues produced shoots and roots in addition to nodule multiplication on the hormone free secondary medium. Nodule multiplication was observed to take place through development and budding of nodules from the surface of existing "mother" nodules (Figure 2). The number of nodules per existing "mother" nodules ranged between 2 and 20. Full strength basal medium supplemented with 30 g/l sucrose was best for long-term maintenance and sub-culture of the nodular tissues of the 3 genetic lines tested. However, occasional transfer to 1/4 strength of the basal medium with 20 g/l of sucrose, after each 3-4 subcultures~ increased the red pigmentation and suppressed the shoots that tend to develop with continual subculture on full strength basal medium. Large numbers of shoots from the maintained cultures were successfully induced and their growth enhanced in a medium with low auxin and cytokinin concentrations: (0. i - 0.2 rag/l) NAA and 0.i mg/l kinetin, similar to that described by Quyang (1981). Rooting efficiencies of 95% were achieved on low concentrations of IBA (0. i 0.3 rag/l). Resultant plant lets were grown in the greenhouse for over six-months with 94% survival with no sign of morphological abnormali ties. These results confirmed previous reports w i t h a number of Eucalyptus species that hypocotyls produce more morphogenic callus, usually nodular and red pigmented, than other types of explants (Kitahara and Caldas 1975; Aneja and Atal 1969; Lakshmi Sita 1979 and 1986; MehraPalta 1982; Oka et al. 1982; Quyang et al. 1982). While few plantlets were produced from such nodular pigmented callus in other studies (Lakshmi sita 1979, Oka et al. 1982) a large number of plantlets were possible in this study (Figure 2) similar to that reported by Quyang et al. (1982) in a system which they described as involving somatic embryogenesis. The higher rates of shoot and root production observed in this study and that of Quyang et al. (1982) compared to the other Eucalyptus reports may be related to the reduction of hormonal concentration in the secondary media. In the other Eucalyptus reports, morphogenic callus was transferred to media with a relatively high cytokinin concentration (0.5 mg/l or more). This study showed that callus production is highly influenced by the kind of explant. Immature inflorescences produced callus in up to i0 mg/l 2,4-D while other explants such as shoot tips and stamen filaments deteriorated at such concentrations. AI though somatic embryogene sis was not observed with the induced calli of the different explants used in this study~ no conclusive judgement is made that these E. grandis hybrids do not have the potential ~ o form somatic embryos. This may be due to missing the right combination of explant and medium or that embryo genic callus was produced but overtaken by other type of calli. Vasil and Vasil (1984) emphasized the importance of early identification, selection and culture of embryogenic callus developed in the primary medium. Further

Table i. E. ~ callus color, mass, and compactnes~ from different types of explants on seven concentrations of 2;4-D and two concentrations of kinetin (K) after 4 weeks. Hormonal con. m~/l 2,4-D K Color

Mass/ Compact./

Immature inflo rescences I 0 White & Yellow 2 0 White & Yellow 3 0 Yellow 4 0 Yellow 6 0 Pale Yellow 8 0 Pale Yellow i0 0 Pale Yellow i i Yellow 2 1 Yellow 3 1 Creamy Yellow 4 1 Creamy Yellow 6 I Creamy Yellow 8 i Creamy Yellow i0 1 Creamy Yellow

2 ~-~ 5 3 3 2 2 I 3 3 3 3 3 i 1

5** 4 2 2 I I 1 5 5 5 4 3 3 2

Coppice *I *2 3 4 6 8 i0 1 2 3 4 6 8 I0

2** 4 3 3 I 0 0 1 4 5 3 i 0 0

4** 3 2 2 i 0 0 1 4 4 3 3 0 0

shoot tips 0 Pale Yellow 0 Pale Yellow 0 Ye llow 0 Yellow 0 Yellow 0 0 0 0 l Pale Yellow 1 Pale Yellow 1 Yellow i Yellow 1 Yellow i 0 i 0

/-Scored in a scale of 1-6 with w i t h "0" indicating not observed. * Only these two treatments showed any root production (each with a root rating of i) **Indicates significant variation between treatments in a column as the 5% level of probability (Kruskal-Wallis nonparametric test).

Figure I. Differentiation of E. grandis callus into shoots, roots, and-nodules on hormone free secondary medium after 4 weeks.

589 experimentation with growth regulators, basal media, explants, and physical environment is needed to identify the appropriate factors and stimuli that may make somatic embryogenesis possible in E. odular tissues have been subcultured for over 3 years so far without losing the ability to regenerate. In order to maintain the nodular tissues and shoot primordia and to suppress further shoot development, reduced basal media was found useful. Such nodular culture could have significant practical applications including a ready source of regenerative material and the potential for long-term conservation of specific genotypes. In addition to the ability to produce large numbers of plantlets, the use of nodule culture for multiplication and shoot enhancement should be appropriate for use with a bioprocessor similar to that described by Levin et al. (1988). This could greatly increase the economic viability of mass micropropagation in this species.

use first as explants to identify embryogenic callus characteristics should precede use of mature tissues. REFERENCES

Aitken-Christie, Jenny, AP Singh and H Davies. 1988. In: Genetic Manipulation of Woody Plants. Hanover JW and DE Keathley (eds) Plenum Press, NY. pp. 413-432. Aneja S, and CK Atal. 1969. Curr. Sci. 38:69. Bennett IJ, and JA McComb. 1982. Aust. For. Res., 12: 121-127. Durnad-Cresswell R, M Boulay, and A Franclet. 1982. In: Tissues Culture in Forestry. JM Bonga, and DJ Durzan (eds). The Hague: Martinus Nijhoff/Dr W. Junk Publisher. pp. 150-181. Kithara EH, and JS Caldas. 21: 242-243.

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Lakshmi Sita G. 1986. In: Plant Tissue Culture and Its Agricult. Implications. LA Withers and PG Alderson (eds). Boston Butterworsths. pp 150-165. Levin R, V Gaha, BS Hirsch, D De Nola, IK Vasil. 1988. Bio/Technology 6:1035-140. McComb~ JA, and IJ Bennett. 1986. In: Biotechnology in Agriculture and Forestry: Trees. Voll. YP Bajaj (ed). Berlin: Springer Verlag. pp. 340-362. McCown BH, EL Zeldin, HA Pinkalla, and RR Dedolph. 1988. In: Genetic Manipulation of Wood Plants.Plen~ Press,NY. pp. 149-166. Mehra-Palta A. 1982. Plant Sci. Let.26:l-ll. Meskimen GR, DL Rockwood, and KV Reddy. 1987. New Forests 3: 197-205. Figure 2. E. grandis scanning monograph of nodules and-nodule multiplication on hormone free secondary medi~. CON CLU S ION Nodule culture initiated from hypocotyl callus of E. grandi__s hybrids could produce large numbe-rs of plantlets when needed. The hypocotyl callus induced on 4 mg/l NAA and i mg/l kinetin confirmed other reports that morphogenic Eucalyptus callus is nodular and red pigmented. Somatic embryogenesis of E. grandis hybrids was not observed under the experimental conditions used in this study, however, explant response to 2,4-D varied with tissues and organs. Floral buds were more tolerant to 2,4-D than other types of explants, producing healthy callus in up to I0 mg/l 2,4-D. Callus color, compactness, and quantity depended on the explant origin. Future studies to induce somatic embryogenesis on this important genus should include more extensive trials with plant growth regulators, especially NAA and IAA. Also, since hypocotyls and juvenile material seem to be more responsive to culture, their

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Oka S, EC Yeung, and TR Thorpe. 1982. Zealand J. For. Sci., 12(3):50-509.

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Quyang Q. Peng Hai-Zang, and Li Qi-quang. 1982. Scientia Sil. Sin., 17:1-7. SAS User Guide: Statistic 1985. Version 5 Edition. By SAS Institute Inc., Cary, NC. Vasil IK, a n d V Vasil. 1980. In: Prospectives in Plant Cell and Tissue Culture. Suppl. IIA Internat. Rev. of Cytology. IK Vasil (ed). Academic Press, NY. pp. 145-173. Vasil V, and IK Vasil. 1984. In: Cell Culture and Somatic Cell Genetics of Plants. Voll. IK Vasil (ed). Orlando. Academic Press. pp. 152-158. Warrag El, MS Lesney, and DL Rockwood. 1990. New Forests (in press).

Nodule culture and regeneration of Eucalyptus grandis hybrids.

Callus of three superior Eucalyptus grandis hybrids was induced from immature inflorescences, floral parts, shoot tips, zygotic embryos, and hypocotyl...
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