For genetic improvement of Leucaena leucocephala, it is necessary to develop genetic transformation procedures for this tree. The purpose of this project is to develop transformation protocols for Leucaena.

OBJECTIVES

1. Develop transformation method for Leucaena leucocephala using Agrobacterium tumefaciens.
2. Develop method for somatic embryogenesis for L. leucocephala and use embryogenic tissue for genetic transformation using particle bombardment.
3. Analyze transformed L. leucocephala plants for insertion and expression of transgenes.
   
   

APPROACH

1. Develop transformation method for Leucaena leucocephala using Agrobacterium tumefaciens. We have already successfully developed the tissue culture regeneration method for L. leucocepahala. Using cotyledon sections as explants for regeneration, we have developed method for nearly 100% shoot and root regeneration. We are going to use the same cotyledon explants procedure for regeneration of transformed plants following transformation using A. tumefaciens. We will use a binary vector construct containing gus gene under the control of a CaMV 35S promoter for Agrobacterium-mediated transformation protocol.
2. Develop method for somatic embryogenesis for L. leucocephala and use embryogenic tissue for genetic transformation using particle bombardment. Besides transforming with Agrobacterium we will use particle bombardment method for transferring genes into Leucaena. For this we will first develop proliferative embryogenic tissues of Leucaena cotyledons on semi-solid medium. The same gus gene construct, described in objective 1, will be used for particle bombardment of somatic embryos. Somatic embryos will be plated on tissue culture medium containing antibiotics for selection of the transgenes. Transformed plants will be regenerated using standard protocol.
3. Analyze transformed L. leucocephala plants for insertion and expression of transgenes. The transformed plants will be screened by Southern hybridization and PCR for the presence of the transgenes. The copy number of the transgenes in the transgenic plants will be determined from the number of the hybridizing bands.
   
   

PROGRESS October 2000 to September 2001

The goal of this research is to develop an efficient plant regeneration method through tissue culture, which is a prerequisite for developing a genetic transformation protocol for Leucaena leucocephala. Two plant regeneration methods applicable to Leucaena were developed. In the first method involving organogenesis via callus formation, cotyledon, hypocotyl and root segments were initiated on MS medium containing different concentrations of N6-benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), and naphthaleneacetic acid (NAA). Both compact (type I) and friable (type II) calli were obtained from the cotyledon and hypocotyl explants treated with different concentrations of the growth regulators. Shoots were generated only from the friable calli formed from the cotyledon explants. The calli formed from the hypocotyl explants did not generate shoot and the root explants died without forming callus. Cotyledon explants from 3-4 day old seedlings showed maximum callus induction compared to those from older seedlings. In a second method involving direct organogenesis, excised cotyledons from which embryos have been removed, were initiated on 1/2 MS medium containing 10-35 mg N6-benzyladenine (BA) per l for 7-14 days. Transfer of the cotyledons to regeneration medium containing low BA resulted in callus formation and subsequent shoot regeneration from the base of the excised cotyledon explants, with up to 100% frequency. Regenerated shoots rooted best on a basal medium containing no growth regulators. To our knowledge, this is the first report of successful tissue culture plant regeneration for this species. The establishment of an efficient tissue culture regeneration system of L. leucocephala will be useful for development of future transformation systems.

IMPACTS October 2000 to September 2001

PUBLICATIONS