AN INTEGRATED IN VITRO AND GREENHOUSE ORTHOTROPIC CLONAL PROPAGATION SYSTEM FOR THEOBROMA CACAO L.
Open Access
- Author:
- Miller, Carter Robert
- Graduate Program:
- Horticulture
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 05, 2009
- Committee Members:
- Mark Guiltinan, Dissertation Advisor/Co-Advisor
Mark Guiltinan, Committee Chair/Co-Chair
Siela Maximova, Committee Member
David Eissenstat, Committee Member
Robert Michael Crassweller, Committee Member
Roy Edward Young, Committee Member - Keywords:
- tissue culture
rooted cutting
vegetative propagation
cocoa - Abstract:
- Two novel methods are described to stimulate intensive and sustained orthotropic (vertical, juvenile) shoot production in softwood and semi-hardwood Theobroma cacao L. stock plants, derived from tissue culture of floral staminodes via primary and secondary somatic embryogenesis. The first stock plant method consisted of development of softwood stock plants in a bench top modified hedge. Harvest of orthotropic shoot material began two months after acclimation in the greenhouse and utilized periodic light pruning of orthotropic softwood shoots (chupons, suckers or water sprouts). This high-intensity propagation method yielded 70 stem cuttings per softwood stock plant per year, approximately 1,250 m-2•year-1. Single and few node orthotropic stem cuttings were rooted under intermittent mist. The propagation efficiency (percentage of plants produced per stuck cutting) from softwood stock plants ranged from 66% to 81%. Under the specific conditions of the current study, the estimated production of orthotropic rooted cuttings from softwood stock plants maintained as a bench top clonal garden was estimated to range between 825 and 1,012 rooted cuttings m-2•year-1. The second orthotropic shoot production method involved the bending to horizontal of the main trunk of semi-hardwood stock plants from 9 to 12 months after acclimation, release of axillary and adventitious orthotropic meristems from the bent trunk, and repeated harvest by periodic light pruning of orthotropic shoots for rooting material. Using the bending method, semi-hardwood stock plants yielded on average over 300 single and few node orthotropic stem cuttings per year, or approximately 500 m-2•year-1 at the stock plant density tested. Single and few node orthotropic stem cuttings were rooted under intermittent fog. The propagation efficiency of rooted cuttings from semi-hardwood stock plants ranged from 68% to 74%. The estimated production of orthotropic rooted cuttings from bent semi-hardwood stock plants was estimated to range between 340 and 370 rooted cuttings m-2•year-1. Overall, the softwood orthotropic stock plants produced 1.5 times more plants per given unit of space and time than the bent semi-hardwood stock plants. Softwood stock plants are compact and have shorter chupons and leaf laminae, as well as smaller stem diameters in comparison to bent semi-hardwood stock plants. Production of softwood stock plants requires a higher level of cultural management and propagation technique than those for bent semi-hardwood stock plants. Utilization of softwood stock plants in the current study significantly shortened the time necessary for greenhouse multiplication of vegetatively propagated orthotropic cacao plants compared to the utilization of bent semi-hardwood stock plants. Comparison of development at the juvenile stage in a climate controlled greenhouse demonstrated that primary somatic embryos (1°SE) and secondary somatic embryos (2°SE) grew true to type as compared to seedlings. As well, orthotropic rooted cuttings from softwood stock plants (RC-SW) and orthotropic rooted cuttings from bent semi-hardwood stock plants (RC-SHW) grew true to type. All four orthotropic vegetatively propagated trees had the characteristic jorquette (whorl of plagiotropic fan branches) at the phase change from the juvenile orthotropic trunk to the mature plagiotropic canopy morphology. RC-SW had a relatively longer juvenile phase, while RC-SHW had a more rapid transition to mature phase that was demonstrated by earlier and lower formation of the jorquette. All orthotropic cacao trees had one or more tap roots (seedlings and SE plants) or their analog, orthogeotropic framework roots, (from an adventitiously -derived root system of rooted cuttings). SE plants and orthotropic rooted cuttings had lower leaf area (LA) per gram of dry weight (DW) of root and higher root to shoot ratio than seedlings, although LA per stem DW did not differ among plants. Photosynthesis measurements at 12 months in the greenhouse showed no difference between 2°SE and seedlings. These orthotropic clonal propagation methods may be adopted by germplasm banks, international cacao research centers, universities, private laboratories, as well as regional and local extension services and nurseries for use in international, regional, and local distribution schemes for elite or other newly developed clonal varieties of cacao or other instances where original stock plant material is limited or the orthotropic seminal tree architecture is desired. The use of the bending technique for semi-hardwood stock plants may be of particular utility to nurseries and growers interested in robust, on-farm propagation of clonal varieties that have orthotropic tree architecture and an orthogeotropic adventitious framework root system similar to that of a seminal tree. A single line of somatic embryo plants that were genetically transformed by cocultivation with Agrobacterium tumefaciens strain AGL1 and containing the EGFP and NPTII genes were acclimated to the greenhouse. Non-destructive growth measurements were taken to compare the transgenic line and non-transgenic 2°SE plants during 18 months. Growth analysis of plant height, stem diameter, number of leaves and leaf area, as well as photosynthesis and excised root respiration measurements did not indicate any differences in these parameters between transgenic and non-transgenic SE-derived plants. EGFP expression was analyzed in seeds from two genetic crosses. One to one segregation of EGFP expression in the seeds indicated that the T-DNA was inserted at a single locus in this line.