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Summary of full report
by Dr M Panaia
September 2006
RIRDC Publication No 06/058 RIRDC Project No KPW-2A
Executive Summary
Background
Many desirable Australian
plants produce poor quality seeds or seeds that are deeply dormant.
Successful plant establishment can often be difficult and sometimes impossible. Research into new propagation technology is essential for meeting the present and future demands for land restoration and horticultural utilisation. Somatic embryogenesis represents a major technology for the low cost, rapid, large-scale production of plant species that respond poorly to conventional methods of propagation.
Somatic embryogenesis was first described for carrot (Steward et al. 1958) and has the potential to produce up to 60,000 plants from one gram of parent tissue (Senaratna 1992). It is a process by which somatic (i.e. non-sexual) cells undergo a developmental sequence, similar to that seen in zygotic embryos, without the need for sexual reproduction. Under normal circumstances it is necessary for the pollen (male) to make contact with the ovary (female) of the flower to fertilise the egg and thus produce a seed. In somatic embryogenesis, a variety of plant growth regulators and environmental conditions are applied in vitro to a tissue source (such as shoot material), which causes the cell to produce an embryo that eventually develops into an entire plant. The embryo is formed without genetic recombination and is therefore a clone of the parent plant. The term "synthetic" or "artificial seed" is used to describe somatic embryos (SE) either in a hydrated or desiccated form, with or without encapsulation. The production of synthetic seeds has enormous potential in the agricultural, horticultural and mining industries and the basis for synthetic seed technology is the phenomenon of somatic embryogenesis.
During the study, a detailed examination of various factors including plant growth regulators and media nutrients on somatic embryogenesis for Baloskion tetraphyllum (Koala Fern), Macropidia fuliginosa (Black Kangaroo Paw), Stirlingia latifolia (Blue Boy), and Lepidosperma squamatum (a sedge) were investigated. The first three species are highly desirable in the horticultural industry and Stirlingia and Lepidosperma are vital species for the rehabilitation of disturbed landscapes.
Objectives
The objective of the project
was to develop a process for the large-scale production of SE for selected
Australian native species to be used as synthetic seeds for the rehabilitation
of disturbed landscapes and for horticultural utilisation. More specifically,
the project examined the following stages in the development of SE for
the above species (1) initiation of SE from primary explants (2) proliferation
of embryogenic cultures (3) conversion of SE into functioning plantlets
and (4) preliminary investigation into the desiccation ability of SE as
a pre-requisite to encapsulation and production of synthetic seeds.
Methodology
While theoretically every cell has the potential to be "totipotent" (develop into an entire plant), the signalling process is not common for all species. The success of this technique is dependent on the tissue source, physiological status, environmental conditions and the cells response to the exogenous application of plant growth regulators. To that end, specific protocols must be developed for each species of interest.
Trials were undertaken to evaluate the impact of selected plant growth regulators (e.g. auxins, cytokinins and thidiazuron) on the ability of different tissue sources (shoot explants, basal portions and zygotic embryos) to express somatic embryogenic potential. Nutritional conditions for embryo induction including different basal salts and variation in the carbohydrate source were also investigated. Once SE had been successfully stimulated, several experiments were undertaken to improve their ability to withstand desiccation (as a precursor to coating, storage, transportation to site and mechanised sowing) by including abscisic acid (ABA) in the media.
Results/Key Findings
Somatic embryogenesis was
successfully achieved for B. tetraphyllum, M. fuliginosa,
S.
latifolia, and L. squamatum. Conversion of SE into functioning
plantlets was successful for M. fuliginosa, S.
latifolia and B. tetraphyllum with plants successfully transferred to soil (70-100% survival) without the addition of plant growth regulators and minimum acclimatisation (1-2 weeks in the glasshouse). As is the case with all species investigated, the stimulation of SE appears to be closely linked to the age of the explant and the younger the material used as the explant, the more successful the process became.
Desiccation of SE for L. squamatum and B. tetraphyllum demonstrated encouraging results. A number of SE displayed germination events (after being dried for 2-3 hours) including re-hydration and production of root hairs. These preliminary results clearly demonstrate that drying (desiccation) of SE is possible pending further research to optimise this protocol.
The key findings of this study are summarised below: 1. Somatic embryogenesis was successfully achieved for Baloskion tetraphyllum (Koala Fern) with more than 1,500 SE produced in 6 weeks from 0.02 g of parent material. This has the potential to produce ~75,000 SE from 1 g of original parent material.
2. Somatic embryogenesis for Macropidia fuliginosa (Black Kangaroo Paw) varied depending on the tissue source:
Callus that remained in culture for a further 6 weeks continued to produce SE. At the end of 12 weeks, there were a total of 278 SE produced from ~1.235g of callus.
4. Significant differences in the response of S. latifolia selections to various plant growth regulators were observed. During experiments investigating 2,4-D and TDZ on shoot explants, only Selection 1 (Deep Red) produced entire plantlets (~ 449) after an extensive treatment regime as follows: callus produced on ½ MS + 10 ?M 2,4-D was transferred to ½ MS + 5 ?M TDZ (for shoot development) and then ½ MS + 5 ?M IAA (for root development).
5. Primary SE were produced for three selections of S. latifolia (Deep Red, Dark Red and CY) when cultured on TDZ, IAA and a combination of these two plant growth regulators. Deep Red responded with the highest number of 170 SE from ~ 0.3 g of shoot material which equates to a possible 566 SE from 1 g of original parent material.
6. There were significant numbers of shoot meristems produced that could be excised and rooted separately for S. latifolia (organogenesis). Selection 4 (CY) had the highest number with 454 shoot meristems produced in 6 weeks from ~0.03 g of shoot material cultured on ½ MS + 10 ?M TDZ and 5 ?M IAA.
7. SE were successfully stimulated for Lepidosperma squamatum using zygotic embryos as the explant source. A total of ~470 SE were produced from ~0.05 g which is estimated could produce ~9,400 SE from 1 g of zygotic tissue.
8. Proliferation of secondary somatic embryogenesis was achieved for Lepidosperma squamatum with a ~ 3-fold increase when primary SE were subcultured onto fresh media.
9. Callus stimulated the production of secondary SE when cultured on a variety of media. The best treatment for Lepidosperma squamatum was a combination of ½ MS + 1 ?M 2,4-D, followed by transfer to ½ MS + 5 ?M TDZ with a total of 148 secondary SE from ~1.2 g of callus.
10. Conversion of SE into functioning plantlets was achieved for M. fuliginosa, S. latifolia and B. tetraphyllum with plants being transferred to soil with 70-100% survival.
11. Desiccation of SE for B. tetraphyllum demonstrated encouraging results with ~ 50 % displaying germination events (after being dried for 2-3 hours) including re-hydration and the production of root hairs when cultured on ½ MS + 20 ?M ABA for one week prior to drying.
12. Desiccation of L. squamatum primary SE was also successful with the best treatment being ½ MS + 50 ?M ABA for one week prior to the drying treatment. Although the number of rehydrated SE is small, these preliminary results clearly demonstrate that drying (desiccation) of SE is possible pending optimisation of this protocol.
Implications for relevant
stakeholders
The mining of native ecosystems
(including the successful restoration of such sites) and the development
of horticultural industries largely depend on the development of an efficient
and cost effective propagation technique that will provide acceptable levels
of biodiversity replacement. The estimated cost of producing a single plant
under normal micropropagation methods can vary from less than $1 to over
$5 depending on the species and how difficult it is to initiate into tissue
culture. In general, 15-20 plantlets can be produced in one tissue culture
vessel compared to approximately 200 SE per petri dish. Somatic embryogenesis
is therefore at least 10 times more efficient, even with nonoptimal protocols,
and, as such, the cost can conservatively be estimated at 10 to 50 cents
per plant.
This is a significant saving over current production costs. Mining in biodiverse regions or highly diverse floras such as the southern parts of Western Australia often means that significant components of the biome are not effectively rehabilitated. For example, southern rushes (Restionaceae), sedges (Cyperaceae) and native heaths (Ericaceae) represent up to 30 % of pre-mined diversity and biomass, yet current rehabilitation technology returns barely 1 – 2 % (Willyams 2005a). In addition, the horticultural potential of unique Australian plants has not yet been fully exploited. The Black Kangaroo Paw (Macropidia fuliginosa) and Koala Fern (Baloskion tetraphyllum) are in heavy demand in the horticultural industry, but are slow to multiply in sufficient numbers to satisfy this demand. A considerable amount of native plant material is still wild sourced, which leads to significant problems in quality and guaranteed supply as well as the environmental damage and loss of biodiversity. The use of somatic embryogenesis as a technique to deliver a low cost, superior, mass propagation method for these native plant taxa will effectively reduce the cost of production, reduce loss of biodiversity, increase the efficiency of rehabilitation programmes and boost the supply of these species to the horticultural industry.
Recommendations
This project has established
a sound foundation to continue work on these species to improve the process
of somatic embryogenesis. It was clear that significant variation occurred
between different tissue types and this phenomenon requires further investigation
to optimise protocols and consistently produce large numbers of SE with
a wide range of selected genotypes.
One of the challenges associated with somatic embryogenesis is that germination and growth of plantlets is often not as vigorous as that of their zygotic counterparts. This is, in part, due to the lack of storage reserves in the SE. As with other characteristics of somatic embryogenesis, the amendment of the initiation media through the addition of plant growth regulators may increase the storage proteins in SE, however, this is yet to be determined for the species investigated in this study and it is recommended that this be a priority research area in the future.
Once synchronous production of SE has been established, it is important that SE be dried for storage purposes without the loss of viability. While preliminary desiccation experiments were encouraging, follow-up research is required investigating factors such as heat shock, non-lethal chilling or osmotic stress to promote desiccation tolerance. Low levels of ABA are present at the onset of reserve accumulation in zygotic seeds, but rise with the onset of desiccation tolerance. ABA is thought to be involved in the accumulation of storage reserves and it is important therefore that its role in promoting desiccation tolerance does not interfere with the deposition of storage reserves. Investigations are therefore required to determine the levels and timing of application to induce desiccation tolerance without negatively influencing reserve accumulation.
One of the final stages is to establish a protocol for the encapsulation of SE to create synthetic seeds.
Encapsulation provides protection
from mechanical damage as well as delivering plant growth regulators, nutrients
and other chemical or biological components required for rapid germination
and plant establishment. This aspect of the development of synthetic seeds
remains to be done. However, the present study has provided significant
advances in production of SE from recalcitrant species that will facilitate
progression to synthetic seed production.
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