Rainforest seed conservation project

Collecting seed from rainforest plants has a number of challenges.

Rainforest seed tends to ripen over a long period, so our collectors need to be able to recognise when fruits are ripe enough to collect. Multiple trips may also be required to ensure enough seeds are collected. In addition, a large number of rainforest fruits are fleshy, have a high moisture content and rot easily, so they need to be handled with care and processed quickly before the seed is damaged.

Extensive planning is needed to prepare for a field trip

Having a vehicle that is up to the task is just the first step. You then need to pack it with all the items needed for collecting different sorts of seeds, processing and storing them while away, along with things for documenting the process as you go. 

Collecting trips are usually aimed at obtaining seeds or plant material of specific species and therefore our collectors go to locations with records of their target species, at a time of year when plants should be in fruit. These means a lot of trips happen in summer and autumn. 

Once a target species is identified, an assessment of seed quality and maturity is made with a cut test. If seeds are suitable, the collection is made and will include a section of plant material including leaves and (where possible) fruit and flowers. The plant material will be used to help to confirm the identification of the plant once it is returned to PlantBank.

To ensure fruit and seeds are returned safely, collections will be packaged and stored appropriately

For fleshy rainforest fruit this will often involve aerated plastic bags and the storage of seeds in cool (but not cold) conditions. Sometimes collections must be made from immature fruits in which case fruit will often be maintained on stems and branches, and seeds will be held in warm humid conditions to help promote ripening. This process must be managed very carefully so that seeds do not go mouldy.

Out and about: seed collecting trips

On the collecting trips not only do our collectors get to see the wonderful flowers and fruits of these rainforest plants they often get glimpses of birds and animals that use these plants for food and habitat.

In December 2014 the PlantBank seed collectors were collecting in the Tweed Valley. They were fortunate to see the endangered Grevillea hilliana, known only from less than 100 plants in NSW, in flower. Some of the remaining specimens are over mature and don’t produce seed but our collectors did find one plant with a good crop of seed developing. 

Another spectacular rainforest flower, Pararistolochia laheyana, was found in the Border Rangers National Park. This species, has become quite common in the areas of Antarctic beech rainforest that had been recently disturbed by storms. While working in the higher areas of the Tweed Valley on 14 December 2014, in the Border Ranges, the PlantBank seed collectors saw the spectacular flower of Trichosanthes subvelutina. There were no fruits at the time, but collector returned the following August when they were able to make a small collection. 

A seed collection trip in January 2017 resulted in the discovery of new populations of two vulnerable species. Macadamia integrifolia, which was believed to be absent from natural areas in NSW, and M. integrifolia were both found growing in Mount Pikapene National Park. 

Depending on what we know about the biology of the species, seeds might be processed straight away and deposited into the seedbank. Others might need to be tested for their response to drying before we can store them appropriately.

Seeds returned to PlantBank usually need some post-collection processing to ensure we have a good quality, clean collection that is ready for research, transport or storage. If we are unsure of the most appropriate conditions for seeds to be stored, a subset will be tested, while the bulk of the collection remains in temporary storage.

Processing fleshy fruits

Rainforest species often have fleshy fruits, so there are a number of different techniques we can try to remove the flesh without damaging the seeds.

Processing hard seeds

In other circumstances rainforest seeds are in a hard covering or within a hard capsule that needs to be cracked open to extract the seeds. Depending on the nature of the covering, one of the following techniques may be applied. 

Final cleaning to remove debris

Once seeds have been released, a final round of cleaning may be required to remove any debris - frass (insect droppings), leaves, twigs, dust etc - that will reduce the quality of the collection. Two simple ways this can be done include separating seeds by their size through a series of sieves, or by weight, in which case seeds are passed through an air current that 'blows' away the rubbish, allowing the heavier seeds to drop into a collection container. Larger debris may also be removed by hand.

Once the collection contains pure seeds, it’s time to package the seeds up ready for transport or storage. However, if we don't know the best way to store the seeds, then this is the time to start testing. 
 

Rainforests are not naturally dry habitats and rainforest seeds are often not used to drying out. This can make seed storage difficult.

For many plants, seedbanking is a cost effective way of conserving vulnerable species outside of their natural habitat but not all species can survive the seedbanking procedure that requires seeds to be tolerant of desiccation. 

Seeds that can tolerate drying are 'orthodox'

Seeds of plants from drier habitats are adapted to being dried out and can therefore be stored in seedbanks at low temperatures for decades without losing viability. Such seeds are termed ‘orthodox’.

Seeds that cannot tolerate drying are 'recalcitrant'

Of particular concern is the probability that many hundreds of rainforest species have seeds that are sensitive to drying out (desiccation sensitive) and are termed ‘recalcitrant’ to seedbanking, meaning they are killed when dried and frozen. This behaviour has been identified in perhaps as much as 50% of the rainforest species growing in equatorial regions1, however drier rainforests or rainforest which experience a dry season tend to have higher numbers of orthodox species.

As at September 2017, research conducted as part of the Rainforest Seed Conservation Project has identified that of 166 species fully assessed, 80 species are likely to be recalcitrant, 52 species are orthodox, with an additional 34 species likely to have intermediate seed storage behaviour.

1. Baskin, C. and Baskin, J. (1998) Seeds: Ecology, biogeography, and, evolution of dormancy and germination. Academic Press, San Diego USA.

Are seeds alive and what do they need to germinate?

Although they might seem like the same thing, when we are talking about seed viability and germination, we are referring to two different things. Viability means whether or not a seed is alive, while germination refers to whether or not a seed can germinate. The number of seeds viable in a collection doesn’t necessarily equal the number of seeds that can germinate, the difference being that some seeds are immature, or dormant (‘asleep’).

Viability

Whenever we make seed collections we try to ensure that seeds are viable and mature (ripe). This is very important because if seeds are not viable, they will not germinate anyhow! If they are immature they will not survive as well in storage as they would if they were mature. Sometimes we can help seeds to mature after collection by the way they are stored. Also, the way seeds are handled during collection and transport will influence the proportion of seeds that remain viable.

When it comes to testing seed viability, there are a number of different techniques that can be used, some of which actually result in the death of the seed! This means we have to choose a test carefully and ensure there are plenty of seeds left for other tests and storage. As such, viability is assessed on a portion of seeds (perhaps 20 seeds for small collections, 100 seeds for large collections), and this is taken to represent the viability of the entire collection.

Dormancy

In the field

Seeds use dormancy to hold off germination until the environment becomes suitable, that is, when the surrounding conditions are going to give the seed the best chance at life. This might mean, for example, that there is water present, that a sunlight gap has formed in the canopy, that nutrients are suddenly available such as after a fire or when the seed is defecated after being eaten by an animal. Sometimes dormancy occurs to prevent all the seeds from germinating at once! Dormancy may reduce the proportion of viable seeds that germinate at any one time, increase how long germination takes to begin, or prevent the seeds from germinating entirely. We need to understand what type of dormancy a seed has so that when we want to germinate seeds we know how to break it. 

In the laboratory

In the laboratory, we use a number of different techniques to break seed dormancy which include scarifying (cutting/scratching) the seed coat, temporary cold or warm stratification (storage), repeatedly rinsing seeds, soaking the seed in hot water or concentrated acid, soaking the seeds or germinating them in the presence of the plant hormone gibberellic acid or smoke water. One technique alone may be suitable, or two or three may be used together, but multiple things are tried until the highest proportion of germination is found. Below is a graph of germination results from an experiment in which groups of seeds of Hibbertia glabrescences subsp puberula were each treated differently. 

Germination

Our viability and germination testing hopefully results in the production of new rainforest seedlings. As seeds germinate they are removed from experiments, potted up and grown on in the nursery. These plants can then serve new roles, forming the basis for tissue culture material or living collections. Material can be planted into our Gardens or even donated to other organisations. 

To dry or not to dry?

When describing the desiccation sensitivity of a seed, we are talking about the ability of a seed to handle being dried down (as you would dry a grape to make a sultana), and still germinate afterward. Understanding this is important because it determines the way seeds are stored.

Categories of desiccation sensitivity

A seed is considered to be desiccation tolerant if it can be successfully dried to approximately 4-7% moisture content (ie, 4-7% of the weight of the seed is water) and still germinate upon rewetting. A desiccation sensitive seed cannot germinate after drying below 15-25%1. However, all seeds do not fit so discreetly into either category, as desiccation sensitivity (or tolerance) is a continuum, in which some seeds can handle a degree of drying, perhaps to 10-21% before loss of viability occurs. These seeds are usually called ‘intermediate’. 

Further to the classification of how much drying seeds can handle, we test whether or not dried seeds can be subsequently frozen. If a seed can be dried and stored at -18 or -20°C (the same temperature as your freezer at home), it is classified as 'orthodox'. Conversely, if a seed cannot be dried (and therefore cannot be frozen), this seed is classified as 'recalcitrant'. 

Continuum of seed storage behaviour in which orthodox seeds can be dried (5-7% moisture content/10-15% equilbrium relative humidity) then cold stored, compared to recalcitrant seeds which cannot survive any degree of artificial drying at all.

Why is desiccation sensitivity, orthodoxy and recalcitrance important?

Well, it dictates how we can and can’t store seeds. Orthodox seeds can be easily stored using standard seed banking methods, and we expect them to remain viable in storage for at least 10 years. For recalcitrant and even intermediate seeds we need to find other ways to store the seeds such as cryopreservation or as living collections. 

So why can some seeds tolerate being dried and not others?

It is thought that desiccation sensitivity in seeds has occurred as a response to environmental factors2. A high proportion of seeds that come from wet environments (eg rainforest) have desiccation sensitive seeds, even as high as 50%2, while seeds from harsh environments such as those that are cold or arid, tend to be desiccation tolerant. This makes sense as plants need water to grow and if it is not available when seeds are mature in the desert, then seedlings would not survive, so being able to survive drying until water is available ensures seeds (and subsequent plants) live. Conversely, seeds in wet humid environments are not going to survive very long on the forest floor before they get consumed or rot, so it’s important that they germinate quickly. Interestingly, this is in direct contrast to desiccation sensitivity in plants, whereby seeds need to be desiccation tolerant in dry environments but plants struggle, while in rainforests, plants thrive with copious amounts of moisture, yet their seeds don’t necessarily do quite as well.

How do we know the best way to store each rainforest species?

When it comes to determining desiccation sensitivity and suitable storage methods for seeds, the best way to do this is by actually testing representative seed samples from each collection. A portion of seed is germinated fresh, a portion is dried and germinated, and a final portion is dried, frozen then germinated. The difference between total germination for each set of seeds gives an indication of desiccation sensitivity. 

Indicators of desiccation sensitivity

There are also other aspects to the seed that give an indication of desiccation sensitivity including the seed size, the ratio of the seed coat to the whole seed, the seed moisture content and metabolic rate at time of maturity, the plant habit and the climatic conditions in which the seed developed. Desiccation sensitive seeds may also have a very limited endosperm, that is they only have a small energy supply and proportionally large cotyledons (seed leaves). If they are going to germinate quickly, they will soon have functioning leaves and can make their own energy from photosynthesis, doing away with the need for a stored energy supply. 

1. Prichard, H. and Dickie, J. 2003 Predicting seed longevity: the use and abuse of seed viability equations. In: Seed Conservation: Turning science into practice. The Cornwell Press, Great Britan, UK. 
2. Tweedle, J., Dickie, J., Baskin, C., Baskin, J. 2003 Ecological aspects of seed desiccation sensitivity. Journal of Ecology 91, 294-304.

Determining how long seeds survive in storage

The ultimate goal of ex situ conservation by seed banking is to hold seed in storage, indefinitely. Unfortunately, even once seeds can be successfully dried and frozen, they may not remain viable in storage for a particularly long time.

Understanding how long seeds will remain alive in storage is therefore essential to ensure the integrity of the collection. As seeds approach the time when viability has decreased by a specific amount (often 50%), it’s time to collect fresh seeds to replenish the older seeds in storage.

But how do we know when seeds have reach 50% viability? Do we just keep testing the collection? What if the seeds take a really long time to germinate? The answer to these questions is quite simple. We estimate how long seeds will survive in storage by completing an accelerated aging or 'comparative longevity' experiment that allows us to rapidly estimate the rate at which seeds in the collection die.

A comparative longevity experiment usually requires the use of 500 seeds and therefore it is only suitable for large collections. If enough seed is available, then seeds, in groups of 50, are placed in an oven at 45°C, at about 100% relative humidity. These conditions are intentionally used to age the seeds thereby reducing their quality and viability. Over a period of about three months, groups of 50 seeds are removed and germinated. Each group should have slightly less germination then the last and by tracking this proportion of germination against time, we can follow the loss of viability. The results are then compared to species for which we have real aging data and this is used to identify how the number of days in the aging conditions corresponds to the number of months (or years) the seeds would be expected to survive under cold storage. Seeds that loose all viability within a few days of the aging experiment might be expected to only live a few months in cold storage while seeds that survive several months in the aging conditions would be expected to remain viable for many years.

There is very little information available on the longevity of rainforest species, but it does appear that many rainforest seeds only remain viable for short periods of time. The naturally high moisture content of rainforest seeds means that they are often ready to germinate once shed and this is believed to be the reason why they may survive as little as one week in the soil seed bank1. Tropical species generally loose viability rapidly to extremely fast in cold storage even when using modified storage methods, while species from temperate environments may remain viable for a year2.

Research on the longevity of a selection of native Australian plants has been gathered together by Merritt et al., (2014).The methodology applied at the Australian PlantBank to test seed longevity has been developed into a technical handout by the Royal Botanic Gardens Kew (PDF, 565 KB).

1. Vazquez-Yanes, C. and Orozco-Segovia, A. (1993) Patterns of seed longevity and germination in the tropical rainforest. Annual Review of Ecology, Evolution and Systematics. 24, 69-87.
2. Berjak, P. and Pammenter, N. (2001) Seed recalcitrance - current perspectives. South African Journal of Botany 67, 79-89.

Super-cool storage!

Cryopreservation is the storage of living material in liquid nitrogen at around -196° C. At such low temperatures, all metabolic activity stops. This greatly increases the amount of time seeds can be held in storage before viability substantially decreases and a new collection must be made.

Cryopreservation for orthodox species

Cryopreservation may be utilised to store orthodox seed collections to extend the period of seed viability in storage. It is often used for intermediate and recalcitrant species that cannot tolerate cold (-20oC) storage, or desiccation, although in this case a lot of perfecting needs to occur to ensure successful preparation, storage and recovery.

Cryopreservation for rainforest species

Cryopreservation has been successfully applied to several rainforest species, however this often requires the dissection of seeds so that just the embryo remains. By dissecting the seeds the material to be stored is smaller which means it freezes a lot faster as well as the added advantage of taking up less space is the storage tank. Upon removal, the embryos need to be carefully grown on using tissue culture techniques to provide a better opportunity for survival.

Conservation in a seedbank requires seeds to be tolerant of both drying and freezing.

Some rainforest seeds don’t tolerate the drying necessary for seed banking. We’ve found others will tolerate the drying process but then don’t survive freezing! We now need to look inside these rainforest seeds1 to see what is happening as they freeze and thaw.

To do this, we’re using a differential scanning calorimeter (DSC)2. This machine can be used to lower the temperature of a seed sample to -150°C and then raise it back to room temperature. The machine records any sudden changes in energy of the seed as it freezes and thaws and the exact temperatures at which those energy changes happen. This information will help us to determine the critical temperatures at which seed damage occurs for a given species and will allow us to customise storage conditions for that species.

One familiar native plant that falls in this ‘freezing sensitive’ category is the macadamia. All four species of macadamia – Macadamia integrifolia, M. jansenii, M. ternifolia and M. tetraphylla – are native to Australian rainforest and all are threatened in the wild. We’re now using cultivated nuts supplied by the Macadamia Conservation Trust, private growers, and the Department of Primary Industries at Alstonville, to see exactly what happens to the seed as it freezes and thaws. We’ll then use that information to develop storage methods that can be used to conserve the wild species.

Determining whether rainforest seeds can be stored in the seedbank usually requires a series of time-consuming and sometimes inconclusive germination tests. To reduce the time and labour involved, and fast-track our conservation efforts, we’re also trialling use of the DSC to rapidly screen species for freezing sensitivity. Some of the seeds for this part of the project are being provided through a 5-yr collaboration to conserve tropical mountain rainforest plants led by the Australian Tropical Herbarium and funded by The Ian Potter Foundation.

1. ‘Looking inside rainforest seeds’ is a 3-yr project funded by The Ian Potter Foundation.
2. The differential scanning calorimeter was kindly donated by the Foundation and Friends of the Botanic Gardens.

Learn more about the DSC and seedbanking in our Branch Out podcast:

Tissue culture is a collection of techniques used to grow whole plants from very small pieces of plant tissue.

While we investigate alternate storage methods for recalcitrant species, plants are often maintained in tissue culture. This process involves taking small sections of plant material and growing them in agar gel enriched with nutrients and hormones which keeps the cuttings growing.

The cuttings typically grow very quickly in tissue culture (faster than they might if growing in a pot) and all the plants are 'the same age'. Additionally the cuttings are grown under sterile conditions which is required to prevent them from being killed by bacterial or fungal contamination. Each of these conditions actually makes plant material generated under tissue culture ideal for further experimentation and storage.

Fortunately, because we have germinated fresh seeds as part of our normal seed testing protocols, living material is often already available to transfer into tissue culture. For other species that we were unable to germinate or obtain seeds, cuttings can alternatively be taken from plants and transferred into tissue culture. Once the material has been placed in culture, it can be maintained there indefinitely, by simply removing the cuttings, trimming them and placing them into new agar.

The best way, by far, to conserve rainforest habitats is to stop clearing them.

According to analysis completed on the Food and Agricultural Organisation of the United Nations (FAO) Global Forest Resources Assessment 2015, global rates of deforestation halved in the period 2010-2015 compared to the 1990s, but still occurs most often in Tropical Forest, with greater frequency in poorer countries1,2. Many of these countries depend on rainforest resources and the land cleared for agriculture to support their GDP. Further pressure is added to poorer countries as wealthier countries continue to demand wood and paper products obtained from rainforest, or demand supplies of products such as coffee and palm oil which has led the conversion of rainforest habitats into ‘more profitable’ plantations.

In situ (on site) conservation

Protection of rainforest habitats is of global importance and countries around the world are moving to protect areas through the establishment of reserves and national parks. This helps to protect natural undisturbed forest, and may allow damaged rainforest to recover and be rehabilitated.

The reality is however, that the establishment of reserves does not guarantee that areas will remain intact and continue to function, nor can they guarantee that species will actually be protected within them. As such, other methods of conservation are still required to ensure the survival of all species and as much of their genetic diversity as possible. 

Ex situ (off site) conservation

The conservation of many rainforest species will utilise methods that protect material away from their natural habitat, an approach known as ex situ (off site) conservation. Methods of ex situ conservation include seedbanking, tissue culture, cryopreservation, transplanting and living collections. In the case of seedbanking, for some rainforest seeds the standard methods of seedbanking will not be appropriate and require modification to successfully stores seeds. This might include maintaining collections at a higher moisture content than would normally be used, or it might include storing seeds not in the freezer (-20°C), but in the fridge (4°C). 

1. Keenan, R., Ream, G., Achard, F., de Freitas, J., Grainger, A., Lindquist, E. 2015 Dynamics of global forest area: Results from the FAO Global Resource Assessment 2015. Forest Ecology and Management 352, 9-20.
2. Sloan, S and Sayer, J. 2015 Forest Resource Assessment 2015 shows positive global trends but forest loss and degredation persist in poor tropical countries. Forest Ecology and Management 352, 134-145.

During 2016, the Royal Botanic Gardens became a recognised authority on rainforest conservation in Australasia. This included hosting the 2016 Seed Science Forum in May, the opportunity for Dr Cathy Offord to travel interstate to facilitate the development of international standards for seed research as well as a role as editor of a seed-focused issue of the Australian Journal of Botany. Dr Karen Sommerville was also awarded another scholarship, this time to attend the National Center for Genetic Resource Preservation in Colorado USA, increasing the capacity of the Project to work with desiccation sensitive species.

Besides a continuation of rainforest seed collection and processing, activities also included hosting a rainforest researcher from Malaysia as well as an honours student and supported two PhD students, both of whom completed their research in 2016. Further developments were also made in the construction of an online rainforest seed biology course, which will complement further capacity building activities in 2017. 

Concluding research at the end of the fourth year of the Rainforest Conservation Project has also allowed for the reconciliation of all research outcomes and the identification of those goals to be targeted in 2017.

In 2015 we consolidated our capacity to collect and conserve seeds of rainforest species. The excellent field collecting conditions noted in 2014 continued this year, allowing us to exceed collection targets.

An expansion of our contacts with external seed collectors, restoration practitioners and interested public also led to the collection of several threatened species that would otherwise have been next to impossible to obtain.

Public awareness of the rainforest project and additional funding for collecting were raised through the ‘Save A Species’ walk – over 30 Gardens’ staff participated in walking a total distance of 300 km in an effort to draw attention to the plight of threatened plant species and raise money to protect them from extinction by seedbanking.

Work on understanding the seed storage behaviour of the collected species also continued. These investigations can take some time to complete for each individual species; however, we are gradually building up our knowledge of which species can be successfully stored in the seedbank and which species require alternative or supplementary measures.

We can now direct our efforts to the tissue culture and cryopreservation of difficult-to-store species and this will be a focus of our research over the next two years. In anticipation of this work, a number of species have already been successfully initiated into tissue culture. Conditions for successfully growing, rooting and transferring plants back into pots were established for seven species this year; with these conditions established, work on cryopreservation of the species can now commence.

The second year of the project has seen the expansion of activities to encompass the full range needed to reach our final goals. Collection of seeds of rainforest species has increased and a better season in some rainforest areas, especially due to increased rainfall, has enabled our scientists to focus on the most vulnerable species.

The Rainforest Seed Conservation Project continues to exceed our original expectations in terms of the species collected and the quality of the information generated. This has created great interest across a number of sectors, including the rainforest restoration industry and ecological researchers, and has firmly established the Royal Botanic Gardens & Domain Trust at the forefront of this research internationally.

The Rainforest Seed Conservation Project was launched in October 2012 at the Royal Botanic Gardens.

The project has been given a tremendous boost by the opening in October 2013 of the new conservation facility, the Australian PlantBank, located at the Australian Botanic Garden, Mount Annan. The co-location of the seed bank, tissue culture, cryostorage and other science facilities within Australia’s largest botanic garden, enables greater efficiency and outcomes from this world-class facility. 

Collection and seed analysis for the project is led by Graeme Errington, while Dr Amelia Martyn was appointed to develop and deliver the communication strategy for the project. Dr Karen Sommerville was appointed to oversee the development of alternative conservation techniques - tissue culture and cryostorage with Amanda Rollason providing technical support in the laboratory and nursery. 

By October 2013, 52 seed collections were analysed for their storage potential enabling the seedbanking of 24 species that would not otherwise have been collected nor stored. 

One group of Myrtaceae species were intensively studied for an honours year project and the results are being prepared for publication.

A list of species requiring alternative conservation techniques was developed and work began on 18 species in the living collection and 8 species in tissue culture. The new tissue culture and cryogenic facilities became operational in October 2013 and will greatly enhance this component of the project.

Training was provided to a range of sectors including local government, NGO’s, academia and Aboriginal land councils. This included workshops on the conservation of rainforest species to representatives of the mining industry Australian Network for and the Science Teachers Association. Intensive training was provided over one month to two staff from Bidoup Nuba National Park (BNNP) in Vietnam.