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Improved Plant Culture Methods Using a Modified Auxin Treatment Step

Field of the Invention

The present invention relates to an improved method of culturing plants involving a modified auxin pulsing step and the use of an aerobic rooting medium. The present invention also provides a method of defining a plant tissue culture method for a plant.

Background

Plant tissue culture techniques have been used to successfully propagate a number of plant species. However, some species have been shown to be particularly difficult to strike roots and hence cultivate using traditional plant tissue culture techniques.

Whilst there can be a number of reasons why plant tissue culture fails or is only partly successful when applied to certain plants, one reason is that at certain stages of the process, such as root development, the environment provided to the plant is not conducive to the optimal growth and differentiation of the plant. This can result in the plant either failing to survive the process or surviving the process in a weakened state that affects its later development when transferred to the nursery or planted out.

Newell et al (2002) "A culture system that gives improved root strike on Australian plant micro-cuttings" [in Taji A, and Williams R (eds) The Importance of Plant Tissue Culture and Biotechnology in Plant Science, University of New England Publication Unit, Australia pp159-164], Newell et al (2003) "The influence of medium aeration on in vitro rooting of Australian plant micro cuttings" [Plant Cell, Tissue and Organ Culture 75: 131-142] and Newell et al (2005) "A Novel in vitro rooting method employing aerobic medium with wide application to Australian flora [Aust Journal of Botany 53: 81-89] describe a method for culturing a number of plant species involving an initial pulsing step for seven days using high auxin agar solidified medium and the use of an aerobic

propagation medium during the rooting phase. The pulsing treatment with auxin is carried out to initiate the rooting process.

The present invention seeks to provide an improved plant tissue culture method that overcomes or at least partially alleviates problems with prior art techniques. At the very least the present invention seeks to provide an attractive alternative to prior art methods.

Summary of the Invention

The present invention provides a method of culturing a plant comprising the steps of:

(i) contacting tissue from the plant for less than 7 days with a first medium containing an auxin capable of initiating rooting; and

(ii) culturing the tissue from step (i) to the stage of mature plantlet in a porous second medium.

The present invention also provides a method of culturing a plant comprising the steps of:

(i) contacting tissue from the plant with a first medium containing an auxin capable of initiating rooting;

(ii) culturing the tissue from step (i) to the stage of mature plantlet in a porous second medium having an air filled porosity of at least 30%; and

(iii) transferring the plantlet from step (ii) to a nursery environment whereby the plantlet can become acclimatised to the environment.

The present invention also provides a method of defining a plant tissue culture method for a plant comprising the steps of:

(i) contacting a plurality of tissue samples of the plant for less than 7 days with a first medium containing an auxin capable of initiating rooting;

(ii) culturing the plurality of tissue samples from step (i) to the stage of mature plantlet in a porous second medium; and

(iii) comparing at least one of: percentage rooting, average total root length and root number per rooted plant; of the plantlets from step (ii) with at least one comparator plantlet of the same species of plant that has been cultured from tissue that was contacted with said first medium for longer than the period in step (i).

The present invention also provides a method of defining a plant tissue culture method for a plant comprising the steps of:

(i) contacting a plurality of tissue samples of the plant for less than 7 days with a first medium containing an auxin capable of initiating rooting;

(ii) culturing the plurality of tissue samples from step (i) to the stage of mature plantlet in a porous second medium; and

comparing at least one of: percentage rooting, average total root length and root number per rooted plant; of the plantlets from step (ii) with at least one comparator plantlet of the same species of plant that has been: (a) cultured from tissue that was contacted with said first medium for longer than the period in step (i); and (b) cultured to the stage of mature plantlet in a porous second medium with a different porosity than the medium in step (ii).

Detailed Description of the Invention

Method of culturing a plant

The present invention provides a method of culturing a plant comprising the steps of:

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(i) contacting tissue from the plant for less than 7 days with a first medium containing an auxin capable of initiating rooting; and

(N) culturing the tissue from step (i) to the stage of mature plantlet in a porous second medium.

The present invention is based on the surprising discovery that viable mature plantlets, that are capable of being successfully acclimatised and subsequently planted out, can be produced using a method including a pulsing step of shorter duration than the prior art methods and porous media. A shorter pulsing step reduces the time taken to carry out the entire process with obvious advantages, at least in respect of time saved. Furthermore, and whilst not wishing to be bound to any particular mode of action, it is believed that the shorter pulsing step places less stress on the plant tissue and thus enables the production of healthier more vigorous plants due to improvements in at least one of percentage rooting, average total root length and root number per rooted plant.

The method may be applied to a range of plants including any member of the following families: Vitaceae, Lauraceae, Myrtaceae, Verticordiidae, Thymelaeaceae, Rutaceae, Proteaceae, Haemodoraceae, Goodeniaceae, Vitaceae, Rosaceae, Santalaceae and Capparaceae.

More particularly the method may be applied to any member of the following genera: Eucalyptus including E. marginata, Santalum including S. spicatum and

S. album, Capparis, Cleome, Isomeris, Malus including Malus angustifolia, Malus asiatica, Malus baccata, Malus coronaria, Malus domestica, Malus doumeri,

Malus florentina, Malus fusca, Malus halliana, Malus honanensis, Malus hupehensis, Malus ioensis, Malus kansuensis, Malus mandshurica, Malus micromalus, Malus neidzwetzkyana, Malus niedzwetzkyana, Malus ombrophilia,

Malus orientalis, Malus prattii, Malus prunifolia, Malus sargentii, Malus sieboldii,

Malus sieversii, Malus sylvestris, Malus toringoides, Malus transitoria, Malus trilobata, Malus tschonoskii, Malus yunnanensis, M. communis and M. pumila,

Persea including P. americana, P. gratissima, P. drymifolia, P. nubigena and P. guatemalensis, Vitis, Parthenocissus, Anthotium, Anigozanthos, Conospermum,

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Dampeira, Goodenia, Grevillea, Lechenaultia, Scaevola, Verticordia, Pimelea, Eriostemon, and Geleznowia.

The tissue from the plant can be varied provide it is capable of being cultured. Preferably the tissue is a micro-cutting. Even more preferably it is a stable stage 2 micro-cutting.

The first medium may be varied provided it is able to support the tissue during the pulsing stage. Preferably, the first medium is an agar medium and more particularly a solidified agar medium.

The auxin can be varied and is preferably an auxin adapted to promote the formation and/or growth of roots. Examples of auxins for use in the present invention include indole butyric acid (IBA) and naphthalene acetic acid (NAA).

The duration of the auxin treatment in step (i) may be varied provided it is less than 7 days. The duration of the auxin treatment step may be 1-6 days (i.e. 24 - 144 hours) or 1-5 days (i.e. 24-120 hours) but it is preferably 1-3 days (i.e. 24-72 hours).

At the conclusion of step (i) root growth has been initiated and the tissue is ready for step (ii). Thus, when the tissue treated with auxin in step (i) is a micro-cutting the tissue cultured in step (ii) is an initiated micro-cutting.

The porous second medium may be varied and includes a medium having at least 2% air filled porosity, more preferably at least 5-10% air filled porosity and even more preferably at least 15-29% air filled porosity. In one form of the invention the air filled porosity of the porous medium is at least 30-75% or 30- 45%. The porous medium may be formed from one or more of a variety of components to deliver the required porosity.

Preferably, the porous second medium comprises at least one of: partially carbonized vegetable matter e.g. peat or sphagnum peat, sand e.g. coarse river sand and siliceous rock e.g. perlite. In one particular form, the porous second medium comprises a combination of sphagnum peat, coarse river sand and

- - perlite. One example of a porous medium for use in the present invention is one comprising sphagnum peat, coarse river sand and perlite (grade P500) in a ratio of 0.5:2:2.

For the purposes of the present invention the term "mature plantlet" means a plantlet that is capable of being acclimatised and planted out successfully. This is largely determined by the level of root development in the mature plantlet.

The method of the present invention may be used to prepare large numbers of homologous plants for planting out in into large scale commercial nurseries where the plants can then be on sold to the market for planting in a desired location. Thus, the present invention also provides a method of culturing a plant comprising the steps of:

(i) contacting tissue from the plant with a first medium containing an auxin capable of initiating rooting;

(ii) culturing the tissue from step (i) to the stage of mature plantlet in a porous second medium having an air filled porosity of at least 30%; and

(iii) transferring the plantlet from step (ii) to a nursery environment whereby the plantlet can become acclimatised to the environment.

The plantlet can be sustained in the nursery environment for as long as necessary prior to sale to the market.

Method of customising a plant tissue culturing method for a plant

The present invention is based on the surprising discovery that the duration of the auxin treatment step can be reduced with concomitant advantages. Thus, the present invention identifies a parameter within a plant tissue culture method that can be varied to generate customised methods for culturing plants. Thus, the present invention also provides a method of defining a plant tissue culture method for a plant comprising the steps of:

(i) contacting a plurality of tissue samples of the plant for less than 7 days with a first medium containing an auxin capable of initiating rooting;

(ii) culturing the plurality of tissue samples from step (i) to the stage of mature plantlet in a porous second medium; and

(iii) comparing at least one of: percentage rooting, average total root length and root number per rooted plant; of the plantlets from step (ii) with at least one comparator plantlet of the same species of plant that has been cultured from tissue that was contacted with said first medium for longer than the period in step (i).

This method may be used to optimise an important step in the culture method that may vary for different plants. Preferably, the comparator plantlet is cultured in said first medium for at least 7 days.

Another important parameter is the porosity of the porous second medium. In this regard, to optimise the culture of plants using the method of the present invention it may be necessary to also customise the porosity of the porous second medium. Thus, the present invention also provides a method of defining a plant tissue culture method for a plant comprising the steps of:

(i) contacting a plurality of tissue samples of the plant for less than 7 days with a first medium containing an auxin capable of initiating rooting;

(ii) culturing the plurality of tissue samples from step (i) to the stage of mature plantlet in a porous second medium; and

(iii) comparing at least one of: percentage rooting, average total root length and root number per rooted plant; of the plantlets from step (ii) with at least one comparator plantlet of the same species of plant that has been: (a) cultured from tissue that was contacted with said first medium for longer than the period in step (i); and (b) cultured to the stage of mature plantlet in a porous second medium with a different porosity than the medium in step (ii).

Preferably, the comparator plantlet is cultured in said porous second medium with a porosity of about 29%.

General

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention as described herein.

The entire disclosures of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are hereby incorporated by reference. No admission is made that any of the references constitute prior art or are part of the common general knowledge of those working in the field to which this invention relates.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

Examples

Example 1 - Improved method for the in vitro culture of soft and woody plants using a shorter period of pulsing

Materials and methods

Microcuttings from established shoot cultures were pulsed for 1-3 days on a high auxin (40μM IBA), agar-solidified medium (RM 40 (0.5MS for soft plants) or (0.5MS Modified for Woody plants) +4OuM IBA+10gm/sucrose, pH 7, set with 8gm/l agar - sterilised at 121 ⁰ C for 20 minutes) in the dark.

The microcuttings were then transferred to porous media (air filled porosity of 29%) in the form of sphagnum peat/coarse river sand and perlite grade P500 (ratio of 0.5:2:2), pH 6.8 sterilised at 121 ⁰ C for 40 minutes and then placed in the culture room under standard light and temperature regimes (22 ⁰ C 16hr/8hr light dark at 50umo!em ^"2 s ^"1 ).

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Media Formulations

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Results

The plants in Table 1 hereunder have been successfully cultured using this methodology.

Example 2 - Improved method for the in vitro culture of soft and woody plants using a shorter period of pulsing and high porosity media

Materials and methods

Microcuttings from established shoot cultures were pulsed for 1-3 days on a high auxin (40μM IBA), agar-solidified medium (RM 40 (0.5MS for soft plants) or (0.5MS Modified for Woody plants) +4OuM IBA+IOgm/sucrose, pH 7, set with 8gm/l agar - sterilised at 121 ⁰ C for 20 minutes) in the dark.

The microcuttings were then transferred to highly porous media (air filled porosity of 30-45%) in the form of sphagnum peat/coarse river sand and perlite grade

P500 (ratio of 0.5:2:2), pH 6.8 sterilised at 121 ⁰ C for 40 minutes and then placed in the culture room under standard light and temperature regimes (22 ⁰ C 16hr/8hr light dark at 50umolerτϊ ² s ^"1 ).

Results

The plants in Table 1 above have also been successfully cultured using this methodology. However, the results obtained are generally superior using this methodology than the methodology in Example 1.

Example 3 - Improved plant tissue culture methods using a modified Auxin treatment step

Materials and Methods

Micro cuttings of established stock plants Grevillea thelmanniana, Red Leschenaultia formosa, Pimelea physodes, Geleznowia verrucosa, Goodenia scapigera, Chamelucium υncinatum were taken from the DAFWA in vitro tissue culture laboratories located at South Perth.

Each culture line was divided and pulsed with auxin. This was achieved by inserting micro cuttings into agar media comprising 1/2 Ms (Murashige and Skoog, 1962) with 10 g/L sucrose and 8 g/L agar (pH adjusted to 7 before autoclaving) and containing 4OmM of IBA (indole-3-butyric acid) in 250 ml clear plastic tubes. These were then placed in the dark in a constant temperature room 20-22 ⁰ C for different lengths of time: 24 h (1d), 48h (2d), 72 h (3d), 96 (4d), 168 h (7d).

At the completion of the pulsing period, shoots were transferred aseptically to porous media for root initiation and growth. Porous media was prepared containing 1 part course river sand, ^λ A Sphagnum Moss (Canadian) and 2 parts perlite 500 (large and sieved), with an air filled porosity (AFP) of 24%. 1.25 g/L of powdered lime was added to bring the pH of the media to pH 6. The porous media was added to seedling punnets (Masrac™ 720 MK8LL punnet) and was placed in a steam enclosure for 5 hours and then autoclaved for 40 min at 121 ⁰ C.

Sterilised water (121 ⁰ C for 1 h) was added to bring the porous mix to field capacity. Under sterile conditions, ten pulsed micro cuttings were individually placed, stem ends first, into the porous mix in the punnet. The punnet was then placed in a sterilised 1L plastic take-away vented food container with a second unvented inverted food container as a "lid" and the joint sealed with Parafilm™ or cling wrap. Cuttings in food containers were maintained at 20-22° C with a 16h photo period at a light intensity of 50μmol m ^'2 s ^"1 during root growth phase. There were 3 punnets (reps) containing 10 cuttings per treatment for the 5 treatment pulsing times.

At the end of the growth phase plants were harvested and scored in relation to: plant health (1 dead, 5 healthy), root strike (ie number of plants that produced roots) main root number, and root length (measured using the Assess computer image based program). Plant dry weight, plant top dry weight and root dry weight were used to calculate % of root per plant on a dry weight basis. All results were analysed using Genstat VII statistical package.

After 6 weeks and/or sufficient root development had occurred, the plastic food containers were placed in a greenhouse under a douche and, after seven days, the inverted plastic food container "lids" were removed. Plants were left under the douche for a further 3 weeks and then removed to outside the douche in the greenhouse. They were then pricked out and potted on in sand: peat: perlite (1 : >.>: 1) potting mix. Plants were then made ready for planting out in the field or potted on into larger display pots for pot plants

Results

Large difference were observed between species in root length measured at harvest, with Goodinea scapigera having by far the most root length and Pimelea physodes and Geleznowia verrucosa the least amount of roots. Analysed across all species tested, the percentage weight of roots per total plant weight was significantly higher for auxin treatment times of 1 , 2, 3 and 4 days compared auxin treatment time of 7 days. There were no significant effects on root length between treatments whereas root numbers per plant were 60% higher following auxin treatment of 1 day compared to other treatment times. There was variation

- - in the response between species to time of auxin treatment. Red Leschenaultia produced the highest percentage root weight following auxin treatment of 1 day and the least percentage following auxin treatment of 7 days. Goodenia scapigera produced the greatest number of roots following auxin treatment of 1 day, with a two fold increase over other auxin treatment times. Grevillea thelmanniana root length and percentage weight of roots was highest after 3 or 4 days auxin treatment. For Geleznowia verrucosa, root length was 4 to 7 fold longer than using 2, 3 and 4 days auxin treatment compared to 1 or 7 days auxin treatment, although this effect was not significant. For Pimelea physodes there was a higher percentage weight of roots following auxin treatment times of 1 , 4 and 7 days.

It appears that timing of auxin pulse treatment affected root growth. Of the range of times for auxin treatment tested, 1 day appears to give a sufficient pulse to induce good root growth for a range of species. Increasing the auxin exposure time up to 4 days could be beneficial in terms of root numbers for some species. For very difficult species such as Pimelea physodes it may be beneficial to increase pulse times to 7 days, but this was not necessary for most species.