FI80185C - Procedure for cutting plant material - Google Patents

Description

1 80185

Method for cutting plant material In the 1960s, it was found that seedlings could be produced from parts of plants or undifferentiated callus cells. This technique is called micro-propagation, which is the asexual propagation of plants in the laboratory. The goal of micro-propagation is to produce genetically identical, valuable champion individuals. Thus, the selection of the parent plant is an essential step because the seedlings produced are replicas of it.

Micropropagation can be started from the cut point of the parent plant, a loop or, for example, a leaf stalk. Growing takes place aseptically on a medium with the main and micro-nutrients needed by the plants, vitamins and hormones to help regulate growth. The medium is usually solidified on agar.

Initially, a shoot is created from the plant part, which is transferred from the test tube to a larger glass vessel to replicate. Hormones, mainly cytokines, induce new shoots from awkward silyls or, for example, from post-silyls formed on the leaf of a plant. After about four weeks of growth, the propagated shoots are cut apart and transferred to new media to propagate. Propagation is continued until the desired number of seedlings has been produced.

For root formation, the shoots are usually transferred to a medium containing auxin. After root development, the seedlings are transferred to the ground in a greenhouse with high humidity. Gradually increasing the lighting will start the connection of the plant.

The most important cost factor for micropropagation is the abundant skilled work, which consists mainly of manual cutting and grafting of plants from one substrate to another.

2 80185

When cut, sensitive plant material can also be easily damaged. Cutting with a knife is slow, especially when working with easily contaminated material, in which case special attention should be paid to asepsis.

It has now surprisingly been found that the above-mentioned problems can be reduced by using a laser beam to cut plant material.

The essential features of the invention are set out in the appended claims.

Living tissue has previously been cut with a laser beam only in connection with surgery. In surgical surgeries, the use of a laser beam can be considered to have the advantage of burning the blood vessels of the tissue, whereby the surgery is facilitated when blood flow is prevented, especially from small vessels. In contrast, in the context of the present invention, it was surprisingly found that the conductive tendons of the plant material are not adversely damaged during laser cutting, but the cell retains its water and nutrient uptake through the cutting surface and the cell close to the cutting area retains its totipotency.

In addition, the advantages of using a laser to cut plant material are its ease of use and speed. Due to the high asepsis requirement, when working with plant material, it is usually necessary to sterilize the knife when cutting with a knife, always by dipping it in ethanol and flaming it. This slows down cutting and ethanol may enter the plant material, which, even in small amounts, delays the onset of growth or may cause plant death. Sterilization can also be performed by heating the instrument. Instead, the laser beam is naturally sterile, greatly improving asepsis. Likewise, the shear rate increases when the sterilization step is omitted.

Il 3 80185

Automation can also be combined with laser surgery. In this case, the proportion of manual work is small and cutting is considerably faster.

In laser surgery, damage to plant material is mainly due to heating. To improve the surgical result, inert shielding gases such as nitrogen, carbon dioxide or argon are used in the surgery. The shielding gas is directed to the object to be cut in an open state by means of a nozzle or alternatively the cutting is performed in a chamber filled with shielding gas. The amount of shielding gas is chosen so that carbonization is kept to a minimum.

In connection with the present invention, laser equipment was used to cut the plant material, the main parameters of which are described below.

The mode of a laser device indicates the distribution pattern of its beam. When performing shear experiments, the TEM 00 mode was continuously used as the operating mode, whereby the intensity of the beam is distributed according to the Gaussian clock curve.

The cutting power of a laser device indicates how much energy the device can transfer to the object to be cut in a unit of time. Often not all power is absorbed by the object to be cut but is reflected from the surface of the object and / or absorbed by the vapors and gases leaving the object. The shear power can also be indicated by the intensity, which indicates the shear power per radius area. Because the focusing lens focuses the beam on a very small area at the focal point, high intensity values can be achieved even with low shear power. Experiments have shown that it is possible to cut plants with a CO laser with a power of only 20 W, but the cutting speed 2 is then not high enough. It has been shown in the literature that a power of about 40 W is sufficient to cut a living animal cell. On the other hand, the price of the laser device rises in proportion to the second power of almost 4,80185 power, so the maximum economically justifiable power of the laser device is about 100 W. Based on the above, the power of a suitable laser device in the present invention should be between 30-100 W.

When pulsed with a laser beam, the values of the pulse length and the time between pulses should be chosen so that the material to be cut heats up as little as possible. Suitable values are between 0.1 and 10 ms.

The diameter of the beam at the focal point also affects the shear result, but usually this parameter is fixed and on the order of 0.2 mm or less.

In the following examples, a longitudinal flow CO (Coherent) laser with a beam mode of 2 TEM 00 and a resonator equipped with an ECQ module that allows the beam to be pulsed was used as the shear laser. The continuous power of the laser device was in principle controllable in the range of 90-350 W, but for the cutting test it was desired to reduce the power with a special gas mixture, where the fixed continuous power was 61 W. The pulse frequency was selectable from about 10-2500 Hz and a single laser pulse from 0 , 1 ms - 10 s. The diameter of the beam at the focal point was about 0.2 mm. Nitrogen with a purity of 99.998% was used as the shielding gas. The workstation consisted of an xy table measuring 600 x 600 mm.

Example

The experiments were performed on birch. The effects of surgery were monitored through an amplification experiment and subsequent greenhouse cultivation.

Because the laser beam heats the object to be cut, the plant cell may dry out or burn to char. Cutting surface damage was monitored with a microscope. If too much power was applied to the cutting surface at point 5 80185, it was often manifested as carbonization and constriction of the cord tendons, i.e., "melting" of the cell. By changing the parameters of the device, the aim was to improve the cut-off.

For cutting, plant parts were fixed in place using sterile small petri dishes filled with agar (9 g / l).

Birch, Betula pendula

Birch shoots (from in vitro cultivation) were laser cut into pieces containing a single abrasion loop. The control experiment was performed by cutting the shoots with a knife. The buds were placed on an amplification medium and grown in a growth cabinet (23 C, humidity 50%, light 2000 lux, 16/8 h). Two parallel experiments were performed (3-7 shoot pieces per experiment).

The growth onset of inocula was monitored and the amplification factor was calculated twice, 4 and 6 weeks after inoculation. The parameters according to Table 1 were tested in laser surgery.

Table 1. Parameter alternatives tested in birch laser cutting.

Shear Pulse parameters Max speed Medium mode No. Tp / ms Te / ms T / ms f / Hz% 1 m / s __________ power / w 1 0.1 0.4 0.5 2000 0.5-0, 6 15.5 2 0.1 0.7 0.8 1250 0.4-0.5 10.5 3 0.1 1.0 1.1 909 0.2 8.5 5 1.0 1.0 2 .0 500 2.0 33 6 1.0 10 11 91 0.2 12 7 10 10 20 50 0.6 32 8 continuous power 2.4 61 6 80185

Laser-cut inoculations replicated well. The multiplication of birches cut in different ways after 4 and 6 weeks of cultivation is shown in Figure 1.

The pulse length (Tp) was kept at 0.1 ms in sections 1-3, but the recovery phase (Te) was varied. When cut with method No. 1, the resting phase between pulses was 0.4 ms and with method No. 2 it was 0.7 ms. The shear rate could be kept almost the same in both. The prolonged recovery phase appears to improve cell viability, which was reflected in an increased proliferation factor. Amplification appeared to be more efficient when using cutting methods 5, 6, and 7. For the fifth experimental member, a long pulse (1.0 ms) and a long pause (1.0 ms) when running fast (2% / ms) yielded good results.

Extending the pause to 10 ms at the expense of driving speed gave the same amplification result in Experiment No. 6. Surprising Member No. 7 produced surprisingly the best growth result: it combined a very long pulse (10 ms) and a very long pulse interval (10 ms) with a reasonable driving speed (0.6 % / ms).

At constant power, a cell cut at 61 W (experimental member No. 8) appeared to multiply less on average than a cell cut at a pulsed beam.

Based on the experiment, it can be considered that the laser is well suited for chopping birch. Reproduction was at least as effective as with knife cutting. Birches were normally rooted after amplification following laser surgery. The best amplification was achieved by pulsing Experiment No. 7, where a long recovery time is assumed to have prevented the cell from overheating and burning. From the experiment, it can also be concluded that a low-frequency oscillating laser causes the least damage to plants (Method 7).

Il

Claims (5)

7 80185 1. A method for microstillary material for microtillers, which can be used for cleaning the genome by means of a pulsed laser field. A method for cutting plant material for micro-propagation of plants, characterized in that the cutting is performed using a pulsed laser beam. 2. A method according to claim 1, comprising a laser laser CO. 2 Method according to Claim 1, characterized in that a CO laser is used as the laser. 2 3. A patent according to claims 1 or 2, which can be performed under pulsating genomic effects with variations between 5 and 40 W. Method according to Claim 1 or 2, characterized in that the average power during pulsation varies between 5 and 40 W. 4. The subject-matter of the patent claims, which is based on the subject matter of the patent application, is indicated. Method according to one of the preceding claims, characterized in that a shielding gas is used around the object to be cut. Method according to Claim 4, characterized in that an inert gas, preferably nitrogen or carbon dioxide, is used as the shielding gas. 5. A method according to claim 4, which comprises the use of an inert gas, preferably a solution or a monoxide.