Method and equipment for cryopreservation of garlic callus

Abstract

The application belongs to the technical field of super-low temperature storage of agricultural products, and discloses a method and a device for super-low temperature storage of garlic callus, wherein callus with good growth potential is subjected to pre-culture; the callus is loaded, dehydrated and vitrified by using a cryoprotective agent; the callus is warmed after being treated by the cryoprotective agent; the callus is recovered after being treated by the cryoprotective agent; and the recovered callus is inoculated into a culture medium for primary culture and propagation. The method can effectively improve the super-low temperature storage effect of garlic callus, and improve the survival rate of the callus after super-low temperature storage. The pre-culture time is 2 days, the loading medium loading time is 20 minutes, and the PVS2 vitrification solution treatment time is 15 minutes, so that the survival rate of the callus is obviously improved. The method can be applied to super-low temperature storage of garlic callus, and is simple in operation process and easy to apply to production, and can be used for efficient and safe storage of garlic germplasm resources.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of super-low temperature preservation of agricultural products, and particularly relates to a method for super-low temperature preservation of garlic callus and a super-low temperature preservation device. BACKGROUND

[0002] At present, garlic (English name: Garlic; Latin name: Allium sativum L.) is the underground bulb of the Liliaceae Allium plant. China is the largest garlic planting and exporting country in the world. Garlic is one of the important crop varieties, and garlic and its products are the traditional seasoning foods that people like.

[0003] At the same time, garlic is a very seasonal agricultural product. Fresh garlic can be preserved by refrigeration, but it can only be stored until the next year's new garlic is on the market, otherwise it will lose its commercial value. China is a big garlic exporter, and the profit it brings each year is particularly considerable. Therefore, it is very important to study different excellent varieties of garlic, prolong the preservation time, and improve the yield of high-quality varieties.

[0004] Super-low temperature preservation technology is currently the only method for long-term effective preservation of plant resources, which is a biological technology for preserving planting resources in super-low temperature below-80℃, and liquid nitrogen is most commonly used. Super-low temperature preservation generally means that the metabolic and growth activities of the preserved living cells are almost completely stopped in a super-low temperature environment of-196℃ (liquid nitrogen). This method can maintain the genetic stability of biological materials and also does not lose the potential of morphogenesis. Super-low temperature preservation technology can preserve and rescue species and avoid the occurrence of species mutation, and is an important method for long-term stable preservation of plant species resources and precious experimental materials. So far, more than 100 plants have been used for super-low temperature preservation. In the early stage of super-low temperature preservation, freezing preservation technology was mainly used, which is mainly divided into fast and slow methods. With the continuous maturity of technology, there are vitrification method, dry freezing method, embedding dehydration method, etc. The vitrification method of super-low temperature preservation technology is currently an ideal method for long-term stable preservation of plant germplasm resources.

[0005] Traditional super-low temperature preservation of garlic is achieved by manually stripping the stem tip under a microscope. This method not only requires mature stripping stem tip operation technology, but also needs to be strictly carried out under sterile conditions during the stripping process. It is not only time-consuming and labor-intensive, but also easy to be contaminated, which brings great challenges to the large-scale super-low temperature preservation of garlic resources. Callus is a tissue block formed by the totipotent characteristics of cells, which not only has good detoxification effect, but also is easy to realize large-scale propagation. If a super-low temperature preservation method for garlic through callus can be invented, it will provide a new idea and method for the safe preservation of garlic resources.

[0006] Through the above analysis, the problems and defects of the prior art are:

[0007] (1) There is no report on garlic callus, and no reference method.

[0008] (2) In the prior art, the survival rate of garlic other tissues after cryopreservation is not high.

[0009] (3) In the prior art, the growth rate of other tissues of garlic is slow.

[0010] The difficulty of solving the above problems and defects is:

[0011] (1) There is no method for cryopreservation of garlic callus.

[0012] (2) Related research started late in China, and there is little data in the prior art and no system.

[0013] (3) Different garlic explants, even different garlic varieties, have different basic culture media and hormone ratios suitable for in vitro induction of adventitious buds. However, in the current research, the exploration of wide adaptability of garlic in vitro regeneration system is less involved.

[0014] The significance of solving the above problems and defects is:

[0015] (1) A method for cryopreservation of garlic callus is invented.

[0016] (2) Callus can be used as an important carrier tissue for garlic resource preservation, and has wide application value.

[0017] (3) Improve the cryopreservation effect of garlic tissue and improve the survival rate.

[0018] (4) Establish a cryopreservation system suitable for garlic resources.

[0019] (5) Efficient and safe preservation of garlic germplasm resources. SUMMARY

[0020] In order to solve the problems of the prior art, the present application provides a method for cryopreservation of garlic callus and a cryopreservation device.

[0021] The present application is realized in that a method for cryopreservation of garlic callus comprises:

[0022] S1, the callus with good growth potential is taken and inoculated in a culture dish containing Gamborg B5 basic medium+NAA+2iP, and the inoculated culture dish is placed in darkness for pre-culture;

[0023] S2, PVS2 is loaded as a cryoprotectant, dehydrated and vitrified;

[0024] S3, warming of the PVS2-treated calli;

[0025] S4, recovery of the PVS2-treated calli;

[0026] S5, subculture of the recovered calli into fresh medium for primary culture and propagation.

[0027] Further, the implementation process of the step S1 is as follows:

[0028] Step S11, configuration of Gamborg B5 culture dish containing 2iP and NAA: add Gamborg B5 basal medium (G398 2 ) 3.21 g / L, 2iP 0.05 mg / L, NAA 0.01 mg / L, sucrose 30 g / L, agar 6.5 g / L to distilled water;

[0029] Step S12, inoculation: inoculate the calli in the culture dish under the condition of 5℃ in the dark for 2 days of pre-culture respectively.

[0030] The 2iP is stored in frozen state at minus 20 degrees, and needs to be pre-packaged to avoid repeated freezing and thawing of 2iP.

[0031] Further, the implementation process of the step S2 is as follows:

[0032] Step S21, loading: transfer the pre-cultured calli to the culture dish of loading medium, and soak the calli for twenty minutes;

[0033] Step S22, dehydration: take out the calli after twenty minutes, replace it with frozen (0℃) PVS2 vitrification solution, and place the calli in the PVS2 solution on ice for fifteen minutes;

[0034] Step S23, vitrification: after fifteen minutes, use tweezers to place 4-5 calli on a sterile foil strip (0.5*15 mm). There is no need to remove excess PVS2 from the aluminum foil, and immediately insert the foil strip into a shallow container containing liquid nitrogen.

[0035] The culture dish of the loading medium is MS basal salt mixture 5 (M524 2 ) 2.165 g / L, glycerol 184.2 g / L, sucrose 205.4 g / L, MS-G 6 25 ml and MS-vitamins 6 10 ml, wherein MS-G 6 and MS-vitamins 6 need to be prepared in advance.

[0036] The MS-G 6 The solution is prepared from thiamine 0.08 g / L, myo-inositol 20 g / L and is stored frozen. MS-vitamins 6 The solution is prepared from glycine 0.2 g / L, myo-inositol 10 g / L, nicotinic acid 0.05 g / L, pyridoxine hydrochloride 0.05 g / L and thiamine 0.01 g / L and is stored at 5°C.

[0037] The PVS2 vitrification solution is prepared from glycerol 300 g / L, ethylene glycol 135 ml, reagent grade sucrose 137 g / L and DMSO 136 ml and needs to be filtered in a clean bench.

[0038] Further, the specific operation process of the step S3 is as follows:

[0039] Once the lid is heated to be opened, the frozen foil strip is taken out from the cryovial. The foil strip is soaked in 1.2M sucrose + 1 / 2MS medium for twenty minutes, and the new solution is changed after ten minutes. Once the callus is heated to be shed, the foil strip is removed from the medium. After twenty minutes, the tissue is transferred to a new petri dish containing Gamborg B5 basal medium + NAA + 2iP, and the petri dish is wrapped with aluminum foil (to prevent exposure to light) and placed in a growth chamber for 24 hours.

[0040] The 1.2M sucrose + 1 / 2MS medium is MS basal medium w / vitamins 5 (M519 2 ) 4.43 g / L, sucrose 410.76 g / L.

[0041] Further, the specific operation process of the step S4 is as follows:

[0042] After 24 hours, the tissue is moved to a new petri dish containing Gamborg B5 basal medium + NAA + 2iP, wrapped with tin paper, and the petri dish is placed in a growth chamber for four days, then the foil paper is removed, and the tissue is placed under dim light for another five days. After five days, the tissue is moved to full light, and the tissue is placed in a petri dish until it grows beyond the space of the petri dish, and then the tissue is moved to a petri dish containing Gamborg + NAA + 2iP.

[0043] Another object of the present application is to provide a garlic callus cryopreservation device for implementing the garlic callus cryopreservation method.

[0044] In combination with all the technical solutions above, the method for garlic callus cryopreservation provided by the application has the advantages and positive effects that: the callus with good growth potential is inoculated on a Gamborg B5 culture dish containing 2iP and NAA, and the inoculated culture dish is placed in darkness for pre-culture; PVS2 is used as a cryoprotectant for loading, dehydration and vitrification; the callus after PVS2 treatment is rewarmed; the callus after PVS2 treatment is recovered; and the recovered callus is inoculated in a culture medium for primary culture and propagation. The pre-culture time of the culture dish containing Gamborg B5 basic medium+NAA+2iP in darkness is 2 days, the loading time of 2M glycerol+0.6M sucrose+1 / 2MS loading medium is 20 minutes, and the PVS2 vitrification solution treatment time is 15 minutes, so that the problem of low survival rate of garlic callus after cryopreservation is solved.

[0045] Through the method of the application, the cryopreservation of garlic callus is realized; through condition optimization, the survival rate of garlic callus after cryopreservation is effectively improved; the callus selected by the application saves the tedious work of peeling the stem tip in the traditional stem tip cryopreservation, has the advantages of time saving, labor saving and cost saving, has popularization significance and effectively reduces the pollution rate.

[0046] The pre-culture time is 2 days, the loading time of the loading medium is 20 minutes, the PVS2 vitrification solution treatment time is 15 minutes, and the survival rate of the callus is obviously improved.

[0047] The method of the application is simple and easy to apply to production, and can be applied to the cryopreservation of garlic callus.

[0048] The method of the application can be used for the preservation of garlic germplasm resources, effectively preserves the germplasm, and effectively improves the utilization of the germplasm resources.

[0049] The effects and advantages obtained by combining experimental or test data and the prior art are as follows:

[0050] (1) The callus of garlic used in the application is a creative invention.

[0051] (2) According to the literature, the survival rate of the stem tip cryopreserved by the garlic stem tip vitrification method is about 70% to 80%, and the survival rate of the application is about 75% to 90%, which greatly improves the survival rate of garlic cryopreservation.

[0052] (3) The experimental materials such as stem tips are not easy to obtain, and must be prepared through complicated disinfection, etc. The disinfection time is too long to affect the physiological condition of the stem tip, and the disinfection time is insufficient to cause pollution of the material itself. The cryopreservation of callus material is convenient to use at any time, so the cryopreservation steps are simplified, the material pollution is reduced, and the advantages of time saving, labor saving and cost saving are obtained, which has popularization significance. BRIEF DESCRIPTION OF DRAWINGS

[0053] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required to be used in the embodiments of the present application will be briefly introduced as follows. Obviously, the drawings described below are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0054] Figure 1 is the main effect diagram of the callus ultra-low temperature preservation orthogonal experiment analysis provided by the embodiments of the present application.

[0055] Figure 2 is the comparison diagram of the traditional method and the method of the present application.

[0056] In the figure: A, the effect diagram of the traditional stem tip method; B, the effect diagram of the callus method of the present application. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical solutions and advantages of the present application more clear, the present application will be further described in detail in combination with embodiments. It should be understood that the specific embodiments described here are only used to explain the present application, and are not used to limit the present application.

[0058] In the prior art, there is no method for realizing the ultra-low temperature preservation of garlic resources through callus.

[0059] In the prior art, the effect of the ultra-low temperature preservation of garlic bulbs is poor. In the prior art, the survival rate of garlic varieties after the ultra-low temperature preservation of callus is low.

[0060] In view of the problems in the prior art, the present application provides a method for the ultra-low temperature preservation of garlic callus and an ultra-low temperature preservation equipment, which will be described in detail in combination with the drawings.

[0061] The method for the ultra-low temperature preservation of garlic callus provided by the embodiments of the present application comprises:

[0062] S101, the callus with a good growth trend is taken and inoculated on a culture dish containing Gamborg B5 basic medium+NAA+2iP, and the inoculated culture dish is placed in darkness for pre-culture.

[0063] S102, PVS2 is used as a low temperature protective agent for loading, dehydration and vitrification.

[0064] S103, the callus after PVS2 treatment is thawed.

[0065] S104, the callus after PVS2 treatment is recovered.

[0066] S105, the recovered callus is inoculated into a culture medium for primary culture and propagation.

[0067] The implementation process of step S101 is as follows:

[0068] Step S11, configure Gamborg B5 culture dish containing 2iP and NAA: add Gamborg B5 basal medium (G398 2 ) 3.21 g / L, 2iP 0.05 mg / L, NAA 0.01 mg / L, sucrose 30 g / L, agar 6.5 g / L to distilled water.

[0069] Step S12, inoculation: inoculate the callus in the culture dish under the condition of 5℃ in the dark, and carry out pre-culture for 2 days respectively.

[0070] The 2iP is stored at minus 20 degrees for freezing, and should be pre-packaged to avoid repeated freezing and thawing of 2iP.

[0071] The implementation process of step S102 is as follows:

[0072] Step S21, loading: transfer the pre-cultured callus to the culture dish of loading medium, and soak the callus for twenty minutes;

[0073] Step S22, dehydration: after twenty minutes, take out the callus, replace it with frozen (0℃) PVS2 vitrification solution, and place the callus in PVS2 solution on ice for fifteen minutes;

[0074] Step S23, vitrification: after fifteen minutes, use tweezers to place 4-5 calli on a sterile foil strip (0.5*15 mm). There is no need to remove excess PVS2 from the aluminum foil, and immediately insert the foil strip into a shallow container containing liquid nitrogen.

[0075] The culture dish of loading medium is MS basal salt mixture 5 (M524 2 ) 2.165 g / L, glycerol 184.2 g / L, sucrose 205.4 g / L, MS-G 6 25 ml and MS-vitamins 6 10 ml, wherein MS-G 6 and MS-vitamins 6 need to be prepared in advance.

[0076] The MS-G 6 solution is prepared by thiamine 0.08 g / L, inositol 20 g / L, and stored in the freezer. MS-vitamins 6The solution is prepared from glycine 0.2 g / L, myo-inositol 10 g / L, nicotinic acid 0.05 g / L, pyridoxine hydrochloride 0.05 g / L and thiamine 0.01 g / L and stored at 5°C.

[0077] The PVS2 vitrification solution is prepared from glycerol 300 g / L, ethylene glycol 135 ml, reagent grade sucrose 137 g / L and DMSO 136 ml and filtered on a clean bench.

[0078] The specific operation process of the step S103 is as follows: once the lid is heated to be opened, the frozen foil strip is taken out from the cryogenic bottle immediately. The foil strip is soaked in 1.2M sucrose+1 / 2MS medium for twenty minutes, and the new solution is changed after ten minutes. Once the callus is heated to fall off, the foil strip is removed from the medium. After twenty minutes, the tissue is transferred to a new culture dish containing Gamborg B5 basic medium+NAA+2iP, the culture dish is wrapped with aluminum foil (to prevent exposure to light), and is placed in a growth chamber for 24 hours.

[0079] The 1.2M sucrose+1 / 2MS medium is MS basal medium w / vitamins 5 (M519 2 )4.43 g / L, sucrose 410.76 g / L.

[0080] The specific operation process of the step S104 is as follows:

[0081] After 24 hours, the tissue is moved into a new culture dish containing Gamborg B5 basic medium+NAA+2iP, is wrapped with tin paper, and is placed in a growth chamber for four days, then the foil paper is removed, and is placed under dim light for another five days. After five days, the tissue is moved to full light, is placed in a culture dish, and is grown until it exceeds the space of the culture dish, and then is moved to a culture dish containing Gamborg+NAA+2iP.

[0082] The application will be further described in connection with specific examples and experiments.

[0083] Examples

[0084] The method for cryopreservation of garlic callus provided by the embodiments of the application comprises the following steps:

[0085] In the step S1, the callus with a good growth potential is inoculated on a culture dish containing Gamborg B5 basic medium+NAA+2iP, and the inoculated culture dish is placed in darkness for pre-culturing.

[0086] Select full no damage garlic as experimental material, configuration contains 2iP and NAA Gamborg B5 culture dish: to distilled water in the addition Gamborg B5 basal medium (G398 2 ) 3.21g / L, 2iP 0.05mg / L, NAA 0.01mg / L, sucrose 30g / L, agar 6.5g / L, take the better growth potential callus access in containing Gamborg B5 basal medium+NAA+2iP culture dish, with sealing film, the inoculated culture dish is placed in the dark, pre-culture, callus inoculation in culture dish is placed in the dark 5 ℃ conditions, respectively, pre-culture 2 days.

[0087] Step S2, PVS2 as cryoprotectant loading, dehydration, vitrification.

[0088] Preparation of loading medium culture dish for MS basal salt mixture 5 (M524 2 ) 2.165g / L, glycerol 184.2g / L, sucrose 205.4g / L, MS-G 6 25ml and MS-vitamins 6 10ml, wherein MS-G 6 And MS-vitamins 6 . The pre-culture callus is transferred to the loading medium culture dish, and the callus is soaked for twenty minutes. After twenty minutes, the callus is removed, and the callus is replaced with a frozen (0 ℃) PVS2 vitrification solution. The callus is placed in the PVS2 solution and placed on ice for fifteen minutes. After fifteen minutes, 4-5 calli are placed on a sterile foil strip (0.5*15mm) using tweezers. There is no need to remove excess PVS2 from the aluminum foil, and the foil strip is immediately inserted into a shallow container containing liquid nitrogen.

[0089] Step S3, the callus after PVS2 treatment is warmed up.

[0090] Once the lid is heated to open, the frozen foil strip is removed from the cryovial. The foil strip is immersed in 1.2M sucrose+1 / 2MS medium for twenty minutes, and the solution is replaced after ten minutes. Once the callus is heated to fall off, the foil strip is removed from the medium. After twenty minutes, the tissue is transferred to a new culture dish containing Gamborg B5 basal medium+NAA+2iP, and the culture dish is wrapped with aluminum foil (to prevent exposure to light) and placed in a growth chamber for 24 hours.

[0091] Step S4, the callus after PVS2 treatment is recovered.

[0092] After 24 hours, the tissue heads are moved into new Petri dishes containing Gamborg B5 basal medium + NAA + 2iP, wrapped with tin foil, and placed in the growth chamber for four days, then the foil is removed and placed under dim light for another five days, after which the tissue is moved to full light and placed in Petri dishes until it outgrows the space, then it is moved to Petri dishes containing Gamborg + NAA + 2iP.

[0093] Step S5, the recovered callus is inoculated into the medium for primary culture and propagation.

[0094] The method for cryopreservation of callus is determined by orthogonal experiment, B5 medium is used as the basic medium, and three components, i.e., pre-culture time, loading time and PVS2 treatment time, are designed at three levels, then the experiment is carried out through orthogonal experiment design, the callus survival rate is counted, and the experimental settings at different levels are shown in Table 1.

[0095] Table 1 Orthogonal experiment data of cryopreservation of callus of variety 8N1279

[0096]

[0097]

[0098] Table 2 Orthogonal experiment data of cryopreservation of callus of variety 8N1279

[0099]

[0100] According to the experimental data, the survival rate of variety 8N1279 is the best when the pre-culture time is at level 3, the loading time and the PVS2 treatment time are at level 2, and the survival rate is the worst when the pre-culture time is at level 1, the loading time and the PVS2 treatment time are at level 1.

[0101] Table 3 Orthogonal experiment data of cryopreservation of callus of variety T141

[0102]

[0103]

[0104] According to the experimental data, the survival rate of variety T141 is the best when the pre-culture time is at level 3, the loading time and the PVS2 treatment time are at level 3, and the survival rate is the worst when the pre-culture time is at level 1, the loading time and the PVS2 treatment time are at level 1.

[0105] Table 4 Orthogonal experiment data of cryopreservation of callus of variety T167

[0106]

[0107] From the experimental data, it is known that the survival rate of the variety T167 is the best when the pre-culture time is level 3, the loading time and the PVS2 treatment time are level 2; the survival rate is the worst when the pre-culture time is level 2, the loading time and the PVS2 treatment time are level 1.

[0108] Table 5 orthogonal experimental data of cryopreservation of callus

[0109]

[0110] Through the summary of Table 2, Table 3 and Table 4, the average value of the survival rate of each variety under different treatment time is obtained, and the experimental data is arranged as shown in Table 5. It can be concluded that the average survival rate is the best when the pre-culture time is level 3, the loading time and the PVS2 treatment time are level 3; the average survival rate is the worst when the pre-culture time is level 1, the loading time and the PVS2 treatment time are level 3.

[0111] In the present application, Figure 1 is the main effect diagram of the orthogonal experiment analysis provided by the embodiment of the present application. Figure 2 is a comparison diagram of the traditional method and the method of the present application. In the diagram: A, the effect diagram of the traditional stem tip method; B, the effect diagram of the callus method of the present application.

[0112] The following is a comparison table of the traditional stem tip method and the method of the present application.

[0113]

[0114]

[0115] The above merely provides a specific implementation of the present application, but the protection scope of the present application is not limited to this. Any modification, equivalent replacement and improvement made by any person skilled in the art within the technical range disclosed by the present application, as long as it is within the spirit and principle of the present application, should be covered within the protection scope of the present application.

Claims

1. A method of cryopreservation of garlic callus, characterized by, The method for cryopreservation of garlic callus comprises the following steps: Step one, taking callus with good growth potential for pre-culture; Step two, loading, dehydrating and vitrifying the cryoprotectant; Step three, rewarming the callus after cryoprotectant treatment; Step four, recovery of the callus after cryoprotectant treatment; Step five, inoculating the recovered callus into culture medium for primary culture and propagation: The step one specifically comprises the following steps: Step 1, preparing Gamborg B5 culture dish containing 2iP and NAA: the culture dish contains Gamborg B5 basal medium 3.21 g / L, 2iP 0.05 mg / L, NAA 0.01 mg / L, sucrose 30 g / L and agar 6.5 g / L; Step 2, inoculation: inoculating the callus into the culture dish and placing it in darkness at 5 DEG C for pre-culture for 2 days; The step two specifically comprises the following steps: Step S21, loading: transferring the pre-cultured callus into a culture dish of loading medium, soaking for 20-30 minutes; the culture dish of loading medium is MS 2.165 g / L, glycerol 184.2 g / L, sucrose 205.4 g / L, MS-G 6 25ml and MS-vitamins 6 10ml; the MS-G 6 solution is prepared from thiamine 0.08 g / L, myo-inositol 20 g / L; MS-vitamins 6 solution is prepared from glycine 0.2 g / L, myo-inositol 10 g / L, nicotinic acid 0.05 g / L, pyridoxine hydrochloride 0.05 g / L and thiamine 0.01 g / L; Step S22, dehydrating: after 20-30 minutes, removing the callus and replacing it with 0 DEG C frozen PVS2 vitrification solution, and placing the callus in the PVS2 solution on ice for 15-20 minutes; Step S23, vitrifying: after 15-20 minutes, placing 4-5 calli on a sterile foil strip; and inserting the foil strip into a shallow container containing liquid nitrogen; The step three specifically comprises the following steps: immersing the foil strip in 1.2 M sucrose+1 / 2MS medium for 20 minutes, replacing the solution after 10 minutes, heating to remove the callus, and removing the foil strip from the medium; and after 20 minutes, transferring the callus to a new culture dish containing Gamborg B5 basal medium+NAA+2iP, wrapping the culture dish with aluminum foil, and placing it in a growth chamber for 24 hours; The step four specifically comprises the following steps: after 24 hours, transferring the callus to a new culture dish containing Gamborg B5 basal medium+NAA+2iP, wrapping the culture dish with tin foil, placing it in a growth chamber for four days, then removing the foil and placing it in dim light for another five days, and after five days, transferring it to full light, placing the callus in a culture dish until it grows beyond the space of the culture dish, and then transferring it to a culture dish containing Gamborg B5+NAA+2iP.

2. The method of cryopreservation of garlic callus according to claim 1, wherein, The 2iP is stored at minus 20 DEG C and is divided into small portions.

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