VACUUM BASED ANTHER CULTURE DEVICE AND METHOD OF DOUBLED HAPLOID PRODUCTION

Abstract

The present disclosure relates to vacuum based anther culture device (100) comprising a vacuum generator (110), a negative pressure gauge (120), a control chamber (130), a non-collapsible rubber pipe (140), an anther collection container (150), a non-collapsible rubber pipe (160), a collecting nozzle (170) and a non-collapsible rubber pipe (180). The present disclosure also relates to a method of doubled haploid production of a crop. The present disclosure provides an efficient and non-laborious method for the production of anther based doubled haploids of rice which is time and cost efficient.

Specification

Description:FIELD OF THE INVENTION
[0001] The present disclosure pertains to the field of agriculture biotechnology. More particularly, the present disclosure relates to a vacuum based anther culture device. The present disclosure also relates to a method of doubled haploid production of a crop by using the vacuum based anther culture device.

BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Doubled haploid technology is a useful research tool for obtaining complete homozygosity in a crop within a single season. In the sphere of agriculture, developing doubled haploid lines in rice cultivars is a beneficial practice. The procedure is guided by the success of two integral steps: one, induction of the callus from the cultured explants (microspore or whole anther); second, development of green shoots from the developed calli. Under sterile in vitro conditions, a callus is a clump of undifferentiated plant cells that forms as a stress response by the cultured cells. Callus induction in cultured microspores (anthers) is achieved by using mixture of auxin (2,4-D) and cytokinin (BAP) at precise ratio. Callus-mediated regeneration can be improved by adjusting macro and micronutrients, vitamins, and plant growth regulators. Anther culture is often used to produce haploids and homozygous doubled haploids on a regular basis. After the relevance of doubled haploids in plant breeding was recognised, substantial attempts were made to induce haploid embryogenesis and improve the frequency of doubled haploid recovery in rice.
[0004] Importance of rice is well established; it has been the major source of carbohydrate for over half of world's population. Since, the first report of haploid production in rice using anther culture (Niizeki and Oono, 1968), several attempts have been made to achieve sufficiency for its demand. There are several ways to make haploids and then by the use of chromosomal doubling, get doubled haploids, including pollination of irradiated pollens, wide hybridization, and in vitro culture of anther or microspore. Of all the methods in vitro anther culture or androgenesis is the most efficient method to obtain haploids or doubled haploids in a crop species. Under androgenesis the anthers containing the microspores are enforced to abandon its gametophytic cycle and follow the somatic pathway to produce a regenerant. The formation of regenerant from the microspores can be attained either by direct embryogenesis or by intermediary callus formation.
[0005] The successful androgenesis will result in the formation of a green plant which could fall into one of the three categories, haploids, diploids/candidate doubled haploids and polyploids. Haploids can be identified by their weak stature and sterility. By doubling the chromosome number of such plants at early stage one can easily obtain the doubled haploids. Second are the diploids or doubled haploids are having all the characters similar to the donor parent. Third, are the polyploids which have gigantic growth high number of tillers but often found to be sterile or very less fertile. The doubled haploid technology allows us to obtain stable lines within a very short span of time, which is the limit factor for a varietal development. Also, the lines obtained using this technology are true homozygous (100% homozygosity) which is not possible to obtain using the traditional breeding techniques. Recent popularity has allowed the use of doubled haploid population in QTL/gene identification, stabilizing the genetically improved and transgenic lines.
[0006] The success of androgenesis primarily depends upon the quantity and quality of anthers cultured. Since the quality of anthers is in situ condition of the plants, the quantity requires manual labour and expertise. Limiting the wide of use androgenesis in crop improvement strategies.
[0007] Thus, developing a new tissue culture device can overcome the limitations indicated above with quick and easy production of doubled haploids in crops.

OBJECTS OF THE INVENTION
[0008] An objective of the present invention is to develop an efficient vacuum based anther culture device which can reduce the labour-intensive process of culturing anthers.
[0009] Another objective of the present invention is to provide a quick and easy method of doubled haploid production of a crop.
[0010] Still another objective of the present invention is to provide a method of doubled haploid production of rice.
[0011] Yet another objective of the present invention is to provide a protocol for the anther culture of rice to obtain true doubled haploids of rice.

SUMMARY OF THE INVENTION
[0012] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description section. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
[0013] An aspect of the present disclosure is to develop a vacuum based anther culture device (100) comprising: a vacuum generator (110) for creating vacuum is configured with a negative pressure gauge (120) for measuring the vacuum; a control chamber (130) is connected to the vacuum generator (110) via a non-collapsible rubber pipe (140), wherein the control chamber (130) is for pressure modulation and prevention of anthers from entering the vacuum generator (110); an anther collection container (150) is connected to the control chamber (130) via a non-collapsible rubber pipe (160) for anther capture or culture, wherein the anther collection container (150) contains a callus induction medium for anther culture and for only collection of anthers for crossing purposes; and a collecting nozzle (170) is connected to the anther collection container (150) via a non-collapsible rubber pipe (180) for collecting the anthers and transfer it to the anther collection container (150).
[0014] Another aspect of the present disclosure is to develop a method of doubled haploid production of a crop comprising: a) identifying explants source and collecting and sterilizing the boots followed by cold-treatment to obtain a culture ready boots; b) dispensing a callus induction media, a regeneration media and a root induction media in the anther collection container (150); culture tubes and glass culture tubes respectively; c) surface-sterilizing the culture ready boots of step a) under condition to obtain a surface sterilized boots which are open and spikelets are separated; d) separating the each spikelet containing maximum florets at proper microspore stage of the anthers from rest of the bunch followed by cutting each floret; e) collecting anthers from each floret by the vacuum based anther culture device (100) and cultured by tabbing onto a callus induction media in anther collection container (150); f) incubating the culture of step e) under condition and examining the tubes periodically over 4-6 weeks for callus induction; g) initiating the callus marked by browning of the cultured anthers which appears swollen after 4-6 weeks of incubation to obtain a calli with compact pale-yellow texture sizing 1-2 mm; h) transferring the calli of step g) to shoot regeneration medium and incubated the culture under condition to obtain a green shoots after 2-4 weeks of incubation; i) transferring the green and healthy shoots of step h) to the root induction medium followed by incubation under condition to obtain a plantlets with well-developed roots; and j) transferring the plantlets with well-developed roots of step i) to tube containing water for 2-3 days for a acclimatization under condition followed by the hardened plantlets are potted in the net house for necessary growth and development.
[0015] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments.

DESCRIPTION OF THE FIGURES
[0016] The accompanying drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary embodiments of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.
[0017] Figure 1 illustrates representative diagram of newly designed vacuum based anther culture/collection device.
[0018] Figure 2 illustrates anther culture using the vacuum-based anther culture device. a. Cutting of surface sterilized florets using sterile scissors; b. collection of anthers using the collecting nozzle; c. collected anther in the collection apparatus containing the callus induction media.
[0019] Figure 3 illustrates anther culture using manual tabbing method. a. Cutting of florets; b. collected florets; c. Tabbing florets using Bayonet's forceps; d. Cultured anthers in callus induction media; e & f. removal of florets from the culture tube.

DETAILED DESCRIPTION OF THE INVENTION
[0020] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.
[0021] Unless the context requires otherwise, throughout the specification which follow, the word "comprise" and variations thereof, such as, "comprises" and "comprising", are to be construed in an open, inclusive sense that is as "including, but not limited to."
[0022] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.
[0023] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in the light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.
[0024] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it is individually recited herein.
[0025] All processes described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
[0026] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[0027] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[0028] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
[0029] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description that follows, and the embodiments described herein, is provided by way of illustration of an example, or examples, of particular embodiments of the principles and aspects of the present disclosure. These examples are provided for the purposes of explanation, and not of limitation, of those principles and of the disclosure.
[0030] Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.
[0031] The present invention focuses on the quick and easy production of rice doubled haploids from rice anthers. To be specific, the invention concern with easy and quick isolation of rice anther, using the proposed device along with an efficient callus induction and green shoot regeneration medium. The invention could also be used in different crops also making it one of its kind inventions.
[0032] An embodiment of the present disclosure is to develop a vacuum based anther culture device (100) comprising: a vacuum generator (110) for creating vacuum is configured with a negative pressure gauge (120) for measuring the vacuum; a control chamber (130) is connected to the vacuum generator (110) via a non-collapsible rubber pipe (140), wherein the control chamber (130) is for pressure modulation and prevention of anthers from entering the vacuum generator (110); an anther collection container (150) is connected to the control chamber (130) via a non-collapsible rubber pipe (160) for anther capture or culture, wherein the anther collection container (150) contains a callus induction medium for anther culture and for only collection of anthers for crossing purposes; and a collecting nozzle (170) is connected to the anther collection container (150) via a non-collapsible rubber pipe (180) for collecting the anthers and transfer it to the anther collection container (150).
[0033] In an embodiment, the anther collection container (150) allows the user for autoclaving the same with least exposure of media to the open air reducing the chances of contamination.
[0034] In an embodiment, the anther collection container (150) also prohibits the diploid contaminants such as calli developed from floret wall in the media providing true double haploids.
[0035] In an embodiment, the collection nozzle (170) is adjusted according to the target anther size and allows the collection of anthers of variable sizes from variable crop systems for culture and crossing.
[0036] In an embodiment, the control chamber (130), the non-collapsible rubber pipe (140), anther collection container (150), non-collapsible rubber pipe (160), collecting nozzle (170) and non-collapsible rubber pipe (180) are autoclavable and are autoclaved before the setup of the device.
[0037] Another embodiment of the present disclosure is to provide a method of doubled haploid production of a crop comprising: a) identifying explants source and collecting and sterilizing the boots followed by cold-treatment to obtain a culture ready boots; b) dispensing a callus induction media, a regeneration media and a root induction media in the anther collection container (150); culture tubes and glass culture tubes respectively; c) surface-sterilizing the culture ready boots of step a) under condition to obtain a surface sterilized boots which are open and spikelets are separated; d) separating the each spikelet containing maximum florets at proper microspore stage of the anthers from rest of the bunch followed by cutting each floret; e) collecting anthers from each floret by the vacuum based anther culture device (100) and cultured by tabbing onto a callus induction media in anther collection container (150); f) incubating the culture of step e) under condition and examining the tubes periodically over 4-6 weeks for callus induction; g) initiating the callus marked by browning of the cultured anthers which appears swollen after 4-6 weeks of incubation to obtain a calli with compact pale-yellow texture sizing 1-2 mm; h) transferring the calli of step g) to shoot regeneration medium and incubated the culture under condition to obtain a green shoots after 2-4 weeks of incubation; i) transferring the green and healthy shoots of step h) to the root induction medium followed by incubation under condition to obtain a plantlets with well-developed roots; and j) transferring the plantlets with well-developed roots of step i) to tube containing water for 2-3 days for a acclimatization under condition followed by the hardened plantlets are potted in the net house for necessary growth and development.
[0038] In an embodiment, the collection of boots in step a) includes selection of correct stage of boots in early morning containing maximum numbers of uni-nucleate microspores validated using acetocarmine stain followed by the cold treatment of boots in step a) is carried out at a temperature of 10? for a period in the range of 1-8 days which ensures the maximum callus induction.
[0039] In an embodiment, the callus induction media comprising with auxin in the range of 6.79µM-9.05µM, cytokinin in the range of 2.22µM-3.33µM and maltose in 87.64mM.
[0040] In a preferred embodiment, the callus induction media comprising N6 supplemented with 9.05µM 2,4-dichlorophenoxyaceticacid, 2.22µM 6-benzyl-amino purine as growth regulator and 87.64mM(30gm/l) Maltose as carbon source.
[0041] In an embodiment, the regeneration media comprising auxin in the range of 2.68µM-5.37µM, cytokinin in the range of 4.65µM -11.1µM), proline in the range of 21.71µM-43.43µM and sucrose in 87.64mM.
[0042] In a preferred embodiment, the regeneration media comprising minimal MS media fortified exactly with 43.43µM Proline, 2.68µM ?-naphthalene acetic acid, 4.65µM kinetin, 8.88µM 6-benzyl-amino purine as growth regulators with 87.64mM Sucrose as sole carbon source.
[0043] In an embodiment, the root induction media comprising media supplemented with auxin (5.3704µM) + cytokinin (0.465µM) + sucrose (146.07mM) along with 0.8% agar.
[0044] In a preferred embodiment, the root induction media comprising MS, NAA (5.3704µM), Kinetin (0.465µM), sucrose (146.07mM) along with 0.8% agar.
[0045] In a preferred embodiment, the callus induction media, the regeneration media and the root induction media are stream-sterilized at a temperature of 121°C at a pressure of 15 psi for a period of 15 min.
[0046] In an embodiment, the culture tube of the regeneration media is slanted at 45 ° for increased surface area of contact.
[0047] In an embodiment, the surface-sterilization in step c) is carried out by rinsing boots in 70% (v/v) ethanol for a period of 4min followed by 4% sodium hypochlorite (w/v) for a period of 2 min and rinsed three times with sterile double-distilled water, before culturing of the anthers.
[0048] In an embodiment, the condition in step f) includes incubation in the dark at a temperature of 25±2ºC (static culture).
[0049] In an embodiment, the culture in step h) is incubated 3000 lux cool white fluorescence light with 16h light/8h dark at 25±2°C and 50-60% Relative Humidity.
[0050] In an embodiment, the green shoots in step i) are subjected to MS based root induction media ensuring proper root development.
[0051] In an embodiment, the incubation in step i) includes 3000 lux cool white fluorescence light with 16h light/8h dark at 25±2°C and 50-60% Relative Humidity.
[0052] In an embodiment, the acclimatization condition includes temperature in the range of 25-30°C.
[0053] The present disclosure provides an efficient anther culture device and method for easy and reliable anther culture of rice. The present disclosure includes a unique anther culture device along with an efficient media combination which could produce rice doubled haploids in shorter period of time. In this invention, a vacuum generator, moderator chamber, anther collection chamber/media chamber and collecting duct connected in series, one for capturing the anthers and other as a pressure modulator, which also prevents the anthers form entering the vacuum generator. For this devise plant materials bearing the anthers of proper microspore stage are selected which is amenable to androgenesis. The isolated anthers are easy to culture with zero diploid contamination by anther wall or palea-lemma (floret) of any such for calli induction. The first barrel of the device could be kept empty for containing the anthers for various crossing purposes or can be filled with media for anther culture. Also, the nozzle could be modified for capturing the anthers for other crops which made this device useful in a wide range of crops. As per its commercial value, the device is very cheap to produce and reduces the labour-intensive procedure of culturing the anthers by tabbing. Using the device, anthers are collected individually which place them directly onto the medium stored in the collection chamber. Callus induction media fortified with auxin (6.79µM-9.05µM), cytokinin (2.22µM-3.33µM) and maltose (87.64mM) is used for achieving the anther derived callus. Subsequently, the compact calli of size 2-3mm are transferred to the shoot regeneration medium containing auxin (2.68µM-5.37µM), cytokinin (4.65µM -11.1µM), proline (21.71µM-43.43µM) and sucrose (87.64mM) for development of green shoots. The fully elongated shoots are thereafter transferred to a different media supplemented with auxin (5.3704µM) + cytokinin (0.465µM) + sucrose (146.07mM) along with 0.8% agar for supporting the root development. This method provides a heterozygote free pure doubled haploid regenerants which is essential for the success of rice androgenesis which will be utilized for various rice improvement programmes including QTL/gene identification, QTL/gene introgression and varietal development. The device can be customized to work with different crop systems other than rice and has its wider application in crossing and pollination by breeders to produce hybrids.
[0054] The device according to the present invention is a vacuum-based anther collector/culture system capable of quick and easy collection of anthers for culturing to produce doubled haploids or for crossing purposes.
[0055] Referring to the Fig.1 of vacuum-based anther collector/culture system, comprising of anther collecting nozzle (170) (length 70.5mm, opening dia 2mm (adjustable), connection dia 8mm) with adjustable opening to work with the anthers of variable sizes allowing user to use the devise with various crop systems. The collecting nozzle (170) is connected to a non-collapsible rubber pipe (180) (inner dia x outer dia: 6.35mm x 15.875mm) connecting the anther collection container (150) also called collector/media container for anther capture or culture. It is a glass container (85mm x 145mm; Neck Diameter: 34mm) contains the callus induction media for anther culture. The container itself is autoclavable reducing the chances of microbial contamination and making it ideal for culture purposes. The narrow neck of the container reduces the anther dryness making it suitable for anther storage for longer period for crossing purposes. The anther collection container (150) is connected to the control chamber (130) also called pressure modulator container via non-collapsible rubber pipe (160) (inner dia x outer dia: 6.35 mm x 15.875 mm). The control chamber (130)/ or pressure modulator container made up of polypropylene (1000ml) allowing autoclave sterilization making it suitable for culture purposes. The pressure modulator has two openings one connected to the anther collection container (150) and other to the vacuum generator (110) via non-collapsible rubber pipe (140). The vacuum pump creates the suction pressure of 700-650psi (max). The vacuum pressure was adjusted to 350-300psi measured at the exit valve of the pressure modulator container. The final vacuum pressure at the opening of the collection nozzle was measured to be 350-300psi.
[0056] The success of rice doubled haploid production via androgenesis depends upon the large amount of anthers cultured. While the traditional manual tabbing Fig.3 method only allows culture of few anthers (50-60 anthers/min), the said device can culture 500-600 anthers/min increasing the success of rice doubled haploid production and bringing this technology to the main-stream commercialization form the ongoing lab scale production.
[0057] The invention also refers to an efficient anther culture method Fig.2 under which a process is standardized for achieving the efficient callus induction and green shoot regeneration using this device. The method includes selection of correct stage of boots in early morning containing maximum numbers of uni-nucleate microspores. The stages of microspores were validated using acetocarmine stain. After which the boots are subjected to Cold stress treatment under 10? for 1-8 days which ensures the maximum callus induction.
[0058] Culture as used herein may be defined as placing the anthers on the callus induction media to obtain callus followed by green shoot production from the callus when transferred the callus into regeneration media and subsequently transfer of shoots to the root induction media for the development of proper plantlet with roots.
[0059] Initiation of androgenesis is marked by the formation of a compact pale-yellow callus after 4-5 weeks of culture incubated in dark. The second phase of the androgenesis comprises the regeneration of shoots from the calli incubated in 48hrs dark followed by 3000 lux cool, white fluorescent lamp under 16h photoperiod at 25±2?C and Relative Humidity 50 -60% for 2-4 weeks.
[0060] According to the invention, the callus induction medium comprises the N6 minimal salts, a carbohydrate source (87.64mM maltose), an effective-mixture of amino acid (glycine), vitamins and mixture of plant growth regulators. Similarly, the regeneration medium comprises the MS minimal salts, a carbohydrate source (87.64mM sucrose) and an additional amino acid (proline 43.43µM).
[0061] The preferred concentration of plant growth regulators were 9.05µM 2,4-dichlorophenoxyaceticacid, 2.22µM 6-benzyl-amino purine as growth regulator along with 87.64mM Maltose as the only carbon source in the media. For effective regeneration medium the basal MS was fortified with 43.43µM Proline, 2.68µM ?-naphthalene acetic acid, 4.65µM kinetin, 8.88µM 6-benzyl-amino purine as growth regulators with 87.64mM sucrose as sole carbon source.
[0062] While the foregoing describes various embodiments of the disclosure, other and further embodiments of the disclosure may be devised without departing from the basic scope thereof. The invention is not limited to the described embodiments, versions, or examples, which are included to enable a person having ordinary skill in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art.
EXAMPLES
[0063] The present invention is further explained in the form of the following examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention.
Example 1:
[0064] The steps involved for doubled haploid production of rice are outlined as below:
(A) Identifying explant source:
[0065] Seedlings of rice after 30 days of sowing were transplanted and grown in fields following the standard agronomic practices. Selection of boots was done monitoring the position of anthers in the spikelets, preference was given to the boots containing the anthers at the mid position of the spikelet (~ 8-10 after the boot formation). The microspore stage was confirmed using the acetocarmine staining which conferred the mid position of the anthers in the spikelet with the maximum percentage of the microspore in mid-late uninucleate stage. The boots were collected early in the morning and wrapped in the moist absorbent cotton and sealed in the polyethene bags. The boots were then surface sterilized using a cotton dipped in 70% (v/v) ethanol and stored in a separate polyethene bag in 10? for 1-8 days for cold pre-treatment.
(B) Media preparation:
[0066] Nutrient media for callus induction, regeneration and root induction were prepared using the separate stock solutions based on N6 and MS minimal media with varying concentrations of plant growth regulators and 2 different carbohydrate sources (sucrose and maltose). All the media were maintained an optimum pH of 5.7-5.8 using 0.1N HCl and 0.1N NaOH before the addition of 0.7% agar. 50 ml of callus induction media and 10-15 ml of regeneration media was dispensed equally in the glass conical flask (250ml) and culture tubes (25x150mm) respectively, while the root induction media (10-12ml) was dispensed in 25x200mm glass culture tubes and plugged with nonabsorbent cotton. All the media were steam-sterilized at 121? (15psi) for 15min. After the sterilization the tubes of regeneration media were slanted (45º) for increased surface area of contact.
(C) Anther culture device setup:
[0067] All the parts of the said device except the vacuum generator was wipe-cleaned with 70% (v/v) ethanol and autoclaved at 121? (15psi) for 15min. After sterilization the device is assembled in the laminar. The vacuum generator is connected to the first container via non-collapsible rubber pipe which acts as the control chamber. This container is further connected to the anther collector or in this case to the conical flask containing callus induction medium. The anther collector is connected to a disposable tip (with desired opening) through another non-collapsible pipe which collects the anthers and transfer it to collection chamber.
(D) Culturing anthers:
[0068] After the cold pre-treatment the boots were again surface sterilized by rinsing in 70% (v/v) ethanol for 4min followed by 4% sodium hypochlorite (w/v) for 2min and rinsed three times with sterile double-distilled water, before culturing of the anthers. All the steps were carried under the sterile environment of laminar airflow.
[0069] After surface sterilization the boots were open and spikelets were separated. Each spikelet containing maximum florets at appropriate microspore stage of the anthers were separated from rest of the bunch. Each floret is then cut open manually using a sterile steel scissors Fig.2a. With the help of the anther culture device anthers from each floret is collected and cultured Fig.2b. The said device can collect 500-600 anthers/min Fig.2c as compared to the manual tabbing method which only allows collection of 50-60 anthers/min. The manual tabbing method includes cutting of florets Fig.3a just below the anthers without damaging the anther wall. The cut florets were collected on a sterile petri-dish Fig.3b. If the cut florets are kept for longer period it will lead to anthesis and anther drying reducing the chance of callus induction. The florets were picked using and Bayonet's forceps and anthers were tabbed Fig.3c onto a callus induction media. Sometimes the florets along with the anther drops into the culture tube Fig.3d leading to culture contamination. The florets are removed using the red-hot Bayonet's forceps Fig.3e & f. The cultures were incubated in the dark at 25+2ºC (static culture) and the tubes were periodically examined over 4-6 weeks for callus induction.
(E) Callus induction and shoot regeneration:
[0070] The callus initiation is marked by browning of the cultured anthers which appears swollen after 4-6 weeks of incubation. N6 supplemented with 9.05µM 2,4-dichlorophenoxyaceticacid, 2.22µM 6-benzyl-amino purine as growth regulator and 87.64mM(30gm/l) Maltose as carbon source was optimum for callus induction in rice. Calli with compact pale-yellow texture sizing 1-2mm were transferred to shoot regeneration medium. The said regeneration medium contains minimal MS media fortified exactly with 43.43µM Proline, 2.68µM ?-naphthalene acetic acid, 4.65µM kinetin, 8.88µM 6-benzyl-amino purine as growth regulators with 87.64mM Sucrose as sole carbon source. The cultures were incubated 3000 lux cool white fluorescence light with 16h light/8h dark at 25±2°C and 50-60% Relative Humidity. Green shoots appear after 2-4 weeks of incubation.
(F) Root induction, hardening and development of tentative doubled haploids:
[0071] Developed green regenerants with healthy shoots were transferred to the root induction medium containing MS + NAA (5.3704µM) + Kinetin (0.465µM) + sucrose (146.07mM) along with 0.8% agar. After 4-5 weeks of incubation under 3000 lux cool white fluorescence light with 16h light/8h dark at 25±2°C and 50-60% Relative Humidity, plantlets with well-developed roots were transferred to tubes containing tap water for 2-3days for acclimatization in room temperature. The hardened plantlets were potted in the net house for necessary growth and development.
[0072] The foregoing examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the scope of the invention.

ADVANTAGES OF THE PRESENT INVENTION
[0072] The present invention provides a method for the production of anther-derived doubled haploids in rice, which eliminates diploid contamination by preventing the introduction of diploid florets into the culture medium. Unlike the conventional manual tabbing method, which is laborious and requires specialized skills, the present device for anther culture offers significantly improved efficiency. The device allows for the collection of approximately 500-600 anthers per minute, in contrast to the manual method, which collects only 50-60 anthers per minute. This makes the invention time-efficient, cost-effective, which works to increase the productivity in anther culture for generation of doubled haploids. , Claims:
1. A vacuum based anther culture device (100) comprising:
a vacuum generator (110) for creating vacuum is configured with a negative pressure gauge (120) for measuring the vacuum;
a control chamber (130) is connected to the vacuum generator (110) via a non-collapsible rubber pipe (140), wherein the control chamber (130) is for pressure modulation and prevention of anthers from entering the vacuum generator (110);
an anther collection container (150) is connected to the control chamber (130) via a non-collapsible rubber pipe (160) for anther capture or culture, wherein the anther collection container (150) contains a callus induction medium for anther culture and for only collection of anthers for crossing purposes; and
a collecting nozzle (170) is connected to the anther collection container (150) via a non-collapsible rubber pipe (180) for collecting the anthers and transfer it to the anther collection container (150).
2. The vacuum based anther culture device (100) as claimed in claim 1, wherein the anther collection container (150) allows the user for autoclaving the same with least exposure of media to the open air reducing the chances of contamination.
3. The vacuum based anther culture device (100) as claimed in claim 1, wherein the anther collection container (150) also prohibits the diploid contaminants such as calli developed from floret wall in the media providing true double haploids.
4. The vacuum based anther culture device (100) as claimed in claim 1, wherein the collection nozzle (170) is adjusted according to the target anther size and allows the collection of anthers of variable sizes from variable crop systems for culture and crossing.
5. The vacuum based anther culture device (100) as claimed in claim 1, wherein the control chamber (130), the non-collapsible rubber pipe (140), anther collection container (150), non-collapsible rubber pipe (160), collecting nozzle (170) and non-collapsible rubber pipe (180) are autoclavable and are autoclaved before the setup of the device.
6. A method of doubled haploid production of a crop comprising:
a) identifying explants source and collecting and sterilizing the boots followed by cold-treatment to obtain a culture ready boots;
b) dispensing a callus induction media, a regeneration media and a root induction media in the anther collection container (150); culture tubes and glass culture tubes respectively;
c) surface-sterilizing the culture ready boots of step a) under condition to obtain a surface sterilized boots which are open and spikelets are separated;
d) separating the each spikelet containing maximum florets at proper microspore stage of the anthers from rest of the bunch followed by cutting each floret;
e) collecting anthers from each floret by the vacuum based anther culture device (100) and cultured by tabbing onto a callus induction media in anther collection container (150);
f) incubating the culture of step e) under condition and examining the tubes periodically over 4-6 weeks for callus induction;
g) initiating the callus marked by browning of the cultured anthers which appears swollen after 4-6 weeks of incubation to obtain a calli with compact pale-yellow texture sizing 1-2 mm;
h) transferring the calli of step g) to shoot regeneration medium and incubated the culture under condition to obtain a green shoots after 2-4 weeks of incubation;
i) transferring the green and healthy shoots of step h) to the root induction medium followed by incubation under condition to obtain a plantlets with well-developed roots; and
j) transferring the plantlets with well-developed roots of step i) to tube containing water for 2-3 days for a acclimatization under condition followed by the hardened plantlets are potted in the net house for necessary growth and development.
7. The method as claimed in claim 6, wherein the collection of boots in step a) includes selection of correct stage of boots in early morning containing maximum numbers of uni-nucleate microspores validated using acetocarmine stain followed by the cold treatment of boots in step a) is carried out at a temperature of 10? for a period in the range of 1-8 days which ensures the maximum callus induction.
8. The method as claimed in claim 6, wherein the callus induction media comprising N6 supplemented with 9.05µM 2,4-dichlorophenoxyaceticacid, 2.22µM 6-benzyl-amino purine as growth regulator and 87.64mM(30gm/l) Maltose as carbon source.
9. The method as claimed in claim 6, wherein the regeneration media comprising minimal MS media fortified exactly with 43.43µM Proline, 2.68µM ?-naphthalene acetic acid, 4.65µM kinetin, 8.88µM 6-benzyl-amino purine as growth regulators with 87.64mM Sucrose as sole carbon source.
10. The method as claimed in claim 6, wherein the root induction media comprising MS, NAA (5.3704µM), Kinetin (0.465µM), sucrose (146.07mM) along with 0.8% agar.

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