NL2028061A - Method for rapidly breeding new rice variety by combining anther culture with in vitro chromosome doubling - Google Patents

Abstract

The present invention discloses a method for rapidly breeding new rice variety by combining 5 anther culture with in vitro chromosome doubling, comprising the following steps: a. hybridization of different rice varieties to obtain hybrids; b. hybrid planting and anther culture to get materials ; c. low-temperature pre-treatment of materials; d. anther culture to induce calluses; e. in vitro chromosome doubling of calluses; f. recovery culture of chromosome doubled calluses; g. shoots regenerating from calluses; h. rooting culture of shoots; i. plantlet transplanting; j. ploidy 10 identification; k. comprehensive character identification. Compared with the traditional breeding method comprising steps of anther culture to form calluses, callus differentiation to form plants, and in vivo chromosome doubling of identified haploid plants, the method disclosed by the present invention has the advantages of short consumed time and high efficiency. In the present invention, in vitro chromosome doubling is conducted directly by using calluses from anther culture, and 15 plants are obtained by callus differentiation, so new homozygous rice varieties with required characters can be obtained efficiently and rapidly. A new way for breeding new rice varieties by using anther culture is provided.

Description

METHOD FOR RAPIDLY BREEDING NEW RICE VARIETY BY COMBINING ANTHER

CULTURE WITH IN VITRO CHROMOSOME DOUBLING Technical Field The present invention relates to a method for rapidly breeding new rice variety by combining anther culture with in vitro chromosome doubling, which is a method in that calluses is obtained by anther culture of hybrid plants, chromosome doubling is directly conducted on the calluses, doubled haploids are rapidly obtained, and a new homozygous rice variety is obtained by identification and selection, and belongs to the technical field of crop genetic breeding in modern agriculture. Background Haploid is a sporophyte having gametophyte chromosome number. Haploid plays an important role in genetic breeding and basic genetic research, specifically: (1) shortening breeding time and improving breeding efficiency; {2} mutant selection; (3) obtaining chromosome alien addition lines and substitution lines by anther culture of distant hybrids; (4) being used for basic genetic research such as gene function research, construction of genetic mapping population, genome sequencing and the like; (5) being used as a genetic engineering acceptor (Li Shuxian, 2008; Gai Junyi, 2008; Xie Conghua and Liu Jun, 2004). Especially in terms of "shortening breeding time and improving breeding efficiency", there are many elite new varieties bred by haploid breeding in crops such as rice, wheat, corn, rape, tobacco and the like, which play an important role in production (Zhang Xianlong and Tang Kexuan, 2004; Zhang Tianzhen, 2011). Methods to obtain haploid mainly include: (1) unisexual reproduction , i.e. spontaneous or induced parthenogenesis, patrogenesis, apogamy and semigamy; (2) chromosome elimination, for example, Hordeum bulbosum hybridization method and maize hybridization method; (3) in vitro culture of cells or tissue, wherein in vitro culture of cells or tissue is the main method to obtain haploid at present, which includes anther or pollen culture, and unpollinated ovary or ovule culture, the former becomes the main method for inducing haploid due to more haploid cells and material acquisition conveniently (Sun Qixin, 2018; Xi Zhangying et al, 2014).

At present, the main procedure for rice haploid breeding includes: hybridization of two varieties to obtain F: first, anther culture of the F: plants to induce haploid plants, chromosome doubling of the haploid plants to obtain doubled haploid (homozygous diploid), identification on the characters of the doubled haploid, and selecting the elite new varieties that meets the breeding objective (Zhang Tianzhen, 2011). In specific operation, the following steps need to be executed: (1) anther culture of hybrids to induce calluses; (2) callus differentiation to form plants; (3) identification of haploid plants; (4) in vivo chromosome doubling of the haploid plants to obtain doubled haploid; and (5) selection of the doubled haploid. It takes a long time for the calluses regenerating to plants and then to double the identified haploid plants. In addition, the traditional method of chromosome doubling is in vivo doubling, which has the defects of low efficiency and chimera formation. In the present invention, anther culture of hybrid plants is conducted to induce calluses, in vitro chromosome doubling is conducted directly by using calluses from anther culture, plants are obtained by callus differentiation, and then new homozygous rice varieties with required characters can be obtained through identification. The present invention has the main innovation that: (1) chromosome doubling is conducted directly on the calluses from anther culture of hybrid plants; (2) by adopting the method of in vitro chromosome doubling, the doubling efficiency is greatly improved as compared with that of the traditional in vivo chromosome doubling, and chimera is seldom formed. Therefore, the new varieties which meet the breeding objective can be bred more efficiently and rapidly. Summary A technical problem to be solved by the present invention is to provide a method for rapidly breeding new rice variety by combining anther culture with in vitro chromosome doubling, which solves the problems of long consumed time and low efficiency of rice haploid breeding at present and provides a material foundation for genetic breeding and basic genetic research of rice. The technical solution of the present invention is: a. hybridization of different rice varieties to obtain hybrids; b. hybrid planting and anther culture to get materials; c. low-temperature pre- treatment of materials; d. anther culture to induce calluses; e. in vitro chromosome doubling of calluses; f. recovery culture of chromosome doubled calluses; g. shoots regenerating from calluses; h. rooting culture of shoots; i. plantlet transplanting; j. ploidy identification; k. comprehensive character identification.

The specific steps of the present invention are as follows: a. Hybridization of different rice varieties to obtain hybrids Selecting two rice varieties having different favourable characters, or one rice variety having good comprehensive characters but having target characters required to be reformed and the other rice variety having target characters, and conducting sexual hybridization on the two rice varieties to obtain hybrid F4. b. Hybrid planting and anther culture to get materials Planting the hybrid F+, at the booting stage of plants, selecting the rice panicles of which the pulvinus distance is 5-15 cm, the glumes are light green in the upper part and faint yellow in the lower part and the stamen length is close to 1/2 of the glume length, and picking the rice panicles together with leaf sheathes and leaves; taking spikelets from different parts of the rice panicles and picking out anthers, dyeing pollens with 1% Is-KI solution, and observing under microscope, to guarantee that most pollens of the rice panicles taken according to the above- mentioned external morphological standard are at the late-uninucleate stage. c. Low-temperature pre-treatment of materials Cutting off leaves from the rice panicles, reserving leaf sheathes of the last three leaves, wrapping with gauze soaked by distilled water and then putting into a zip lock bag, and sealing and placing in a refrigerator for pre-treatment at 4°C for 7-10d. d. Anther culture to induce calluses Peeling out the rice panicles after low-temperature pre-treatment in an ultraclean workbench, after disinfection treatment, picking anthers in spikelets and transferring to the anther callus induction medium, culturing at 26-28°C in the dark until calluses form, wherein a formula of the anther callus induction medium is as follows: SK3 medium + 1.0-2.0mg/L 2,4-D +

0.5-1.0mg/L NAA + 0.5-1.0mg/L KT + 200mg/L Hydrolysed casein + 2.5% (w/v) Sucrose + 2.5% (w/v) Maltose + 0.75% (w/v) Agar, pH 6.0. e. In vitro chromosome doubling of calluses Transferring vigorous calluses into the doubling medium, placing in an orbital shaker with a temperature of 26-28°C, and conducting shaking culture at 100-110rpm for 48-60h, wherein a formula of the doubling medium is as follows: SK3 medium + 1.0-2.0mg/L 2,4-D + 0.5-1.0mg/L NAA + 0.5-1.0mg/L KT + 300-500 mg/L Colchicine + 200mg/L Hydrolysed casein + 2.5% (w/v) Sucrose + 2.5% (w/v) Maltose + 0.75% (w/v) Agar, pH 6.0. f. Recovery culture of chromosome doubled calluses Rinsing the chromosome doubled calluses with sterile distilled water for 3-5 times, transferring to the recovery medium, and conducting recovery culture at 26-28°C in the dark for 7-10d, wherein a formula of the recovery medium is as follows: SK3 medium + 1.0-2.0mg/L 2,4- D + 0.5-1.0mg/L NAA + 0.5-1.0mg/L KT + 200mg/L Hydrolysed casein + 2.5% (w/v) Sucrose +

2.5% (w/v) Maltose + 0.75% (w/v) Agar, pH 6.0. g. Shoots regenerating from calluses Transferring the calluses after recovery culture to the differentiation medium, culturing at 25°C in the dark for 3-5d, and then culturing in 12-14 h/d illumination condition until shoots regenerate from calluses, wherein a formula of the differentiation medium is as follows: MS medium + 1.0-1.5mg/L 6-BA + 1.0-2.0mg/L KT + 0.2-0.3mg/L NAA + 3% (w/v) Sucrose + 0.75% (w/v) Agar, pH 6.0. h. Rooting culture of shoots When shoots differentiated from the calluses grow to 3-5cm, transferring the shoots to the rooting medium, and culturing at 25°C in an illumination condition, until the shoots root and form plantlets, wherein a formula of the rooting medium is as follows: 1/2MS medium + 0.1-0.3mg/L 6-BA + 0.3-0.5mg/L NAA + 0.02% (w/v) Activated Carbon + 2% (w/v) Sucrose + 0.75% (w/v) Agar, pH 8.0.

i.

Plantlet transplanting When the root number of plantlets is 3-5 and the root length is 2-3cm, uncovering the sealing film of the culture bottle, adding 0.5-1cm of sterile water, and acclimatizing the plantlets at 25°C for 2-3d; washing the agar on the roots of the plantlets with clear water, then transplanting the plantlets to the paddy field or rice pot, and paying attention to shading and preventing waterlog in the first few days after transplanting. j.

Ploidy identification Determining the ploidy of the plants from morphology and fertility, wherein compared with the haploid plant, doubled haploid is diploid (DH), is high in plant, thick in culm, and can normally seed; on the contrary, haploid is small in plant, slim in culm, weak in growth, and cannot seed; meanwhile, detecting cell DNA content by a ploidy detector or flow cytometry, or verifying chromosome number by chromosome preparation of root tips can be performed to confirm the ploidy. k.

Comprehensive character identification Observing and identifying the comprehensive characters of the diploid plants DH: confirmed through ploidy identification or DH; line formed by DH: plant selfing, selecting plants having favourable characters of the two parents or plants having both good comprehensive characters and target characters, thereby obtaining a new homozygous rice variety.

Description of Drawings Fig. 1 Anther culture to induce calluses Fig. 2 In vitro chromosome doubling of calluses Fig. 3 Shoots regenerating from chromosome doubled calluses Fig. 4 Brown rice of new salt-resistant and selenium-rich red rice variety and its parents thereof Left: 9311; Middle: new salt-resistant and selenium-rich red rice variety; Right: sea rice 86 Detailed Description The present invention will be further described below in combination with the creation of a new salt-resistant and selenium-rich red rice variety in an embodiment. a.

Hybridization of different rice varieties to obtain hybrids Selecting rice variety 9311 and sea rice 86 as crossing parents, and hybridizing the two varieties to obtain hybrid F+, where in 9311 is a famous indica rice variety, is a male parent of super hybrid rice Liangyoupeijiu, and is excellent in comprehensive characters; and sea rice 86 is a salt-resistant rice variety, is red in rice grain, is rich in mineral nutrients such as calcium, magnesium, iron, manganese, zinc, selenium and the like, and is especially much higher than common rice varieties in selenium content.

b. Hybrid planting and anther culture to get materials Planting the hybrid F+, at the booting stage of plants, selecting the rice panicles of which the pulvinus distance is 10 cm, the glumes are light green in the upper part and faint yellow in the lower part and the stamen length is close to 1/2 of the glume length, and picking the rice 5 panicles together with leaf sheathes and leaves; taking spikelets from different parts of the rice panicles and picking out anthers, dyeing pollens with 1% Iz-KI solution, and observing under microscope, to guarantee that most pollens of the rice panicles taken according to the above- mentioned external morphological standard are at the late-uninucleate stage. c. Low-temperature pre-treatment of materials Cutting off leaves from the rice panicles, reserving leaf sheathes of the last three leaves, wrapping with gauze soaked by distilled water and then putting into a zip lock bag, and sealing and placing in a refrigerator for pre-treatment at 4°C for 7-10d. d. Anther culture to induce calluses Peeling out the rice panicles after low-temperature pre-treatment in an ultraclean workbench, after disinfection treatment, picking anthers in spikelets and transferring to the anther callus induction medium, culturing at 26-28°C in the dark until calluses form, wherein a formula of the anther callus induction medium is as follows: SK3 medium + 1.0-2.0mg/L 2,4-D +

0.5-1.0mg/L NAA + 0.5-1.0mg/L KT + 200mg/L Hydrolysed casein + 2.5% (w/v) Sucrose + 2.5% (w/v) Maltose + 0.75% (w/v) Agar, pH 6.0. e. In vitro chromosome doubling of calluses Transferring vigorous calluses into the doubling medium, placing in an orbital shaker with a temperature of 26-28°C, and conducting shaking culture at 100rpm for 48h, wherein a formula of the doubling medium is as follows: SK3 medium + 1.0-2.0mg/L 2,4-D + 0.5-1.0mg/L NAA +

0.5-1.0mg/L KT + 300-500 mg/L Colchicine + 200mg/L Hydrolysed casein + 2.5% (w/v) Sucrose + 2.5% (w/v) Maltose + 0.75% (w/v) Agar, pH 6.0. f. Recovery culture of chromosome doubled calluses Rinsing the chromosome doubled calluses with sterile distilled water for 3-5 times, transferring to the recovery medium, and conducting recovery culture at 26-28°C in the dark for 7-10d, wherein a formula of the recovery medium is as follows: SK3 medium + 1.0-2.0mg/L 2,4- D+ 0.5-1.0mg/L NAA + 0.5-1.0mg/L KT + 200mg/L Hydrolysed casein + 2.5% (w/v) Sucrose +

2.5% (w/v) Maltose + 0.75% (w/v) Agar, pH 6.0. g. Shoots regenerating from calluses Transferring the calluses after recovery culture to the differentiation medium, culturing at 25°C in the dark for 3-5d, and then culturing in 12-14 h/d illumination condition until shoots regenerate from calluses, wherein a formula of the differentiation medium is as follows: MS medium + 1.0-1.5mg/L 6-BA + 1.0-2.0mg/L KT + 0.2-0.3mg/L NAA + 3% (w/v) Sucrose + 0.75% (w/v) Agar, pH 6.0.

h.

Rooting culture of shoots When shoots differentiated from the calluses grow to 3-5cm, transferring the shoots to the rooting medium, and culturing at 25°C in an illumination condition, until the shoots root and form plantlets, wherein a formula of the rooting medium is as follows: 1/2MS medium + 0.1-0.3mg/L 6-BA + 0.3-0.5mg/L NAA + 0.02% (w/v) Activated Carbon + 2% (w/v) Sucrose + 0.75% (w/v) Agar, pH 6.0. i.

Plantlet transplanting When the root number of plantlets is 3-5 and the root length is 2-3cm, uncovering the sealing film of the culture bottle, adding 0.5-1cm of sterile water, and acclimatizing the plantlets at 25°C for 2-3d; washing the agar on the roots of the plantlets with clear water, then transplanting the plantlets to the paddy field, and paying attention to shading and preventing waterlog in the first few days after transplanting. j.

Ploidy identification In terms of morphology and fertility, haploid is small in plant, slim in culm, weak in growth, and cannot seed; while the double haploid (diploid) is high in plant, thick in culm, and can normally seed.

It is found by the ploidy detector that the cell DNA content of the diploid plants is twofold of the haploid plants.

It is found through chromosome preparation of root tips that the chromosome number of haploid plants is 2n=x=12, and the chromosome number of diploid plants is 2n=2x=24. k.

Comprehensive character identification Observing the identified diploid plants (DH4), selecting plants that having identical or similar plant type with 9311, and numbering each plant; hulling after rice is mature to observe the color of rice grains, and selecting and reserving the plants that grains are red; detecting the selenium content of each plant using a part of the grains, and reserving the remaining grains containing high selenium according to the detection result.

Planting the seeds of DH: plants selected through above process in single plant to obtain DH: lines, and harvesting the seeds of each single plant and numbering the seeds; taking a part of the seeds of each number for salt tolerance test: soaking the seeds with distilled water for 2d first, then transferring the sprouting seeds to a culture dish lined with 3-5 layers of sterilized filter paper, adding 0.6% NaCl solution prepared by 1/4 Hoagland nutrient solution into the culture dish, treating for 10-12d, replacing new NaCl solution every day, and treating the seeds of sea rice 86 as control at the same time to screen salt-resistant lines; and breeding a new salt-resistant and selenium-rich red rice variety with excellent comprehensive characters finally.

Claims (2)

CONCLUSIONS A method for rapidly cultivating new varieties of rice by combining anther culture with in vitro chromosome doubling, the method comprising the steps of: a. hybridizing different rice varieties to obtain hybrids; b. planting hybrids and cultivating stamens to obtain material; c. pre-treatment of the material at low temperature; d. cultivating anthers to form callus; e. doubling of callus chromosomes in vitro; f. growing callus with duplicated chromosomes; g. regeneration of shoots from callus; h. growing roots from the shoots; i. transplanting the plants; j. determining the ploidy; k. detailed description of the properties. The method for rapidly cultivating new rice varieties by combining anther culture with in vitro chromosome doubling according to claim 1, which method comprises the following steps: a.: hybridization of different rice varieties to obtain hybrids selecting two rice varieties with different favorable characteristics , or one rice variety with good general characteristics but to which target characteristics must be introduced and where the other rice variety has the intended characteristics, and sexual hybridization of the two rice varieties to obtain a hybrid F1; b. planting hybrids and cultivating stamens to obtain material planting the hybrid F1, in the germination stage of the plants, selecting the rice panicles whose pulvinus distance is 5 - 15 cm, the germ layers are light green at the top and light yellow below and the stamen length is almost is half the length of the chaff, and the plucking of the rice panicles together with the leaf sheaths and the leaves; taking spikelets from different parts of the rice panicles and picking out the anthers, staining the pollen with 1% Iz - Kl solution and observing under a microscope, to ensure that most of the pollen from the rice plumes that according to the above-mentioned external morphological standard are in the late-uncleaved stage; c. pre-treatment of the material at low temperature cutting the leaves from the rice panicles, preserving the leaf sheaths of the last three leaves, wrapping with gauze soaked in distilled water and then placing in a slip-lock bag, sealing and in a place in refrigerator for pretreatment at 4°C for 7-10 d; d. cultivating anthers to form callus peeling the rice panicles after pretreatment at low temperature in an ultra-clean fume hood, after disinfection treatment, picking the anthers in the spikelets and transferring to the induction medium to form anther callus, culturing in 26 - 28°C in the dark until callus forms, the composition of the medium for the formation of anther callus being as follows: SK3 - medium + 1.0 -2.0 mg/L 2,4-D + 0, 5- 1.0 mg/L NAA + 0.5 - 1.0 mg/L KT + 200 mg/L hydrolysed casein + 2.5% (w/v) sucrose + 2.5% (w/v) maltose + 0.75% (w/v) agar, pH 6.0; e. doubling the callus chromosomes in vitro transferring vigorous callus into the doubling medium, placing in a shaker with circular shaking movements at a temperature of 26 - 28°C, and growing a shake culture at 100 - 110 rpm for 48 - 60 hours, where the composition of the doubling medium is as follows: SK3 - medium + 1.0 - 2.0 mg/L 2,4-D + 0.5 - 1.0 mg/L NAA + 0.5 - 1.0 mg/L KT + 300 - 500 mg/L colchicine + 200 mg/L hydrolysed casein + 2.5% (w/v) sucrose + 2.5% (w/v) maltose + 0.75% ( w/v) agar, pH 6.0; f. culture of callus with duplicated chromosomes rinse 3 to 5 times with sterile distilled water from the callus in which the chromosomes duplicated, transfer to recovery medium and grow a recovery culture at 28 - 28°C in the dark for 7 to 10 d, keeping the composition of the recovery medium is as follows: SK3 - medium + 1.0 - 2.0 mg/L 2,4-D + 0.5 - 1.0 mg/L NAA + 0.5 - 1.0 mg/L RT + 200 mg/L hydrolyzed casein + 2.5% (w/v) sucrose + 2.5% (w/v) maltose + 0.75% (w/v) agar, pH 6.0; g. regeneration of shoots from callus after recovery cultures transfer the callus to differentiation medium, grow 3-5 d in the dark at 25°C and then light for 12-14 hours per day until shoots emerge from the callus, adjusting the composition of the differentiation medium is as follows: MS medium + 1.0 - 1.5 mg/L 6-BA + 1.0 - 2.0 mg/L KT + 0.2 - 0.3 mg/L NAA + 3% (w /v) sucrose + 0.75% (w/v) agar, pH 6.0; h. grow roots from the shoots when the shoots emerging from the callus are 3 to 5 cm in size, transfer the shoots to rooting medium and grow at 25°C and grow under lighting until the shoots take root and form plantlets, the composition of the rooting medium is as follows: ¥2 MS medium + 0.1 - 0.3 mg/L 6-BA + 0.3 - 0.5 mg/L NAA + 0.02% (w/v) activated carbon + 2% (w/v) sucrose + 0.75% (w/v) agar, pH 6.0; i. transplant the plants when the number of roots of the plants is 3 - 5 and the root length is 2 - 3 cm, remove the sealing film from the culture bottle, add 0.5 - 1 cm sterile water and allow the plants to acclimatize for 2 - 3d at 25°C; washing the agar on the roots of the plants with clear water and then transplanting the plants to the paddy field or a rice pot, taking care to shade them during the first days after transplanting and to avoid water saturation ; j. determining the ploidy determining the ploidy of the plants on the basis of morphology and fertility, where compared to the haploid plant, the doubled haploid is diploid (DH1), the plant is high, the culm is thick, and normal seed can yield; the haploid, on the other hand, is a small plant, has slender culms, grows weakly, and cannot produce seed; wherein the DNA content of the cells is detected by a ploidy detector or by flow cytometry, or the chromosome number can be verified by taking chromosome preparations from root tips to confirm the ploidy; k. comprehensive characterization of the traits observe and identify the general characteristics of the diploid DH1 - plants, confirmed by establishing the ploidy or a DH2 - line formed by self-pollination of DH1 - plants, selecting plants with favorable characteristics of the two parents or plants with both good general characteristics and target characteristics, yielding a new homozygous rice variety.