NL2032537B1 - Method for tissue culture of ornithogalum caudatum - Google Patents

Abstract

The present invention relates to a method for tissue culture of Ornithogalum caudatum, including the following steps: medium preparation, explant selection and sterilization, primary culture, adventitious bud induction, adventitious bud multiplication, adventitious bud rooting induction, tissue culture seedling domestication and transplantation. The minimal media select MS media, where sucrose is used in an amount of 20—40 g/L, and agar powder, which is used as the coagulating agent, is used in an amount of 8—9 g/L, and the pH of the media is adjusted to 5.7—5.8 before subpackaging; the medium for primary culture is MS+6—BA 0.1—0.5 mg/L+NAA 0.05—O.2 mg/L; the medium for adventitious bud induction is MS+6—BA—2.0—4.0 mg/L+NAA 0.05—0.2 mg/L; the medium for adventitious bud multiplication is MS+6—BA O.5—3.0 mg/L+NAA 0.05—0.2 mg/L; and the medium for rooting is MS+IBA 0—0.5 mg/L or 1/2MS+IBA 0—0.5 mg/L

Description

P1480 /NLpd

METHOD FOR TISSUE CULTURE OF ORNITHOGALUM CAUDATUM

TECHNICAL FIELD The present invention relates to plant tissue culture tech- nology and plant shoot tip culture technology, and is mainly a method for tissue culture of Ornithogalum caudatum.

BACKGROUND ART Ornithogalum caudatum, also called Sea onion, is commonly known as Sea squill, False sea onion, etc. It belongs to Liliace- ae, Ornithogalum, and is a perennial bulbous herbaceous flower na- tive to southern Africa. It likes sunlight, but direct sunlight in summer should be avoided. It is also resistant to cold in semi shade condition. It likes moist environment, and after heavy frost in winter the leaf cluster remains green. The plant height of Or- nithogalum caudatum is about 30-40 cm when it is not flowering, and the height of flower stem can reach 50-60 cm when it is flow- ering. Ornithogalum caudatum flowers from the middle ten-day of April to the first ten-day of May each year. Ornithogalum caudatum has a panicle with white, orange and double petal flowers. Orni- thogalum caudatum is gentle and elegant, while it is also plain, with a long flowering period. It is an excellent material for ar- rangements of natural gardens and rock gardens, as well as for cut flowers and potted ornamental. Meanwhile, Ornithogalum caudatum also has a certain medicinal value. Its leaves and the underlying bulb can be cut into slices or blocks and soaked in liquor, which may be useful relaxing the muscles and stimulating the blood cir- culation. It can be used for internal and external application, and has a good recovery effect on bruises, sprains and excessive muscle fatigue. At present, the propagation of Ornithogalum caudatum is main- ly carried out by seed and bulb-dividing propagation. However, the bulbs cannot be used as bulbs for propagation until August and September every year, while the seedlings need to be breeded for 3-4 years to flower. The production of bulbs and seeds is severely limited by season and years, while the propagation amount is lim- ited each year, which is not conducive to the promotion of Orni- thogalum caudatum. Meanwhile, as bulbs of Ornithogalum caudatum are in the underground for a long time, it is easily infected by various viruses. Where the bulbs are used for asexual propagation, once the bulbs are infected by viruses, it is difficult to eradi- cate them by chemical drugs, which will seriously reduce the orna- mental value of Ornithogalum caudatum.

Plant tissue culture technology is considered to be the most efficient means for rapid propagation of plant seedlings current- ly. At present, there is no literature report on rapid propagation of Ornithogalum caudatum using plant tissue culture technology at home and abroad.

SUMMARY An object of the present invention is to overcome the short- comings in the prior art and provide a method for tissue culture of Ornithogalum caudatum, solve the problem of establishing a good tissue culture regeneration system of Ornithogalum caudatum, and provide technical support for Ornithogalum caudatum seedling pro- duction, new variety creation and quality genetic improvement.

The object of the present invention is completed by the fol- lowing technical solutions: a method for tissue culture of Orni- thogalum caudatum includes the following steps: 1), explant selection and treatment: selecting an underground bulb and a flower spike which is not flowering of Ornithogalum caudatum as a starting material, peeling the outer 1-2 layers of scales from the bulb, cutting the flower spike to a length of 1 cm, and using them as the explant for tissue culture; 2), primary culture: transferring the surface-sterilized ex- plant to a sterile inoculation plate on an ultra-clean workbench, sucking off the surface moisture of the explant with sterile fil- ter paper, and inoculating the explant in a medium for primary culture which is MS+6-BA 0.1-0.5 mg/L+NAA 0.05-0.2 mg/L for prima- ry culture, the culture temperature being 25+2°C, the illumination time being 12-14 h/d, and the illumination intensity being 45-60 pmol ‘mF es;

3), adventitious bud induction: inoculating the clean and vi- able material obtained from the primary culture in a medium for adventitious bud induction which is MS+6-BA 2.0-4.0 mg/L+NAA 0.05-

0.2 mg/L on a sterile workbench for adventitious bud induction, the culture temperature being 25+2°C, the illumination time being 12-14 h/d, and the illumination intensity being 45-60 pmol m™~-s*; 4}, adventitious bud multiplication: dividing 2-3 adventi- tious buds with a height of 2-3 cm obtained from adventitious bud induction culture into a cluster on the sterile workbench, gently creating some wounds on the bases of the adventitious buds using a sterile scalpel, and then inoculating the adventitious buds in a medium for adventitious bud multiplication which is MS+6-BA 0.5-

3.0 mg/L+NAA 0.05-0.2 mg/L for multiplication culture, the culture temperature being 25+2°C, the illumination time being 12-14 h/d, and the illumination intensity being 45-60 u ‘mol ‘ms; 5), adventitious bud rooting induction: separating the clus- tered adventitious buds growing to a height of 3-5 cm into indi- vidual adventitious buds from the base, and then inoculating the adventitious buds in a medium for rooting which is MS+IBA 0-0.5 mg/L or 1/2MS+IBA 0-0.5 mg/L for adventitious root induction, the culture temperature being 25+2°C, the illumination time being 12-14 h/d, and the illumination intensity being 45-60 pu ‘mol ms"; 6), tissue culture seedling domestication and transplanta- tion: after 3-5 adventitious roots of 1-2 cm grow from the bases of the adventitious buds, moving the tissue culture to a green- house, opening the bottle cap after culture with scattered light for 5 days, and then the culture continues for another 1-2 days to complete the domestication of the tissue culture seedlings before the transplantation, then taking out the tissue culture seedlings from the culture bottle, washing out the agar on the surface of the tissue culture seedlings, planting the seedlings in a matrix for culture for 15 days with a moisture content maintained above 903, and then culturing the seedlings normally in the greenhouse.

The minimal media for primary culture, adventitious bud in- duction and multiplication, and adventitious bud rooting induction are all MS media. Sucrose is used in an amount of 20-40 g/L, and agar powder, which is used as the coagulating agent, is used in an amount of 8-9 g/L, and the pH of the media is adjusted to 5.7-5.8 before subpackaging.

Beneficial effects of the present invention are as follows: 1, tissue culture technology is used for rapid propagation of Ornithogalum caudatum seedlings. The production is not limited by region, season, climate and growing years of mother plants, which is convenient for industrialized seedling culture. Production can be carried out according to the order. The production time and scale can be controlled. It provides sufficient high-quality seed- lings for promotion and application of Ornithogalum caudatum.

2, propagation coefficient is 3-5, rooting rate is 100%, and transplanting survival rate is 100%, which meets the requirements of industrialized seedling culture production for flowers. At pre- sent, there is no literature report on tissue culture of Ornitho- galum caudatum.

3, the adventitious bud induction of Ornithogalum caudatum adopts direct organogenesis without callus induction and callus differentiation induction, so as to reduce the occurrence of prog- eny variation in the process of plant tissue culture. The progeny seedlings have a consistent genetic background, which may keep the excellent traits of female parent to the maximum.

4, it is beneficial to promote the varieties with low seed setting rate or even no seed setting.

5, it establishes a good tissue culture and rapid propagation system which lays foundation for future research work on new vari- ety breeding and genetic improvement of Ornithogalum caudatum by plant biotechnology.

DETAILED DESCRIPTION OF THE EMBODIMENTS The present invention will be further illustrated below through detailed description, and the present invention will be better understood from the embodiments. However, the present in- vention is not limited to the following embodiments.

A method for tissue culture of Ornithogalum caudatum accord- ing to the present invention includes the following steps: 1, media and culture conditions: the minimal media for prima-

ry culture, adventitious bud induction and multiplication, and ad- ventitious bud rooting induction are all MS media, where sucrose is used in an amount of 20-40 g/L, and agar powder, which is used as the coagulating agent, is used in an amount of 8-9 g/L, and the 5 pH of the media is adjusted to 5.7-5.8 before subpackaging; the medium for primary culture is MS+6-BA 0.1-0.5 mg/L+NAA 0.05-0.2 mg/L; the medium for adventitious bud induction is MS+6-BA 2.0-4.0 mg/L+NAA 0.05-0.2 mg/L; the medium for adventitious bud multipli- cation is MS+6-BA 0.5-3.0 mg/L+NAA 0.05-0.2 mg/L; and the medium for rooting is MS+IBA 0-0.5 mg/L or 1/2MS+IBA 0-0.5 mg/L; culture conditions for primary culture, adventitious bud induction and multiplication, and adventitious bud rooting are as follows: the culture temperature being 25+2°C, the illumination time being 12-14 h/d, and the illumination intensity being 45-60 u ‘mol miss"; 2, explant treatment and sterilization: selecting an under- ground bulb and a flower spike which is not flowering of Ornitho- galum caudatum as a starting material, peeling the outer 1-2 lay- ers of scales from the bulb, cutting the flower spike to a length of 1 cm, washing the treated explant with a detergent solution for 20 min, during which it is gently stirred continuously, then rins- ing the detergent-washed explant material for 20-30 min with run- ning water, transferring the explant rinsed with running water to a sterile conical flask on an ultra-clean workbench which has been sterilized, soaking the explant in 75% alcohol for 60 s, during which the conical flask is gently shaken continuously to remove air bubbles attached on the surface of the explant, pouring out the 75% alcohol, then soaking the material in 0.1% HgCl, solution for 8-10 min or soaking the material in a NaClO solution with an available chlorine concentration of 1% for 10-15 min, during which the conical flask is gently shaken continuously to remove air bub- bles attached on the surface of the explant, pouring out the 0.1% HgCl: solution or the 1% NaClO solution, washing the sterilized explant with sterile water for 4-5 times, and soaking the explant in sterile water for later use.

3, primary culture: transferring the surface-sterilized ex- plant to a sterile inoculation plate on an ultra-clean workbench, sucking off the surface moisture of the explant with sterile fil-

ter paper, and inoculating the explant in a medium for primary culture which is MS+6-BA 0.1-0.5 mg/L+NAA 0.05-0.2 mg/L for prima- ry culture, the culture temperature being 25+2°C, the illumination time being 12-14 h/d, and the illumination intensity being 45-60 u-mol ms; 4, adventitious bud induction: inoculating the clean and via- ble material obtained from the primary culture in a medium for ad- ventitious bud induction which is MS+6-BA 2.0-4.0 mg/L+NAA 0.05-

0.2 mg/L on a sterile workbench for adventitious bud induction, the culture temperature being 25+2°, the illumination time being 12-14 h/d, and the illumination intensity being 45-60 u ‘mol mss"; 5, adventitious bud multiplication: dividing 2-3 adventitious buds with a height of 2-3 cm obtained from adventitious bud induc- tion culture into a cluster on the sterile workbench, gently cre- ating some wounds on the bases of the adventitious buds using a sterile scalpel, and then inoculating the adventitious buds in a medium for adventitious bud multiplication which is MS+6-BA 0.5-

3.0 mg/L+NAA 0.05-0.2 mg/L for multiplication culture, the culture temperature being 25+2°C, the illumination time being 12-14 h/d, and the illumination intensity being 45-60 pmol m™-s*; 6, adventitious bud rooting induction: separating the clus- tered adventitious buds growing to a height of 3-5 cm into indi- vidual adventitious buds from the base, and then inoculating the adventitious buds in a medium for rooting which is MS+IBA 0-0.5 mg/L or 1/2MS+IBA 0-0.5 mg/L for adventitious root induction, the culture temperature being 25+2°C, the illumination time being 12-14 h/d, and the illumination intensity being 45-60 u ‘mol ‘ms, the rooting rate being 100%; 7, tissue culture seedling domestication and transplantation: after 3-5 adventitious roots of 1-2 cm grow from the bases of the adventitious buds, moving the tissue culture to a greenhouse, opening the bottle cap after culture with scattered light for 5 days, and then the culture continues for another 1-2 days to com- plete the domestication of the tissue culture seedlings before the transplantation, then taking out the tissue culture seedlings from the culture bottle, washing out the agar on the surface of the tissue culture seedlings, planting the seedlings in a matrix for culture for 15 days with a moisture content maintained above 90%, and then culturing the seedlings normally in the greenhouse, the transplanting survival rate being 100%.

Finally, it should be noted that the examples above are only representative examples of the present invention. It is clear that the technical solutions according to the invention is not limited to the examples described above, but that many variants are possi- ble, and all variants being directly derivable or conceivable by a person skilled in the art from the disclosure of the invention shall be considered to be within the protection scope of the in- vention.

Claims (2)

CONCLUSIONS A method for tissue culture of Ornithogalum caudatum, characterized in that the method comprises the following steps: 1), explant selection and treatment: selecting a subterranean bulb and a non-flowering flower stem of Ornithogalum caudatum as starting material, the outer 1 -peel off 2 layers of scales from the bulb, cut the flower stalk to a length of 1 cm, and use them as an explant for tissue culture; 2), primary culture: transfer the surface-sterilized explant to a sterile inoculation plate on an ultra-clean bench, aspirate the surface moisture of the explant with sterile filter paper, and inoculate the explant in a primary culture medium containing MS +6-BA is 0.1 to 0.5 mg/L+NAA is 0.05 to 0.2 mg/L for primary culture; 3), adventitious bud induction: inoculating the clean and viable material obtained from the primary culture in a medium for adventitious bud induction, namely MS+6-BA 2.0 to 4.0 mg/L+NAA 0.05 to 0 .2 mg/L on a sterile bench for accidental bud induction; 4), adventitious bud propagation: dividing 2 to 3 adventitious buds with a height of 2 to 3 cm obtained from adventitious bud induction culture in a cluster on the sterile bench, making few wounds carefully on the base of the adventitious buds using a sterile scalpel and then inoculating the adventitious buds in a adventitious bud propagation medium containing MS+6-BA 0.5 to 3.0 mg/L+NAA 0.05 to 0.2 mg/L L is for multiplication culture; 5), induction of adventitious bud rooting: separating the clustered adventitious buds growing to a height of 3 to 5 cm into individual adventitious buds from the base, then inoculating the adventitious buds in a rooting medium containing MS+IBA 0 to 0.5 mg/L or 1/2MS+IBA 0 to 0.5 mg/L is for adventitious root induction; 6), domestication and transplantation of tissue culture seedlings: after growing 3 to 5 adventitious roots of 1 to 2 cm from the base of the adventitious buds, moving the tissue culture to a greenhouse, opening the bottle cap after cultivation with straw - light for 5 days, and then the culture continues for another 1 to 2 days to complete the domestication of the tissue culture seedlings before transplantation, then take out the tissue culture seedlings from the culture flask, wash out the agar on the surface of the tissue culture seedlings, planting the seedlings in a matrix for culture for 15 days with a moisture content maintained above 90%, and then growing the seedlings normally in the greenhouse. The tissue culture method of Ornithogalum caudatum according to claim 1, characterized in that the minimum media for primary culture, adventitious bud induction and multiplication, and adventitious bud root induction are all MS media.