CN102726293A

CN102726293A - Method for in vitro preservation of betel nut genetic resources

Info

Publication number: CN102726293A
Application number: CN201210213429XA
Authority: CN
Inventors: 黄丽云; 周焕起; 范海阔; 李�杰; 刘立云
Original assignee: Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences
Current assignee: Coconut Research Institute of Chinese Academy of Tropical Agricultural Sciences
Priority date: 2012-06-27
Filing date: 2012-06-27
Publication date: 2012-10-17

Abstract

The invention belongs to the plant cells engineering technical field, and relates to a method for in vitro preservation of betel nut genetic resources. The method is characterized in that germplasm resource betel nut trees with 15 years growth are selected for planting, embryo is removed to place in a pre-culture medium for culturing, the embryo is germinated to complete plants, when the plant is grown to the height with 1.5-2 cm and placed in a preservation medium for preserving and culturing to realize the in vitro preservation of the betel nut genetic resources. The method has the advantages of simple technology and strong operationality, by raising the sugar amount preserved in the medium, the growth speed of the betel nut plant can be prolonged, The method is in favor of preservation of germplasm resources, and is in favor of exchange of germplasm resources at home and abroad, the generation of spread of insect diseases in the germplasm exchange process can be avoided, and the problems of large land occupation, high cost and the like can be solved, and a theoretical support is provided for in vitro rapid propagation technology. The method of the invention can provide an effective approach for collection and preservation of the betel nut germplasm resources and industrial production of sprout.

Description

A kind of method of betel nut germ plasm resource {in vitro} conservation

Technical field

The invention belongs to field of plant cell engineering technology, relate to a kind of resource in-vitro conservation method of babassu, especially a kind of method of betel nut germ plasm resource {in vitro} conservation.

Background technology

Betel nut (Areca catechu L.) belongs to Palmae Areca plant, is China's torrid areas main economic crops and tropical afforestation seeds, also is one of important southern medicine resource of China, is classified as first of the four Da Nan medicines, has very high economic worth.In the inferior state and the Pacific region, betel nut mainly as the local masses' the preference of chewing, cultivate gradually to commercialization, large-scale development by betel nut.Betel nut mainly is distributed in Hainan Province in China, and gross yield accounts for more than 99% of continent betel nut output.Cultivated area in Hainan has reached 1,030,000 mu at present, and output reaches 14.36 ten thousand tons, and the fresh fruit annual value of production is more than 2,000,000,000 yuan, and processed output reaches 50～6,000,000,000 yuan, is the main sources of finance of nearly 2,000,000 peasants in east, Hainan, middle part and southern areas.

Tissue culture is the effective method of micropropagation of plants; The important means that embryo culture is preserved breeding as germ plasm resource is widely used; The embryo that utilizes embryo culture to obtain is trained seedling, and not only cost of transportation is low; Also can avoid the damage by disease and insect in the germplasm exchange process to propagate, utilize embryo or embryo training seedling can solve the land for growing field crops and preserve problems such as floor space is big, expense height as quality saving.

Betel nut is the single pole babassu, and conventional method of nourishing and generating such as cuttage, grafting, overhead layering etc. are not suitable for the betel nut breeding, and mainly lean on seminal propagation, the insect pest but this method spreads disease easily.To so far, though the betel nut tissue culture has report, do not set up comparatively perfect cultivating system as yet at home and abroad, the technical research of betel nut tissue culture quick breeding also is in the starting stage.

Summary of the invention

The method that the purpose of this invention is to provide a kind of betel nut germ plasm resource {in vitro} conservation; Through in preserving medium, improving the growth rate that sugared consumption can delay plant well; Help the exchange of domestic and international germ plasm resource, avoid in the germplasm exchange process, taking place damage by disease and insect and propagate, also can solve the land for growing field crops simultaneously and preserve problems such as floor space is big, expense height; For the rapid propagation in vitro technology provides theory support, the batch production production of betel nut germ plasm resource being collected preservation and seedling provides an effective way.

The technical scheme that the present invention adopted:

A kind of method of betel nut germ plasm resource {in vitro} conservation, its step is following:

1, chooses 15 years above germ plasm resource bearing-age trees of the age of tree, gather 7～8 months betel nut kind fruit, excise most of pericarp and endosperm, leave and take the endosperm piece that is wrapped in complete embryo; The endosperm piece is cleaned the back sterilization, place suck dry moisture on the sterilization filter paper, take out embryo then.

2, place pre-culture medium to cultivate 40～50 days embryo, treat that embryo germination forms bud and stem, culture of rootage forms whole plant, when plant strain growth to 1.5～2cm height, carries out {in vitro} conservation.Said pre-culture medium is to be basal medium with the MS medium, and adds AC 1g/L, sucrose 50 g/L.

3, plant that betel nut 1.5～2cm is high is transferred to preserve in the preservation medium and cultivates, and can realize betel nut germ plasm resource {in vitro} conservation.Said preservation medium is to be basal medium with the 1/2MS medium, and adds BA 2mg/L, NAA 0.5mg/L, sucrose 120g/L, AC 1g/L, agar 8g/L.

Technology of the present invention is simple; Through in preserving medium, improving the growth rate that sugared consumption can delay the betel nut plant well; Help the preservation of germ plasm resource than not using the switching of preserving medium to cultivate long 6～8 months its vegetative period, helps the exchange of domestic and international germ plasm resource; Avoiding in the germplasm exchange process, taking place damage by disease and insect propagates; Also can solve the land for growing field crops simultaneously and preserve problems such as floor space is big, expense height, for the rapid propagation in vitro technology provides theory support, the batch production production of betel nut germ plasm resource being collected preservation and seedling provides an effective way.

Embodiment

Below in conjunction with embodiment, specific embodiments of the invention describes in further detail.Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.

Embodiment one

1, the acquisition of embryo and cultivation in advance

Choose 15 years above germ plasm resource bearing-age trees of the age of tree and carry out follow-up investigation; Confirming after the target strain in time to gather when beginning by fruit 7-8 month after female flower is pollinated, (maturity of betel nut embryo influence the germination rate of embryo, and embryo maturity height then emergence rate is also high, and betel nut kind fruit generally maturation time is 12 months; But 12 months betel nut kind really is unfavorable for obtaining of embryo; Because endosperm as hard as flint when maturation is difficult for getting embryo, generally embryo obtains and easy the cultivation easily in the time of 7-8 month).Collect the back earlier planting fruit with the flowing water flushing, leave the endosperm piece that is encapsulating embryo behind the excision pericarp, size is about 2 * 2cm; Stay the endosperm piece that contains embryo after running water is cleaned; Use 70% alcohol surface sterilization 30S again, with 0.1% mercuric chloride solution sterilization 8min, use aseptic water washing at last 3～5 times again.The endosperm piece of cleaning is put suck dry moisture on the sterilization filter paper; Taking out tender embryo with scalpel places pre-culture medium to cultivate 40～50 days; Treat that embryo germination forms bud and stem, culture of rootage forms whole plant, when plant strain growth to 1.5～2cm height, carries out {in vitro} conservation; (sterilization is earlier got embryo again and can be prevented that sterilized solution from directly contacting embryo idiosome is damaged, influence the later stage embryo germination and grow).Pre-culture medium is to be basal medium with the MS medium, and adds AC 1g/L, sucrose 50 g/L.

2, preserve cultivation

2.1, the design of preserving medium: at first adopt three kinds maybe retardings of growing mode: reduce temperature and cultivate, add chlormequat, improve sugared concentration level; In first experiment, through mensuration, confirm its storage life to growth indexes such as root, bud and basal part of stem, the result is with table 1.Result of study shows, improves the growth that sugared concentration level can obviously delay betel nut embryo seedling.

Table 1 different disposal method is to the preservation influence of betel nut resource

Annotate: (M:1/2MS+BA2mg/L+NAA 0.5 mg/L+AC 1g/L)

2.2, preserve confirming of sugared concentration in the medium: to the design that experimentizes of sugared concentration level, the result sees table 2, and the result shows; Plant strain growth is slow when sugared concentration is 120g/L, and the holding time can delay 6-8 month, and plant is shorter; But basal part of stem is sturdy, and root growth is healthy and strong.

Table 2 different sugar concentration is to the preservation influence of betel nut resource

Culture medium prescription	Holding time	Upgrowth situation
			M+ sucrose 30 g/L	3-4 month	Plant grows fine
M+ sucrose 60 g/L	3-4 month	Plant grows fine
			M+ sucrose 90 g/L	4-5 month	The plant growing way is normal
M+ sucrose 120g/L	6-8 month	The plant growing way is normal
			M+ sucrose 150 g/L	6-8 month	The partial blade deformity, growing way is undesired

Annotate: (M:1/2MS+BA2mg/L+NAA 0.5 mg/L+AC 1g/L, cultivation temperature is 25 ± 2 ℃)

2.3, the preservation of different betel nut kinds: sugared concentration is preservation medium and the ordinary culture medium comparative trial of 120g/L, and survival rate drops to 90% o'clock subculture cycle that recovers to cultivate, and its storage life is seen table 3.

The sugared concentration of table 3 is the preservation medium of 120g/L and the storage life table of comparisons of ordinary culture medium

Title	Preserve and cultivate	Common cultivation
			No. 2, happy east	6-7 month	1.5-2 individual month
The Qionghai kind	8 months	1.5-2 individual month
			1 kind of Dingan County	7-8 month	1.5-2 individual month
No. 1, the Wuzhi Mountain	6 months	1.5-2 individual month
			Protect booth No. 2	6-7 month	1.5-2 individual month
Prosperous No. 9	8 months	1.5-2 individual month

Effective and the safety of the method for betel nut resource {in vitro} conservation provided by the present invention, it is good to preserve material recovery growing state, preserves survival rate up to more than 90%.

Be inoculated on the aseptic medium after the betel nut germ plasm resource disinfection that the present invention's handle is collected; Utilize nutrient in the medium to promote the sprouting of rataria; Be through with in advance cause embryo on maternal plant because growth time prolongs to the recuperate consume of branch of tree; Help setting the recovery of body and the maintenance of time annual production, workable, simple and practical.

The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from know-why of the present invention; Can also make some improvement and suggestion, these improvement ideas also should be regarded as protection scope of the present invention.

Claims

1. the method for a betel nut germ plasm resource {in vitro} conservation is characterized in that, its step is following:

1), choose the above germ plasm resource bearing-age tree of 15 years age of trees, gathers 7～8 months the betel nut kind really, excise most of pericarp and endosperm, leave and take the endosperm piece that is wrapped in complete embryo; The endosperm piece is cleaned the back sterilization, place suck dry moisture on the sterilization filter paper, take out embryo then;

2), place pre-culture medium to cultivate 40～50 days embryo, treat that embryo germination forms bud and stem, culture of rootage forms whole plant, when plant strain growth to 1.5～2cm height, carries out {in vitro} conservation; Said pre-culture medium is to be basal medium with the MS medium, and adds AC 1g/L, sucrose 50 g/L;

3), the plant that betel nut 1.5～2cm is high transfers to preserve in the preservation medium and cultivates, and can realize betel nut germ plasm resource {in vitro} conservation; Said preservation medium is to be basal medium with the 1/2MS medium, and adds BA 2mg/L, NAA 0.5mg/L, sucrose 120g/L, AC 1g/L, agar 8g/L.