| Andrographis paniculata（Burm.f.）Nees is a kind of clinical traditional medicinal herb in China.It has long been known as the“natural antibiotics”.Because it is a kind of cryptanthous plant,the separation of species resulted in many problems of the quality of them,such as degeneration of variety,decline of production and reduction of effective constituent.It is extremely important to cultivate new species of varieties of high yield and good quality.The traditional method of breeding new breeds usually needs five to seven years of self-service,which can produce stable varieties.But with the development of the male nucleus in vitro,the haploid plant is cultivated more rapidly and the homozygous diploid can be obtained by chemical treatment.It not only can be shorten the breeding time,but also the recessive gene could be expressed,the germplasm resources could be expand,and the new varieties with higher quality and higher yield could be produced.（1）The anatomical study on the flower of Andrographis paniculata（Burm.f.）Nees for the identification and breeding of Andrographis paniculata（Burm.f.）Nees to provide information.By observing the whole flower,oberserving under the stereoscope and morphological anatomy,studied the microscopic identification of.the flower of Andrographis paniculata（Burm.f.）Nees.The result shows,racemes of flowers,1.2cm1.6cm,white or pale purple flowers,full of glandular hairs and puberulent.Five calyx,lanceolate,labiate corolla,upper lip petals two,with one lobed,white petals connate;the lower have two deep cleft,deep purple markings.Two stamens,intergrowth,filaments white woolly;one pistil stigma,up to two of anther,ovary,two carpels,two rooms,axile.The pollen grain have oblong or spherical surface,shallow wrinkles,longitudinal groove,three germ pores.The observation of developmental stage of microspores,there are nedium uninucleate,late uninucleate,binucleate and Mature stage.Butit is unclear to the corresponding relationship of bud length and period of microspore.These characteristics not only can be used for identification of Andrographis paniculate（Burm.f.）Nees,but also can provide data for the breeding of Andrographis paniculata（Burm.f.）Nees.（2）In order to pollen as experimental materials,clear the relationship between growth stages of flowers and the acquisition time of pollen viability.And establishment of in vitro cultivation method.By the I2-KI staining,TTC staining to determinate the activity of pollen in different periods and acquisition time from Andrographis;the in vitro culture of different medium components and the effect of pretreatment on pollen tube germination.Determination of pollen viability of I2-KI dyeing,the rate of anthers and filaments for 1:4and at the 15:00 collecting taining rate of pollen is the highest;using TTC staining method,the rate of anthers and filaments for 1:6 and at the 15:00 collecting pollen staining rate was the highest,TTC method for the determination of the pollen viability and pollen tube germination trend are the same.The optimal medium of pollen germination was 10%sucrose+0.3g·L-11 calcium chloride+1g·L-11 boric acid+15%PEG,and the germination rate was 82.5%.The pretreatment conditions had a significant effect on the pollen germination in vitro.Cold treatment at 4℃could significantly increase the germination rate of pollen（P<0.05）.Heat treatment of 24 h and cold treatment 48h at 4℃and 30℃through pollen tube germination rate had no significant difference with control group.These characteristics can provide data for the breeding of Andrographis paniculata（Burm.f.）Nees.（3）Taking the flowers of Andrographis paniculata as experimental material,the preparation conditions of pollen microspores suspension andand differentiation conditions of Andrographis paniculata callus were determined.Under aseptic conditions,the pollen microspore suspension and anthers were used as the experimental materials.In the orthogonal experiment with the MS medium as the basic medium,the induction rate was 0.The pollen microspore suspension and anthers could not produce the visible callus with MS as the basic medium.The N6 medium as the basic medium,the induction rate was12.96%.The optimal level of combination is A2B1C2D3,that is,the concentration of PP333 is 1 mg·L-1,the concentration of KT is 1.0 mg·L-1,the concentration of 2,4-D is0.5mg·L-1,and the concentration of NAA is 0.5mg·L-1.The highest induction rate of20.34%on the Nistch medium,higher than the induction rate of 12.96%on the N6 medium.However,the induction time of anther on Nistch medium was long,and it was easy to brownish during subculture.And Nistch medium was not suitable for the haploid induction of Andrographis paniculata.The differentiation medium could not induce embryogenic callus differentiation,but induced embryogenic callus could be used as the material for the next experiment.（4）chromosome ploidy and flow cytometry were used to identify the ploidy of induced embryogenic callus,and compared with normal tetraploidy and lab induced autotetraploid plants.The best method of chromosome preparation is:2000 mg·L-1colchicine solution,treatment time 2h,refrigerator at 4℃.Distilled water was rinsed for 5times,5min at a time,and the pre treated root tip materials were transferred to Kano’s fixative[ethanol:acetic acid（3:1）]to kill cells quickly,immobilized for more than 24hours,and refrigerators were treated at 4℃.Rinsed in the distilled water for 5 times,each time 5min.The fixed apex was taken into 1mol L-11 hydrochloric acid solution at 60℃at constant temperature water bath,dissociated 20 min,and distilled water was rinsed 5 times,each time 5min.The dissociation of good material on the slide,with a blade to cut out the elongation zone,leaving only 12 mm and a drop of meristem carbolfuchsin staining was improved after 5 min with dilute acetic improved carbolfuchsin pickling to remove excess stain,then press,microscope,photographic recording.Andrographis haploid callus chromosome is X=12,the diploid chromosome is 2X=24,the tetraploid chromosome4X=48.In vitro androgenesis of Andrographis callus chromosome was 1/2 of the original diploid plants.The autotetraploid chromosome number is 2 times of the original diploid plants.（5）The content of andrographolide and dehydrandrographolide in different ploidy Andrographispaniculataplantswasdeterminedbyhighperformanceliquid chromatography.The correlation between the ploidy types of Andrographis paniculata chromosome and the content of andrographolide and dehydrated andrographolide were identified.The content of andrographolide Andrographis haploid embryogenic callus(0.660 mg·g-1)lower than the diploid and tetraploid plants(8.836、7.299 mg·g-1),dehydroandrographolide in Andrographis tetraploid(2.893 mg·g-1)higher than the diploid(0.659 mg·g-1).The dehydrated andrographolide was not accumulated in the haploid embryogenic callus of Andrographis paniculata.The dehydrated andrographolide was not accumulated in the haploid embryogenic callus of Andrographis paniculata.Dehydration andrographolide mainly came from the leaves of Andrographis paniculata.Haploid callus of Andrographis paniculata had not yet differentiated into leaves and failed to accumulate dehydrated andrographolide.In the process of differentiation,a very small amount of andrographolide(0.659 mg·g-1)was accumulated during the differentiation of the haploid embryogenic callus of Andrographis paniculata.This method can be used to evaluate the quality of the haploid plant of Andrographis paniculata after differentiation. |
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