| Pinellia ternata（Thunb.）Breit.is a medicinal herb belonging to the genus Pinellia of the family Araceae.For many years,the industrialization development of P.ternata has been restricted by uneven germplasm and unstable provenance.The artificial seed technology of P.ternata has become an important way for the sustainable development of P.ternata provenance industry.Loose callus is an ideal material for obtaining high-quality artificial seed embryos.The relationship between cell wall components and callus texture was investigated in this study after selecting culture conditions and establishing a callus induction system for P.ternata,combining microscopic observation of tissue morphology and cell structure with chemical component determination.Pectinase was discovered to play an important role in the formation of P.ternata loose callus.To obtain P.ternata loose callus,the induction conditions of pectinase in the formation of P.ternata loose callus were investigated at the same time.Following that,investigate the effects of various plant hormones and polyamines on the subculture proliferation of P.ternata loose callus,improve the proliferation rate of P.ternata loose callus while maintaining strong cell division and proliferation ability,in order to facilitate the subsequent production of P.ternata artificial seed embryos by cell suspension culture,and provide strong support for solving P.ternata technical problems.The main results are as follows:1.With the help of a single factor test along with an orthogonal test,the effects of various explants,light intensity,sucrose concentration,and the types and concentrations of auxin and cytokinin on the callus induction rate of P.ternata were examined.The findings demonstrated that when the leaves at 30 days of seedling age were taken as explants under light intensity of 900～1200 lx and 1.0 mg·L-1 2,4-D+0.5 mg·L-1 6-BA+40 g·L-1 sucrose were added,the callus induction rate of P.ternata was increased.The callus displayed a healthy level of yellow,loose,and voluble development.2.The four forms of cultured callus were white loose callus III,transparent loose callus II,yellow embryonic loose callus I,and yellow dense callus IV.Different calluses were found to have drastically diverse cell wall compositions and structures.I and IV stood out among them for having the highest levels of callus porosity,and their distinctions could also be seen in the cell wall’s pectin concentration and pectinase activity.The water-soluble pectin content was likewise much larger than that of type IV callus,and type I callus had the highest pectinase activity among the four callus.Type IV dense callus included more protopectin,less water-soluble pectin,and less pectinase activity.According to these findings,a difference in cell wall pectinase activity led to the degradation of protopectin and the production of a significant amount of water-soluble pectin,which caused the primary wall and intercellular layer of callus cells to lose their structural integrity and had an impact on the texture of the callus.The results of using P.ternata loose callus as a material and directly adding exogenous pectinase solution showed that when the enzyme activity was higher than 20.0 U·m L-1,the callus texture became more loose and grew more vigorously,proving that the texture of the P.ternata callus was closely related to changes in cell wall pectinase activity.Polyamine combination treatment induced changes in pectinase activity in the callus via different light intensities,sucrose concentrations,and plant hormones.When the pectinase activity was too high,the callus was prone to albinism and wet softening,whereas when the enzyme activity was too low,the callus became solid and agglomerated,making it difficult to form a loose callus.The loose callus of P.ternata is prone to loose and readily dispersed particles,retaining a healthy development state,when the pectinase activity is maintained in the range of 4～5U·g-1.Pectinase activity increased with increases in light intensity,sucrose,refined ammonium,2,4-D and IAA concentrations,whereas putrescine and 6-BA concentrations showed a negative link with pectinase activity,and spermidine had essentially no effect.While polyamines typically have a lower effect on the pectinase activity,the pectinase activity of P.ternata loose callus cultured under them is about 1.44～3.24 U·g-1.Among these,light,sucrose,and plant hormones can all significantly increase the pectinase activity of P.ternata loose callus,maintaining between 4-7 U·g-1.As a result,we preliminarily selected the main conditions for inducing endogenous pectinase activity in P.ternata,namely,the appropriate light intensity is 1800～2000 lx,sucrose concentration is 40 g·L-1,plant hormones are 2.0mg·L-1 2,4-D and 1.0 mg·L-1 IAA.3.The material for proliferation culture was taken from the callus of P.ternata.The growth curve demonstrated that the proliferation peaked on day 15,and at this time,cell growth was active,making it ideal for the second culture cycle.The effects of various light intensities,plant hormones,and polyamines on the early growth of the loose callus of P.ternata were examined using a single factor test.The results demonstrated that with higher light intensity of 1800～2000 lx,the loose callus could form at a faster proliferation rate.The addition of 0.5 mg·L-1 IAA and 0.5 mg·L-1 6-BA can improve the callus’s quality while preserving its loose,easily disseminated texture.During polyamine culture,it was discovered that adding 5.0 mg·L-1 of ammonium had the best results.The callus of P.ternata loose had good growth and a loose texture.It was discovered by comparing P.ternata loose callus’cell suspension development with various times of subgeneration that the fewer times of subgeneration,the more vigorous the cell division.The peak fresh weight of P.ternata loose callus’short-term subgeneration could reach 1.197 g,the density of live cells was 5.67×105per milliliter,and the time of embryonic formation was approximately 30 days. |
