Study On The Growth Characteristics And Protoplast Instantaneous Transformation Of Amorphophallus Bulbifer And Amorphophallus Konjac

Konjak is a perennial monocotyledonous plant of Amorphophallus Blume in Araceae.Its bulb contains a large amount of glucomannan,which can be used for medicine,food,health care and other purposes.At present,the main varieties of konjac cultivated in production are Amorphophallus konjac（A.konjac）and Amorphophallus bulbifer（A.bulbifer）.Nowadays,it is generally believed that the content of glucomannan in A.konjac is high,but due to its weak disease resistance and low reproductive coefficient,the actual yield is not satisfied.However,the A.bulbifer also contains high glucomannan,and has strong disease resistance and high reproductive coefficient.Compared to A.konjac,planting and control are simple,and the yield is higher.There are relatively few reports on the comparative study of the growth characteristics between A.konjac and A.bulbifer as well as genetic transformation of konjac protoplasts.This study analyzed the content of glucomannan,starch,soluble sugar,and soluble protein in the roots and stems of A.bulbifer and A.konjac seeds and annual small konjac seeds at various stages of growth,as well as the changes in the content of four hormones,zeatin（ZT）,gibberellin（GA）,auxin（IAA）,and abscisic acid（ABA）,to understand the physiological and biochemical differences between the growth of A bulbifer and A.konjac;Provide theoretical basis for adopting different cultivation measures for planting different konjac varieties.Protoplasts were isolated from young leaves of A.bulbifer,and green fluorescent gene was transferred into the isolated protoplasts by PEG/Mg²⁺method.A preliminary study was conducted on the transient transformation system of protoplasts from leaves of A.bulbifer.The main results are as follows:1.Analysis of the difference in nutrient content between A.bulbifer and A.konjac:The content of glucomannan in the corms of A.konjac,regardless of whether it is a seed or a small seed,is not significantly different from that of A.bulbifer at various stages,but the content of glucomannan in the A.konjac seeds will decrease at maturity,However,the glucomannan content of the A.bulbifer seeds was significantly higher than that of the A.konjac seeds.The starch content in the corms of A.konjac at various stages is slightly higher than that of A.bulbifer,especially in the seed of A.bulbifer.However,there is no significant difference between the starch content in the corms of A.konjac at the mature stage and the starch content in the corms of A.bulbifer at the mature stage when the seed of A.bulbifer sprouts and grows.During the germination and full seedling stages,the content of soluble sugar and soluble protein in the seeds and small taros was significantly higher A.bulbifer than A.konjac,but there was no difference between the content of soluble sugar and soluble protein in the A.bulbifer and A.konjac at the late growth stage.The research results showed that the physiological metabolism of A.bulbifer was stronger than A.konjac at the early growth stage,which was conducive to the formation and growth of multi leaf of A.bulbifer.2.Analysis of the difference in hormone content between A.bulbifer and A.konjac:The Zeatin（ZT）content of konjac seedlings grown from A.bulbifer seeds and small konjac seeds increased during the expansion stage,while the growth of A.konjac seedlings and small taro seeds increased during the germination and full seedling stages.This indicates that a higher ZT content promotes the growth of A.konjac seedlings,while for A.bulbifer,it promotes the growth of corms.At the early stage of germination of both A.bulbifer seeds and A.konjac seeds,the change in the content of gibberellin（GA）was not significant,but the content of GA in A.konjac seeds and small taros increased significantly when the bulbs began to increase during the expansion stage,while the content of GA in A.bulbifers seeds and small taros increased significantly during the mature stage,indicating that GA has a promoting effect on konjac bulb enlargement.The content of auxin（IAA）in the seed of A.bulbifer and A.konjac is significantly higher than these small taros,but it tends to decrease during the growth and development process.However,in the seed of A.bulbifer and A.konjac,the content of IAA increases during the germination period,and then decreases with the growth of konjac,indicating that IAA has the effect of promoting the germination of A.bulbifer and A.konjac;The content of abscisic acid（ABA）was the highest in the dormant stage of seedling seeds and small taros in the cold storage.With germination and growth,the content of ABA gradually decreased,but significantly increased at the mature stage,indicating that ABA promoted the maturation of konjac.These results indicate that the difference in hormone content between A.bulbifer and A.konjac is mainly reflected in the high content of ZT in A.bulbifer in the early stage,which promotes the growth of early germination and full seedling stage,while A.konjac has a high content of ZT in the later stage of expansion,which promotes the expansion and growth of A.konjac in the later stage..3.Optimization of the optimal enzymatic hydrolysis conditions for the preparation of protoplasts from A.bulbifer leaves:Cellulase and dissociative enzymes have important effects on the preparation of protoplasts.Insufficient enzymatic hydrolysis,insufficient destruction of the cell wall,and inability to properly separate the cytoplasmic wall,resulting in reduced protoplast production;If the enzyme is hydrolyzed for too long,the protoplast will be damaged,resulting in a decrease in the integrity of the protoplast.The optimal conditions for protoplast preparation were as follows:adding 0.1 g/m L of leaves to the enzymatic hydrolysate when the cellulase R-10 was 2.0%and the dissociating enzyme R-10 was 0.1%,and obtaining high-quality protoplasts in a dark environment at a temperature of 30℃for 4 hours.4.Study on the transient expression system of A.bulbifer protoplasts:Construct a p RI101 Ab ZFP48-GFP overexpression vector,and establish a transient expression system of A.bulbifer protoplasts using PEG/Mg2+method.Observe and take photos under a laser confocal microscope to show that GFP is expressed in the entire protoplast,obtaining the transient transformation results of A.bulbifer protoplasts.The research shows that there is no significant difference in the content of glucomannan and starch between the seed and the small taros of Konjac,while the content of soluble sugar and soluble protein in A.bulbifer is significantly higher than A.konjac,which is beneficial to the growth of A.bulbifer with multiple leaves;The difference in ZT content between A.bulbifer and A.konjac indicates the promoting effect of ZT on the early growth of A.bulbifer.A method for instantaneous transformation of A.bulbifer protoplasts was established by preparing and optimizing protoplasts from the leaves of A.bulbifer seedlings,laying the foundation for future biological research such as genetic transformation of A.bulbifer protoplasts.