Study On Tissue Culture And Bulbous Enlargement Of Bletilla Striata In Vitro

In this study,the technique of bulbous enlargement was established on the basis of the germination of seeds and the aseptic culture of Bletilla striata seedlings in vitro.The purpose was to explore the method of promoting the bulbous enlargement of B.striata in vitro,improve the existing rapid propagation technique and screen out the medium formula suitable for bulbous enlargement of B.striata in vitro.Furthermore,the seedlings were replaced by bulbs for transplanting,which provided a reference for in vitro rapid propagation,breeding and industrial production of B.striata.The conclusions were described as follows:1.Establishment a aseptic culture system of B.striata seedlingsBased on the germination of seeds,the aseptic culture system of B.striata seedlings adopted the rapid propagation method of“inducing protocorm→adventitious bud differentiation→adventitious bud proliferation→rooting culture”.Subculture with adventitious buds was superior to subculture with protocorms.The medium for seed germination and protocorm induction was MS+0.01 mg/L NAA+1.0 mg/L 6-BA;The adventitious bud differentiation and proliferation medium were MS+0.1 mg/L IBA+1.0mg/L 6-BA;The medium of rooting was 1/2MS+0.5 mg/L IBA+1.0 mg/L GA_3.2.The technique of bulbous enlargement of B.striata in vitroThe seedlings in the rooting medium were cultured for 40 days in vitro,and then the leaves and roots were cut out.The bulbs of similar size were inoculated into the enlarged medium to study the effects of various factors on the bulbous enlargement of B.striata in vitro.The orthogonal test results showed that the effects of NAA and basal medium on bulbous enlargement was greater than that of 6-BA.When the concentration of NAA was0.1 mg/L,the value of each index of bulb enlargement were the largest.1/2MS was more beneficial to the rooting and bulbous enlargement of B.striata in vitro.However,6-BA had no significant effect on each index of bulbous enlargement.In addition,sucrose was an important factor affecting the bulbous enlargement of B.striata in vitro.With the increase of sucrose content,the bulbs gradually expanded.Situation of the roots,plant growth and bulb proliferation were better when sucrose content was 20⁴0 g/L.Therefore,the medium formula for the initial bulbous enlargement of B.striata was 1/2MS+0.1 mg/L NAA+1.0mg/L 6-BA+20 g/L sucrose.Further research found that the weight gain rate was the highest（33.41%）with 0.5 g/L activated carbon.The suitable concentration of PP₃₃₃ was 10mg/L.The effect of SA on bulbous enlargement was not obvious and was not suitable for rooting of B.striata.The photoperiod has no significant effect on bulbous enlargement of B.striata.The short-term dark culture was not conducive to the rooting and leaves,but promoted the bulbous enlargement of B.striata.Liquid medium can promote nutrient absorption of bulbs more effectively in vitro.10 mmol/L of NH₄⁺and 0.625 mmol/L of H₂PO₄^-were suitable for the bulbous enlargement of B.striata.Correlation analysis showed that the growth of root promoted bulbous enlargement and the differentiation of bud inhibited bulking.And the proliferation rate of bulbs was inversely proportional to the initial value of bulbs.The smaller the initial values of bulbs,the higher the enlargement rate.In addition,the dynamic results of the bulbous enlargement time of B.striata in the initially selected enlarged medium formula showed that the bulbs increased slowly during the first 90 days of the enlargement culture.The difference between the indicators of bulbous enlargement and the corresponding indicators at the time of inoculation was not significant.The weight gain rate increased linearly from the 90¹50 d.The diameter and high proliferation rate also increased the fastest at the 90¹20 d,and still increased significantly at 120¹50 d.Therefore,it was recommended that the bulbous enlargement time of B.striata in vitro was at least 120 d.3.Bulb transplanting of B.striata in vitroThe bulbs from the enlargement medium were classified to three grades on the basis of diameter:3.5⁵ mm,5⁷ mm,>7 mm,and transplanted with rooting seedlings of B.striata in vitro.The results showed that the survival rate of transplanted bulbs（80⁹8.33%）was higher than that of rooted seedlings（66.67%）.The larger the bulbs,the higher the survival rate of transplanting,and the survival rate of bulb with diameter>7 mm could reach over 98%.