红松成熟胚愈伤组织诱导外植体选择及培养条件优化

【目的】通过培养条件的优化和适宜外植体类型的筛选,解决红松成熟胚愈伤组织诱导中存在的愈伤组织诱导率低、生长状态差、褐化率较高等问题,为建立红松成熟胚体胚发生体系奠定基础。【方法】用5种培养基类型、3种蔗糖浓度和9种植物生长调节剂组合,选择8种来源于成熟种子不同部位的外植体,对愈伤组织诱导的培养条件和适宜外植体类型进行优化和筛选。【结果】(1)在5种培养基中,DCR培养基上愈伤组织诱导率高于其他培养基、生长状态好于其他培养基;(2)添加在DCR中的3种蔗糖浓度中,35 g/L时的愈伤组织诱导率最高(92.01%)、褐化率最低(17.01%);(3)DCR添加35 g/L蔗糖情况下,9种植物生长调节剂组合中NAA 2.0 mg/L+6-BA 1.5 mg/L组合的愈伤组织诱导率最高、褐化率最低、生长状态最佳;(4)在选出的优化培养条件下,8种来源外植体中,从裸胚上切离的下胚轴的愈伤组织诱导率高、褐化率低,且增殖能力强,具备胚性愈伤组织的形态解剖特征。【结论】红松成熟胚愈伤组织诱导的优化培养条件为DCR+蔗糖35 g/L+NAA 2.0 mg/L+6-BA 1.5mg/L,适宜外植体裸胚上切离的下胚轴。

Optimization of culture conditions and selection of suitable explants for callus induction from mature embryo of Pinus koraiensis

Object] Through optimizing culture conditions and determining suitable explant sources,our goal was to resolve the problems of low induction rates,poor growth and high browning rates in callus induction from mature Pinus koraiensis embryos.Our study could provide insights into cstablishing an effective system of somatic embryogenesis.Methods]We used eight different parts of mature P.koraiensis seeds (cotyledon slices from naked embryo,hypocotyl slices from naked embryo,radicle slices from naked embryo,intact naked embryo,intact kernel with embryo and megagametophyte,kernel with 0.5 cm slices at the ovule hole end,half kernel with the ovule hole end,and two-third longitudinally kernel slices of intact embryo) as explants.To find the optimal conditions for callus induction,we individually cultured these explants in five types of basic media (DCR,GLH,MSG,LM and EM),supplemented with sucrose at three different concentrations (30,35 and 40 g/L) and nine combinations of 6-BA (1,1.5 and 2 mg/L) and NAA (1,2 and 4 mg/L).Results] ① Of the five tested media,the DCR medium supported the highest callus induction rate and the optimum callus growth status,with 27.03％ callus browning rate,which was significantly lower than those in LM and EM media,but not significantly different from those in GLH and MSG media.The callus derived from the hypocotyl of the intact embryo explant was semi-transparent and white in color with a tiny protuberance on the surface,and exhibited strong growth capability.② DCR medium supplemented with 35 g/L sucrose led to the highest callus induction rate (92.01％)and the lowest browning rate (17.01％) among the three tested sucrose concentrations.Furthermore,the callus exhibited a more healthy growth status and stronger proliferation capability under this condition than with the other two sucrose concentrations.③ DCR medium with 35 g/L sucrose,2.0 mg/L NAA and 1.5 mg/L 6-BA led to the highest callus induction rate,lowest browning rate,and optimum growth status among the nine tested combinations of plant growth regulators.NAA exhibited a stronger influence on callus induction rate than 6-BA,but they displayed comparable effects on callus browning rates.④ In addition,under these optimized culture conditions of DCR with 35 g/L sucrose,2.0 mg/L NAA,and 1.5 mg/L 6-BA,the sliced hypocotyl explant of naked P.koraiensis mature embryo showed higher callus induction rate (98.00％),lower browning rate (20.00％),and stronger proliferation capacity than the other seven explants,and the morphological and histological characteristics of the induced callus also conformed to those of embryogenic calli.On the other hand,sliced cotyledon from naked embryo showed a relatively low induction rate (56.92％) and high browning rate (47.69％) without any embryogenic callus morphological and histological characteristics.Sliced radicle from naked embryos and intact naked embryos exhibited both high callus induction rates (88.57％ and 100％,respectively) and significant browning rates (67.14％ and 91.43％,respectively).The other explants,including intact kernel with embryo and megagametophyte,kernel with 0.5 cm slices on the ovule hole end,half kernel with ovule hole end,and longitudinal two-third slices of the kernels of instant embryos showed high callus induction rates (＞80％) and markedly low browning rates (＜5％),but the callus originated from a megagametophyte that could not proliferate in subculture.Conclusion] The optimal combination of culture conditions and explant type for callus induction from mature seeds of P.koraiensis was the use of DCR medium supplemented with 35 g/L sucrose,2.0 mg/L NAA and 1.5 mg/L 6-BA to culture sliced hypocotyls of naked mature embryos.