邓恩桉微体快繁技术和耐寒性的研究
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摘要
耐寒桉树研究是中国桉树研究一个新的起点。邓恩桉种源和家系试验已经取得一定的进展,但邓恩桉的种子一直依赖于进口。这是由于邓恩桉开花结实力(原产地)较差,无法自产种子推广造林,从澳大利亚进口种子,价格昂贵,致使该树种在生产推广上受到影响。本文以澳大利亚进口的邓恩桉种子进行容器苗培育新技术研究,并以邓恩桉种子作为外植体进行器官发生、愈伤组织发生的研究;对邓恩桉在福建省北部6个县(市)、区造林受冻害情况进行调查,并以幸存的抗寒品种的不同材料作为外植体,探讨不同因素对建立邓恩桉稳定、高效的快速繁殖体系的影响,旨在为工厂化生产优质、高度抗寒的邓恩桉试管苗提供技术支持,同时也为邓恩桉基因工程和分子育种提供技术平台。其主要研究结果如下:
以邓恩桉种子在室内平盘垫滤纸当发芽床,与在室外黄心土98%+过磷酸钙2%的发芽床及黄心土50%+火烧土48%+过磷酸钙2%的发芽床进行比较。结果表明:以室内用平盘垫滤纸作发芽床,其发芽率与实验室发芽率相同。种子播后发芽天数少,发芽整齐,及时移栽于容器袋内,有利于苗木的生长。比在黄心土98%+过磷酸钙2%的发芽床上,平均发芽率提高了37.58%;比在黄心土50%+火烧土48%+过磷酸钙2%的发芽床上,提高了24.88%。在平盘垫滤纸上生长的芽苗平均移栽成活率达63.3%,而在黄心土98%+过磷酸钙2%发芽床的芽苗平均移栽成活率只有32.7%,在黄心土50%+火烧土48%+过磷酸钙2%发芽床的芽苗平均移栽成活率为37.7%。以室内用平盘垫滤纸作发芽床培育的苗木,在秋冬季培育3个月,苗高达10 cm以上的占70%以上,可以比较早出圃上山造林。在室内用平盘垫滤纸作发芽床播种移栽培育容器苗,可节省费用和用工18%～47%。在工厂化育苗中,在测定了种子的品质以后,就可准确地计算和安排种子用量。
以邓恩桉的种子作为外植体,进行愈伤组织的直接诱导;将种子播在培养基上萌芽,再将芽、子叶、下胚轴和根作为外植体进行诱导。结果表明:MS培养基是较适合邓恩桉种子萌芽和生长的基本培养基。MS+KT1.0 mg/L+2,4-D2.0 mg/L+半胱氨酸30 mg/L是诱导种子直接脱分化成愈伤组织的理想培养基配方。邓恩桉实生苗的芽来繁芽的较理想培养基配方为MS+ Ad 2.0mg/L+IAA0.2mg/L。邓恩桉实生苗根脱分化的最佳培养基配方为B5+6-BA2.0mg/L+NAA0.2mg/L。邓恩桉实生苗下胚轴诱导芽分化的最佳培养基配方是MS+6-BA2.0mg/L +2,4-D0.2mg/L。B5+Ad2.0mg/L+IAA0.2mg/L培养基是诱导邓恩桉下胚轴脱分化为可形成正常芽苗愈伤组织的较佳培养基配方。
对经2005年遭20年一遇的灾害性极端严重冻害的闽北各地邓恩桉进行调查。统计数据显示,冻害程度依次顺序为:浦城县>邵武市>建阳市>顺昌县>建瓯市>延平区。邓恩桉的冻害程度与其林龄有关,2004年造>2003年造>2002年造。另外冻害的严重程度与栽培技术措施有关,当年5月份对邓恩桉施第一次肥,8月之前施第二次肥,则可促进高生长,提高梢部的木质化水平,不易遭受冻害;而若施肥推迟到10月,则到了冬季梢部还在生长,木质化程度低,则易受冻害影响。提出邓恩桉在闽北地区种植的北限应为建阳市。
以2005年遭冻害幸存的邓恩桉抗寒品种的不同材料作为外植体进行诱导,结果显示:采用半木质化的带芽茎段(即顶芽以下第3～6个芽位),酒精消毒30 s、升汞消毒10 min的方法来消毒,可以使外植体的褐化率最低,萌芽率最高,污染率中等。采用MS基本培养基、蔗糖用量为40g/L,邓恩桉芽茎段出芽时间为5d,出芽率为78%,出芽指数为2.6,30d芽均高为0.7cm,有效芽为79%,芽的长势正常。其所有的生长因子的综合指标均为最佳。通过细胞分裂素种类和浓度的选择和调配,在KT浓度采用0.5 mg/L与6-BA浓度采用1.0 mg/L二者均为较佳的种类和浓度进行比较,采用KT,无论是出芽率、出芽指数、芽均高、有效芽的比率等几个生长因子均比采用6-BA更佳。通过生长素种类和浓度的选择和调配,发现采用NAA 0.05 mg/L是诱导邓恩桉芽茎段的较佳生长素种类和浓度。在MS+KT1.0 mg/L+NAA0.05 mg/L+4%蔗糖培养基上,芽的长势正常,芽基部的愈伤组织较小,邓恩桉芽的诱导分化能力强,出芽率和芽均高以及有效芽比率高。通过不同的光处理试验表明:邓恩桉的芽茎段在接种后,应对外植体进行暗培养10d,可以在降低外植体褐化率的同时,提高邓恩桉的出芽率。邓恩桉继代的最佳基本培养基是改良MS培养基。改良MS+KT0.8 mg/L+NAA1.0 mg/L+半胱氨酸20 mg/L对邓恩桉芽的继代增殖影响最大,效果最好。继代培养条件中尽量采用太阳光照或照度大约为300LUX的白炽灯光照,温度调整在26℃～28℃,对邓恩桉的继代苗高生长和今后的生根培养均有利。在以改良MS+KT0.8 mg/L+NAA1.0 mg/L+半胱氨酸20 mg/L培养基为对照,加入AC、VH和1.5倍的有机成分,试验证明:无论AC采用何种浓度,邓恩桉的继代苗叶片上均产生大量愈伤组织,最后苗均萎蔫死亡;无论VH添加哪种浓度,对邓恩桉继代苗生长没有明显的影响。而对邓恩桉壮苗的影响最大的是有机成分,当其用量为1.5倍时,继代苗生长健壮,半木质化程度高,对下一步的生根培养具有很重要的作用。
将邓恩桉抗寒品种进行生根试验,结果表明:ABT生根粉能有效诱导邓恩桉组培苗生根,在0.5～3.0mg/L范围内,生根数随处理浓度的增加而增加,生根率最高为78%,其浓度超过1.5mg/L时,明显抑制组培苗的长高。ABT与IBA配合使用可提高根的诱导率,其生根率最高为96%,当ABT浓度超过0.5mg/L时,IBA浓度超过0.05mg/L时,诱导组培苗产生愈伤组织,并影响苗的长高。在培养基中添加GA0.05mg/L,可以明显抑制生根进程中愈伤组织的产生,并促进苗的生长。1/2MS+ABT1.0～1.5mg/L+IBA0.05mg/L+GA0.05是促进邓恩桉组培苗生根的最佳培养基配方。采用二步生根法,可以明显抑制愈伤组织的形成,根系直接从茎段基部长出,生根率提高。
邓恩桉组培苗的下地移栽使用轻基质网袋容器育苗比有土栽培效果好。而在轻基质各种原料配比中,以在6号的基质原料配比(树皮20%+谷壳30%+木屑40%+泥炭土28%+复合肥2%)中,邓恩桉组培苗的各生长因子为最佳,其高生长为23.9 cm,地径为0.25 cm,地上部份鲜重为2.39g,干重为0.65g,移栽成活率为100%。
采用2005年遭冻害幸存的邓恩桉抗寒品种的组培苗造林,经过2008年初特大冰雪灾害考验的、1年多生的组培苗的抗寒能力比同龄的实生苗强,冻害等级只有Ⅰ级,冻死株数<10%。
Study on hardy Eucalyptus is a new research starting point in China. Eucalyptus dunnii seed source and family selection have been made some progress, but its seeds have been relied on imports. Because Eucalyptus dunnii flowering and fruitification (origin) are so poor that there are no homegrown seeds for afforestation. Seed import from Australia is too expensive to be promoted in the production of the species. In this paper, a new technological research will be be introduced that the Eucalyptus seeds from Australia have been used to nurture its container seedlings and been used as explants to induce organ and callus. A survey how Eucalyptus dunnii planting was damaged on the northern six counties (cities) in Fujian is conducted. And different materials from the survivors of the cold hardiness have been used as explants so as to consider which factor is the most important to establish a stable and effect system of Eucalyptus dunnii rapid propagation. By this way, it can provide not only a technical support to produce a high degree of quality and cold hardiness of Eucalyptus dunnii but also a technology platform for genetic engineering and molecular breeding. Its main findings are as follows.
It is compared using flat pad filter as germination bed indoor with using yellow subsoil 98% + SSP 2% outdoor and with using yellow subsoil 50% + burned soil 48% + SSP 2% outdoor for Eucalyptus dunnii seeds. The result shows that when using flat pad filter as germination bed indoor, the germination rate is the same as that in laboratory. The germination is only a few days after Seeds are sown. Germination time is the just the same. And if the seedlings are transplanted into container bags in time, it is conducive to the growth of seedlings. Compared using yellow subsoil 98% + SSP 2% as germination bed, the average germination rate with using flat pad filter as germination bed indoor increases by 37.58%.And compared in the subsoil 50% + burned soil 48% + SSP 2%, 24.88 % increases. The average transplant survival rate is 63.3%, when the shoots used are cultured in the flat filter pad. And the average transplant survival rate is only 32.7%, when the shoots used are cultured in the yellow subsoil 98% + SSP 2%. While the average transplant survival rate is only 37.7%, when the shoots used are cultured in the yellow subsoil 50% + burned soil 50% + SSP 2%. After the seedlings cultured indoor on the flat filter pad have been cultivated for 3 months in autumn and winter, they can be accounted for more than 70% at height up to 10 cm, and can be relatively early reforested uphill. It will be able to reduce labor costs from 18% to 47% when transplanting the nurturing container seedlings cultured indoor on the flat filter pad indoor. In the nurserying, after determination of the seed quality, we can accurately calculate and arrange the seed amount.
One way to induct callus is using Eucalyptus dunnii seeds directly as explants. The other way is sowing seeds in the medium, then using the shoots, cotyledons, and roots as explants to induct, after the seeds sprout.The results show that: MS is a suitable basic medium for Eucalyptus dunnii seed germination and growth. MS + KT1.0 mg / L +2,4-D2 .0 mg / L + cysteine 30 mg / L is the ideal medium to induce seeds to dedifferentiate directly into callus. MS+ Ad 2.0mg / L + IAA0.2mg / L is respectively an ideal medium for its shoots to shoot. B5+ 6- BA2.0mg/L + NAA0.2mg / L is the best medium for its seedling roots to dedifferentiate. MS + 6-BA 2.0 mg/L +2,4-D0.2 mg/L is the best medium to induce its seedling’s hypocotyls into buds. B5+Ad2.0 mg/L +IAA0.2mg/L is a better medium to induce its seedling’s hypocotyls into a callus which can differentiate and form a normal shoots.
An investigation was taken to extremely serious damaged Eucalyptus dunnii throughout the northern Fujian after the disaster was taken in 2005 which happens about one time in 20 years. The statistics shows that the damage degree order follows: Pucheng County> Shaowu City> Jianyang City> Shunchang County> Jianou City> Yanping District. Eucalyptus dunnii damaged extent is related to their age. Afforestation in 2004> afforestation in 2003 > afforestation in 2002. And the damage severity is related to the cultivating techniques. If the first fertilizer application was done to Eucalyptus dunnii on May this year, then the second one before August, Eucalyptus dunnii will promote higher growth, improve the shoot lignified level, and not easy to be damaged.If fertilization was postponed to October, the shoots still grow in the winter and have the low lignified level of, then are vulnerable to be damaged. It is proposed that its cultivation in northern Fujian be limited to Jianyang City.
Different materials of the survivors of Eucalyptus dunni by cold damage in 2005 were used as explants to induce. The results show that using the stem with bud and half-lignification (terminal bud following 3 ~ 6-bud), alcohol disinfection for 30 s and mercuric chloride for 10 min to disinfect can make the explants brown at the lowest rate, the germination at the highest rate, the pollution at the middle rate. When MS medium and sucrose consumption of 40 g / L are used, it takes the Eucalyptus dunnii stems with buds 5 days to sprout. And sprouting rate is 78%. Budding index is 2.6. Buds growing for 30 days are as high as 0.7 cm. The effective buds are 79%. And Buds are in a fair condition. The all composite indicators of its growth factors are the best. In selection and deployment of the type and concentration of cytokinin, it is found that when 0.5 mg / L KT and 1.0 mg / L 6 - BA are used, both of them are good. But if KT is used, several growth factors, such as sprouting rate, budding index, bud height and ratio of effective bud, are much better than those when 6– BA is used. By selecting and deploying the auxin types and concentrations, it is found that 0.05 mg / L NAA is the best auxin types and concentrations to induce Eucalyptus dunni buds. On the medium of MS + KT1.0 mg / L + NAA0.05 mg / L +4% sucrose, the buds grow normally. The callus grown at buds base is small. The buds have differentiation ability. And the sprouting rate of buds, the average height and the effective shoots rate are high. MS medium is the best medium for Eucalyptus dunni to subculture. Improved MS + KT0.8 mg / L + NAA1.0 mg / L + cysteine 20 mg / L is the best medium for buds to subculture and proliferation.It is as far as possible to use solar light or illumination of incandescent about 300 Lux as subculture conditions. It is beneficial for Eucalyptus dunni subcultures to grow high and root in future when temperature is adjusted from 26℃to 28℃. When modified MS + KT0.8 mg / L + NAA1.0 mg / L + cysteine 20 mg / L medium is used as a control, then added AC, VH and the organic composition as 1.5 times as that in MS, the test shows that whatever AC concentration is, a lot of callus on the leaves of subculture seedlings are produced, the seedlings are wilting and die at last, and no matter what VH concentration added in is, there is no significant effect on the subculture seedlings growth. But the largest effect on sturdy seedlings is organic ingredients. When organic ingredients are used as 1.5 times as that in MS, the subculture seedlings grow vigorously and have a high degree of half-lignification. It will play a very important role to next phase of rooting culture.
Using cold resistance varieties of Eucalyptus dunni to do the rooting test, the results showed that ABT can effectively induce Eucalyptus dunni bud to root. The rooting number is increasing with concentration varied from 0.5 to 3.0 mg / L. The maximum rooting rate is 78%. When the concentration is more than 1.5 mg / L, the seedlings height is significantly limited. ABT can be used in conjunction with IBA can increase the rooting rate, and the maximum rooting rate is 96%. When ABT concentration used is more than 0.5 mg / L and IBA concentration used is more than 0.05 mg / L, the seedlings will be induced to produce callus, and be affected its high growth. The callus will be limited to produce in its rooting process and the growth of seedlings will be promoted, when GA 0.05mg / L is added in the medium. 1/2MS+ABT1.0～1.5mg/L+IBA0.05mg/L+GA0.05 is the best medium formula for Eucalyptus dunni seedlings to promote rooting. Using two-step rooting method, the callus formation can be inhibited. And root systems can directly grow from the base of seedlings and rooting rate will increase.
Container seedlings in light matrix containers grow much better than that in soil ones. While in various raw material proportions, every growth factor of E. dunni is the best when it is planted in Matrix 6 which ratio of raw materials is bark 20% + hull 30% + sawdust 40%+ peat 28% + fertilizer2%. Its high growth is 23.9 cm,diameter is 0.25 cm, fresh weight on the part is 2.39g, dry weight is 0.65g and survival rate after being transplanted is 100%.
以邓恩桉种子在室内平盘垫滤纸当发芽床,与在室外黄心土98%+过磷酸钙2%的发芽床及黄心土50%+火烧土48%+过磷酸钙2%的发芽床进行比较。结果表明:以室内用平盘垫滤纸作发芽床,其发芽率与实验室发芽率相同。种子播后发芽天数少,发芽整齐,及时移栽于容器袋内,有利于苗木的生长。比在黄心土98%+过磷酸钙2%的发芽床上,平均发芽率提高了37.58%;比在黄心土50%+火烧土48%+过磷酸钙2%的发芽床上,提高了24.88%。在平盘垫滤纸上生长的芽苗平均移栽成活率达63.3%,而在黄心土98%+过磷酸钙2%发芽床的芽苗平均移栽成活率只有32.7%,在黄心土50%+火烧土48%+过磷酸钙2%发芽床的芽苗平均移栽成活率为37.7%。以室内用平盘垫滤纸作发芽床培育的苗木,在秋冬季培育3个月,苗高达10 cm以上的占70%以上,可以比较早出圃上山造林。在室内用平盘垫滤纸作发芽床播种移栽培育容器苗,可节省费用和用工18%～47%。在工厂化育苗中,在测定了种子的品质以后,就可准确地计算和安排种子用量。
以邓恩桉的种子作为外植体,进行愈伤组织的直接诱导;将种子播在培养基上萌芽,再将芽、子叶、下胚轴和根作为外植体进行诱导。结果表明:MS培养基是较适合邓恩桉种子萌芽和生长的基本培养基。MS+KT1.0 mg/L+2,4-D2.0 mg/L+半胱氨酸30 mg/L是诱导种子直接脱分化成愈伤组织的理想培养基配方。邓恩桉实生苗的芽来繁芽的较理想培养基配方为MS+ Ad 2.0mg/L+IAA0.2mg/L。邓恩桉实生苗根脱分化的最佳培养基配方为B5+6-BA2.0mg/L+NAA0.2mg/L。邓恩桉实生苗下胚轴诱导芽分化的最佳培养基配方是MS+6-BA2.0mg/L +2,4-D0.2mg/L。B5+Ad2.0mg/L+IAA0.2mg/L培养基是诱导邓恩桉下胚轴脱分化为可形成正常芽苗愈伤组织的较佳培养基配方。
对经2005年遭20年一遇的灾害性极端严重冻害的闽北各地邓恩桉进行调查。统计数据显示,冻害程度依次顺序为:浦城县>邵武市>建阳市>顺昌县>建瓯市>延平区。邓恩桉的冻害程度与其林龄有关,2004年造>2003年造>2002年造。另外冻害的严重程度与栽培技术措施有关,当年5月份对邓恩桉施第一次肥,8月之前施第二次肥,则可促进高生长,提高梢部的木质化水平,不易遭受冻害;而若施肥推迟到10月,则到了冬季梢部还在生长,木质化程度低,则易受冻害影响。提出邓恩桉在闽北地区种植的北限应为建阳市。
以2005年遭冻害幸存的邓恩桉抗寒品种的不同材料作为外植体进行诱导,结果显示:采用半木质化的带芽茎段(即顶芽以下第3～6个芽位),酒精消毒30 s、升汞消毒10 min的方法来消毒,可以使外植体的褐化率最低,萌芽率最高,污染率中等。采用MS基本培养基、蔗糖用量为40g/L,邓恩桉芽茎段出芽时间为5d,出芽率为78%,出芽指数为2.6,30d芽均高为0.7cm,有效芽为79%,芽的长势正常。其所有的生长因子的综合指标均为最佳。通过细胞分裂素种类和浓度的选择和调配,在KT浓度采用0.5 mg/L与6-BA浓度采用1.0 mg/L二者均为较佳的种类和浓度进行比较,采用KT,无论是出芽率、出芽指数、芽均高、有效芽的比率等几个生长因子均比采用6-BA更佳。通过生长素种类和浓度的选择和调配,发现采用NAA 0.05 mg/L是诱导邓恩桉芽茎段的较佳生长素种类和浓度。在MS+KT1.0 mg/L+NAA0.05 mg/L+4%蔗糖培养基上,芽的长势正常,芽基部的愈伤组织较小,邓恩桉芽的诱导分化能力强,出芽率和芽均高以及有效芽比率高。通过不同的光处理试验表明:邓恩桉的芽茎段在接种后,应对外植体进行暗培养10d,可以在降低外植体褐化率的同时,提高邓恩桉的出芽率。邓恩桉继代的最佳基本培养基是改良MS培养基。改良MS+KT0.8 mg/L+NAA1.0 mg/L+半胱氨酸20 mg/L对邓恩桉芽的继代增殖影响最大,效果最好。继代培养条件中尽量采用太阳光照或照度大约为300LUX的白炽灯光照,温度调整在26℃～28℃,对邓恩桉的继代苗高生长和今后的生根培养均有利。在以改良MS+KT0.8 mg/L+NAA1.0 mg/L+半胱氨酸20 mg/L培养基为对照,加入AC、VH和1.5倍的有机成分,试验证明:无论AC采用何种浓度,邓恩桉的继代苗叶片上均产生大量愈伤组织,最后苗均萎蔫死亡;无论VH添加哪种浓度,对邓恩桉继代苗生长没有明显的影响。而对邓恩桉壮苗的影响最大的是有机成分,当其用量为1.5倍时,继代苗生长健壮,半木质化程度高,对下一步的生根培养具有很重要的作用。
将邓恩桉抗寒品种进行生根试验,结果表明:ABT生根粉能有效诱导邓恩桉组培苗生根,在0.5～3.0mg/L范围内,生根数随处理浓度的增加而增加,生根率最高为78%,其浓度超过1.5mg/L时,明显抑制组培苗的长高。ABT与IBA配合使用可提高根的诱导率,其生根率最高为96%,当ABT浓度超过0.5mg/L时,IBA浓度超过0.05mg/L时,诱导组培苗产生愈伤组织,并影响苗的长高。在培养基中添加GA0.05mg/L,可以明显抑制生根进程中愈伤组织的产生,并促进苗的生长。1/2MS+ABT1.0～1.5mg/L+IBA0.05mg/L+GA0.05是促进邓恩桉组培苗生根的最佳培养基配方。采用二步生根法,可以明显抑制愈伤组织的形成,根系直接从茎段基部长出,生根率提高。
邓恩桉组培苗的下地移栽使用轻基质网袋容器育苗比有土栽培效果好。而在轻基质各种原料配比中,以在6号的基质原料配比(树皮20%+谷壳30%+木屑40%+泥炭土28%+复合肥2%)中,邓恩桉组培苗的各生长因子为最佳,其高生长为23.9 cm,地径为0.25 cm,地上部份鲜重为2.39g,干重为0.65g,移栽成活率为100%。
采用2005年遭冻害幸存的邓恩桉抗寒品种的组培苗造林,经过2008年初特大冰雪灾害考验的、1年多生的组培苗的抗寒能力比同龄的实生苗强,冻害等级只有Ⅰ级,冻死株数<10%。
Study on hardy Eucalyptus is a new research starting point in China. Eucalyptus dunnii seed source and family selection have been made some progress, but its seeds have been relied on imports. Because Eucalyptus dunnii flowering and fruitification (origin) are so poor that there are no homegrown seeds for afforestation. Seed import from Australia is too expensive to be promoted in the production of the species. In this paper, a new technological research will be be introduced that the Eucalyptus seeds from Australia have been used to nurture its container seedlings and been used as explants to induce organ and callus. A survey how Eucalyptus dunnii planting was damaged on the northern six counties (cities) in Fujian is conducted. And different materials from the survivors of the cold hardiness have been used as explants so as to consider which factor is the most important to establish a stable and effect system of Eucalyptus dunnii rapid propagation. By this way, it can provide not only a technical support to produce a high degree of quality and cold hardiness of Eucalyptus dunnii but also a technology platform for genetic engineering and molecular breeding. Its main findings are as follows.
It is compared using flat pad filter as germination bed indoor with using yellow subsoil 98% + SSP 2% outdoor and with using yellow subsoil 50% + burned soil 48% + SSP 2% outdoor for Eucalyptus dunnii seeds. The result shows that when using flat pad filter as germination bed indoor, the germination rate is the same as that in laboratory. The germination is only a few days after Seeds are sown. Germination time is the just the same. And if the seedlings are transplanted into container bags in time, it is conducive to the growth of seedlings. Compared using yellow subsoil 98% + SSP 2% as germination bed, the average germination rate with using flat pad filter as germination bed indoor increases by 37.58%.And compared in the subsoil 50% + burned soil 48% + SSP 2%, 24.88 % increases. The average transplant survival rate is 63.3%, when the shoots used are cultured in the flat filter pad. And the average transplant survival rate is only 32.7%, when the shoots used are cultured in the yellow subsoil 98% + SSP 2%. While the average transplant survival rate is only 37.7%, when the shoots used are cultured in the yellow subsoil 50% + burned soil 50% + SSP 2%. After the seedlings cultured indoor on the flat filter pad have been cultivated for 3 months in autumn and winter, they can be accounted for more than 70% at height up to 10 cm, and can be relatively early reforested uphill. It will be able to reduce labor costs from 18% to 47% when transplanting the nurturing container seedlings cultured indoor on the flat filter pad indoor. In the nurserying, after determination of the seed quality, we can accurately calculate and arrange the seed amount.
One way to induct callus is using Eucalyptus dunnii seeds directly as explants. The other way is sowing seeds in the medium, then using the shoots, cotyledons, and roots as explants to induct, after the seeds sprout.The results show that: MS is a suitable basic medium for Eucalyptus dunnii seed germination and growth. MS + KT1.0 mg / L +2,4-D2 .0 mg / L + cysteine 30 mg / L is the ideal medium to induce seeds to dedifferentiate directly into callus. MS+ Ad 2.0mg / L + IAA0.2mg / L is respectively an ideal medium for its shoots to shoot. B5+ 6- BA2.0mg/L + NAA0.2mg / L is the best medium for its seedling roots to dedifferentiate. MS + 6-BA 2.0 mg/L +2,4-D0.2 mg/L is the best medium to induce its seedling’s hypocotyls into buds. B5+Ad2.0 mg/L +IAA0.2mg/L is a better medium to induce its seedling’s hypocotyls into a callus which can differentiate and form a normal shoots.
An investigation was taken to extremely serious damaged Eucalyptus dunnii throughout the northern Fujian after the disaster was taken in 2005 which happens about one time in 20 years. The statistics shows that the damage degree order follows: Pucheng County> Shaowu City> Jianyang City> Shunchang County> Jianou City> Yanping District. Eucalyptus dunnii damaged extent is related to their age. Afforestation in 2004> afforestation in 2003 > afforestation in 2002. And the damage severity is related to the cultivating techniques. If the first fertilizer application was done to Eucalyptus dunnii on May this year, then the second one before August, Eucalyptus dunnii will promote higher growth, improve the shoot lignified level, and not easy to be damaged.If fertilization was postponed to October, the shoots still grow in the winter and have the low lignified level of, then are vulnerable to be damaged. It is proposed that its cultivation in northern Fujian be limited to Jianyang City.
Different materials of the survivors of Eucalyptus dunni by cold damage in 2005 were used as explants to induce. The results show that using the stem with bud and half-lignification (terminal bud following 3 ~ 6-bud), alcohol disinfection for 30 s and mercuric chloride for 10 min to disinfect can make the explants brown at the lowest rate, the germination at the highest rate, the pollution at the middle rate. When MS medium and sucrose consumption of 40 g / L are used, it takes the Eucalyptus dunnii stems with buds 5 days to sprout. And sprouting rate is 78%. Budding index is 2.6. Buds growing for 30 days are as high as 0.7 cm. The effective buds are 79%. And Buds are in a fair condition. The all composite indicators of its growth factors are the best. In selection and deployment of the type and concentration of cytokinin, it is found that when 0.5 mg / L KT and 1.0 mg / L 6 - BA are used, both of them are good. But if KT is used, several growth factors, such as sprouting rate, budding index, bud height and ratio of effective bud, are much better than those when 6– BA is used. By selecting and deploying the auxin types and concentrations, it is found that 0.05 mg / L NAA is the best auxin types and concentrations to induce Eucalyptus dunni buds. On the medium of MS + KT1.0 mg / L + NAA0.05 mg / L +4% sucrose, the buds grow normally. The callus grown at buds base is small. The buds have differentiation ability. And the sprouting rate of buds, the average height and the effective shoots rate are high. MS medium is the best medium for Eucalyptus dunni to subculture. Improved MS + KT0.8 mg / L + NAA1.0 mg / L + cysteine 20 mg / L is the best medium for buds to subculture and proliferation.It is as far as possible to use solar light or illumination of incandescent about 300 Lux as subculture conditions. It is beneficial for Eucalyptus dunni subcultures to grow high and root in future when temperature is adjusted from 26℃to 28℃. When modified MS + KT0.8 mg / L + NAA1.0 mg / L + cysteine 20 mg / L medium is used as a control, then added AC, VH and the organic composition as 1.5 times as that in MS, the test shows that whatever AC concentration is, a lot of callus on the leaves of subculture seedlings are produced, the seedlings are wilting and die at last, and no matter what VH concentration added in is, there is no significant effect on the subculture seedlings growth. But the largest effect on sturdy seedlings is organic ingredients. When organic ingredients are used as 1.5 times as that in MS, the subculture seedlings grow vigorously and have a high degree of half-lignification. It will play a very important role to next phase of rooting culture.
Using cold resistance varieties of Eucalyptus dunni to do the rooting test, the results showed that ABT can effectively induce Eucalyptus dunni bud to root. The rooting number is increasing with concentration varied from 0.5 to 3.0 mg / L. The maximum rooting rate is 78%. When the concentration is more than 1.5 mg / L, the seedlings height is significantly limited. ABT can be used in conjunction with IBA can increase the rooting rate, and the maximum rooting rate is 96%. When ABT concentration used is more than 0.5 mg / L and IBA concentration used is more than 0.05 mg / L, the seedlings will be induced to produce callus, and be affected its high growth. The callus will be limited to produce in its rooting process and the growth of seedlings will be promoted, when GA 0.05mg / L is added in the medium. 1/2MS+ABT1.0～1.5mg/L+IBA0.05mg/L+GA0.05 is the best medium formula for Eucalyptus dunni seedlings to promote rooting. Using two-step rooting method, the callus formation can be inhibited. And root systems can directly grow from the base of seedlings and rooting rate will increase.
Container seedlings in light matrix containers grow much better than that in soil ones. While in various raw material proportions, every growth factor of E. dunni is the best when it is planted in Matrix 6 which ratio of raw materials is bark 20% + hull 30% + sawdust 40%+ peat 28% + fertilizer2%. Its high growth is 23.9 cm,diameter is 0.25 cm, fresh weight on the part is 2.39g, dry weight is 0.65g and survival rate after being transplanted is 100%.
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