葡萄种质离体保存中低温和矿物油覆盖延长继代间隔的效应

doi:10.16420/j.issn.0513-353x.2020-0304

摘要/Abstract

摘要：

以‘巨峰’等14个葡萄品种试管苗为试材,研究不同保存温度和培养基表面覆盖矿物油对其生长的影响。结果表明:低温可有效延长试管苗的继代间隔时间,转入常温培养后恢复生长良好,但不同品种适宜的温度不同。设定存活率降至30% ~ 40%时的保存时间为最长继代时间,‘红斯威特’‘莫丽莎’‘皇家夏天’‘克瑞森无核’葡萄最适低温9 ℃,可保存850 d以上,部分可至1 060 d;‘巨峰’‘巨玫瑰’‘皇家秋天’‘火焰无核’‘夏黑’‘公主’‘粒粒特’‘赤霞珠’‘美人指’‘魏可’最适低温12 ~ 15 ℃,可保存350 ~ 600 d。葡萄试管苗低温保存前需常规培养一段时间（预培养）,并根据培养温度及品种决定适宜的预培养时间,一般为7 ~ 21 d。接种不带叶片的单芽茎段并立即在培养基表面覆盖6 ~ 8 cm厚矿物油能将多个葡萄品种试管苗在常温下的继代间隔时间由150 d延长至420 d,将矿物油倒出,试管苗即可恢复生长。

关键词: 葡萄, 种质保存, 试管苗, 延缓生长, 继代间隔, 低温, 预培养, 矿物油覆盖

Abstract:

Fourteen grape cultivars were used as plant materials to reveal the influences of low culture temperature and mineral oil covering on medium of plantlets in vitro. The results showed that the low temperature could significantly prolong the subculture period,but the optimal low temperature varied according to the different cultivars. Plantlets cultured in the low temperature could resume growth well after being transferred to routine temperature culture. When the survival rate of 30%-40% was a concern,the optimal temperature was 9 ℃ for‘Sweet Scarlet’,‘Melissa’,‘Summer Royal'and‘Crimson Seedless’cultivars,and the subculture period could reach more than 850 days,and maximum 1 060 days for certain cultivar;the optimal temperature was 12-15 ℃ for‘Kyoho’,‘Jumeigui’,‘Autumn Royal’,‘Flame Seedless’,‘Summer Black’,‘Princess’,‘Lilit’,‘Cabernet Sauvignon’,‘Manicure Finger'and‘Wink’,and the subculture period could last 350 to 600 days. Prior to the low temperature culture,the plantlets needed to be cultured under routine condition for a period of time（preculture）. The duration depended on the culture temperature and the varieties,and it was usually 7 to 21 days. By inoculating shoots with single bud and without leaf,as well as adding 6-8 cm mineral oil on the top of the medium,the subculture period could be up to 420 days in routine temperature culture condition while the control was 150 days. The plantlets resumed growth after the mineral oil was removed.

Key words: grape, germplasm preservation, plantlet in vitro, retard growth, subculture interval, low temperature, preculture, mineral oil covering

中图分类号:

S663.1

成晓华, 徐志微, 王莉, 师校欣, 杜国强. 葡萄种质离体保存中低温和矿物油覆盖延长继代间隔的效应[J]. 园艺学报, 2021, 48(3): 477-486.

CHENG Xiaohua, XU Zhiwei, WANG Li, SHI Xiaoxin, DU Guoqiang. Efficacy of Low Temperature and Mineral Oil Covering in Prolonging Subculture Interval During Grape Germplasm Preservation in Vitro[J]. Acta Horticulturae Sinica, 2021, 48(3): 477-486.

图/表 10

参考文献 17

[1]	Ahmed M, Anjum M A, Shah A H, Hamid A. 2010. In vitro preservation of Pyrus germplasm with minimal growth using different temperature regimes. Pakistan Journal of Botany, 42 (3),1639-1650.
[2]	Bridgen M P, Staby G L. 1981. Low pressure and low oxygen storage of Nicotiana tabacum and Chrysanthemum × morifolium tissue cultures. Plant Science Letters, 22 (2):177-186. doi: 10.1016/0304-4211(81)90140-1 URL
[3]	Caplin S M. 1959. Mineral oil overlay for conservation of plant tissue cultures. American Journal of Botany, 46 (5):324-329. doi: 10.1002/ajb2.1959.46.issue-5 URL
[4]	Engelmann F. 1997. In vitro conservation methods. Biotechnology in Agriculture Series,(19):119-162.
[5]	Hassan N S. 2004. Storage of in vitro banana shoot cultures at low temperature or under mineral oil layer. International Journal of Agriculture & Biology, 6 (2):303-306.
[6]	Liu Yue-xue, Liu Xiao-jun, Wang Jia-fu, Lin Shun-quan. 2004. Effects of low temperature and another condition on Eriobotrya japonica germplasm preservation in vitro. Journal of Plant Resources and Environment,(1):28-31. (in Chinese)
	刘月学, 刘小军, 王家福, 林顺权. 2004. 低温等因素对枇杷种质离体保存的影响. 植物资源与环境学报,(1):28-31.
[7]	Lonardo C S D. 2013. In vitro conservation of chestnut( Castanea sativa)by slow growth. In Vitro Cellular & Developmental Biology-Plant, 49:605-610.
[8]	Moriguchi T, Kozaki I, Matsuta N, Yamaki S. 1988. Plant regeneration from grape callus stored under a combination of low temperature and silicone treatment. Plant Cell Tissue and Organ Culture, 15 (1):67-71. doi: 10.1007/BF00039890 URL
[9]	Niu Ai-guo, Zhang Kai-chun, Zhang Xiao-ming, Yan Guo-hua, Li Wen-sheng, Jiang Li-jie. 2005. Preservation of in vitro grown shoot of cherry germplasm. Acta Horticulturae Sinica, 32 (2):298-300. (in Chinese)
	牛爱国, 张开春, 张晓明, 闫国华, 李文生, 姜立杰. 2005. 樱桃种质资源试管苗保存方法. 园艺学报, 32 (2):298-300.
[10]	Ozudogru E. A, Benelli C, Dradi G, Lambardi M. 2017. Effect of culture container and carbohydrate content on in vitro slow growth storage of the cherry rootstock‘Gisela^® 5’. Acta Physiologiae Plantarum, 39 (4):94. doi: 10.1007/s11738-017-2372-2 URL
[11]	Pan X J, Zhang W E, Li X. 2014. In vitro conservation of native Chinese wild grape( Vitis heyneana Roem. & Schult)by slow growth culture. Vitis, 53 (4):207-214.
[12]	Sharma N, Satsangi R, Pandey R, Singh R, Kaushik N, Tyagi R K. 2012. In vitro conservation of Bacopa monnieri(L.)using mineral oil. Plant Cell Tissue and Organ Culture, 111 (3):291-301. doi: 10.1007/s11240-012-0194-x URL
[13]	Sota V, Kongjika E. 2014. Slow growth in vitro conservation of Zizyphus jujuba Mill. Agriculture & Forestry, 60 (1):27-37.
[14]	Wang Chen. 2008. Studies on growth characteristics and rhythm regulation of apple plantlets repeatedly subcultured in vitro[M. D. Dissertation]. Baoding:Agricultural University of Hebei. (in Chinese)
	王晨. 2008. 苹果组培苗多代保存过程中生长特性及生长节奏调控技术研究[硕士论文]. 保定:河北农业大学.
[15]	Wu Yong-jie, Li Yu-sheng, Wu Ya-qin, Chen Long, Cheng He-he, Zhao Yan-hua. 2019. Advances in conservation of plant germplasm resources. Hebei Fruits,(1):1-3. (in Chinese)
	吴永杰, 李玉生, 吴雅琴, 陈龙, 程和禾, 赵艳华. 2019. 植物种质资源保存研究进展. 河北果树,(1):1-3.
[16]	Xu Gang-biao. 2000. Plant germplasm conservation in vitro. Journal of Central South Forestry University,(4):81-87. (in Chinese)
	徐刚标. 2000. 植物种质资源离体保存研究进展. 中南林学院学报,(4):81-87.
[17]	Xu Zhi-wei, Yang Li-li, Du Guo-qiang, Shi Xiao-xin. 2014. Efficacy of osmotic pressure and plant growth regulators in media on conservation of grape plantlets in vitro. Molecular Plant Breeding, 12 (4):720-725. (in Chinese)
	徐志微, 杨丽丽, 杜国强, 师校欣. 2014. 培养基渗透压和生长调节剂对葡萄种质离体保存的效应. 分子植物育种, 12 (4):720-725.

温度/℃ Tempera- ture	红斯威特 Sweet Scarlet	莫丽莎 Melissa	皇家夏天 Summer Royal	克瑞森无核 Crimson Seedless	巨峰 Kyoho	巨玫瑰 Jumeigui	皇家秋天 Autumn Royal	火焰无核 Flame Seedless	夏黑 Summer Black	粒粒特 Lilit	公主 Princess	赤霞珠 Cabernet Sauvignon	美人指 Manicure Finger	魏可 Wink
9	1 060	1 060	850	850	510	450	270	380	270	330	280	350	180	170
12	650	650	650	650	550	600	510	550	500	460	430	370	260	300
15	330	330	320	400	390	330	390	340	400	360	340	390	350	360
25	140	140	140	160	150	140	150	150	130	130	160	150	140	140

温度/℃ Tempera- ture	红斯威特 Sweet Scarlet	莫丽莎 Melissa	皇家夏天 Summer Royal	克瑞森无核 Crimson Seedless	巨峰 Kyoho	巨玫瑰 Jumeigui	皇家秋天 Autumn Royal	火焰无核 Flame Seedless	夏黑 Summer Black	粒粒特 Lilit	公主 Princess	赤霞珠 Cabernet Sauvignon	美人指 Manicure Finger	魏可 Wink
9	1 060	1 060	850	850	510	450	270	380	270	330	280	350	180	170
12	650	650	650	650	550	600	510	550	500	460	430	370	260	300
15	330	330	320	400	390	330	390	340	400	360	340	390	350	360
25	140	140	140	160	150	140	150	150	130	130	160	150	140	140

保存天数 Preserved days	红斯威特 Sweet Scarlet			莫丽莎 Melissa			克瑞森无核 Crimson Seedless
保存天数 Preserved days	株高/cm Plantlet height	叶片数 Leaf number	叶面积/cm² Leaf area	株高/cm Plantlet height	叶片数 Leaf number	叶面积/cm² Leaf area	株高/cm Plantlet height	叶片数 Leaf number	叶面积/cm² Leaf area
238	4.38 ± 0.33 a	3.33 ± 0.68 a	2.05 ± 0.41 a	8.20 ± 0.20 a	6.17 ± 0.14 a	1.72 ± 0.22 a	6.75 ± 0.20 a	6.33 ± 0.14 a	1.72 ± 0.05 a
600	3.27 ± 0.32 a	2.87 ± 0.35 a	0.98 ± 0.10 ab	5.53 ± 0.57 b	5.73 ± 0.58 ab	1.40 ± 0.29 a	4.42 ± 0.24 b	5.45 ± 0.18 ab	1.25 ± 0.20 a
843	2.84 ± 0.39 a	2.01 ± 0.44 a	0.65 ± 0.10 b	4.03 ± 0.44 b	3.83 ± 0.59 b	0.80 ± 0.03 a	3.58 ± 0.65 b	3.17 ± 0.49 b	1.05 ± 0.20 a

保存天数 Preserved days	红斯威特 Sweet Scarlet			莫丽莎 Melissa			克瑞森无核 Crimson Seedless
保存天数 Preserved days	株高/cm Plantlet height	叶片数 Leaf number	叶面积/cm² Leaf area	株高/cm Plantlet height	叶片数 Leaf number	叶面积/cm² Leaf area	株高/cm Plantlet height	叶片数 Leaf number	叶面积/cm² Leaf area
238	4.38 ± 0.33 a	3.33 ± 0.68 a	2.05 ± 0.41 a	8.20 ± 0.20 a	6.17 ± 0.14 a	1.72 ± 0.22 a	6.75 ± 0.20 a	6.33 ± 0.14 a	1.72 ± 0.05 a
600	3.27 ± 0.32 a	2.87 ± 0.35 a	0.98 ± 0.10 ab	5.53 ± 0.57 b	5.73 ± 0.58 ab	1.40 ± 0.29 a	4.42 ± 0.24 b	5.45 ± 0.18 ab	1.25 ± 0.20 a
843	2.84 ± 0.39 a	2.01 ± 0.44 a	0.65 ± 0.10 b	4.03 ± 0.44 b	3.83 ± 0.59 b	0.80 ± 0.03 a	3.58 ± 0.65 b	3.17 ± 0.49 b	1.05 ± 0.20 a

处理 Treatment	存活率/% Survival rate	株高/cm Plantlet height	生根率/% Rooting rate	生根数 Number of roots
未加矿物油（对照） Without mineral oil adding（Control）	23.41 b	≥ 8.50 a	100.00 a	3.33 a
接种后覆盖矿物油 Covering mineral oil while inoculation	100.00 a	2.36 b	66.67 b	1.11 b
培养15 d后覆盖矿物油 Covering mineral oil at 15 d after inoculation	0 b	—	100.00 a	3.44 a