Preliminary Studies on the Tissue Culture of Cannabis sativa L.(Industrial hemp)

Ying JIANG,Zunmin XIA,Yan TANG,Qiang HAN,Chengwei HAN

1.Daqing Branch of Heilongjiang Academy of Sciences,Daqing 163319,China;

2.Heilongjiang University,Harbin 150080,China

Responsible editor:Xiaoxue WANG Responsible proofreader:Xiaoyan WU

Cannabis sativa L.is the Cannabinaceae Cannabis and annual herbaceous plant,and Cannabis sativa L.can be divided into industrial hemp and marijuana hemp.Now,industrial hemp is widely used at home and abroad.Specifically,industrial hemp refers to the hemp with content of tetrahydrocannabinol(THC)lower than 0.3%,which called Han-ma(Industrial hemp)in China[1-2].Industrial hemp is full of treasure and has higher application value.For example,indus trial hemp seeds have good medicinal value and commercial value[3].In recent years,people pay more attention to environmental and health concept and have a deep understanding on the product characteristics of industrial hemp with people returning to nature.The countries of the world pay more and more attention to the use and development of industrial hemp resources.

At present,most domestic hemp varieties are farm varieties,mixed and degraded,whose yield of fiber is low,and the rate of long fiber is only around 10%.This leads to lower income of farmers and the benefit of raw material processing,which affects the revitalization of hemp industry directly.The technical bottleneck of the development of hemp industry is to spread and plant the new varieties of hemp with high fiber,high resistance,low toxicity,high-quality,and high yield.Breeding of new hemp varieties mainly uses the methods of introduction,selection,hybridization,heterosis,physical mutation and so on.However,some problems still exist,such as long cycle,difficult field selection,insufficient resistant sources and so on.Biological engineering breeding is advanced technology that innovates hemp resources and cultivates highquality varieties.But breeding researches of hemp haploid,polyploidy,and cell engineering are still blank.Therefore,the establishment of hemp efficient regeneration system is of significance for hemp genetic transformation and haploid culture,etc.The establishment of hemp efficient regeneration system also can lay foundation for cultivating the new varieties of hemp with high fiber,high resistance,low toxicity,and high-quality using biological engineering breeding.

Materials and Methods

Seeds of Cannabis sativa L.were obtained from Daqing Branch of Heilongjiang Academy of Sciences:Longma No.1.The test selected ripe,full,and glossy hemp seeds,sterilized in 75% alcohol for 2 min and rinsed in sterile water for 3-4 times.Then they were sterilized in 1‰HgCl2for (S1)0 min,(S2) 3 min,(S3) 5 min,(S4) 8 min,(S5) 10 min,and thoroughly rinsed in sterile water for 5-6 times.Subsequently,the seeds (20 seeds per treatment,3 replications) were inoculated in MS (no hormone) solid medium after soaking in water for 16-24 h under sterile conditions.Seedlings measuring 3-5 cm,grown in a growth chamber at (24±1)℃under a 16 h photoperiod,were counted the situation of germination,pollution and aseptic seedling.Explants of young internodes were implanted in Petri dishes containing MS basal medium supplemented with various concentration of hormones:6-BA,KT and NAA (Table1).The explants (20 explants per treatment,3 replications)were kept in darkness and callus was inducted at 24 ℃for 2-3 weeks.Then the callus (20 callus per treatment,3 replications) was transferred to the same fresh medium and incubated in a growth chamber at 24 ℃under a 16 h photoperiod (1 800-2 300 lux).For root formation,when grew to 2-3 cm,the adventitious buds were cut off from callus and cultured on MS medium supplemented with 1/2MS+ 0.1 mg/L IBA+0.05 mg/L NAA[4].

Table1 Four combinations of 6-BA,KT and NAA in MS medium used for callus formation and adventitious bud differentiation of Cannabis sativa L

Table2 Effect of 1‰HgCl2 with different treatment time on hemp seed disinfection

Results

The study used 1‰ HgCl2as disinfectant,combined with 75% ethanol,followed by disinfection of hemp seeds.The results showed that the seedling rate reached 83.33% when hemp seeds were sterilized in 75%alcohol for 2 min and sterilized in 1‰HgCl2for 3min.But the growth of seeds would be polluted by this step that would influence the progress of test.And the seedling rate reached 70.04% when hemp seeds were sterilized in 75%alcohol for 2 min and sterilized in 1‰ HgCl2for 5min,suggesting that this step was relatively good.(Table2) (The germination of seeds is 90.00%±1.00%).

論教育科學(xué)的本性……………………………………………………………………………………………………李 軍（4.63）

When the seeds grew to seedling after 10 days,using the stems as explants,the different hormone combinations would affect the rate of callus induction.The results (Table3)showed that the highest hormone combination of callus production was 1.0 mg/L 6-BA + 0.5 mg/L NAA(88.33%).The callus transferred into MS medium with different plant growth regulator combinations for differentiating adventitious bud.After the adventitious bud cultured in a period of time,the differentiation rate of adventitious bud was compared.Besides,the results (Table4) also showed that the hormone combinations,containing 1.0 mg/L 6-BA + 0.5 mg/L NAA and 1.0 mg/L 6-BA+1.0 mg/L NAA didn’t differentiate,and the highest hormone combination of callus production was 1.0 mg/L KT+0.5 mg/L NAA(65.00%).

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Table3 Effect of different plant growth regulators on the induction of callus

Table4 Effect of different plant growth regulators on differentiation rate of adventitious bud

Discussions

Aseptic seedlings are the key factor in the process of hemp tissue culture.So the selection of appropriate disinfectants and processing time is very important.Not only do that have good sterilizing effect and reduce the rate of pollution,but also that obtain better seedlings[5].HgCl2has strong bactericidal effect,1‰ HgCl2can kill vegetative cells of bacterium within a few minutes.Therefore,hemp seed disinfection used 75% alcohol for 2 min and sterilized in 1‰ HgCl2for 5 min.Medium is the material basis of plant tissue culture.Statistically the medium of hemp regeneration system at home and abroad is MS medium that is the most widely applicative medium[5].So,this study chose MS medium as the basal medium.Although the amount of plant growth regulators is small,but essential.That can regulate the growth and development,differentiation and organogenesis of the culture[6].NAA is a spectral plant growth regulator that can promote cell division and growth,induce the formation of adventitious root and so on.The regeneration condition of the new cultivar Long-ma No.1 of Cannabis sativa L.:1.0 mg/L 6-BA and 0.5 mg/L NAA for the induction of callus,1.0 mg/L KT and 0.5 mg/L NAA for differentiation rate of adventitious bud.

[1]JANICK J,WHIPKEY A.Trends in new crops and new uses [M].Alexandria:ASHS Press,2002.

[2]AVICO U,PACIFICI R,ZUCCARO P.Variations of tetrahydro-cannabinol content in Cannabis plants to distinguish the fiber-type from drugtype plants[J].Bull Narc,1985,37(4):61-65.

[3]JIANG Y,HAN CW,et al.Exploitation and utilization of hemp seeds [J].Heilongjiang Science,2014,5(2):6-7.

[4]TONG JF,GAO N,et al.Optimization of rooting medium for tube seedling of Cannabis sativa L.[J].Journal of Anhui Agri.Sci.2008,36(33):14438-14440.

[5]JIANG Y,HAN CW,et al.The development of studies on the tissue culture of Cannabis sativa L.(industrial hemp)[J].Journal of Anhui Agri.Sci.2014,42(1):7-8.

[6]JIA WQ.Optimization of flax efficient regeneration system and bar genetic transformation[C].2011.1-4.