Research Progress on Tissue Culture and Genetic Transformation of Kenaf (Hibiscus cannabinus)

Xia An; Guanrong Jin; Jingyu Zhang; GuangYing Ma; LunjinDai; Liang Jin; Xiahong Luo; Changli Chen; Xiaohua Shi; Jun Zhou; Wei Wei; Wenlue Li; Cong Chen; Gang Deng; Guanlin Zhu

doi:10.1515/biol-2017-0055

Article Open Access

Research Progress on Tissue Culture and Genetic Transformation of Kenaf (Hibiscus cannabinus)

Xia An , Guanrong Jin , Jingyu Zhang , GuangYing Ma , LunjinDai , Liang Jin , Xiahong Luo , Changli Chen , Xiaohua Shi , Jun Zhou , Wei Wei , Wenlue Li , Cong Chen , Gang Deng and Guanlin Zhu

Published/Copyright: December 29, 2017

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From the journal Open Life Sciences Volume 12 Issue 1

Abstract

Recent research progresses on tissue culture (e.g. fast reproduction, another culture, protoplast culture and organogenesis) and genetic transformation of kenaf were reviewed and summarized in this paper. Existing problems were discussed, aiming to provide scientific references for promoting tissue culture and genetic transformation of kenaf.

Keywords: bast fiber crops; kenaf; genetic engineering; genetic transformation

1 Introduction

Kenaf (Hibiscus cannabinus), or known as ambary, belongs to annual economic crops of Hibiscus L., Malvaceae. Kenaf originates in Africa and owns extensive distribution in the world, mainly concentrating in tropical and subtropical zones, Asia and Latin America. Kenaf is characteristic of short growth cycle, tall strong plants, high stress resistance, soft bast fiber, strong tensile force of fiber, high fiber yield, good hygroscopicity and quick water dispersal. Biological yield of kenaf is about 3-4 times that of forest and CO₂ assimilation capacity is about 4-5 times that of trees [1]. Full-pole reproduced pulp quality is equal to that prepared by broadleaf forest. It is widely accepted as a new raw material for papermaking and is highly appreciated in developed countries. Wood pulp has lost advantages in papermaking due to massive deforestation behaviors and intensifying environmental problems (e.g. global warming and desertization) since 1990s. Kenaf is viewed as high-quality papermaking raw materials in view of fast growth, high yield, suitable growth in river and lake bottomlands and compatibility with grains and cottons. Rapid development of kenaf production is of important significance to protect forest resources and develop national economy. In addition, kenaf is an important industrial crop of traditional hemp spinning industry. With good bacteriostasis, ventilation and moisture retention as well as quick water dispersal and degradation of natural fiber, fibrous materials of kenaf are widely applied in R&D and production of automobile lining, plastics, agricultural paper films, light plate material, fluff pulp, purification material of sewage, soil conditioner, plastic filler, active carbon and environmental-friendly adsorption materials in recently years except for traditional products (e.g. hemp rope, hemp bag, geotechnical cloth, carpet backing, textile wall covering, canvas and curtain cloth). Kenaf is titled as the “potential dominant crop of the 21^st century” and “futurist crop” [1].

Great changes on production of bast fiber crop and its scientific research have taken place. These are consequences of extensive use of chemical fiber materials since 1980s. With economic development, people’s consciousness of environmental protection is enhanced. They put forward higher and higher requirements on quality of life. Chemical fiber materials which are widely used nowadays can’t meet people’s requirements on environmental protection and quality of life, thus bringing traditional hemp products to the public again. Additionally, kenaf products are environmental-friendly. For example, the Japanese Dongli wallpaper made of kenaf uses aqueous solvent which is safe to human health. Compared to other wallpapers in the market, Japanese Dongli wallpaper contains no harmful volatile organic compounds like methylbenzene and xylene. Kenaf wallpaper is made of front materials of nontimber materials and recycled paper, so it won’t produce waste gas at combustion. Moreover, it is degradable in soil. Therefore, developing kenaf production can be used as one of countermeasures to protect forest resources and prevent air pollution and global warming. In China’s foreign trade, there are no quota restrictions on export of textile products which contain over 50% fiber. Textile export can be increased by hemp cotton and hemp-fiber blended products. According to statistics of FAO, the global demands for natural fiber are increasing at an annual rate of 8% and domestic demands are increasing at an annual rate of 15%. Developing kenaf production is an important way to solve increasing demands of natural fiber. Germplasm innovation of kenaf is a crucial strategy to realize this goal.

Despite of advantages of traditional bast fiber crops, their quality and output still couldn’t meet market demands. Therefore, developing kenaf varieties with high quality and high yield is an effective guarantee to satisfy natural fiber demands and promote harmonious development of agricultural ecology. Plant tissue culture has been developed for over 100 years and has become an indispensable technology in bioscience. It plays an important role in crop breeding and genetic transformation. So far, more than 600 varieties of plants have been regenerated successfully through cell tissue culture [2]. Great progress in tissue culture research (e.g. rapid reproduction, anther culture, protoplast culture and organogenesis) and genetic transformation of kenaf have been achieved. Anther (pollen) and ovary culture are conducive to accelerate isozygoty of crop offsprings, combine multiple properties quickly, shorten breeding process, simplify the breeding procedure and realize rapid propagation of varieties [3,4]. Breeding methods are improved by developing tissue culture technologies. For instance, aneuploid and polyploidy plants are gained through chromosome variation in anther and cell tissue culture, and then used for breeding of crops by chromosome engineering. Distant hybrid plants were acquired by distant hybrid embryo culture, thus enabling to screen new varieties or distant hybrid species of new materials for breeding. Intraspecific and interspecific somatic cell hybrid plants were acquired by protoplast fusion. The expected somatic cell hybrid of new species was gained. Transgenic plant was cultured by using protoplast as the target gene receptor, so the expected transgenesis species were screened. With tissue culture technology, existing conventional breeding technique can be reformed and new breeding technique will be developed. These are conducive to modernization and high-efficiency of crop improvement. Transgenic breeding is the most effective way for new variety breeding and variety improvement. Scholars attach high attention to transgenic technology to improve breeding of kenaf species and resistance to external environmental factors. In this paper, existing research on tissue culturing and genetic transformation of kenaf were reviewed systematically. Research conclusions provide some scientific references for further development of tissue culture and genetic transformation of kenaf.

2 Research progress of kenaf tissue culture

2.1 Fast reproduction of terminal bud and axillary bud

Reproduction which uses terminal bud and axillary bud of plants as the explants is a highly-efficient and a fast method for reproduction of non-toxic seedlings. Since the regeneration of plants does not requirecallus culture, it shortens the expanding reproduction period and reduces mutations in tissue cultures [5]. Srivatanakul et al. [6] inoculated terminal buds of five kenaf varieties (Ev71, SF459, Tainung 1, Tainung 2 and 7N) into MS culture media with different concentrations of 2,4-D (0-2.3μmol/L) and TDZ (0-20μmol/L). They cultivated multiple shoots, accompanied with basically same shooting rates of different test varieties. Ayadi et al. [7] used the axillary bud of Guangdong 743-2 kenaf as explant and induced shooting on the culture media containing MS + BAP (0-2mg/L) + NAA (0-1mg/L) + IBA (0-1mg/L). They reported the highest shooting rate (90.5%) in MS culture media without any hormone and the lowest shooting rate in the culture media with MS + 0. 5mg/L BAP + 1mg/L NAA. However, shooting rate of kenaf wouldn’t increase by adding BAP, NAA or IBA alone. Besides, adding exogenous hormone in MS culture media will cause dwarf of adventitious bud and easy formation of callus. The survival rate of transplanted test-tube plantlet in room temperature reaches as high as 70%. Zapata et al. [8] used terminal buds of Tainung 1, Tainung 2 and EV71 which contained some cotyledons as explants and induced germination of adventitious bud in MS and 1/2MS media with different concentrations of NAA (0, 0.1, 1mg/L) and 6-BA (0, 0.1, 1mg/L). Research results demonstrated that the highest shooting rate of adventitious bud was achieved in MS + 0.1mg/L 6-BA and there’s no evident difference among different genotypes in term of average shooting rates of adventitious bud. The shooting rates of adventitious bud in MS and 1/2MS were basically similar and auxin couldn’t influence shooting rate of kenaf significantly. High-concentration of 6-BA and NAA is disadvantageous to induction of regeneration bud and shooting, but can form abundant callus.

2.2 Protoplast culture and suspension cell culture

Vasil et al. [9] established the suspension system by embryonic callus of pearl millet and separated protoplast with suspension cells. They also achieved protoplast regeneration plants of the grass family for the first time. Subsequently, protoplast separation based on embryonic suspension cells was applied extensively in protoplast culture of grass family. Yan et al. [10] and Rhodes et al. [11] acquired protoplast regeneration plants from wheat and corn, respectively. As the ideal receptor of cell fusion, organelle transplantation and gene transformation, protoplast can form a complete plant through tissue culture. With respect to previous research on protoplast culture of kenaf, Niu et al. [12] gained high-quality protoplast by processing hypocotyledonary axis of kenaf 722 aseptic seedlings with 0.4M mannitol and dissociation in 5.0mg/L cellulase and 5.0mg/L pectinase (pH5.5). KM₈P + 0.5mg/L 2,4-D + 0.5mg/L 6-BA + 0.45M mannitol + 2% saccharose is the optimal medium for protoplast division. Microdroplet culture is superior to liquid superficial culture and agarose plate culture. Wang et al. [13] acquired relatively complete protoplast from 6h treatment of kenaf callus with 0.55M mannitol and 2% cellulase + 0.1% pectinase enzymolysis. The best pH for enzymolysis was 5.8. Besides, Wang et al. [14] gained stable heterozygotic cells by the same method after fusion of flax and kenaf protoplasts in 40% PEG 6000 and 0.3M CaCl₂ solution (containing 9% mannitol, pH 9.5).

2.3 Culture of anther and hybrids embryo

Anther culture refers to the process of changing the development pathway of pollen in synthetic media to prevent the formation of gamete, but allowing to develop full plants through division and differentiation like somatic cells [15]. Many countries have studied anther culture since the successful in vitro culture of haploid plant based on anther of Daturainoxia [16]. As a result, anther culture becomes more and more mature. It can accelerate breeding practice, increase selection effect and save manpower and material resources. Anther culture is a widely used biotechnology in breeding at present. Anther culture changes division method of pollen nucleus selectively and thereby producing callus or embryoid. In this process, different culture conditions and genetic factors can influence induction frequency of pollen plants independently or collaboratively [17]. Chen et al. [18] reported that anther culture of kenaf dramatically amongdifferent varieties of genotype. Researches on anther culture of kenaf started early. Bast Fiber Plant Institute of Chinese Academy of Agricultural Sciences implemented anther culture based on 71-33 and 722 varieties in 1978-1984. It found that the best induction medium for pollen callus is MS + 2,4-D (2mg/L) + KT (1mg/L) + NAA (1mg/L), and frequency of anther callus induction is highly sensitive to material type, year and seasonal factors. During callus culture proliferation and differentiation, adding 300-5000mg lactoalbumin hydrolysate or 0.1% yeast leachate and increasing sucrose concentration to 8% are conducive to anther culture of kenaf. 1/2 MS + 4-6mg/L GA₃ + 500mg /LLH (lactoalbuminhydrolysate) was used as tumour-body differential medium. In hybrid young embryo culture of kenaf, Rhododendron westlandii was used as the male parent, while kenaf722 and Pericarpium citri reticulatae viride 3# were used as the female parent. The young embryo was used for the in vitro culture. MS + KT 0.5mg/L + IAA 1.0mg/L + 2,4-D 0. 4mg/L was the best culture medium. The ovule gained from hybridization of kenaf KB11 as the female parent and roselle 85-122 as the male parent germinated axillary buds in MS (sucrose concentration=10%) + KT 1.0mg/L + IAA 0.2mg/L + GA₃ 0.4mg /L + LH 500mg/L, MS + 6-BA 2.0mg/L + IBA 0.1mg/L +GA₃ 0.4mg/L + LH 500mg/L and MS + NAA 0.2mg/L + IAA 0.1mg/L. Finally, axillary buds were collected for rooting culture into complete plants [1].

2.4 Organogenesis

Plant organogenesis refers to the process that in vitro plant tissues or cells form rootless seedling, root and bud under tissue culture conditions [19-20]. There are two ways for explants to produce regeneration plants through organogenesis: one is to produce adventitious bud directly from explants. The other is to induce callus from explants firstly and then induce regeneration plant from callus.

2.4.1 Direct regeneration of adventitious bud

In researches on genetic transformation of plants, plant regeneration of explants based on induction pathways of adventitious bud is beneficial to maintain excellent agricultural properties of original variety or material. On this basis, the goal of breeding improvement can be realized by controlling one target genes and successful expression of a specific property [21].

Viewed from explants, existing researches have successfully induced adventitious bud from roots, stems, plumular axis, cotyledon and euphylla of different plants. Most induced adventitious bud from cotyledon and euphylla (e.g. soybean [22] and eggplant [23]). Compared to cotyledon, hypocotyledonary axis of soybean can induce more multiple shoots [22]. Cotyledon and euphylla of eggplant are significantly superior to other explants in term of adventitious bud induction [23].

Viewed from the perspective of hormone, TDZ and BA are common hormones for induction of adventitious bud. TDZ is superior to other cytokinin with respect to induction adventitious bud of many plants. It shows highefficiency of cell division activity and plays different roles in induced differentiation from other cytokinins. TDZ can increase endogenous cytokinin level, which is attributed to inhibition of oxidase for cytokinin degradation [24]. TDZ might regulate endogenous hormones in plants. Effects of TDZ on high-efficiency induction of in vitro regeneration of plants has been proved by many plants, such as kiwi fruit [25], apple [26], grape [27], pear [28], soybean [24] and several woody plants [29]. BA is better than other cytokinins in term of micropropagation of several Euphorbiaceae [30,31]. Multiple shoots induced by TDZ will continue to proliferate in the subculture process. This has been observed in several woody plants [29].

Instead of forming callus, direct differentiation and germination of explants is a high-efficiency way of plant regeneration. There are few researches on tissue culture of kenaf. Liu et al. [32] realized direct differentiation of adventitious buds by culturing cotyledon and euphylla of kenaf varieties (917, 722, BG155, F76, GM26 and Iran Begonia caciniata) in MS + TDZ( 3.5mg/L) + NAA (0.1mg/L). Different genotypes have significantly different induction rates of adventitious buds. Among all kenaf varieties, 722, BG155 and F76 had the highest induction rates, while GM26 and Begonia caciniata had relatively lower induction rates, and the 917 presented the lowest induction rate. Moreover, the induction rate of cotyledon is higher than that of euphylla. Both cotyledon and euphylla with poor induction effect produced abundant callus.

2.4.2 Adventitious bud regeneration based on callus

Bast Fiber Plant Institute of Chinese Academy of Agricultural Sciences inoculated cotyledon into spheroplast induction medium MS + 6-BA (2.0-3.0mg/L) and then into MS + 6-BA (2.0mg/L) + IBA (0.1mg/L) + LH (500mg/L) to form tumor body [1], and finally into MS + 6-BA (0.5-1.0mg/L) + IBA (0.1-0.2mg/L) + GA₃ (0.5-1.0mg/L) to develop a complete plant. However, this is a conclusion of early studies. Later, experiments proved it having poor repeatability [5]. No new research on somatic cell regeneration of kenaf has been reported yet. A new somatic cell regeneration technology of kenaf is needed urgently.

3 Research progress on genetic transformation of kenaf

3.1 Agrobacterium-mediated method

Agrobacterium tumefaciens belongs to Agrobacterium, Rhizobiaceae pribram. Agrobacterium is one type of gram negative bacteria and spreads widely in soil [33]. It can infect injured parts of most dicotyledonous under natural conditions and induce crown gall nodule or hairy roots. Agrobacterium which induces crown gall nodule is called Agrobacterium tumefaciens. Agrobacterium tumefaciens contains Ti plasmid. Agrobacterium which induces stem gall is called Agrobacterium rhizogenes. Agrobacterium rhizogenes contains Ri plasmid. Ti plasmid and Ri plasmid not only can induce diseases of plant cells, but also are ideal carriers for genetic engineering. There’s one segment of T-DA on plasmid. Agrobacterium enters into cells by infecting plant injuries and move exogenous genes on T-DNA to plant genomes, thus producing transgenic plants [33].

Srivatanakul et al. [34] explored parameters to upregulate expression in T-DNA zone of kenaf. They found that the best agrobacterium strain was LBA4404, and the optimal expression temperature of the T-DNA zone was 28°C or 25°C. The toxic zone virG/virE and adding TDZ could increase survival rate of terminal bud. The optimal concentration of AS during Agrobacterium co-culture was 200μmol/L. Herath et al. [35] discussed influencing factors of integration and expression of T-DNA zone of kenaf. Results showed that transformation of explants with callus can upregulate transient expression of GUS genes and prolonging preincubate time can increase transient expression of GUS to one threshold, but the transient expression of GUS has no direct relationship with its fixed expression. Xiong [1] summarized the Agrobacterium tumefaciens method of Yong et al. and introduced two target genes (Npt II and GUS) into kenaf EV41 and EV71, achieving transgenic plants. Based on kenaf 7804 and KB11, Zang introduced BT and chitinase-dextranase bivalent genes into kenaf by agrobacterium-mediated method.

3.2 Particle bombardment

Particle bombardment, or known as microprojectile bombardment, is to coat exogenous DNA onto micro gold or tungsten particle surface and then inject microparticles into recipient cells or tissues under high pressure [36]. Exogenous DNA on particles are integrated onto plant chromosome and then expressed. Particle bombardment has been widely used in rice [37,38,39,40], cotton [41], wheat [42,43,44,45], soybean [46,47,48,49] and corn [50,51,52].

Xiong [1] summarized particle bombardment of Yong et al. to transform Npt II and GUS into leaves of kenaf EV41 and EV71. Nex, leaves were cultured in MS + TDZ 0.3-0.5mg/L + NAA 0.1mg/L. Both two kenaf varieties have gained positive plants.

3.3 Pollen tube path way

The main principle of pollen tube path is to transfer exogenous genes by using exogenous DNA into plant embryo sac through germ cells of plants and pollen tube path formed after fertilization. [53-54]. This method makes an effective use of germ cells (e.g. pollen grain, egg cell and ovary) of plants and allows exogenous genes to participate in fertilization process of plants. It avoids regeneration of plant tissues in common transformation methods. Transformation of the fertilization process is the key. Pollen tube path has been used successfully in development and application of cotton [55], rice [56], wheat [57], corn [58], soybean [59], watermelon [60] and other agricultural crops and vegetables.

Qi et al. [61] introduced insect resistant DNA into the receptor kenaf variety - Fuhong 952 by using the pollen tube path. They gained expression of target genes related with Bt insect resistance in the receptor. Molecular verification of four generations of transgenic strains was accomplished by PCR and Southern hybridization technique. Gene segments of Bt inset resistance were detected in all four generations. This reveals that exogenous genes have been integrated into genotypes of kenaf and stable inheritance has been acquired. Cao [62] introduced bar genes with exogenous herbicide resistance into kenaf through the pollen tube path. Influences of different introduction ways (ovary injection method and stigma dripping method) and technological parameters on setting percentage of the current generation as well as rate of emergence and transformation rate of the T1 generation were analyzed. They concluded that introduction way is an important influencing factor. Specifically, the ovary injection method achieves higher setting percentage, but lower rate of emergency compared to the stigma dropping method. Concentration and dose of exogenous DNA influence setting percentage of the current generation and rate of emergence of the T1 generation slightly. Later, PCR and Southern hybridization proved that exogenous genes have been transformed into kenaf plants [63]. Wu et al. [64] transformed the salt-resistance gene SaNHX of Spartina anglica into kenaf plants through the pollen tube path. In indoor and outdoor salt-resistance observations and PCR test of the T1 generation, they found that SaNHX is integrated into kenaf genomes. Xiong [1] summarized the research results of Jin who introduced chitinase genes and Basta-resistance genes into Zhejiang Xiaohongma 1# by stigma dripping method.

3.4 Shoot transformation method

Kojima et al. [65] infected apical meristem and axillary bud of kenaf plants by Agrobacterium LBA4404 containing binary vector pBI-res and M-21 mutant, getting taller positive plants with thicker stems compared to the control group.

4 Problems and prospects

Tissue culture of kenaf starts later than staple crops, such as rice, corn, wheat, cotton and rapeseed. The genetic transformation takes place in the beginning stage.

Although some research progress has been achieved, further explorations are still needed. Research techniques of tissue culture and genetic transformation of other crops shall be learned to further improve technological system.

4.1 Protoplast culture and hybridization of interspecific cells

Protoplast is a good material of cell hybridization and an effective way to realize interspecific cell hybridization. Currently, protoplast culture of kenaf can only get callus, but it is difficult to develop a complete plant. The corresponding technological system hasn’t been established and perfected yet. China’s super hybrid breeding of kenaf has achieved outstanding results. However, fiber quality is hardly improved. Establishing and perfecting the technological system for protoplast culture of mature kenaf can realize hybridization of ramie and kenaf cells, facilitate improvement of fiber quality effectively, and achieve breakthroughs in quality breeding. Although complete ramie plant has been gained from protoplast culture, it has poor stability. No stable and high-efficiency protoplast regeneration system has been established yet. In a word, there’s no universal protoplast culture system for all bast fiber crops. This deserves high attentions from related scientific researches.

4.2 Establishment of the high-efficiency anther culture system

Anther culture provides an effective way for artificial massive production of haploid, which makes regenerated plants into haplobionts. Homozygous and fertile diploid plants can be gained by spontaneous doubling or artificially induced doubling. Hence, people can choose satisfying gene combinations and provide useful breeding materials for further breeding and genetic studies. Although some research on anther culture of kenaf have been reported, no outstanding results were achieved so far. So far, there’s no mature and high-efficiency anther culture system. Existing researches of anther culture are only early trial studies. Due to low start point and insufficient talents, anther culture is underestimated. Therefore, establishing a high-efficiency technological system for anther culture of kenaf is a technical bottleneck that has to be solved urgently.

4.3 Organogenesis

Direct adventitious bud production through somatic cells is a common technique used in tissue culture of kenaf. However, it is still immature. Only few researches concerning regeneration of adventitious bud based on callus are available due to long period and low differentiation rate of callus. There are disagreements on hormones for differentiation of adventitious bud. Moreover, research results have poor experimental repetition. It is necessary to develop a new method with mature system, good repetition and high rate of emergency.

5 Conclusion

Genetic transformation is the most effective mean to gain new excellent varieties. Nevertheless, research on genetic transformation of kenaf is still in the early stage and no obvious breakthrough has been achieved. It still has some disadvantages, such as low transformation efficiency and poor genetic stability. In existing studies, genetic transformation has been achieved through pollen tube path and stem tip infection method, but few involved Agrobacterium-mediated exogenous genetic transformation. Besides, Agrobacterium-mediated genetic transformation, the major way of transgenic crops, is significantly more efficiency than pollen tube path and stem tip infection method. Therefore, deep studies on Agrobacterium-mediated genetic transformation of kenaf are needed. Some outstanding research progresses about Agrobacterium-mediated genetic transformation of ramie have been achieved. The corresponding research methods and influencing factors shall be discussed [65,66]. Furthermore, particle bombardment-mediated genetic transform is another effective way to realize transgenic plants. However, some new genetic transformation methods, such as chloroplast transformation method, silicon carbide fiber-mediated method, ultrasonic-assisted Agrobacterium-mediated transformation, vacuum penetration method and Alginate beads-mediated method, are hardly used. These methods can be tried in future studies to expand genetic transformation ways of kenaf and construct a set of complete, mature and highefficiency regeneration system for transgenic breeding of kenaf.

Acknowledgement

This paper receives financial supports from Youth Talent Program of Zhejiang Academy of Agricultural Sciences (2016R25R08E01), Hangzhou Science and Technology Plan Guidance program (20163501Y79), China Agriculture Research System (CARS-16-S05) and Zhejiang Science and Technology Planning Project (2017C32022).

Conflict of interest: The authors state no conflict of interest.

References

[1] Xiong H.P., Breeding of Bast Fiber Crops, First Edition, Beijing: Agricultural Science and Technology Press China, 2008,10-208Search in Google Scholar

[2] Jiao C.H., Present situation and Countermeasures of agricultural biotechnology development in Hubei Province, Hubei Agricultural Sciences, 2001, 2, 3-5Search in Google Scholar

[3] Yan P.M., The principle of tissue culture applied to crops, Journal of Taiyuan Normal College, 1999, 4, 34-35Search in Google Scholar

[4] Niu Y., Organ culture and protoplast culture of Kenaf, Master’s thesis, Huazhong Agricultural University, 2004Search in Google Scholar

[5] Qin X.C., Qi J.M., Fang P.P., Research progress in tissue culture and genetic transformation of hemp crops, Journal of plant genetic resources, 2012, 13(4), 589-595Search in Google Scholar

[6] Srivatanakul M., Park S.H., Sanders J.R., Salas M.G., Smith R.H., Multiple shoot regeneration of kenaf (Hibiscus cannabinus L.) from a shoot apex culture system, Plant Cell Reports, 2000,19(12), 1165-117010.1007/s002990000256Search in Google Scholar PubMed

[7] Ayadi R., Hamrouni L., Hanana M., Bouzid S., Trifi M., Khouja M.L., In vitro propagation and regeneration of an industrial plant kenaf (Hibiscus cannabinus L.), Industrial Crops and Products, 2011, 33(2), 474-48010.1016/j.indcrop.2010.10.025Search in Google Scholar

[8] Zapata C., Srivatanakul M., Park S.H., Lee B.M., Salas M. G., Smith R.H., Improvements in shoot apex regeneration of two fiber crops: cotton and kenaf, Plant Cell, Tissue and Organ Culture, 1999, 56(3), 185-19110.1023/A:1006238924439Search in Google Scholar

[9] Vasil V., Vasil I.K., Isolation and culture of cereal protoplasts, Theoretical and Applied Genetics, 1980, 56(3), 97-9910.1007/BF00265079Search in Google Scholar PubMed

[10] Yan Q.S., Zhang X.Q., Shi J.B., Li J.M., The green plant regeneration from Protoplasts of barley, 1990Search in Google Scholar

[11] Rhodes C.A., Pierce D.A., Mettler I.J., Mascarenhas D., Detmer J.J., Genetically transformed maize plants from protoplasts, Science, 1988, 240(4849), 204-20710.1126/science.2832947Search in Google Scholar PubMed

[12] Niu Y., Liu H.W., Zhou R.Y., Tian H.Z., Isolation and culture of protoplasts from kenaf Hypocotyls, Hubei Agricultural Sciences, 2006, 45(2), 149-152Search in Google Scholar

[13] Wang L.S., Zhang Z.S., Zheng J.L., Optimization of the conditions for protoplast isolation from Kenaf, Journal of Dalian Institute of Light Industry, 2010, 29(4), 243-247Search in Google Scholar

[14] Wang L.S., Yi J.C., Zhang Z.S., Protoplast fusion of flax and Kenaf, Guangdong Agricultural Sciences, 2010( 4), 191-193Search in Google Scholar

[15] Germanà M.A., Anther culture for haploid and doubled haploid production, Plant Cell, Tissueand Organ Culture, 2011, 104, 283-30010.1007/s11240-010-9852-zSearch in Google Scholar

[16] Guha S., Maheshwari S.C., In vitro production of embryos from anthers of Datura, Nature, 1964, 204, 49710.1038/204497a0Search in Google Scholar

[17] Wang W.G., Gao Y.K., Research progress in Anther Culture, Journal of Mongolia University: Natural Science Edition, 2005, 3(20), 280-284Search in Google Scholar

[18] Chen X.Y., Li S.C., Studies on pollen plant induction in jute, Plant Fiber Science in China, 1985(3), 1-4Search in Google Scholar

[19] Cuo K.R., Plant hormones on induction and regulation of somatic embryogenesis, Genetic, 2000, 22(5), 349-354Search in Google Scholar

[20] Fang J.B., Huang H.W., Li S.H., CPPU symmetric monkey peach fruit development of sugar, effects of acid content change, Journal of Fruit Science, 2002, 19(4), 235-239Search in Google Scholar

[21] Zhou Y., Zhang Z.Q., Zhong W.J., Effects of 3 Growth Regulators on the direct regeneration of adventitious buds in lettuce, Journal of Jilin Agricultural University, 1998, 20(3), 31-34Search in Google Scholar

[22] Kaneda Y., Tabei Y., Nishimura S., Harada K., Akihama T., Kitamura K., Combination of thidiazuron and basal media with low salt concentrations increases the frequency of shoot organogenesis in soybeans [Glycine max (L.) Merr.], Plant Cell Reports, 1997, 17(1), 8-1210.1007/s002990050342Search in Google Scholar PubMed

[23] Magioli C., Rocha A.P.M., De Oliveira D.E., Mansur E., Efficient shoot organogenesis of eggplant (Solanum melongena L.) induced by thidiazuron, Plant Cell Reports, 1998, 17(8), 661-66310.1007/s002990050461Search in Google Scholar PubMed

[24] Hare P.D., Van Staden J., Inhibitory effect of thidiazuron on the activity of cytokinin oxidase isolated from soybean callus, Plant and Cell Physiology, 1994, 35(8), 1121-112510.1093/oxfordjournals.pcp.a078704Search in Google Scholar

[25] Suezawa K., Matsuta N., Omura M., Yamaki S., Plantlet formation from cell suspensions of kiwifruit (Actinidiachinensis Planch. var. chinensis), Scientiahorticulturae, 1988, 37(1-2), 123-12810.1016/0304-4238(88)90155-0Search in Google Scholar

[26] Fasolo F., Zimmerman R.H., Fordham I., Adventitions shoot formation on excised leaves of in vitro grown shoots of apple cultivars, Plant Cell, Tissue and Organ Culture, 1989, 16(2), 75-8710.1007/BF00036516Search in Google Scholar

[27] Matsuta N., Hirabayashi T., Embryogenic cell lines from somatic embryos of grape (Vitisvinifera L.), Plant Cell Reports, 1989, 7(8), 684-68710.1007/BF00272061Search in Google Scholar

[28] Leblay C., Chevreau E., Raboin L., M. Adventitious shoot regeneration from in vitro leaves of several pear cultivars (Pyruscommunis L.), Plant Cell, Tissue and Organ Culture, 1991, 25(2), 99-10510.1007/BF00042180Search in Google Scholar

[29] Huetteman C.A., Preece J.E., Thidiazuron: a potent cytokinin for woody plant tissue culture, Plant Cell, Tissue and Organ Culture, 1993, 33(2), 105-11910.1007/BF01983223Search in Google Scholar

[30] Tideman J., Hawker J.S., In vitro propagation of latex-producing plants, Annals of Botany, 1982, 49(2), 273-27910.1093/oxfordjournals.aob.a086251Search in Google Scholar

[31] Ripley K.P., Preece J.E., Micropropagation of Euphorbia lathyris L, Plant Cell, Tissue and Organ Culture, 1986, 5(3), 213-21810.1007/BF00040132Search in Google Scholar

[32] Liu H.W., Niu Y., Zhou R.Y., Cotyledon and leaf of adventitious buds induced directly in kenaf, Journal of Hunan Agricultural University: Natural Science Edition, 2005, 31(3), 245-248Search in Google Scholar

[33] Guanlin W., Yuejian S., Jie N., Progress and problems of commercialprodution to transgenic plants in China, Scientia Agricultura Sinica, 2006, 39(7), 1328-1335Search in Google Scholar

[34] Srivatanakul M., Park S.H., Salas M.G., Smith R.H., Transformation parameters enhancing T-DNA expression in kenaf (Hibiscus cannabinus), Journal of Plant Physiology, 2001, 158(2), 255-26010.1078/0176-1617-00289Search in Google Scholar

[35] Herath S.P., Suzuki T., Hattori K., Factors influencing Agrobacterium mediated genetic transformation of kenaf, Plant Cell, Tissue and Organ Culture, 2005, 82, 201-20610.1007/s11240-004-7289-ySearch in Google Scholar

[36] Liu Z.J., Shan L., Xu P.L., Li G.C., Wang X.L., Gramineous crop gene gun mediated genetic transformation research progress, Journal of Shenyang Agricultural University, 2001, 32(6), 465-468Search in Google Scholar

[37] Li L.C., Qu R.D., Kochko A., An improved transformation of embryogenic grape cell suspensions, Plant Cell Report, 1993, 12, 250-25510.1007/BF00237129Search in Google Scholar PubMed

[38] Yi Z.L., Cao S.Y., Wang L., Chu C.C., Li X., He S.J., et al., Study on improvement of Agrobacterium transformation frequency of rice, Journal of Genetics and Genomics, 2001, 28(4), 352-358Search in Google Scholar

[39] Tian W.Z., Study on regeneration frequency of calli in Indica Rice, Journal of Genetics and Genomics, 1994, 21(3), 215-221Search in Google Scholar

[40] Ye S.Q., Chu C.C., Cao S.Y., Tang Z.S., Wang L., Zhao S.M., et al., Research on improving the conversion rate of several factors in Rice, Journal of Genetics and Genomics, 2001, 28(10), 933-938Search in Google Scholar

[41] Guo Y.L., Lu X.M., Zhu Q.L., Li M.Y., Pei Y., Transgenic cotton was obtained by bombardment with embryogenic calli, Journal of agricultural biotechnology, 2005, 13(2), 162-166Search in Google Scholar

[42] Shi N.N., He G, Y., Li K.X., Jones H.D., Shewry P.R., Transgenic wheat expressing high quality HMW subunit gene by bombardment, Scientia Agricultura Sinica, 2005, 38(5), 874-881Search in Google Scholar

[43] Zhang Y., Yu X.D., Tang K.X., Xia L.Q., Wheat aphid resistance variation of Arisaema AHA gene obtained by Biolistic Method, Acta Agronomica Sinica, 2012, 38(8), 1538-154310.3724/SP.J.1006.2012.01538Search in Google Scholar

[44] Altpeter F., Vasil V., Srivastava V., Vasil I.K., Integration and expression of the high-molecular-weight glutenin subunit 1Ax1 gene into wheat, Nature biotechnology, 1996, 14(9), 1155-115910.1038/nbt0996-1155Search in Google Scholar PubMed

[45] Blechl A.E., Anderson O.D., Expression of a novel high-molecular-weight glutenin subunit gene in transgenic wheat, Nature biotechnology, 1996, 14(7), 875-87910.1038/nbt0796-875Search in Google Scholar

[46] Wang P., Xu N., Wang G., Zhong Y., Ji J., Wang E.X., et al., Effect of hypertonic treatment on the conversion results of different genotypes of soybean immature cotyledon gene gun, Journal of crops, 2013, 6, 008Search in Google Scholar

[47] McCabe D.E., Swain W.F., Martinell B.J., Christou P., Stable transformation of soybean (Glycine max) by particle acceleration, Nature Biotechnology, 1988, 6, 923-92610.1038/nbt0888-923Search in Google Scholar

[48] Finer J.J., McMullen M.D., Transformation of soybean via particle bombardment of embryogenic suspension culture tissue, In Vitro Cellular & Developmental Biology-Plant, 1991, 27P, 175-18210.1007/BF02632213Search in Google Scholar

[49] Hadi M.Z., McMullen M.D., Finer J.J., Transformation of 12 different plasmids into soybean via particle bombardment, Plant Cell Reports, 1996, 15, 500-50510.1007/BF00232982Search in Google Scholar

[50] Vain P., McMullen M.D., Finer J.J., Osmotic treatment enhances particle bombardment-mediated transient and stable transformation of maize, Plant Cell Reports, 1993, 12, 84-8810.1007/BF00241940Search in Google Scholar

[51] Yin X.J., Weng J, F., Xie C.X., Hao Z, F., Wang H.N., Zhang S.H., et al., The research and application of transgenic technology in Maize, Journal of Crops, 2010, 6, 1-9Search in Google Scholar

[52] Rafiq M., Fatima T., Husnain T., Bashir K., Khan M.A., Riazuddin S., Regeneration and transformation of an elite inbred line of maize (Zea mays L.), with a gene from Bacillus thuringiensis, South African Journal of Botany, 2006, 72(3), 460-46610.1016/j.sajb.2005.12.010Search in Google Scholar

[53] Jian C.P., Li K.M., Ou W.J., Advances in transgenic breeding of pollen tube pathway, Chinese Journal of Tropical Crops, 2012, 33(5), 956-961Search in Google Scholar

[54] Hess D., Investigations on the intra-and interspecific transfer of anthocyanin genes using pollen as vectors, Zeitschrift für Pflanzenphysiologie, 1980, 98(4), 321-33710.1016/S0044-328X(80)80262-5Search in Google Scholar

[55] Huang G., Dong Y., Sun J., Introduction of exogenous DNA into cotton via the pollen-tube pathway with GFP as a reporter, Chinese Science Bulletin, 1999, 44(8), 69810.1007/BF02909705Search in Google Scholar

[56] Luo Z.X., Wu R., A simple method for the transformation of rice via the pollen-tube pathway, Plant Molecular Biology Reporter, 1988, 6(3), 165-17410.1007/BF02669590Search in Google Scholar

[57] Zeng J., Wang D., Wu Y., Zhang J., Zhou W., Zhu X., et al., Transgenic wheat plants obtained with pollen-tube pathway method, Science in China, Series B, Chemistry, life sciences & earth sciences, 1994, 37(3), 319-325Search in Google Scholar

[58] Yang A., Su Q., An L., Liu J., Wu W., Qiu Z., Detection of vector-and selectable marker-free transgenic maize with a linear GFP cassette transformation via the pollen-tube pathway, Journal of biotechnology, 2009, 139(1), 1-510.1016/j.jbiotec.2008.08.012Search in Google Scholar PubMed

[59] Zeng J., Wang D., Wu Y., Zhang J., Zhou W., Zhu, X., et al., Transgenic wheat plants obtained with pollen-tube pathway method, Science in China, Series B, Chemistry, life sciences & earth sciences, 2002, 20(4), 325-334Search in Google Scholar

[60] Chen W.S., Chiu C.C., Liu H.Y., Lee T.L., Cheng J.T., Lin C.C., et al., Gene transfer via pollen-tube pathway for anti-fusarium wilt in watermelon, IUBMB Life, 1998, 46(6), 1201-120910.1080/15216549800204762Search in Google Scholar

[61] Qi J.M., Xu J.T., Lin L.H., Molecular analysis of Bt transgenic kenaf Fuhong 952 generations, Journal of Fujian Agriculture And Forestry University: Natural Science Edition, 2008, 1(37), 77-79Search in Google Scholar

[62] Cao D.J., Li A.Q., Effective methods and parameters of pollen tube method for introducing exogenous herbicide gene into Kenaf, Plant Fiber Sciences in China, 2000, 22(1), 1-5Search in Google Scholar

[63] Cao D.J., Cheng B.J., Lin Y., Wan P., Molecular verification of herbicide resistant transgenic Kenaf, Plant Fiber Sciences in China, 2001, 23(3), 1-4Search in Google Scholar

[64] Wu J.M., Yao Y.F., Lin L.H., Xu J.T., Lin R.X., Chi R.M., et al., The salt tolerance of SaNHX gene into preliminary identification of salt tolerance of Kenaf T1 generation, Plant Fiber Sciences in China, 2010, 32(6), 316-322Search in Google Scholar

[65] Kojima M., Shioiri H., Nogawa M., Nozue M., Matsumoto D., Wada A., et al., In planta transformation of kenaf plants (Hibiscus cannabinus var. aokawa no. 3) by Agrobacterium tumefaciens, Journal of bioscience and bioengineering, 2004, 98(2), 136-13910.1016/S1389-1723(04)70256-XSearch in Google Scholar

[65] An X., Wang B., Liu L., Jiang H., Chen J., Ye S., et al., Agrobacterium-mediated genetic transformation and regeneration of transgenic plants using leaf midribs as explants in ramie [Boehmerianivea (L.) Gaud], Molecular biology reports, 2014, 41(5), 3257-326910.1007/s11033-014-3188-4Search in Google Scholar PubMed

[66] An X., Liao Y., Zhang J., Dai L., Zhang N., Wang B., et al., Overexpression of rice NAC gene SNAC1 in ramie improves drought and salt tolerance, Plant Growth Regulation, 2015, 76(2), 211-22310.1007/s10725-014-9991-zSearch in Google Scholar