The development of genetically transformed plants is an elusive landmark in Cannabis sativa L. breeding. Despite its economic interest, at present, protocols for producing transgenic C. sativa plants are scarce. We studied the ability of hypocotyl, cotyledon and meristem explants from six C. sativa hemp varieties for transgenic plant regeneration. For this, we firstly evaluated in vitro regeneration rates of hypocotyls cultured in medium without plant growth regulators, and cotyledons cultured in medium supplemented with 0.4 mg L⁻¹ of thidiazuron (TDZ) and 0.2 mg L⁻¹ of α-naphthaleneacetic (NAA). Subsequently, the effect of different kanamycin concentrations (50, 100, 200, 500 and 750 mg L⁻¹) on hypocotyl regeneration rate was determined. Finally, we assessed transformation rates after hypocotyl, cotyledon and meristem co-culture with Agrobacterium tumefaciens strain LBA4404 carrying the binary plasmid pBIN19 containing the β-glucuronidase (uidA) reporter gene and the kanamycin resistance neomycin phosphotransferase (nptII) genes. Plant transformation was validated through in vitro culture of regenerating shoots in kanamycin-containing selective regeneration medium, by GUS histochemical assay for uidA expression, and by PCR amplification of uidA and nptII genes. Our results showed that hypocotyls reached a higher regeneration rate (53.3 %) than cotyledons (18.1 %) without Agrobacterium co-culture. On the other hand, 100 mg L⁻¹ kanamycin proved to be the best concentration in terms of regeneration rate (63.3 %) and spontaneous rooting rate of hypocotyl regenerating shoots (12.2 %), which displayed a 7.1 % of albinism rate. After co-culture with A. tumefaciens and subsequent culture in antibiotic-containing selective regeneration medium, hypocotyl was the best explant type achieving 23.1 % of regeneration rate, which contrasts with the 1.0 % regeneration rate detected for cotyledons. Transgenic plants were obtained from all explant types evaluated. Although there were significant differences among varieties evaluated, hypocotyls proved to be superior to already-developed meristems, reaching a transformation rate of 5.0 % and 0.8 % respectively. Despite the extremely low regeneration rate of cotyledons after A. tumefaciens co-culture, all cotyledon-derived regenerating shoots analyzed were successfully transformed. Our hormone-free protocol doubles the transformation rate of regenerating shoots, also producing transgenic plants three times faster than other already published protocols. This has relevant implications for C. sativa breeding, enabling not only genetic transformation, but also the use of new plant breeding techniques such as targeted genome editing by using CRISPR/Cas systems. This may foster the development of C. sativa varieties with specific biochemical profiles, or tolerant to biotic and abiotic stresses among others.

转基因植物的发展是大麻育种中一个难以捉摸的里程碑。尽管具有经济利益，但目前用于生产转基因C. sativa植物的协议很少。我们研究了来自六个C. sativa大麻品种的下胚轴、子叶和分生组织外植体用于转基因植物再生的能力。为此，我们首先评估了在不含植物生长调节剂的培养基中培养的下胚轴和在补充有 0.4 mg L ^-1噻二唑隆 (TDZ) 和 0.2 mg L ^{-1 的}培养基中培养的子叶的体外再生率α-萘乙酸 (NAA)。随后，确定了不同卡那霉素浓度（50、100、200、500和750mg L ^-1）对下胚轴再生速率的影响。最后，我们评估了下胚轴、子叶和分生组织与根癌土壤杆菌菌株 LBA4404共培养后的转化率，该菌株携带含有β-葡萄糖醛酸酶( uidA ) 报告基因和卡那霉素抗性新霉素磷酸转移酶( nptII ) 基因的二元质粒 pBIN19 。通过在含有卡那霉素的选择性再生培养基中体外培养再生芽，通过uidA 的GUS 组织化学测定验证植物转化表达，并通过uidA和nptII基因的PCR 扩增。我们的结果表明，与没有农杆菌共培养的子叶 (18.1%) 相比，下胚轴达到了更高的再生率 (53.3%) 。另一方面，就再生率（63.3 %）和下胚轴再生芽的自发生根率（12.2 %）而言，100 mg L ^-1卡那霉素被证明是最佳浓度，白化病率为7.1%。与根癌农杆菌共培养后和随后在含抗生素的选择性再生培养基中培养，下胚轴是最好的外植体类型，可实现 23.1% 的再生率，这与检测到的 1.0% 的子叶再生率形成对比。从所有评估的外植体类型获得转基因植物。尽管评估的品种之间存在显着差异，但下胚轴被证明优于已经发育的分生组织，转化率分别达到 5.0% 和 0.8%。尽管根癌农杆菌后子叶的再生率极低共培养，所有分析的子叶衍生的再生芽均成功转化。我们的无激素方案使再生芽的转化率加倍，并且生产转基因植物的速度也比其他已发表的方案快三倍。这对C. sativa育种具有相关意义，不仅可以实现遗传转化，还可以使用新的植物育种技术，例如使用 CRISPR/Cas 系统进行靶向基因组编辑。这可能会促进具有特定生化特征或耐受生物和非生物胁迫等的C. sativa品种的发展。