Kumar Surender, Thakur Aayushee, Sharma Munish, Thakur Neha, Kumar Sanjeev, Singh Anupama
Department of Biotechnology, College of Horticulture, Dr. Y. S. Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India.
Department of Plant Sciences, School of Life Sciences, Central University of Himachal Pradesh, Shahpur, Kangra, Himachal Pradesh, India.
J Genet Eng Biotechnol. 2025 Jun;23(2):100498. doi: 10.1016/j.jgeb.2025.100498. Epub 2025 May 9.
Bell pepper is an important vegetable crop from Solanaceae family, which is cultivated and consumed globally due to its delicious, pleasant and nutrient-rich nature. Most of the commercially cultivated bell pepper cultivars are however, highly susceptible to P. capsici. They are recalcitrance to in vitro regeneration, which make the crop improvement efforts difficult through plant tissue culture-based approaches such as double haploid production. In this study, in vitro callus regeneration protocol was established from anthers of two Phytophthora resistant-backcross lines derived from crossing of two highly susceptible bell pepper cultivars (California Wonder and Solan Bharpur) with a highly resistant chilli landrace, CM334.The cultures were initially established in full strength MS medium, supplemented with different concentration of auxin and cytokinin. Maximum callus induction was achieved in MS medium CI2 supplemented with IAA (2 mg/L) and BAP (0.3 mg/L) (41.59 %) at pH 5.8, without heat-shock and cold stress treatments. The light-green and white compact calli were obtained which proliferated and maintained in the half strength MS medium containing 0.1 mg/L Kinetin (pH 5.8). The presence of Phytophthora resistance loci and successful crossings were confirmed through PCR amplification with the closely linked (RGA-C) and pungency -specific (Pun2) molecular markers, and phenotypic evaluations against P. capsici. The haploid nature and genetic stability were revealed through squash staining with 2 % acetocarmin. This study standardized the conditions appropriate for double haploid culture and regeneration of whole plants to speed up the development of Phytophthora-resistant bell pepper varieties.
甜椒是茄科的一种重要蔬菜作物，因其美味、宜人且营养丰富而在全球范围内种植和食用。然而，大多数商业种植的甜椒品种对辣椒疫霉高度敏感。它们对离体再生具有抗性，这使得通过基于植物组织培养的方法（如双单倍体生产）进行作物改良变得困难。在本研究中，从两个高度感病的甜椒品种（加利福尼亚奇迹和茄兰布尔布尔）与一个高抗辣椒地方品种CM334杂交得到的两个抗辣椒疫霉回交系的花药中建立了离体愈伤组织再生体系。培养物最初在添加了不同浓度生长素和细胞分裂素的完全强度MS培养基中建立。在添加了IAA（2mg/L）和BAP（0.3mg/L）的MS培养基CI2中，pH值为5.8，无需热激和冷胁迫处理，实现了最大愈伤组织诱导率（41.59%）。获得了浅绿色和白色紧实愈伤组织，它们在含有0.1mg/L激动素（pH值5.8）的半强度MS培养基中增殖并维持。通过使用紧密连锁的（RGA-C）和辣味特异性（Pun2）分子标记进行PCR扩增以及对辣椒疫霉的表型评价，确认了辣椒疫霉抗性位点的存在和成功杂交。通过2%醋酸洋红压片染色揭示了单倍体性质和遗传稳定性。本研究规范了适合双单倍体培养和全株再生的条件，以加速抗辣椒疫霉甜椒品种的培育。
